The Ministry of Health, Labour and Welfare Ministerial Notiˆcation No. 461

In accordance with the provisions of Article 41, Paragraph 1 of the Pharmaceutical AŠairs Law (Law No. 145, 1960), we hereby revise a part of the Japanese Phar- macopoeia (Ministerial Notiˆcation No. 111, 2001) as follows, and the revised Japanese Pharmacopoeia shall come into eŠect on January 1, 2005, [including dele- tion from O‹cial Monographs fro Part II in The Japanese Pharmacopoeia, Four- teenth Edition of the articles of Absorbent Cotton, Puriˆed Absorbent Cotton, Sterile Absorbent Cotton, Sterile Puriˆed Absorbent Cotton and Absorbent Gauze and Sterile Absorbent Gauze (hereinafter referred to as ``sanitary materials'')]. Provi- so: With respect to the drugs which are included in the Japanese Pharmacopoeia (hereinafter referred to as ``the old Japanese Pharmacopoeia'') [limited to those included in the Japanese Pharmacopoeia whose standards are changed with this notiˆcation published (hereinafter referred to as ``the new Japanese Phar- macopoeia'')] and those which are approved as of January 1, 2005 pursuant to the provisions of Article 14, Paragraph 1 of this Law (including cases where it shall apply mutatis mutandis under Article 23 of this Law; the same hereinafter) [including the drugs designated as those exempted from approval (hereinafter referred to as ``the drugs exempted from approval'') among the drugs etc. designated by the Minister of Health, Labour and Welfare as those exempted from manufacturing or import approval pursuant to the provisions of Article 14, Paragraph 1 of the Pharmaceutical AŠairs Law (Ministerial Notiˆcation No. 104, 1994), the standards established in the old Japanese Pharmacopoeia (limited to the standards for the relevant drugs) shall be recognized, up to June 30, 2006, as the standards established in the new Japanese Pharmacopoeia. With respect the drugs which are included in the new Japanese Pharmacopoeia (excluding those which are included in the old Japanese Phar- macopoeia) and those which are approved as of January 1, 2005 pursuant to the provisions of Article 14, Paragraph 1 of this Law (including the drugs exempted from approval), the drugs may be treated, up to June 30, 2006, as those which are not included in the new Japanese Pharmacopoeia. Further, sanitary materials may be treated, up to September 30, 2006, under the previous regulation.

Hidehisa Otsuji The Minister of Health, Labour and Welfare

December 28, 2004

(The texts referred to by the term ``as follows'' are omitted here. All of them are made available for public exhibition at the Evaluation and Licensing Division, Phar- maceutical and Medical Safety Bureau, Ministry of Health, Labour and Welfare, at each Regional Bureau of Health and Welfare and at each Prefectural O‹ce in Japan.)

The term ``as follows'' here indicates the contents from Part I to Ultraviolet-visible Reference SpectraintheSupplementIItotheJapanesePharmacopoeiaFourteenthEdition(pp.1669 – 1866). CONTENTS

Preface ...... i Supplement II to The Japanese Pharmacopoeia, Supplement II to The Japanese Pharmacopoeia, Fourteenth Edition, Part II...... 1755–1778 Fourteenth Edition, Part I...... 1669–1754 General Rules for Crude Drugs ...... 1755 General Tests, Processes and Apparatus ... 1669 O‹cial Monographs ...... 1757 6. Bacterial Endotoxins Test...... 1669 39. Nuclear Magnetic Resonance Infrared Reference Spectra ...... 1779–1789 Spectroscopy ...... 1669 Part I ...... 1779 47. Pyrogen Test ...... 1672 Part II ...... 1789 52. Residue on Ignition Test ...... 1672 53. Speciˆc Surface Area Determination... 1673 Ultraviolet-visible Reference Spectra .... 1791–1799 54. Sterility Test ...... 1673 Part I ...... 1791 70. Reference Standards; Reagents, Test Solu- tions; Standard Solutions for Volumetric General Information Analysis; Standard Solutions; Matching 5. International Harmonization Implemented Fluids for Color; Optical Filters for in the Japanese Pharmacopoeia Fourteenth Wavelength and Transmission Rate Edition ...... 1801 Calibration; and Measuring Instruments, 20. Amino Acid Analysis ...... 1814 Appliances ...... 1677 21. Capillary Electrophoresis...... 1822 (1) Reference Standards ...... 1677 22. Isoelectric Focusing...... 1828 (2) Reagents, Test Solutions...... 1677 23. Peptide Mapping ...... 1830 (3) Standard Solutions for Volumetric 24. Rapid Identiˆcation of Microorganisms Analysis ...... 1685 Based on Molecular Biological 74. Powder Particle Density Method ...... 1833 Determination...... 1685 25. Solid and Particle Densities ...... 1835 O‹cial Monographs ...... 1687 26. Total Protein Assay ...... 1836

Index...... 1841 Index in Japanese...... 1857 Preface

The Fourteenth Edition of the Japanese Phar- and to securing and maintaining international con- macopoeia was promulgated on March 30, 2001 by sistency. Ministerial Notiˆcation No. 111 of the Ministry of It was also agreed that JP articles should cover Health, Labour and Welfare. To keep pace with drugs which are important from the viewpoint of progress in medical and pharmaceutical sciences, in health care and medical treatment, clinical results and November 2001, the Council, at a meeting of the frequency of use, as soon as possible after they reach Committee on Japanese Pharmacopoeia (JP), estab- the market. lished the basic principles for the preparation of the It was also decided to make a deˆnite rule for selec- JP Fifteenth Edition, setting out the characteristics tion of articles by clarifying the standards for selec- and roles of the JP, the deˆnite measures for the tion. The rule was shown in the verdict ``What the revision, the date of the revision, and the organization future Japanese Pharmacopoeia should be'' by the of the Subcommittee on JP. Pharmaceutical AŠairs and Food Sanitation Council At the above meeting, the following ``ˆve pillars'' (PAFSC) on December 2002. The JP Fifteenth were established as the basic principles of the JP: Edition was decided to be slated for completion in Making it more substantial by including all drugs April 2006. which are important from the viewpoint of health care The panels on JP was reorganized into the following and medical treatment; Making prompt partial eleven panels in accordance with the recommendation revision as necessary and facilitating smooth adminis- of the PAFSC: Panel on Planning and Revisions; trative operation; Promoting international harmoni- Panel on Nomenclature for Pharmaceuticals; Panel zation; Ensuring transparency regarding the revision on Excipients; Panel on Physico-chemical Tests; Panel and dissemination to the public of the JP; and on Medicinal Chemicals; Panel on Biologically Positively introducing contemporary analytical tests Derived Drugs; Panel on Biological Tests; Panel on and developing reference standards. It was decided at Antibiotics; Panel on Crude Drugs; Provisional Panel the meeting that each panel set up under the Subcom- on Planning and Revisions and Panel on Phar- mittee on JP should make eŠorts, on the basis of these macopoeial Harmonization (PDG), followed by the principles, to ensure that the JP is used more eŠective- establishment of new panels: Panel on Water for ly in the ˆelds of health care and medical treatment by Pharmaceutical Preparations, Panel on JP Reference taking appropriate measures, including getting the Standards, and three working groups under Panel on understanding and cooperation of other parties con- Medicinal Chemicals to expedite discussion of revision cerned. drafts of drug monographs. The JP should comprise an o‹cial standard being In the Committee on Japanese Pharmacopoeia, required to assure the quality of drugs in this country Mitsuru Uchiyama took the role of chairman from in response to the progress in science and technology January 2001 to December 2002, Tadao Terao from and clinical demands at the time, it should deˆne the January to June 2003, and Takao Hayakawa from standards for speciˆcations as well as the methods of July 2003 to December 2004. tests to assure the overall quality of all drugs in It was decided that the JP will be revised not only principle, and it should have a role in clarifying the every ˆve years, in line with the basic principles for the criteria for quality of drugs which are recognized to be preparation of the JP Fifteenth Edition, but also as important from the viewpoint of medical treatment. necessary to take account of recent progress of science At the same time, it was agreed that the JP should and in the interests of international harmonization. be prepared with the aid of the knowledge and In accordance with the revision principles, the experience of many persons involved in the phar- panels continued discussions on selection of articles, maceuticals, that it should have the characteristics of and revisions for General Notices, General Rules for an o‹cial standard, which might be widely used by all Preparations, General Tests, and monographs on parties concerned, that it should provide information drugs, and the supplement I to JP 14 was promulgated and understanding about the quality of drugs to the on December 2002. After the promulgation of the public, and that it should be conducive to smooth and supplement I, the panels continued discussions to take eŠective government control of the quality of drugs, account of the progress of science and international i ii Preface Supplement II, JPXIV harmonization. Reference Spectra in Part I; Infrared Reference Draft revisions covering subjects in General Rules Spectra and Ultraviolet-visible Reference Spectra in for Crude Drugs, General Tests, and monographs on Part II; General Information, and as an appendix, a drugs, for which discussions were ˆnished between Cumulative Index containing references to the main March 2002 and December 2003, were prepared for a volume, the supplement I and the supplement II. supplement to the book. They were examined by the 2. The articles in General Tests, Processes and Committee on Japanese Pharmacopoeia in September Apparatus, Monographs on Drugs, Infrared Refer- 2004, followed by the PAFSC in December 2004, and ence Spectra and Ultraviolet-visible Reference Spectra then submitted to the Minister of Health, Labour and are respectively placed in alphabetical order. Welfare. 3. The following items in each monograph are put Numbers of discussions in the panels to prepare in the order shown below, except that unnecessary supplement drafts were as follows: Panel on Planning items are omitted depending on the nature of the drug: and Revisions, 9 times; Panel on Nomenclature for (1) English title Pharmaceuticals, 10 times; Panel on Excipients, 11 (2) Commonly used name(s) times; Panel on Physico-chemical Tests, 30 times; (3) Latin title (only for Crude Drugs) Panel on Medicinal Chemicals (including the working (4) Title in Japanese groups), 24 times; Panel on Biologically Derived (5) Structural formula or empirical formula Drugs, 11 times; Panel on Biological Tests, 10 times; (6) Molecular formula and molecular mass Panel on Antibiotics, 19 times; Panel on Crude Drugs, (7) Chemical name 19 times; Provisional Panel on Planning and Revi- (8) Origin sions, 12 times; Panel on Pharmacopoeial Harmoniza- (9) Limits of the content of the ingredient(s) tion (PDG), 9 times; Panel on Water for Pharmaceuti- and/or the unit of potency cal Preparations, 2 times; Panel on JP Reference (10) Labeling requirements Standards, 3 times. (11) Method of preparation It should be noted that in the preparation of the (12) Description drafts for the Supplement I, generous cooperation was (13) Identiˆcation tests given by the Technical Committee of the Pharmaceuti- (14) Speciˆc physical and/or chemical values cal Manufacturer's Association of Tokyo and of (15) Purity tests Osaka, the Crude Drugs Association of Tokyo, the (16) Loss on drying, loss on ignition, and/or water Japan Pharmaceutical Excipients Council, the (17) Residue on ignition, total ash, and/or acid-in- Federation of Crude Drugs Associations of Japan, the soluble ash Japan Antibiotics Research Association, the Japan (18) Tests being required for pharmaceutical prepa- Flavor and Fragrance Manufacturer's Association, rations and other special tests the Japan Medical Plants Federation, the Japan (19) Isomer ratio Pharmaceutical Manufacturer's Association, the (20) Assay or the content of the ingredient(s) Japanese Society of Hospital Pharmacists, the Japan (21) Containers and storage Pharmaceutical Association, and the Japan Oilseed (22) Expiration date Processors Association. (23) Others In consequence of this revision, the JP Fourteenth 4. In each monograph on a drug, the following Edition carries 907 articles in Part I owing to the physical and chemical values representing the proper- addition of 27 articles and the deletion of 1 article; and ties and quality of the drug are given in the order indi- 484 articles in Part II owing to the addition of 12 cated below, except that unnecessary items are omitted articles and the deletion of 9 articles. depending on the nature of the drug: The principles of description and the salient points (1) Alcohol number of the revision in this volume are as follows: (2) Absorbance (3) Congealing point 1. The Supplement II to JP Fourteenth Edition (4) Refractive index comprises the following items, in order: Notiˆcation (5) Osmolarity of the Ministry of Health, Labour and Welfare; (6) Optical rotation Contents; Preface; followed by General Tests, Proc- (7) Viscosity esses and Apparatus; monographs on drugs in Part I, (8) pH and General Rules for Crude Drugs; monographs on (9) Speciˆc gravity drugs in Part II, then followed by Ultraviolet-visual (10) Boiling point Supplement II, JPXIV Preface iii

(11) Melting point (32) Silver (12) Acid value (33) Alkaline earth metals (13) Saponiˆcation value (34) Arsenic (14) Ester value (35) Foreign matter (15) Hydroxyl value (36) Related substances (16) Iodine value (37) Residual solvent (38) Other mixtures 5. Identiˆcation tests comprise the following (39) Readily carbonizable substances items, which are generally put in the order given be- low: 7. The following items of the General Tests, Proc- (1) Coloration reactions esses and Apparatus were partially revised: (2) Precipitation reactions Bacterial Endotoxins Test (3) Decomposition reactions Nuclear Magnetic Resonance Spectroscopy (1H) (4) Derivatives Pyrogen Test (5) Visible, ultraviolet or infrared spectra Residue on Ignition Test (6) Special reactions Speciˆc Surface Area Determination (7) Cations Sterility Test (8) Anions 8. The following test was added to the General 6. Purity tests comprise the following items, which Tests, Processes and Apparatus: are generally put in the order given below, except that Powder Particle Density Determination unnecessary items are omitted depending on the 9. The following Reference Standard was deleted: nature of the drug: Digitalis (1) Color (2) Odor 10. The following Reference Standards were (3) Clarity and/or color of solution added: (4) Acidity or alkalinity Azithromycin (5) Acid Cisplatin (6) Alkali Etoposide (7) Chloride Furosemide (8) Sulfate Methylprednisolone Succinate (9) Sulˆte Nilvadipine (10) Nitrate Thiamylal (11) Nitrite Tranexamic Acid (12) Carbonate Trichlormethiazide (13) Bromide 11. English and Latin titles of drugs are derived, (14) Iodide in principle, from International Nonproprietary (15) Soluble halide Names (INN) for Pharmaceutical Substances recom- (16) Thiocyanide mended by the World Health Organization. Japanese (17) Selenium titles are derived from the Japanese version of this (18) Cationic salts book. The chemical names are based on the rules of (19) Ammonium the International Union of Pure and Applied Chemis- (20) Heavy metals try (IUPAC). (21) Iron (22) Manganese 12. Molecular formulas of organic compounds (23) Chromium begin with C and then H, followed by other involved (24) Bismuth elements in the alphabetical order of the symbols of (25) Tin the elements. (26) Aluminum 13. Structural formulas of drugs represent, as far (27) Zinc as possible, steric conˆgurations. (28) Cadmium (29) Mercury 14. Test procedures in monographs in Part I are, (30) Copper in principle, written in full even in corresponding (31) Lead monographs in Part II, and vice versa. The test proce- iv Preface Supplement II, JPXIV dures in monographs for preparations are also written Lindera Root in full even within the same part, except in the mono- Lonicera Leaf and Stem graphs for preparations having a corresponding Lycium Bark monograph of their principal material substances. Lycium Fruit Processed Aconite Root 15. The following articles were deleted from Powdered Processed Aconite Root O‹cial Monograph Processed Ginger Part I Sappan Wood Santonin Tablets Termeric Part II Tribulus Fruit Absorbent Cotton 17. The following monographs were revised in Puriˆed Absorbent Cotton commonly used name: Sterile Absorbent Cotton Part I Sterile Puriˆed Absorbent Cotton Calcium Folinate Absorbent Gauze Sterile Absorbent Gauze Part II Adhesive Plaster Cellulose Acetate Phthalate Digitalis 18. The following monographs were revised in Powdered Digitalis structural formula and in chemical name: 16. The following articles were newly added to Part I o‹cial monographs: Cefuroxime Axetil Part I Mepivacaine Hydrochloride Alprostadil 19. The part of origin of the following mono- Azithromycin Hydrate graphs were deleted: Benidipine Hydrochloride Part II Benidipine Hydrochloride Tablets Chorionic Gonadotrophin Cefepime Dihydrochloride for Injection Serum Gonadotrophin Cisplatin Eperisone Hydrochloride 20. The following monographs were revised in Etoposide origin: Flomoxef Sodium for Injection Part I Flopropione Capsules Acetohexamide Furosemide Tablets Atropine Sulfate Injection Glutathione Benzylpenicillin Benzathine Methylprednisolone Succinate Clarithromycin Metoclopramide Tablets Colchicine Nicorandil Cytarabine Nilvadipine Digoxin Injection Nilvadipine Tablets Digoxin Tablets Oxytocin Dimorpholamine Pirenzepine Hydrochloride Hydrate Dimorpholamine Injection Piroxicam Ethionamide Serrapeptase Etilefrine Hydrochloride Tablets Tiaramide Hydrochloride Tablets Fosfomycin Calcium Tizanidine Hydrochloride Fosfomycin Sodium Tranexamic Acid Capsules Furosemide Tranexamic Acid Injection dl-Methylephedrine Hydrochloride Tranexamic Acid Tablets Methyltestosterone Trichlormethiazide Tablets Methyltestosterone Tablets Oxytocin Injection Part II Pyridoxine Hydrochloride Cnidium Monnieri Fruit Pyridoxine Hydrochloride Injection Epimedium Herb Sodium Aurothiomalate Supplement II, JPXIV Preface v

Sodium Thiosulfate Heparin Sodium Testosterone Propionate Injection Heparin Sodium Injection Thiamylal Sodium Kallidinogenase Thiamylal Sodium for Injection D-Mannitol Tinidazole Meropenem Trihydrate Tranexamic Acid dl-Methylephedrine Hydrochloride Trichlormethiazide 10z dl-Methylephedrine Hydrochloride Powder Vinblastine Sulfate for Injection Methyltestosterone Methyltestosterone Tablets Part II Oxytocin Injection Benzyl Alcohol Pirenoxine Cellulose Acetate Phthalate Pyrazinamide Corn Starch Pyridoxine Hydrochloride Opium Alkaloids Hydrochlorides Pyridoxine Hydrochloride Injection Powdered Aloe Sodium Aurothiomalate Powdered Gardenia Fruit Sodium Polystyrene Sulfonate Powdered Peony Root Sodium Thiosulfate Powdered Rhubarb Tegafur Powdered Scutellaria Root Testosterone Propionate Rhubarb Testosterone Propionate Injection Uncaria Thorn Thiamine Hydrochloride 21. The following monographs were revised by Thiamylal Sodium addition or change in Method of preparation: Thiamylal Sodium for Injection Part I Tinidazole Oxytocin Injection Tranexamic Acid Trichlormethiazide Part II Vasopressin Injection Chorionic Gonadotrophin Vinblastine Sulfate Serum Gonadotrophin Vinblastine Sulfate for Injection 22. The following monographs were revised by Part II addition or change in Description and in test(s): Benzyl Alcohol Part I Carmellose Calsium Acetohexamide Cellulose Acetate Phthalate Anhydrous Citric acid Chorionic Gonadotrophin Atropine Sulfate Injection Chorionic Gonadotrophin for Injection Benzylpenicillin Benzathine Chrysanthemum Flower Cefuroxime Sodium Corn Starch Ciclosporin Cornus Fruit Clarithromycin Ginger Colchicine Opium Alkaloids Hydrochlorides Cytarabine Potato Starch Deferoxamine Mesilate Powdered Aloe Digoxin Powdered Gardenia Fruit Digoxin Injection Powdered Ginger Digoxin Tablets Powdered Peony Root Dimorpholamine Serum Gonadotrophin Dimorpholamine Injection Serum Gonadotrophin for Injection Ethionamide Uncaria Thorn Etilefrine Hydrochloride Tablets Wheat Starch Flavin Adenine Dinucleotide Sodium Fosfomycin Calcium 23. The following monographs were revised in Fosfomycin Sodium Identiˆcation by addition or change based on adop- Furosemide tion of the Ultraviolet-visible Reference Spectra: vi Preface Supplement II, JPXIV

Part I Eperisone Hydrochloride Alprostadil Ethionamide Benidipine Hydrochloride Etoposide Cisplatin Furosemide Colchicine Glutathione Cytarabine dl-Methylephedrine Hydrochloride Eperisone Hydrochloride Methylprednisolone Succinate Ethionamide Methyltestosterone Etoposide Nicorandil Furosemide Nilvadipine dl-Methylephedrine Hydrochloride Pirenzepine Hydrochloride Hydrate Methylprednisolone Succinate Piroxicam Methyltestosterone Pyridoxine Hydrochloride Nilvadipine Testosterone Propionate Oxytocin Thiamylal Sodium Pirenzepine Hydrochloride Hydrate Tinidazole Piroxicam Tizanidine Hydrochloride Pyridoxine Hydrochloride Tranexamic Acid Testosterone Propionate Trichlormethiazide Thiamylal Sodium Vinblastine Sulfate Tizanidine Hydrochloride Part II Trichlormethiazide Benzyl Alcohol 24. The following monographs were revised in 25. The following monographs were revised in Identiˆcation by addition or change based on adop- Containers and storage: tion of the Infrared Reference Spectra: Part I Part I Ethionamide Alprostadil Vinblastine Sulfate Azithromycin Hydrate Vinblastine Sulfate for Injection Benidipine Hydrochloride Cisplatin Part II Colchicine Corn Starch Cytarabine Potato Starch Digoxin Wheat Starch Dimorpholamine Supplement II, JPXIV Preface vii

Those who were engaged in the preparation of the Michinao Mizugaki Hisakazu Sunada Supplement I to JP Fourteenth Edition are as follows: Taiichi Mizuta Hideyo Suzuki Kaoru Morikawa Senji Suzuki Norio Aimi Yoshiaki Kato Osamu Morita Yoshikazu Takahashi Mitsuo Aoki Noriko Katori Takashi Morita Tadahiro Takeda Kiichi Aonuki Nobuo Kawahara Toshimi Murai Yasushi Takeda** Nobuo Aoyagi Toru Kawanishi Hiroshi Nakamura Toyoshige Tanabe Yoshichika Arakawa Toshiaki Kawanishi Tatsuya Nakano Haruo Tanaka Keiko Arimoto Nana Kawasaki Hiroyuki Nakazawa Toshihiro Tanaka Kazuhide Ashizawa Toshisuke Kawasaki Masaaki Naotsuka Kenichi Tanamoto Shinichiro Aso Yoshiaki Kawashima Masao Nasu Tsuyoshi Tanimoto Yukio Aso Keiji Kijima Koji Nishijima Susumu Terabayashi Hiroyuki Fuchino Takao Kiyohara Motohiro Nishijima Tadao Terao* Shigeyuki Fujikura Takashi Kobayashi Tatsumi Nishiyama Reiko Teraoka Goro Funamoto Toyohiko Kobayashi Takashi Nomoto Hiroshi Tokunaga Akihiro Furukawa Masayoshi Kohase Yasuo Ohno Kiyoshi Tomioka Yoshiharu Geki Shigeo Kojima Yoshiro Ohtani Motowo Tomita Yukihiro Goda Hiroyasu Kokubo Minoru Okada Tatsuru Tomizawa Morio Hamashima Eiichi Kokue Satoshi Okada** Yosuke Tsuji Ruri Hanajiri Katsuko Komatsu Kimiya Okazaki Nobuchika Tsumagari Kouji Hasegawa Akio Komura Tsuneo Okubo Eriko Uchida Ryuichi Hasegawa Toshifumi Konda Haruhiro Okuda Mitsuru Uchiyama* Takao Hayakawa* Seizo Kondo Masami Otsuka Yoshimasa Uehara Masahiro Hayashi Takao Kunisada Eiji Sakai Kazuichi Umemoto Kenji Higuchi Takeshi Kurata Hideki Sasaki Takashi Unno Katsufuto Hiramatsu Masaaki Kurihara Tsuguo Sasaki Haruo Watanabe Fusayoshi Hirayama Haruo Kuriyama Motoyoshi Satake Morimasa Yagisawa Yukio Hiyama Fumiyo Kusu Akihiro Sato Takehiko Yajima Kunimoto Hotta Kumiko Kusuyama Michiko Sekiguchi Teruhide Yamaguchi Takanori Ichikawa Masako Maeda Setsuko Sekita Keiichi Yamamoto Nobukazu Igoshi Midori Makita Yasuo Shimada Keiji Yamamoto Kenichi Inui Keiichi Maruyama Kesamitsu Shimizu Tosuke Yamamoto Shigeru Itai Toshio Masaoka Kyoko Shimura Kenichi Yamazaki Yuji Ito Toshihiko Matsubara Fumitoshi Shincho Takeshi Yamazaki Takashi Itoh Yoshihisa Matsuda Osamu Shirota Hikaru Yoden Shozo Iwagami Norio Matsuki Kouichi Shudo Chikako Yomota Akemi Kai Shigeru Matsuki Hisashi Sonobe Hitoo Yoshida Kazuaki Kakehi Hayashi Matsukura Shoko Sueyoshi Kazumasa Yoshikawa Takemine Kanai Satoshi Minobe Shinji Sugaya Sumie Yoshioka Nahoko Kaniwa Hiroto Miyamoto *Chairman, Committee on JP Motoko Kanke Naoki Miyata **Acting Chairman, Committee on JP General Tests, Processes and Apparatus

loss on drying, total ash, acid-insoluble ash, extract content, Change the introduction to read: essential oil content of crude drugs are performed as directed in the corresponding items under the Crude Drugs Test. General Tests, Processes and Apparatus includes common methods for tests and other articles related to them. Unless otherwise speciˆed, the procedures for absorbance determi- nation, absorbance ratio determination, acid-neutralizing 6. Bacterial Endotoxins Test capacity determination of gastrointestinal medicines, Change the (ii) Interpretation of (2) Limit test alcohol number determination, ammonium determination, under Gel-clot techniques to read: arsenic determination, atomic absorption spectrophotomet- ry, test for bacterial endotoxins, boiling point determina- Gel-clot techniques tion, distilling range determination, chloride determination, (2) Limit test conductivity measurement, congealing point determination, (ii) Interpretation test for content uniformity, determination of bulk and The test is valid when both replicates of solutions B and C tapped densities, digestion test, disintegration test, dissolu- are positive and those of solution D are negative. tion test, endpoint detection in titrimetry, ‰ame coloration, The sample meets the endotoxin limit requirement of the ‰uorometry, foreign insoluble matter test for injections, gas test when a negative result is found for both replicates of chromatography, heavy metals determination, test for glass solution A. containers for injections, infrared spectrophotometry, Repeat the test in duplicate when the test results are posi- insoluble particulate matter test for injections, insoluble tive for one test but negative for the other one. The sample particulate matter test for ophthalmic solutions, iron deter- meets the endotoxin limit requirement of the test when a mination, liquid chromatography, loss on drying determina- negative result is found for both replicates of solution A in tion, loss on ignition determination, mass variation test, the repeat test. melting point determination, test for metal particles in The sample does not meet the endotoxin limit requirement ophthalmic ointments, methanol determination, methoxyl of the test when a positive result is found for both replicates assay, microbial assay for antibiotics, test for microbial of the solution A at a dilution equal to the MVD. If the test limit, test for microbial limit for crude drugs, mineral oil is positive for the sample at a dilution less than the MVD, determination, nitrogen determination, nuclear magnetic the test may be performed at a dilution not greater than the resonance spectroscopy, optical rotation determination, MVD. osmolarity determination, oxygen ‰ask combustion method, paper chromatography, particle size distribution test for preparations, pH determination, test for plastic containers, 39. Nuclear Magnetic Resonance powder particle density determination, powder particle size 1 determination, test for pyrogen, qualitative test, test for Spectroscopy ( H) readily carbonizable substances, refractive index determina- tion, residual solvents test, residue on ignition determina- Change to read: tion, test for rubber closure for aqueous infusions, speciˆc gravity and density determination, speciˆc surface area determination, test for sterility, sulfate determination, 39. Nuclear Magnetic Resonance thermal analysis, thin-layer chromatography, test for total Spectroscopy organic carbon, viscosity determination, vitamin A assay, test for volatile contaminants in ethanol, water determina- Nuclear magnetic resonance (NMR) spectroscopy is based tion, and X-ray powder diŠraction are performed as directed on the phenomenon that speciˆc radio frequency radiation is in the corresponding articles under the General Tests, Proc- absorbed by magnetic nuclei in a sample placed in a magnet- esses and Apparatus. The tests for melting point of fats, ic ˆeld; target nuclei are 1H, 13C, 15N, 19F, 31P, etc. These congealing point of fatty acids, speciˆc gravity, acid value, nuclei have intrinsic spin angular momentum, of which the saponiˆcation value, ester value, hydroxyl value, unsaponiˆ- magnitude is given by I (I＋1)/h/2p,whereI is the spin able matter and iodine value of fats and fatty oils are quantum number and is integral or half-integral (I=1/2 for performed as directed in the corresponding items under the 1Hand13C). When the magnetic nuclei are placed in a mag- Fats and Fatty oils Test, and the tests for foreign matter and netic ˆeld, they are oriented in 2I＋1 possible orientations

1669 1670 General Tests, Processes and Apparatus Supplement II, JPXIV corresponding to 2I＋1 equally spaced energy levels (two Spectrometer energy levels for 1Hand13C). The transition between two There are two types of spectrometers. successive quantized energy levels corresponding to adjacent (1) Fourier transform NMR (FT-NMR) spectrometers orientations can be induced by electromagnetic radiation (Figure 1) with a suitable frequency. The precise relation between the Target nuclei are simultaneously excited in all frequency ˆeld strength and the resonant frequency n is given by range of the nuclei by means of an intense radio frequency H pulse. The FID (free induction decay) after the pulse is n＝g･ 0 2p detected, which is a time domain signal, is converted to a where H0 is the strength of the applied external magnetic frequency domain spectrum by Fourier transformation. ˆeld and g is the gyromagnetic ratio, a constant characteriz- Number of data points suitable for the spectral range, ‰ip ing a particular isotope. The absorption of radiation (NMR angle, acquisition time, delay time and number of scans signal) can occur only when the irradiating radio frequency should be set appropriately. satisˆes the resonance condition. Since the absorption Recently FT-NMR is commonly used because of its high coe‹cient (the transition probability) does not depend on sensitivity and various advanced applications. the environment in which the nuclei are located, the intensity is basically proportional to the number of nuclei. The excess spins shifted to the higher energy levels by the transition process return to the thermal equilibrium state at various rates determined by a characteristic time constant (known as the relaxation time). A nucleus is shielded from the applied magnetic ˆeld by the electrons belonging to its own atom and to the molecule. Therefore nuclei in diŠerent environments are shielded to diŠerent extents and resonate at diŠerent frequencies. The diŠerence in resonance frequencies is deˆned as chemical shift (d), which is independent of the strength of the magnet- ic ˆeld, and is given by

nS－nR d＝ ＋dR nR Fig. 1 FT-NMR spectrometer where,

nS: The resonance frequency of the observed signal, (2) Continuous wave NMR (CW-NMR) spectrometers

nR: The resonance frequency of the reference signal, (Figure 2)

nR: The chemical shift of the reference signal (in the case In the case of the CW method, a spectrum is obtained by of the value not being 0). sweeping the radio frequency or magnetic ˆeld continuously over the frequency range of the nuclei being observed. The chemical shifts are normally expressed in ppm, a dimensionless unit, by assuming the chemical shift of the reference compound as 0 ppm. When the chemical shift of the reference compound is not assumed to be 0 ppm, chemi- cal shifts of samples are corrected accordingly. In addition to the shielding due to electrons, the nucleus is subjected to eŠects due to the spin orientations of other magnetic nuclei through chemical bonds, resulting in an additional splitting of the signal. The spacing between two adjacent components of the signal is known as the spin-spin coupling constant (J). Coupling constants are measured in hertz and are independent of the strength of the external magnetic ˆeld. The increased number of interacting nuclei will make the multiplet pattern more complex. From the NMR spectrum the following four parameters Fig. 2 CW-NMR spectrometer can be obtained: chemical shift, spin-spin coupling constant, resonance intensity (intensities of 1H are proportional to the number of nuclei and those of 13C and others are susceptible Measurement to the nuclear Overhauser eŠect (NOE) and relaxation) and Prior to measurements, the sensitivity and resolution of relaxation time. These parameters are useful for structural the instrument must be adjusted to the optimum levels using determination, identiˆcation and quantitative analysis of a standard sample (ethylbenzene, 1,2-dichlorobenzene or molecules. Spin decoupling, NOE, and two-dimensional acetaldehyde) dissolved in an appropriate NMR solvent. NMR techniques are also available for structural analysis. (1) The sample dissolved in a suitable solvent is trans- Supplement II, JPXIV General Tests, Processes and Apparatus 1671 ferred into an NMR tube. The reference compound can be correlations. As NOE can observe correlations among added directly to the sample solution (internal reference), or spatially proximate protons, the conˆguration and the a sealed capillary tube containing the reference compound conformation can be analyzed. can be inserted into the NMR tube (external reference). The Broadband decoupling, INEPT and DEPT are usually sample solutions should be completely homogeneous. In appliedinone-dimensional13C spectroscopy. The broad- particular, solid contaminants should be removed in order to band decoupling technique simpliˆes a spectrum and obtain good spectra. Various deuterated NMR solvents are achieves enhancement of sensitivity. INEPT (insensitive commonly used for NMR measurement and the following nuclei enhanced by polarization transfer) and DEPT (distor- points should be considered in selecting an appropriate tionless enhancement of polarization transfer) enhance the solvent: (i) The solvent signals do not overlap with the sensitivity of 13C by means of polarization transfer from sample signals. (ii) The sample must be soluble in the solvent directly bonded 1H with a large magnetic moment. They can selected. (iii) The solvent does not react with the sample. be applied to identify primary, secondary, tertiary or Furthermore, it should be noted that chemical shifts can quarternary carbon. depend upon the solvent employed, sample concentration Two-dimensional spectroscopy can observe all correlation and deuterium ion concentration, and that viscous solutions peaks between nuclei through spin-spin coupling or NOE in usually give rather broad, poorly resolved spectra. a single experiment, and there are many techniques for (2) For the reference standards use the reagents for homonuclear and heteronuclear measurements. Representa- nuclear magnetic resonance spectroscopy. For 1Hand13C tive techniques are described below. spectra, tetramethylsilane (TMS) is usually used as the COSY (2D correlation spectroscopy), HOHAHA reference compound for samples dissolved in organic (homonuclear Hartmann-Hahn spectroscopy) or TOCSY solvents. For samples dissolved in deuterium oxide, sodium (total correlation spectroscopy): Correlation between pro- 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) or sodium 3- tons through scalar spin-spin coupling is obtained and (trimethylsilyl)propionate (TSP) is used. For other nuclei, intramolecular connectivities of hydrogen atoms are nitromethane, trichloro‰uoromethane and phosphoric acid revealed. are used as reference compounds for 15N, 19Fand31P, NOESY (2D nuclear Overhauser enhancement and respectively. Furthermore, chemical shifts of residual pro- exchange spectroscopy): NOE is measured by a two-dimen- tons in deuterated solvents and 13C in the solvent instead of a sional technique. Approximate distances between spatially reference compound can be used for 1Hand13CNMR. proximate hydrogen atoms are obtained to analyze the three- dimensional structure. Record of apparatus and measurement conditions INADEQUATE (incredible natural abundance double Type of instrument, frequency, solvent, temperature, quantum transfer experiment): Although this technique is sample concentration, reference compound, experimental insensitive because it involves double quantum transfer by technique, etc. should be recorded to allow appropriate 13C-13C scalar coupling in a sample with natural isotopic comparison of spectra, because NMR spectra depend on the abundance, the connectivities of all neighboring 13Cnuclei measurement conditions. canbeobtainedtoanalyzethecarbonskeleton. Identiˆcation HMQC (heteronuclear multiple quantum coherence): This The sample solution is prepared and tested by the method technique observes correlations between 1Hand13Cwith directed in each monograph. Usually in the case of 1HNMR, direct spin-spin coupling using 1H detection and reveals the sample is identiˆed by the following method. intramolecular chemical bonds between hydrogen and (1) Identiˆcation by the use of chemical shift, multiplic- carbon atoms. ity and relative intensity HMBC (heteronuclear multiple bond connectivity): This When chemical shifts, multiplicities and relative intensi- technique observes correlations between 1Hand13Cwith ties of signals are deˆned, the sample can be identiˆed as long range spin-spin coupling using 1H detection and reveals being the same substance when all chemical shifts, multiplic- intramolecular connectivities of hydrogen and carbon ities and relative intensities are the same as those prescribed. atoms. (2) Identiˆcation by the use of a Reference Standard There are many other techniques such as DQF-COSY Measurement conditions should be the same as those used (double quantum ˆltered COSY) and HSQC (heteronuclear inthecaseoftheReferenceStandard.Whenthespectraofa single quantum coherence). Furthermore, multidimensional sample and the Reference Standard exhibit the same NMR techniques are used to analyze macromolecules. multiplicities and relative intensities of signal at the same chemical shifts, the sample can be identiˆed as being the same substance as the Reference Standard.

Experimental techniques of 1Hand13C NMR spectroscopy NMR spectroscopy includes one-, two- and multi-dimen- sional techniques, which are used for various purposes. Spin decoupling, and NOE are available in one-dimen- sional 1H spectroscopy. Spin decoupling can assign coupling 1672 General Tests, Processes and Apparatus Supplement II, JPXIV

exceeding 10 min. Hypotonic test sample may be made 47. Pyrogen Test isotonic by the addition of pyrogen-free sodium chloride. Record the temperature of each rabbit during a period of Change to read: 3 hours after the injection, taking the measurements at intervals of not more than 30 min. The diŠerence between The Pyrogen Test is a method to test the existence of the control temperature and the maximum temperature of pyrogens by using rabbits. each rabbit is taken to be the rise in body temperature. Test animals Consider any temperature decreases as zero rise. Use healthy mature rabbits, each weighing not less than Interpretation of results 1.5 kg, which have not lost body mass when kept on a The test is carried out on a group of three rabbits and the constant diet for not less than one week. House the rabbits result is judged on the basis of the sum of the three tempera- individually in an area free from disturbances likely to excite ture rises. Repeat if necessary on further groups of three rab- them. Keep the temperature of the area constant between bits to a total of three groups, depending on the results 209Cand279C for at least 48 hours before and throughout obtained. If the summed response of the ˆrst group does not the test. Before using a rabbit that has not previously been exceed 1.39C, the sample is judged to be pyrogen-negative. used for a pyrogen test, condition it 1 to 3 days prior to the If the summed response exceeds 2.59C,thesampleisjudged test by conducting a sham test omitting the injection. Do not to be pyrogen-positive. If the summed response exceed use a rabbit for pyrogen tests more frequently than once 1.39C but does not exceed 2.59C, repeat the test on another every 48 hours, or after it has been given a test sample that group of three rabbits. If the summed response of the ˆrst was adjudged pyrogen-positive or that contained an antigen and second group does not exceed 3.09C,thesampleis present commonly in the test sample to be examined. judged to be pyrogen-negative. If the summed response of Apparatus, instruments the 6 rabbits exceeds 4.29C, the sample is judged to be (1) Thermometer—Use a rectal thermometer or temper- pyrogen-positive. If the summed response exceeds 3.09Cbut ature-measuring apparatus with an accuracy of ±0.19Cor does not exceed 4.29C, repeat the test on one more group of less. three rabbits. If the summed response of the 9 rabbits does (2) Syringe and injection needle—Depyrogenate the not exceed 5.09C, the sample is judged to be pyrogen- syringes and needles in a hot-air oven using a validated proc- negative. If the summed response exceeds 5.09C, the sample ess, usually by heating at 2509C for not less than 30 minutes. is judged to be pyrogen-positive. Sterilized syringes with needles are also available provided When the test sample is judged to be pyrogen-negative, that they have been validated to assure that they are free of the sample passes the pyrogen test. detectable pyrogens and do not interfere with the test.

Test procedures (1) Quantity of injection—Unless otherwise speciˆed, 52. Residue on Ignition Test inject 10 mL of the sample per kg of body mass of each Change the Procedure to read: rabbit. (2) Procedure—Perform the test in a separate area at an Procedure environmental temperature similar to that of the room Previously ignite a suitable crucible (silica, platinum, wherein the animals were housed and free from disturbances quartz or porcelain) at 600±509C for 30 minutes, and weigh likely to excite them. Withhold food from the rabbits for accurately after cooling in a desiccator (silica gel or other several hours before the ˆrst record of the temperature and suitable dessicant). throughout the testing period. The test animals are usually Take the sample of the amount directed in the mono- restrained with loosely ˆtting neck stocks that allow the graph, transfer into the ignited crucible, and weigh accurate- rabbits to assume a natural resting posture. Determine the ly. When the quantity of the sample to be taken is indicated temperature of each rabbit by inserting the thermometer or in a volume, pipet exactly the amount directed in the temperature-measuring probe into the rectum of the test monograph and transfer into the above crucible. When animal to a constant depth within the range of 60 mm to directed as ``after evaporating,'' heat properly to evaporate 90 mm. The ``control temperature'' of each rabbit is the the solution. mean of two temperature readings recorded for that rabbit Moisten the sample with a small amount of sulfuric acid, at an interval of 30 min in the 40 min immediately preceding usually 1 mL, then heat slowly at a temperature as low as theinjectionofthesampletobeexamined.Rabbitsshowing practicable until the sample is completely carbonized, and a temperature variation greater than 0.29C between the two cool. Moisten again with a small amount (usually 1 mL) of successive temperature readings or rabbits having an initial sulfuric acid, heat gently until white fumes are no longer temperature higher than 39.89C are withdrawn from the evolved, and ignite at 600±509C until the residue is com- test. pletely incinerated. Ensure that ‰ames are not produced at Warmthetestsolutiontoatemperatureof37±29C any time during the procedure. Cool the crucible in a desic- before injection, and inject the solution slowly into the cator (silica gel or other suitable dessicant), and reweigh marginal vein of the ear of each rabbit over a period not accurately to calculate the percentage of residue. Supplement II, JPXIV General Tests, Processes and Apparatus 1673

Unless otherwise speciˆed, if the amount of the residue so obtained exceeds the limit speciˆed in the individual mono- 54. Sterility Test graph, repeat the moistening with sulfuric acid, heating and ignition as before until constant mass is attained or until the Change to read: percentage of residue complies with the limit in the individ- Test for sterility is the method to establish the presence or ual monograph. absence of viable microorganisms (bacteria and fungi) using the deˆned culturing method. Unless otherwise speciˆed, the test is carried out by I. Membrane ˆltration method or II. 53. Speciˆc Surface Area Direct inoculation method. Water, reagents, test solutions, Determination equipment, materials and all other requisites for the test should be pre-sterilized. The test for sterility is carried out Change to read the text up to the paragraph under aseptic conditions. In order to achieve such condi- headed ``Method 1 Dynamic Flow Method'' as tions, the test environment has to be adapted to the way in follows: which the sterility test is performed. The precautions taken to avoid contamination are such that they do not aŠect any Speciˆc Surface Area Determination is a method to deter- microorganisms which are to be revealed in the test. The mine the speciˆc surface area (the total surface area of working conditions in which the tests are performed are powder per an unit mass) of a powdered pharmaceutical monitored regularly by appropriate sampling of the working preparation by using the gas adsorption method. The gas area and by carrying out appropriate controls. adsorption method is a method for measuring the amount of gas adsorbed on the surface of a powder sample as a func- Media and rinsing ‰uids tion of the pressure of the adsorbate gas, and the measure- Fluid thioglycolate medium, soybean-casein digest ments are usually performed at the boiling point of liquid medium are used, unless otherwise speciˆed. When it is nitrogen (－1969C). di‹cult to use ‰uid thioglycolate medium due to turbidity or When the gas is physically adsorbed by the powder viscosity of samples, alternative thioglycolate medium can sample, the following relationship holds when P/P0 is in the beused,provideditisheatedonawaterbathjustpriorto range of 0.05 to 0.30 for pressure P of the adsorbate gas in use and incubated under anaerobic conditions. Other equilibrium for the volume of gas adsorbed, Va. products of suitable quality yielding similar formulations maybeusedaccordingtotheindicationsonthelabel. 1 (C－1) P 1 ＝ × ＋ (1) Fluid thioglycolate medium P V C P V C V 0 －1 m 0 m aØ P » L-Cystine 0.5 g Agar 0.75 g P: Partial vapor pressure of adsorbate gas in equilibrium Sodium chloride 2.5 g (kPa) Glucose, monohydrate/anhydrate 5.5/5.0 g P : Saturated vapor pressure of the adsorbate gas at 0 Yeast extract (water-soluble) 5.0 g －1969C(kPa) Pancreatic digest of casein 15.0 g V : Volume of gas adsorbed at equilibrium (mL) a Sodium thioglycolate or 0.5 g V : Volume of gas adsorbed in a monolayer (mL) m Thioglycolic acid 0.3 mL C: Dimensionless constant relating to the enthalpy of Resazurinsodiumsolution(1in1000), adsorption and condensation of the adsorbate gas freshly prepared 1.0 mL The speciˆc surface area, S, is determined from V ,the m Water 1000 mL volume of gas adsorbed in a monolayer on the sample. (pH after sterilization 7.1±0.2) V ×N×a Mix the L-cystine, agar, sodium chloride, glucose, water- S＝ m m×22400 soluble yeast extract and pancreatic digest of casein with the water, and heat until solution is eŠected. Dissolve the S: Speciˆc surface area (m2/g) sodium thioglycolate or thioglycolic acid in the solution and, N: Avogadro constant 6.022×1023/mol if necessary, add sodium hydroxide TS so that, after sterili- a: EŠective cross-sectional area of one adsorbate molecule zation, the solution will have a pH of 7.1±0.2. If ˆltration is (m2) necessary, heat the solution again without boiling and ˆlter N0.162×10－18 2 while hot through moistened ˆlter paper. Add the resazurin Kr: 0.195×10－18 sodium solution, mix and place the medium in suitable m: Mass of the test powder (g) vessels which provide a ration of surface to depth of medium Speciˆc surface area is generally expressed in unit of m2/g. such that not more than the upper half of the medium has Either of the methods described below can be used to undergone a color change indicative of oxygen uptake at the measure the gas adsorption. end of the incubation period. Sterilize using a validated process. Store the medium at a temperature between 2 – 259C. If more than the upper one-third of the medium has acquired a pink color, the medium may be restored once by 1674 General Tests, Processes and Apparatus Supplement II, JPXIV heating the containers in a water-bath or in free-‰owing (2) Growth promotion test steam until the pink color disappears and cooling quickly, Test each batch of ready-prepared medium and each batch taking care to prevent the introduction of non-sterile air into (lot)of medium prepared either from dehydrated medium or the container. from ingredients1. Inoculate a small number (not more than (2) Alternative thioglycolate medium 100 CFU) of microorganism listed in Table 1 or other strains L-Cystine 0.5 g considered to be equivalent to these strains in containers of Sodium chloride 2.5 g each medium. Each of the test organisms should show Glucose, monohydrate/anhydrate 5.5/5.0 g clearly visible growth in all inoculated media within 3 days Yeast extract (water-soluble) 5.0 g for bacteria and within 5 days for fungi. Pancreatic digest of casein 15.0 g Table 1. Microorganisms for growth promotion test and Sodium thioglycolate or 0.5 g the validation test Thioglycolic acid 0.3 mL Incubation Water 1000 mL Medium Test microorganisms conditions (pH after sterilization 7.1±0.2) The methods for preparation follow those of ‰uid Staphylococcus aureus thioglycolate medium. (ATCC 6538, NBRC13276, (3) Soybean-casein digest medium CIP 4.83, NCTC 10788, Casein peptone 17.0 g NCIMB 9518) Soybean peptone 3.0 g Fluid Pseudomonas aeruginosa thioglycolate (ATCC 9027, NBRC 13275, Aerobic Sodium chloride 5.0 g medium NCIMB 8626, CIP 82.1l8) Dipotassium hydrogen phosphate 2.5 g Clostridium sporogenes Glucose, monohydrate/anhydrous 2.5/2.3 g (ATCC 19404, CIP 79.3, Water 1000 mL NCTC 532, or ATCC 11437, (pH after sterilization 7.3±0.2) NBRC 14293) Mix all the ingredients and heat until solution is eŠected. Clostridium sporogenes If necessary, add sodium hydroxide TS so that, after sterili- Alternative (ATCC 19404, CIP 79.3, zation, the solution will have a pH of 7.3±0.2. Filter, if thioglycolate Anaerobic NCTC 532, or ATCC 11437, necessary, to clarify, distribute into suitable vessels and medium NBRC 14293) sterilize using a validated process. Store at a temperature between 2 – 259C in a sterile container. Bacillus subtilis (4) Rinsing ‰uids (ATCC 6633, NBRC 3134, Meat or casein peptone 1.0 g CIP 52.62, NCIMB 8054) Candida albicans Water 1000 mL Soybean-casein (ATCC 10231, NBRC 1594, Aerobic (pH after sterilization 7.1±0.2) digest medium IP 48.72, NCPF 3179) Dissolve animal tissue or casein peptone in water and Aspergillus niger adjust the pH of the solution so that, after sterilization, it (ATCC 16404, NBRC 9455, will show 7.1±0.2. Filter, if necessary, to clarify, distribute IP 1431.83, IMI 149007) into suitable vessels and sterilize using a validated process. Store at a temperature between 2 – 259Cinasterile Seed lot culture maintenance techniques (seed-lot systems) container. are used so that the viable microorganisms used for inocula- tion are not more than ˆve passages removed from the To rinsing ‰uid to be used for antibiotics or pharmaceuti- original master seed-lot. cal products containing an antimicrobial agent, a suitable neutralizer or inactive agent at concentration shown to be EŠective period of media appropriate in the validation of the test can be added. To If prepared media are stored in unsealed containers, they rinsing ‰uid to be used for oils, oily solutions, ointments or can be used for one month, provided that they are tested for creams, suitable emulsifying agent at a concentration shown growth promotion within two weeks of the time of use and to be appropriate in the validation of the test, for example that color indicator requirements are met. If stored in tight polysorbate 80 at a concentration of 10 g/L can be added. containers, the media can be used for one year, provided that they are tested for growth promotion within 3 months Suitability of media of the time of use and that the color indicator requirements The media used comply with the following tests, carried are met. out before or in parallel with the test on the product to be examined. Validation test (1) Sterility of media The validation may be performed simultaneously with the Conˆrm the sterility of each sterilized batch of medium by Test for sterility of the product to be examined in the incubating a portion of the media at the speciˆed incubation following cases. temperature for 14 days. No growth of microorganisms a) When the test for sterility has to be carried out on a new occurs. product. Supplement II, JPXIV General Tests, Processes and Apparatus 1675 b) Whenever there is a change in the experimental condi- Table 2. Number of items to be taken from the lot tionsofthetest. Minimum number of items to Number of items in the lot Carry out the test as described below under Test for sterili- be tested for each medium* ty of the product to be examined using exactly the same methods except for the following modiˆcations. Injections 10z or 4 containers, which- Membrane ˆltration After transferring the content of the Not more than 100 containers ever is greater containerorcontainerstobetestedtothemembraneaddan More than 100 but not more 10 containers inoculum of a small number of viable micro-organisms (not than 500 containers more than 100 CFU) to the ˆnal portion of sterile diluent More than 500 containers 2z or 20 containers, which- used to rinse the ˆlter. ever is less Direct inoculation After transferring the contents of the For large-volume products 2z or 10 containers, which- container or containers to be tested to the culture medium (More than 100 mL) ever is less addaninoculumofasmallnumberofviablemicro-organ- Ophthalmic and other isms (not more than 100 CFU) to the medium. non-injectable products In both cases use the same micro-organisms as those Not more than 200 containers 5z or 2 containers, which- described above under Growth promotion test. Perform a ever is greater growth promotion test as a positive control. Incubate all the More than 200 containers 10 containers containers containing medium for not more than 5 days. If If the product is presented in clearly visible growth of micro-organisms is obtained after the form of single-dose con- the incubation, visually comparable to that in the control tainers, apply the scheme vessel without product, either the product possesses no shown above for preparations antimicrobial activity under the conditions of the test or for parenteral use such activity has been satisfactorily eliminated. The test for Bulk solid products sterility may then be carried out without further modiˆca- Up to 4 containers Each container tion. If clearly visible growth is not obtained in the presence More than 5 containers but 20z or 4 containers, which- of the product to be tested, visually comparable to that in not more than 50 containers ever is greater the control vessels without product, the product possesses More than 50 containers 2z or 10 containers, which- antimicrobial activity that has not been satisfactorily elimi- ever is greater nated under the conditions of the test. Modify the conditions Antibiotic Solids in order to eliminate the antimicrobial activity and repeat the Pharmacy bulk packages (º5g) 20 containers validation test. Pharmacy bulk packages (Æ5g) 6 containers

In the membrane ˆltration, the antimicrobial activity should *If the contents of one container are enough to inoculate the be suppressed by suitable means such as replacement of the two media, this column gives the number of containers need- membrane ˆlters with less adsorptive ones, increase of the ed for both the media together. amount of rinsing ‰uid, or addition of a suitable inactivating Testing methods agent to the rinsing ‰uid. Do not exceed a washing cycle of The test may be carried out using the technique of mem- 5 times 100 mL per ˆlter, even if during validation it has brane ˆltration or by direct inoculation of the culture media been demonstrated that such a cycle does not fully eliminate with the product to be examined. Appropriate negative the antimicrobial activity. controls are included. The technique of membrane ˆltration In the direct inoculation, use a suitable inactivating agent is used whenever the nature of the product permits, that is, which does not aŠect the growth of microorganisms or in- for ˆlterable aqueous preparations, for alcoholic or oily crease the volume of medium irrespective of the prescription preparations and for preparations miscible with or soluble in in II-2 so that no antimicrobial activity remains. aqueous or oily solvents provided these solvents do not have Test for sterility of the products to be examined an antimicrobial eŠect in the conditions of the test. Number of articles to be tested I. Membrane ˆltration Items to be used for the test are taken from the lot accord- By this method, a test article is ˆltered through a mem- ing to an appropriate sampling plan prepared by referring to brane ˆlter, and the ˆlter is rinsed and incubated by being the numbers speciˆed in Table 2. transferred to a medium or by adding a medium to the ˆltra- tion apparatus. Use membrane ˆlter made from suitable material having a nominal pore size of 0.45 mm or smaller. Use a ˆlter funnel sterilizable by the moist heat method or other methods and free from any leakage or back ‰ow when ˆltration is performed with the membrane in place. The technique described below assumes that membranes about 50 mm in diameter will be used. If ˆlters of a diŠerent diameter are used the volumes of the dilutions and the 1676 General Tests, Processes and Apparatus Supplement II, JPXIV washings should be adjusted accordingly. Table 3. Minimum quantity to be used for each medium I-1. Preparation of sample solution Minimum quantity to be Quantity per container a) Liquid medicine: Use as it is, as the sample solution. used for each medium b) Solid medicine: In the case of a solid medicine, to be administered after dissolving or suspending, the sample Liquids solution is prepared with the provided solvent, isotonic Less than 1 mL The whole contents of each container sodium chloride solution or water to give the concentra- 1–40mL Half the contents of each tion of use. container but not less than c) Oils and oily solutions: Oils and oily solutions of 1mL su‹ciently low viscosity may be ˆltered without dilution Greater than 40 mL and 20 mL through a dry membrane. Viscous oils may be diluted as not greater than 100 mL necessary with a suitable sterile diluent such as isopropyl Greater than 100 mL 10z of the contents of the myristate shown not to have antimicrobial activity in the container but not less than conditions of the test. 20 mL d) Ointments and creams: Ointments in a fatty base and Antibiotic liquids 1mL emulsions of the water-in-oil type may be diluted by using Other preparations soluble The whole contents of each sterile isopropyl myristate that has previously been in water or in isopropyl container to provide not less myristate than 200 mg ˆltered through a sterilizing membrane ˆlter or by using other solvents not aŠecting the growth of microorgan- Insoluble preparations, creams Use the contents of each isms. Heat the sample preparation, if necessary, to not and ointments to be suspended container to provide not less more than 409C. In exceptional cases it may be necessary or emulsiˆed than 200 mg to heat to not more than 449C. Solids I-2. Quantities of sample solution to be tested Less than 50 mg The whole contents of each Use for each medium not less than quantity of the product container prescribed in Table 3, unless otherwise speciˆed. If the 50 mg or more but less than Half the contents of each con- contents of one container are insu‹cient to inoculate the 300 mg tainer but not less than 50 mg two media, twice or more containers shown in Table 2 are 300mg–5g 150 mg used. When using the technique of membrane ˆltration, use, Greater than 5 g 500 mg whenever possible, the whole contents of the container, but not less than the quantities indicated in Table 3, diluting I-3. Procedures where necessary to about 100 mL with a suitable sterile Usually complete the ˆltration of the sample solution with rinsing ‰uid. one or two separate ˆlter funnels. Transfer the contents of the container or containers to be tested to the membrane or membranes. If the sample solution is not readily ˆlterable, it may be further diluted with rinsing ‰uid and thereafter ˆltered. Rinse the membrane(s) with each 100-mL of rinsing ‰uid per ˆlter for established cycles in the validation test. Provided the sample does not have antimicrobial activity, the rinsing procedure can be omitted. Employ either of the two methods described below for incubation of the membrane(s). Use the same volume of each medium as in the validation test. (1) The processed membrane is aseptically transferred from the apparatus and cut into two equal parts, or half the volume of sample solution is ˆltered into an entire mem- brane. Transfer each half of the cut membrane, or each whole membrane into the medium. (2) After ˆltration of sample solution into the apparatus to which the membrane ˆlters are ˆtted, each medium is added to the apparatus itself.

II. Direct inoculation of the culture medium This is the method by which the entire content or a portion of the content of a sample container is transferred directly to the culture medium and incubated. Usually, this method is applied for medicines to which the membrane ˆltration method cannot be applied or for which the application of the direct transfer method, rather than the membrane ˆltration Supplement II, JPXIV General Tests, Processes and Apparatus 1677 method, is rational. For products containing a mercurial preservative that 70. Reference Standards; cannot be tested by the membrane-ˆltration method, ‰uid thioglycolate medium incubated at 20 – 259Cmaybeused Reagents, Test Solutions; Standard instead of Soybean-casein digest medium. Solutions for Volumetric Analysis; II-1. Preparation of sample solution Standard Solutions; Matching Usually, proceed as directed for the membrane ˆltration method. In the case of an insoluble medicine, the product is Fluids for Color; Optical Filters for suspended or crushed in a suitable manner and used as a Wavelength and Transmission Rate sample. a) Oily liquids. Use media to which have been added a suita- Calibration; and Measuring ble emulsifying agent at a concentration shown to be Instruments, Appliances appropriate in the validation of the test, for example polysorbate 80 at a concentration of 10 g/L. b) Ointments and creams. Prepare by diluting to about 1 in (1) Reference Standards 10 by emulsifying with the chosen emulsifying agent in a suitable sterile diluent such as a 1 g/L neutral solution of Add the following: meat or casein peptone. Transfer the diluted product to a medium not containing an emulsifying agent. Azithromycin, Cisplatin, Etoposide, Furosemide, Methyl- II-2. Quantities of sample solution to be tested prednisolone Succinate, Nilvadipine, Thiamylal, Tranexam- Transfer the quantity of the preparation to be examined ic Acid, Trichlormethiazide prescribed in Table 3, by using pipette, syringe or other suitable inoculation devices, directly into the culture medium so that the volume of the product is not more than (2) Reagents, Test Solutions 10z of the volume of the medium, unless otherwise prescribed. Shake cultures containing oily products gently Delete the following: each observation day. However when thioglycolate medium Cyclohexylamine for thin-layer chromatography is used for the detection of anaerobic microorganisms keep Dicyclohexylurea for thin-layer chromatography shaking or mixing to a minimum in order to maintain anaerobic conditions. Change the following: Cultivation and observation Calcium hydroxide for pH determination Calcium Fluid thioglycolate medium and Alternative thioglycolate hydroxide prepared for pH determination. medium are to be incubated at 30 – 359C and Soybean- casein digest medium is to be incubated at 20 – 259Cfornot [6]-Gingerol for thin-layer chromatography C17H26O4 less than 14 days. Observe the cultures several times during A yellow-white to yellow, liquid or solid. Freely soluble in the incubation period. If the material being tested renders methanol, in ethanol (99.5) and in diethyl ether, and practi- the medium turbid so that the presence or absence of cally insoluble in water. microbial growth cannot be readily determined by visual Purity Related substances—Dissolve 1.0 mg of [6]-gin- examination, 14 days after the beginning of incubation gerol for thin-layer chromatography in exactly 2 mL of transfer suitable portions of the medium to fresh vessels of methanol. Perform the test with 10 mLofthissolutionas the same medium and then incubate the original and transfer directed in the Identiˆcation under Ginger: any spot other vessels for not less than 4 days. than the principal spot at the Rf value of about 0.3 does not appear. Observation and interpretation of results

If no evidence of microbial growth is found, the product Piperidine hydrochloride C5H11N.HCl A white crys- to be examined complies with the test for sterility. If talline powder. Dissolves in water and in methanol. The pH evidence of microbial growth is found the product examined of the aqueous solution (1 in 20) is between 3.0 and 5.0. does not comply with the test for sterility, unless it can be Melting point:247–2529C clearly demonstrated that the test was invalid for causes Purity Clarity and color of solution—Dissolve 1.0 g of unrelated to the product to be examined. If no evidence of piperidine hydrochloride in 20 mL of water: the solution is microbial growth is found in the repeat test the product clear and colorless. complies with the Sterility Test. If microbial growth is found Residue on ignition: not more than 0.10z (1 g). in the repeat test the product does not comply with the Content: not less than 99.0z. Assay—Dissolve about Sterility Test. 0.25 g of piperidine hydrochloride, accurately weighed, in 50mLofwater,add5mLofdilutednitricacid(1in3),and titrate with 0.1 mol/L silver nitrate VS (potentiometric titra- tion). Perform a blank determination in the same manner, 1678 General Tests, Processes and Apparatus Supplement II, JPXIV and make any necessary correction. sary correction.

Each mL of 0.1 mol/L silver nitrate VS Each mL of 0.1 mol/L perchloric acid VS

＝12.16 mg of C5H11N.HCl ＝18.12 mg of C9H11NO3 Sodium 1-decanesulfonate C H NaO SAwhitepow- 10 21 3 Add the following: der. Purity Clarity and color of solution—Dissolve 1.0 g in 0.05 mol/L Acetic acid-sodium acetate buŠer solution, 20 mL of water: the solution is clear and colorless. pH 4.6 Dissolve 6.6 g of sodium acetate trihydrate in Loss on drying:notmorethan3.0z (1 g, 1059C, 900 mL of water, and add 3 mL of acetic acid and water to 3 hours). make 1000 mL. Content: not less than 98.0z. Assay—Weigh accurately 0.25 mol/L Acetic acid TS To 3 g of acetic acid (100) about 0.45 g of sodium 1-decanesulfonate, dissolve in 50 mL add water to make 200 mL. of water, and pass through a column, about 1.2 cm in inside diameter and about 25 cm in length, packed with about Aconitine for purity C34H47NO11 White, crystals or crys- 20 mL of strongly acidic ion-exchange resin (0.3 to 1.0 mm, talline powder. Sparingly soluble in acetonitrile and in H type) at a ‰ow rate of about 4 mL per minute. Wash with ethanol (99.5), slightly soluble in diethyl ether, and practi- 150 mL of water at a ‰ow rate of about 4 mL per minute. cally insoluble in water. Melting point: about 1859C(with Combine the washing and the elute, and titrate with decomposition). 0.1 mol/L sodium hydroxide VS (potentiometric titration). Identiˆcation—Determine the infrared absorption spec- Perform a blank determination in the same manner, and trum of aconitine for purity as directed in the potassium make any necessary correction. bromide disk method under the Infrared Spectrophotomet- ry: it exhibits absorption at the wave numbers of about Each mL of 0.1 mol/L sodium hydroxide VS 3500 cm－1,1718cm－1, 1278 cm－1,1111cm－1, 1097 cm－1 ＝24.43 mg of C H NaO S 10 21 3 and 717 cm－1. 1z Thioglycolate medium I for sterility test See ‰uid Absorbance E1cm (230 nm): 211 – 243 [5 mg dried for thioglycolate medium. not less than 12 hours in a desiccator (reduced pressure not exceeding 0.67 kPa, phosphorus (V) oxide, 409C), ethanol Thioglycolate medium II for sterility test See alternative (99.5), 200 mL]. thioglycolate medium. Purity Related substances— Turmeric paper Macerate 20 g of powdered dried (1) Dissolve 5.0 mg of aconitine for purity in 2 mL of rhizome of Curcuma longa Linnáe with four 100 mL-portions acetonitrile, and use as the sample solution. Pipet 1 mL of of cold water, decant the supernatant liquid each time, and the sample solution, add acetonitrile to make exactly 50 mL, discard it. Dry the residue at a temperature not over 1009C. and use as the standard solution. Perform the test with these Macerate the dried residue with 100 mL of ethanol (95) for solutions as directed under the Thin-layer Chromatography. several days, and ˆlter. Immerse ˆlter paper in this ethanol Spot 20 mL each of the sample solution and the standard decoction, and allow the ethanol (95) to evaporate spontane- solution on a plate of silica gel for thin-layer chro- ously in clean air. matography, and proceed the test as directed in the Identiˆ- Sensitivity—Dip a strip of turmeric paper, about 1.5 cm cation in Processed Aconite Root: the spot other than the length, in a solution of 1 mg of boric acid in a mixture of principal spot obtained with the sample solution is not more 1 mL of hydrochloric acid and 4 mL of water, after 1 minute intense than the spot with the standard solution. remove the paper from the liquid, and allow it to dry spon- (2) Dissolve 5.0 mg of aconitine for purity in 5 mL of taneouly: the yellow color changes to brown. When the strip acetonitrile, and use as the sample solution. Pipet 1 mL of is moistened with ammonia TS, the color of the strip the sample solution, add acetonitrile to make exactly 50 mL, changes to greenish black. and use as the standard solution. Perform the test with

L-Tyrosine C9H11NO3 White, crystals or crystalline exactly 10 mL each of the sample solution and the standard powder. Odorless and tasteless. Freely soluble in formic solution as directed under the Liquid Chromatography ac- acid, very slightly soluble in water, and practically insoluble cording to the following conditions, and determine each in ethanol (95) and in diethyl ether. It dissolves in dilute peak area by the automatic integration method: the total hydrochloric acid and in dilute nitric acid. area of the peaks other than the peaks of aconitine and the 20 Optical rotation [a]D : －10.5 – －12.59 (after drying, solvent obtained with the sample solution is not larger than 2.5 g, 1 mol/L hydrochloric acid TS, 50 mL, 100 mm). the peak area of aconitine with the standard solution. Loss on drying: not more than 0.30z (1 g, 1059C, Operating conditions 3 hours). Detector, column, and column temperature: Proceed as Content: not less than 99.0z. Assay—Weigh accurately directed in the operating conditions in the Purity under about 0.3 g of L-tyrosine, previously dried, dissolve in 6 mL Processed Aconitine Root. of formic acid, add 50 mL of acetic acid (100), and titrate Mobile phase: A mixture of phosphate buŠer solution for with 0.1 mol/L perchloric acid VS (potentiometric titra- processed aconite root and tetrahydrofuran (9:1). tion). Perform a blank determination, and make any neces- Flow rate: Adjust the ‰ow rate so that the retention time Supplement II, JPXIV General Tests, Processes and Apparatus 1679 of aconitine is about 26 minutes. Ammonium amminetrichloroplatinate for liquid chro-

Time span of measurement: About 3 times as long as the matography Cl3H7N2Pt To 20 g of cisplatin add 600 mL retention time of aconitine. of 6 mol/L hydrochloric acid TS, and heat under a re‰ux System suitability condenserfor4–6hourstoboilwhilestirring.Aftercool- Test for required detectability: Pipet 1 mL of the standard ing, evaporate the solvent, and dry the orange residue at solution, and add acetonitrile to make exactly 20 mL. room temperature under reduced pressure. To the residue so Conˆrm that the peak area of aconitine obtained from 10 mL obtained add 300 mL of methanol, and heat at about 509C of this solution is equivalent to 3.5 to 6.5z of that obtained to dissolve. Filter, separate insoluble yellow solids, and wash from 10 mL of the standard solution. the solids with 10 mL of methanol. Combine the ˆltrate and System performance: Dissolve 1 mg each of aconitine for the washing, heat at about 509C, and add slowly 100 mL of purity, hypaconitine for purity and mesaconitine for purity, ethyl acetate while stirring. Cool the mixture to room and 8 mg of jesaconitine for purity in 200 mL of acetonitrile. temperature avoiding exposure to light, and allow to stand When the procedure is run with 10 mL of this solution under at －109C for 1 hour. Filter the mixture to take oŠ the the above operating conditions, mesaconitine, hypaconitine, formed crystals, wash the crystals with 100 mL of acetone, aconitine and jesaconitine are eluted in this order, and each combine the washing to the ˆltrate, and evaporate to dryness resolution between these peaks is not less than 1.5, respec- to obtain orange crystals. If necessary, repeat the puriˆca- tively. tion procedure described above to take oŠ the insoluble System repeatability: When the test is repeated 6 times crystals. To the orange crystals obtained add 300 to 500 mL with 10 mL of the standard solution under the above operat- of a mixture of acetone and methanol (5:1), and heat at ing conditions, the relative standard deviation of the peak about 509C while stirring to dissolve. Filter while hot to take area of aconitine is not more than 1.5z. oŠ the insoluble crystals, wash the crystals with the mixture, Water: not more than 1.0z [5 mg dried for not less than and combine the ˆltrate and washing. Repeat the procedure 12 hours in a desiccator (reduced pressure not exceeding several times, and evaporate to dryness. Suspense the 0.67 kPa, phosphorus (V) oxide, 409C), coulometric titra- crystals so obtained in 50 mL of acetone, ˆlter, wash the tion]. crystals with 20 mL of acetone, and dry the crystals at room temperature under reduced pressure. It is a yellow-brown Aconitum diester alkaloids standard solution for purity crystalline powder. It is a solution containing 10 mg of aconitine for purity, Identiˆcation—Determine the infrared absorption spec- 10 mg of jesaconitine for purity, 30 mg of hypaconitine for trum of the substance to be examined, previously dried at purity and 20 mg of mesaconitine for purity in 1000 mL of a 809C for 3 hours, as directed in the potassium bromide disk mixture of phosphate buŠer solution for processed aconite method under the Infrared Spectrophotometry: it exhibits root and acetonitrile (1:1). When proceed the test with 20 mL absorption at the wave numbers of about 3480 cm－1, of this solution as directed in the Purity under Processed 3220 cm－1, 1622 cm－1, 1408 cm－1 and 1321 cm－1. Aconite Root, using the detection wavelength, 231 nm, the Related substances—Cisplatin Conduct this procedure peaks of aconitine, jesaconitine, hypaconitine and using light-resistant vessels. Dissolve 10 mg in N,N- mesaconitine are observed, and the ratio of their peak dimethylformamide to make exactly 10 mL, and use this heights is about 10:1:35:30. When proceed the test using the solution as the sample solution. Separately, dissolve 10 mg detection wavelength, 254 nm, the peaks of aconitine, of Cisplatin in N,N-dimethylformamide to make exactly jesaconitine, hypaconitine and mesaconitine are observed, 50 mL. Pipet 5 mL of this solution, add N,N-dimethylfor- and the ratio of their peak heights is about 2:8:7:6. mamide to make exactly 20 mL, and use this solution as the Alternative thioglycolate medium See the Sterility Test standard solution. Perform the test with exactly 40 mLeach under the General Tests, Processes and Apparatus. of the sample solution and the standard solution as directed under the Liquid Chromatography according to the follow- 4-(Aminomethyl)benzoic acid C H NO Awhitepow- 8 9 2 ing conditions, and determine the peak area of cisplatin by der. the automatic integration method: the peak area from the Purity—Dissolve 10 mg of 4-(aminomethyl)benzoic acid sample solution is not more than that from the standard in 100 mL of water, and use this as the sample solution. solution. Pipet 1 mL of the sample solution, add water to make Operating conditions exactly 20 mL, and use this solution as the standard Proceed as directed in the operating conditions in the solution. Perform the test with exactly 20 mLeachofthe Assay under Cisplatin. sample solution and the standard solution as directed under System suitability the Liquid Chromatography according to the operating System performance: When the procedure is run with conditions as directed in the Purity (5) under Tranexamic 40 mL of the standard solution under the above operating Acid, and determine each peak area by the automatic conditions, the number of theoretical plates and the symmet- integration method: each area of the peak other than 4- ry factor of the peak of cisplatin are not less than 2500 and (aminomethyl)benzoic acid obtained from the sample solu- not more than 2.0, respectively. tion is not more than the peak area of 4-(aminomethyl)ben- System repeatability: When the test is repeated 6 times zoic acid from the standard solution. with 40 mL of the standard solution under the above 1680 General Tests, Processes and Apparatus Supplement II, JPXIV operating conditions, the relative standard deviation of the Calibration ball with a known volume prepared for measure- peak area of cisplatin is not more than 5.0z. ment of particle density. The volume of the calibration ball must be accurately determined to the nearest 0.001 cm3. Benidipine hydrochloride C28H31N3O6.HCl [Same as thenamesakemonograph] Caprylic acid CH3(CH2)6COOH A clear and colorless oily liquid, having a slight unpleasant odor. Freely soluble in Benidipine hydrochloride for assay C H N O .HCl 28 31 3 6 ethanol (95) and in chloroform, and very slightly soluble in [Same as the monograph Benidipine Hydrochloride. When water. dried, it contains not less than 99.5z of benidipine 20 Refractive index: nD 1.426 – 1.430 hydrochloride (C28H31N3O6.HCl)] 20 Speciˆc gravity d4 : 0.908 – 0.912

Benzoin C6H5CH(OH)COC6H5 White to pale yellow, Distilling range 238 – 2429C, not less than 95 volz. crystals or powder. Chlorobutanol C H Cl O [Same as the namesake Melting point:132–1379C 4 7 3 monographinPartII] N-Benzoyl-L-isoleucyl-L-glutamyl(g-OR)-glycyl-L-arginyl- Chlorogenic acid for thin-layer chromatography p-nitroanilide hydrochloride An equal amount mixture of C H O .xH O A white powder. Freely soluble in two components, R＝HandR＝CH .Awhitepowder. 16 18 9 2 3 methanol and in ethanol (99.5), and sparingly soluble in Slightly soluble in water. water. Melting point: about 2059C (with decomposition). Absorbance E1z (316nm): 166–184 (10mg, water, 1cm Purity Related substances—Dissolve 1.0 mg of chloro- 300 mL). genic acid for thin-layer chromatography in 2 mL of Benzoylmesaconine hydrochloride for thin-layer chro- methanol, and use this solution as the sample solution. matography C31H43NO10.HCl.xH2O White, crystals or Perform the test with this solution as directed under the crystalline powder. Soluble in water and in ethanol (99.5). Thin-layer Chromatography. Spot 10 mLofthesample Melting point: about 2509C (with decomposition). solution on a plate of silica gel for thin-layer chro- Purity Related substances—Dissolve 1.0 mg of ben- matography, develop the plate with a mixture of ethyl zoylmesaconine hydrochloride for thin-layer chro- acetate, water and formic acid (6:1:1) to a distance of about matography in exactly 10 mL of ethanol (99.5), and use this 10 cm, and air-dry the plate. Examine under ultraviolet light solution as the sample solution. Perform the test with this (main wavelength: 365 nm): no spot other than the principal solution as directed under the Thin-layer Chromatography. spot at around Rf 0.5 appears. Spot 10 mL of the sample solution on a plate of silica gel for Cisplatin [Same as the namesake monograph] thin-layer chromatography. Proceed the test as directed in the Identiˆcation under Processed Aconite Root: no spot Cyclohexylamine C6H11NH2 A clear and colorless other than the principal spot at around Rf 0.4 appears. liquid, having a characteristic amine-like odor. Miscible with water, with N,N-dimethylformamide and with acetone. 4,4?-Bis(diethylamino)benzophenone Purity Related substances—Use cyclohexylamine as the (C H ) NC H ] CO Light yellow crystals. 2 5 2 6 4 2 sample solution. Separately, pipet 1 mL of cyclohexylamine, Content:notlessthan98z. Assay—Weigh accurately add hexane to make exactly 100 mL, and use this as the stan- 0.25 g of 4,4?-bis(diethylamino)benzophenone, dissolve in dard solution. Perform the test as directed in the Thin-layer 50 mL of acetic acid (100), and titrate with 0.1 mol/L Chromatography. Spot 5 mL each of the sample solution and perchloric acid VS (potentiometric titration). Perform a the standard solution on a plate of silica gel for thin-layer blank titration in the same manner, and make any necessary chromatography, develop the plate with a mixture of ethyl correction. acetate, methanol, ammonia water (28) and cyclohexane Each mL of 0.1 mol/L perchloric acid VS (6:2:1:1) to a distance of about 10 cm, and air-dry the plate.

＝16.22 mg of C21H28N2O Allow the plate to stand in iodine vapor: the spot other than the principal spot obtained with the sample solution is not Borate-hydrochloric acid buŠer solution, pH 9.0 Dis- more intense than the spot with the standard solution. solve 19.0 g of sodium borate in 900 mL of water, adjust the pH to exactly 9.0 with 1 mol/L hydrochloric acid TS, and Cyclohexylmethanol C7H14O A liquid having slight add water to make 1000 mL. camphor odor. Soluble in ethanol (99.5). Refractive index n20: about 1.464 Bovine activated blood coagulation factor X Aprotein D Bioling point: about 1859C obtained from bovine plasma. It has an activity to decom- pose prothrombin speciˆcally and limitedly and produce N,N?-Dibenzylethylenediamine diacetate Awhiteto thrombin. It does not contain thrombin and plasmin. It slightly pale yellow crystalline powder. contains not less than 500 Units per mg protein. One unit Identiˆcation—Determine the infrared absorption spec- indicates an amount of the factor X which hydrolyzes trum of the substance to be examined as directed in the 1 mmol of N-benzoyl-L-isoleucyl-L-glutamyl(g-OR)-glycyl-L- potassium bromide disk method under the Infrared Spec- arginyl-p-nitroanilide in 1 minute at 259C. trophotometry: it exhibits absorption at the wave numbers of about 1530 cm－1, 1490 cm－1, 1460 cm－1,1400cm－1 and Calibration ball for particle density measurement Supplement II, JPXIV General Tests, Processes and Apparatus 1681

1290 cm－1. tion between these peaks being not less than 20. Content: not less than 99.0z. Assay—Weigh accurately System repeatability: When the test is repeated 6 times about 25 mg of N,N?-dibenzylethylenediamine diacetate, with 20 mL of the standard solution under the above operat- dissolve in 25 mL of methanol, and add a solution contain- ing conditions, the relative standard deviation of the peak ing 1.02 g of disodium hydrogen phosphate and 6.80 g of area of N,N?-dibenzylethylenediamine is not more than potassium dihydrogen phosphate in 1000 mL of water to 2.0z. make exactly 50 mL. Pipet 5 mL of this solution, add a 1,2-Dichlorobenzene C H Cl A colorless liquid. mixture of the solution containing 1.02 g of disodium hydro- 6 4 2 Speciˆc gravity d20: 1.306 gen phosphate and 6.80 g of potassium dihydrogen phos- 4 Boiling point: 180 – 1819C phate in 1000 mL of water and methanol (1:1) to make exactly 20 mL, and use this solution as the sample solution. 2,6-Dichlorophenol C6H4Cl2O White to purplish white Separately, weigh accurately about 8 mg of acetic acid (100), crystals. add 25 mL of methanol, and add the solution containing Melting point:65–679C 1.02 g of disodium hydrogen phosphate and 6.80 g of Dicyclohexyl C H potassium dihydrogen phosphate in 1000 mL of water to 12 22 Speciˆc gravity d20: about 0.864 make exactly 50 mL. Pipet 5 mL of this solution, add a 20 Boiling point: about 2279C mixture of the solution containing 1.02 g of disodium hydro- Melting point: about 49C gen phosphate and 6.80 g of potassium dihydrogen phos- phate in 1000 mL of water and methanol (1:1) to make Dicyclohexylurea C6H11NHCONHC6H11 Awhitecrys- exactly 20 mL, and use this solution as the control solution. talline powder, having no odor. Perform the test with exactly 20 mL each of the sample solu- Purity Related substances—Dissolve 50 mg of dicyclo- tion and the control solution as directed under the Liquid hexylurea in methanol to make 100 mL. Pipet 10 mL of this Chromatography according to the following conditions, and solution, and add methanol to make 100 mL. Pipet 20 mL of determine each peak area by the automatic integration this solution, add 10 mL of 0.5 mol/L sodium hydroxide method. After making correction for the peak areas based TS, shake, then add 5 mL of diluted hydrochloric acid (1 in on the valiance of the base-line and the peak of acetic acid 10), and shake. Perform the test with 50 mL of this solution on the chromatogram obtained with the sample solution, as directed under the Liquid Chromatography according to calculate the amount of N,N?-dibenzylethylenediamine by the following conditions, determine the area of each peak by the area percentage method. the automatic integration method, and calculate the amount Operating conditions by the area percentage method: the total amount of the Detector: An ultraviolet absorption photometer peaks other than dicyclohexylurea is not more than 3.0z. (wavelength: 220 nm). Operating conditions Column: A stainless steel column 4.6 mm in inside Detector, column, column temperature, mobile phase, diameter and 25 cm in length, packed with octadecyl- and ‰ow rate: Proceed as directed in the operating condi- silanized silica gel for liquid chromatography (5 mmin tions in the Purity (5) (ii) under Acetohexamide. particle diameter). Time span of measurement: About 5 times as long as the Column temperature: A constant temperature of about retention time of dicyclohexylurea after the solvent peak. 409C. System suitability Mobile phase: A mixture of water, methanol and 0.25 Test for required detectability: To exactly 5 mL of the mol/L potassium dihydrogen phosphate TS, pH 3.5 standard solution add water to make exactly 200 mL. (11:7:2). Conˆrm that the peak area of dicyclohexylurea obtained Flow rate: Adjust the ‰ow rate so that the retention time with 50 mL of this solution is equivalent to 1.8 to 3.3z of of N,N?-dibenzylethylenediamine is about 4 minutes. that with 50 mL of the standard solution. Time span of measurement: About 5 times as long as the System performance, and system repeatability: Proceed as retention time of N,N?-dibenzylethylenediamine. directed in the system suitability in the Purity (5) (ii) under System suitability Acetohexamide. System performance: Dissolve an amount of Benzyl- Digoxin C H O [Same as the namesake mono- penicillin Benzathine, equivalent to about 85,000 Units, in 41 64 14 graph] 25 mL of methanol, add a solution containing 1.02 g of disodium hydrogen phosphate and 6.80 g of potassium 0.04 mol/L Disodium dihydrogen ethylenediamine tetraa- dihydrogen phosphate in 1000 mL of water to make exactly cetate TS Dissolve 14.890 g of disodium dihydrogen 50 mL. Pipet 5 mL of this solution, add a mixture of the ethylenediamine tetraacetate dihydrate in water to make solution containing 1.02 g of disodium hydrogen phosphate 1000 mL. and 6.80 g of potassium dihydrogen phosphate in 1000 mL Ethylbenzene C H C H A colorless liquid. Freely of water and methanol (1:1) to make exactly 20 mL. When 6 5 2 5 soluble in acetone and in ethanol (99.5), and practically in- theprocedureisrunwith20mLofthissolutionunderthe soluble in water. above operating conditions, N,N?-dibenzylethylenediamine Speciˆc gravity d20:0.862¿0.872 and benzylpenicillin are eluted in this order with the resolu- 4 1682 General Tests, Processes and Apparatus Supplement II, JPXIV

Boiling point: about 1359C silica gel for thin-layer chromatography, and proceed the test as directed in the Identiˆcation in Processed Aconite Flopropione [Same as the namesake monograph] Root: the spot other than the principal spot obtained with Flopropione for assay [Same as the monograph the sample solution is not more intense than the spot with Flopropione. It contains not less than 99.0z of ‰opropione the standard solution.

(C9H10O4), calculated on the anhydrous basis.] (2) Dissolve 5.0 mg of hypaconitine for purity in 5 mL of acetonitrile, and use as the sample solution. Pipet 1 mL of Fluid thioglycolate medium See the Sterility Test under the sample solution, add acetonitrile to make exactly 50 mL, the General Tests, Processes and Apparatus. and use as the standard solution. Perform the test with ex-

85z Glycerin C3H8O3 [Same as the monograph actly 10 mL each of the sample solution and the standard so- Glycerin] lution as directed under the Liquid Chromatography accord- ing to the following conditions, and determine each peak Human antithrombin III Serine protease inhibition area by the automatic integration method: the total area of factor obtained from normal plasma of health human. It is a the peaks other than the peaks of hypaconitine and the sol- protein, which inhibits the activities of thrombin and vent obtained with the sample solution is not larger than the activated blood coagulation factor X. It contains not less peak area of hypaconitine with the standard solution. than 300 Units per mg protein. One unit indicates an amount Operating conditions of the antithrombin III which inhibits 1 unit of thrombin at Detector, column, and column temperature: Proceed as 259C under the existence of heparin. directed in the operating conditions in the Purity under Dried human normal plasma powder Freeze-dried Processed Aconite Root. normal plasma obtained from healthy human. Mobile phase: A mixture of phosphate buŠer solution for processed aconite root and tetrahydrofuran (9:1). Hydrogen peroxide-sodium hydroxide TS To a mixture Flow rate: Adjust the ‰ow rate so that the retention time of water and hydrogen peroxide (30) (9:1) add 3 drops of of hypaconitine is about 23 minutes. bromophenol blue TS, and then add 0.01 mol/L sodium Time span of measurement: About 3 times as long as the hydroxide TS until a purple-blue color develops. Prepare retention time of hypaconitine. before use. System suitability N-(2-Hydroxyethyl)isonicotinamide nitric ester Test for required detectability: Pipet 1 mL of the standard

C8H9N3O4 A white crystalline powder. solution, and add acetonitrile to make exactly 20 mL. Identiˆcation—Determine the infrared absorption spec- Conˆrm that the peak area of hypaconitine obtained from trum of N-(2-hydroxyethyl)isonicotinamide nitric ester as 10 mL of this solution is equivalent to 3.5 to 6.5z of that directed in the potassium bromide disk method under the obtained from 10 mL of the standard solution. Infrared Spectrophotometry: it exhibits absorption at the System performance: Dissolve 1 mg each of aconitine for wave numbers of about 3270 cm－1, 1653 cm－1, 1546 cm－1 purity, hypaconitine for purity and mesaconitine for purity, and 1283 cm－1. and 8 mg of jesaconitine for purity in 200 mL of acetonitrile. When the procedure is run with 10 mLofthissolutionunder Hypaconitine for purity C H NO White, crystals or 33 45 10 the above operating conditions, mesaconitine, hypaconitine, crystalline powder. Soluble in acetonitrile, sparingly soluble aconitine and jesaconitine are eluted in this order, and each in ethanol (99.5) and in diethyl ether, and practically insolu- resolution between these peaks is not less than 1.5, respec- ble in water. Melting point: about 1759C (with decomposi- tively. tion). System repeatability: When the test is repeated 6 times Identiˆcation—Determine the infrared absorption spec- with 10 mL of the standard solution under the above operat- trum of hypaconitine for purity as directed in the potassium ing conditions, the relative standard deviation of the peak bromide disk method under the Infrared Spectrophotomet- area of hypaconitine is not more than 1.5z. ry: it exhibits absorption at the wave numbers of about Water: not more than 1.0z [5 mg dried for not less than 3500 cm－1,1728cm－1,1712cm－1, 1278 cm－1, 1118 cm－1, 12 hours in a desiccator (reduced pressure not exceeding 1099 cm－1 and 714 cm－1. 0.67 kPa, phosphorus (V) oxide, 409C), coulometric titra- Absorbance E1z (230 nm): 217 – 252 [5 mg dried for 1cm tion]. not less than 12 hours in a desiccator (reduced pressure not exceeding 0.67 kPa, phosphorus (V) oxide, 409C), ethanol Icariin for thin-layer chromatography C33H40O15 Light (99.5), 200 mL]. yellow crystals. Very slightly soluble in methanol and in Purity Related substances—(1) Dissolve 5.0 mg of ethanol (99.5), and practically insoluble in water. Melting hypaconitine for purity in 2 mL of acetonitrile, and use as point: about 2349C (with decomposition). the sample solution. Pipet 1 mL of the sample solution, add Purity Related substances—Dissolve 1.0 mg of icariin acetonitrile to make exactly 50 mL, and use as the standard for thin-layer chromatography in 1 mL of methanol. Per- solution. Perform the test with these solutions as directed form the test with 10 mL of this solution as directed in the under the Thin-layer Chromatography. Spot 20 mLeachof Identiˆcation under Epimedium Herb: no spot other than the sample solution and the standard solution on a plate of the principal spot having Rf value about 0.4 appears. Supplement II, JPXIV General Tests, Processes and Apparatus 1683

L-Isoleucine C6H13NO2 [Same as the namesake mono- the above operating conditions, mesaconitine, hypaconitine, graph] aconitine and jesaconitine are eluted in this order, and each resolution between these peaks is not less than 1.5, respec- Jesaconitine for purity C H NO A white powder. 35 49 12 tively. Freelysolubleinacetonitrile,inethanol(99.5)andindiethyl System repeatability: When the test is repeated 6 times ether, and practically insoluble in water. with 10 mL of the standard solution under the above operat- Identiˆcation—Determine the infrared absorption spec- ing conditions, the relative standard deviation of the peak trum of jesaconitine for purity as directed in the potassium area of jesaconitine is not more than 1.5z. bromide disk method under the Infrared Spectrophotomet- Water: not more than 1.0z [5 mg dried for not less than ry: it exhibits absorption at the wave numbers of about 12 hours in a desiccator (reduced pressure not exceeding 3500 cm－1,1715cm－1,1607cm－1, 1281 cm－1, 1259 cm－1, 0.67 kPa, phosphorus (V) oxide, 409C), coulometric 1099 cm－1 and 772 cm－1. 1z titration]. Absorbance E1cm (258 nm): 270 – 291 [5 mg dried for not less than 12 hours in a desiccator (reduced pressure not Loganin for thin-layer chromatography C17H26O10 exceeding 0.67 kPa, phosphorus (V) oxide, 409C), ethanol White, crystals or crystalline powder. Soluble in water, (99.5), 200 mL]. sparingly soluble in methanol, and very slightly soluble in Purity Related substances—(1) Dissolve 5.0 mg of ethanol (99.5). Melting point: 221 – 2279C. jesaconitine for purity in 2 mL of acetonitrile, and use as the Purity Related substances—Dissolve 1.0 mg of loganin sample solution. Pipet 1 mL of the sample solution, add for thin-layer chromatography in 2 mL of methanol. Per- acetonitrile to make exactly 50 mL, and use as the standard form the test with 10 mL of this solution as directed in the solution. Perform the test with these solutions as directed Identiˆcation under Cornus Fruit: any spot other than the under the Thin-layer Chromatography. Spot 20 mLeachof principal spot at the Rf value of about 0.4 does not appear. the sample solution and the standard solution on a plate of Mesaconitine for purity C H NO White, crystals or silica gel for thin-layer chromatography, and proceed the 33 45 11 crystalline powder. Slightly soluble in acetonitrile and in test as directed in the Identiˆcation in Processed Aconite ethanol (99.5), very slightly soluble in diethyl ether, and Root: the spot other than the principal spot is not more in- practically insoluble in water. Melting point: about 1909C tense than the spot with the standard solution. (with decomposition). (2) Dissolve 5.0 mg of jesaconitine for purity in 5 mL of Identiˆcation—Determine the infrared absorption spec- acetonitrile, and use as the sample solution. Pipet 1 mL of trum of mesaconitine for purity as directed in the potassium the sample solution, add acetonitrile to make exactly 50 mL, bromide disk method under the Infrared Spectrophotomet- and use as the standard solution. Perform the test with ex- ry: it exhibits absorption at the wave numbers of about actly 10 mL each of the sample solution and the standard so- 3510 cm－1,1713cm－1, 1277 cm－1,1116cm－1, 1098 cm－1 lution as directed under the Liquid Chromatography accord- and 717 cm－1. ing to the following conditions, and determine each peak Absorbance E1z (230 nm): 211 – 247 [5 mg dried for area by the automatic integration method: the total area of 1cm not less than 12 hours in a desiccator (reduced pressure not the peaks other than the peaks of jesaconitine and the sol- exceeding 0.67 kPa, phosphorus (V) oxide, 409C), ethanol vent is not larger than the peak area of jesaconitine with the (99.5), 200 mL]. standard solution. Purity Related substances—(1) Dissolve 5.0 mg of Operating conditions mesaconitine for purity in 2 mL of acetonitrile, and use as Detector, column, and column temperature: Proceed as the sample solution. Pipet 1 mL of the sample solution, add directed in the operating conditions in the Purity under acetonitrile to make exactly 50 mL, and use as the standard Processed Aconite Root. solution. Perform the test with these solutions as directed Mobile phase: A mixture of phosphate buŠer solution for under the Thin-layer Chromatography. Spot 20 mLeachof processed aconite root and tetrahydrofuran (9:1). the sample solution and the standard solution on a plate of Flow rate: Adjust the ‰ow rate so that the retention time silica gel for thin-layer chromatography, and proceed the of jesaconitine is about 36 minutes. test as directed in the Identiˆcation under Processed Aconite Time span of measurement: About 3 times as long as the Root: the spot other than the principal spot is not more in- retention time of jesaconitine. tense than the spot with the standard solution. System suitability (2) Dissolve 5.0 mg of mesaconitine for purity in 5 mL Test for required detectability: Pipet 1 mL of the standard of acetonitrile, and use as the sample solution. Pipet 1 mL of solution, and add acetonitrile to make exactly 20 mL. the sample solution, add acetonitrile to make exactly 50 mL, Conˆrm that the peak area of jesaconitine obtained from and use as the standard solution. Perform the test with ex- 10 mL of this solution is equivalent to 3.5 to 6.5z of that actly 10 mL each of the sample solution and the standard so- obtained from 10 mL of the standard solution. lution as directed under the Liquid Chromatography accord- System performance: Dissolve 5 mg each of aconitine for ing to the following conditions, and determine each peak purity, hypaconitine for purity and mesaconitine for purity, area by the automatic integration method: the total area of and 1 mg of jesaconitine for purity in 200 mL of acetonitrile. the peaks other than the peaks of mesaconitine and the sol- When the procedure is run with 10 mL of this solution under vent is not larger than the peak area of mesaconitine with the 1684 General Tests, Processes and Apparatus Supplement II, JPXIV standard solution. practically insoluble in water. Melting point: 83 – 849C. Operating conditions Purity Related substances—Dissolve 1.0 mg of osthole Detector, column, and column temperature: Proceed as for thin-layer chromatography in 1 mL of methanol. Per- directed in the operating conditions in the Purity under form the test with 10 mL of this solution as directed in the Processed Aconite Root. Identiˆcation under Cnidium Monnieri Fruit: on spot ap- Mobile phase: A mixture of phosphate buŠer solution for pears other than the principal spot at around Rf0.3. processed aconite root and tetrahydrofuran (9:1). Oxytocin C H N O S [Same as the namesake Flow rate: Adjust the ‰ow rate so that the retention time 43 66 12 12 2 monograph] of mesaconitine is about 19 minutes.

Time span of measurement: About 3 times as long as the D-Phenylglycine C8H9NO2 White, crystals or crystal- retention time of mesaconitine. line powder. Slightly soluble in water. System suitability Loss on drying:notmorethan0.5z (1 g, 1059C, Test for required detectability: Pipet 1 mL of the standard 3 hours). solution, and add acetonitrile to make exactly 20 mL. Content:notlessthan98.5z. Assay—Weigh accurately Conˆrm that the peak area of mesaconitine obtained from about 0.3 g of D-phenylglycine, previously dried, dissolve in 10 mL of this solution is equivalent to 3.5 to 6.5z of that 3 mL of formic acid, add 50 mL of acetic acid (100), and obtained from 10 mL of the standard solution. titrate with 0.1 mol/L perchloric acid VS (potentiometric System performance: Dissolve 1 mg each of aconitine for titration). Perform a blank determination in the same purity, hypaconitine for purity and mesaconitine for purity, manner, and make any necessary correction. and 8 mg of jesaconitine for purity in 200 mL of acetonitrile. Each mL of 0.1 mol/L perchloric acid VS When the procedure is run with 10 mL of this solution under ＝15.12 mg of C H NO the above operating conditions, mesaconitine, hypaconitine, 8 9 2 aconitine and jesaconitine are eluted in this order, and each Phenylpiperazine hydrochloride C10H14N2.HCl A resolution between these peaks is not less than 1.5, respec- white powder. tively. Melting point: about 2479C (with decomposition). System repeatability: When the test is repeated 6 times Phenylsilanized silica gel for liquid chromatography with 10 mL of the standard solution under the above operat- Prepared for liquid chromatography. ing conditions, the relative standard deviation of the peak area of mesaconitine is not more than 1.5z. 0.05 mol/L Phosphate buŠer solution, pH 3.5 To 1000 Water: not more than 1.0z [5 mg dried for not less than mL of 0.05 mol/L potassium dihydrogen phosphate TS add 12 hours in a desiccator (reduced pressure not exceeding a suitable amount of a solution of phosphoric acid (49 in 0.67 kPa, phosphorus (V) oxide, 409C), coulometric titra- 10,000) to make a solution having pH 3.5. tion]. Phosphate buŠer solution for processed aconite root

Methanol for liquid chromatography CH3OH A clear, Dissolve 19.3 g of disodium hydrogen phosphate 12-water in colorless liquid. Mixable with water. 3660 mL of water, and add 12.7 g of phosphoric acid. Purity Ultraviolet-absorbing substances—Perform the [6]-Shogaol for thin-layer chromatography C H O test as directed in the Ultraviolet-visible Spectrophotometry 17 24 3 A pale yellow oil. Miscible with methanol, ethanol (99.5) using water as the blank: the absorbances at 210 nm, at and with diethyl ether, and practically insoluble in water. 220 nm, at 230 nm, at 240 nm and at 254 nm are not more Purity Related substances—Dissolve 1.0 mg of [6]- than 0.70, 0.30, 0.15, 0.07 and 0.02, respectively. shogaol for thin-layer chromatography in 2 mL of Metoclopramide for assay [Same as the monograph methanol, and perform the test with this solution as directed Metoclopramide.Whendried,itcontainsnotlessthan under the Thin-layer chromatography. Spot 10 mLonaplate

99.0z of metoclopramide (C14H22ClN3O2).] of silica gel for thin-layer chromatography, develop the plate with a mixture of ethyl acetate and hexane (1:1) to a distance 1,3-Naphthalenediol C H O Red-brown crystals or 10 8 2 of about 10 cm, and air-dry the plate. Spray evenly 4- gray-brown powder. Freely soluble in water, in methanol dimethylaminobenzaldehyde TS on the plate, heat at 1059C and in ethanol (99.5). Melting point: about 1249C. for 5 minutes, and allow to cool: no spot other than the 1,3-Naphthalenediol TS Dissolve 50 mg of 1,3- principal spot at around Rf 0.5 appears. naphthalenediol in 25 mL of ethanol (99.5), and add 2.5 mL 0.0375 mol/L Sodium 1-decanesulfonate TS Dissolve of phosphoric acid. 3.665 g of sodium 1-decanesulfonate in 400 mL of water. 3-Nitrophenol C H NO A light yellow crystalline 6 5 3 Sodium dihydrogen phosphate NaH PO Awhite, powder. 2 4 powder or crystalline powder. Freely soluble in water, and Melting point:96–999C very slightly soluble in ethanol (99.5). It has a hygroscopic

Osthole for thin-layer chromatography C15H16O3 A property. white crystalline powder, having no odor. Freely soluble in A solution is acidic. methanol and in ethyl acetate, soluble in ethanol (99.5), and Supplement II, JPXIV General Tests, Processes and Apparatus 1685

Soybean-casein digest medium See the Sterility Test Note: Store protected from light. This solution, if stored under the General Tests, Processes and Apparatus. for a long period, should be restandardized before use.

Thioacetamide C H NS A white crystalline powder or 2 5 Add the following: colorless crystals. Freely soluble in water and in ethanol (99.5). Melting point: 112 – 1159C Sodium Thiosulfate, 0.002 mol/L 1000 mL of this solution contains 0.4964 g of sodium Thioacetamide TS To 0.2 mL of a solution of thioa- thiosulfate pentahydrate (Na S O .5H O: 248.19). cetamide (1 in 25) add 1 mL of a mixture of 15 mL of 2 2 3 2 Preparation—Before use, dilute 0.1 mol/L sodium sodium hydroxide TS, 5 mL of water and 20 mL of 85z thiosulfate VS with freshly boiled and cooled water to make glycerin, and heat in a water bath for 20 seconds. Prepare exactly 50 times the initial volume. before use.

Tiaramide hydrochloride for assay [Same as the monograph Tiaramide Hydrochloride. When dried, it con- Add the following: tains not less than 99.0z of tiaramide hydrochloride (C15H18ClN3O3S.HCl).] 74. Powder Particle Density Trichloroacetic acid TS for serrapeptase Dissolve 1.80 g Determination of trichloroacetic acid and 1.80 g of anhydrous sodium Powder Particle Density Determination is a method to acetate in 5.5 mL of 6 mol/L acetic acid TS and water to determine particle density of powdered pharmaceutical make 100 mL. drugsorrawmaterialsofdrugs,andthegasdisplacement

Trichloro‰uoromethane CCl3F A colorless liquid or pycnometer is generally used. The powder density by this gas. method is determined with an assumption that the volume of 17.2 Speciˆc gravity d4 :1.494 the gas displaced by the powder in a closed system is equal to Boiling point:23.79C the volume of the powder. The bulk density at loose packing or the tapped density at tapping express the apparent Vasopressin C H N O S A white powder. 46 65 15 12 2 densities of the powder, since interparticulate void volume Constituent amino acids—Perform the test as directed in of the powder is assumed to contribute a part of the volume the Constituent amino acids under Oxytocin, and calculate of the powder. On the contrary, the pycnometric particle the respective molar ratios with respect to glycine: 0.9 – 1.1 density expresses the powder density nearly equal to the crys- for aspartic acid, 0.9 – 1.1 for glutamic acid, 0.9 – 1.1 for tal density, since the volume of the powder, that is deducted proline, 0.8 – 1.1 for tyrosine, 0.9 – 1.1 for phenylalanine, with void volume of open pores accessible to gas, is counted. 0.9–1.1forarginineand0.8–1.1forcystine,andnotmore Powder particle density is expressed in mass per unit than 0.03 for other amino acids. volume (kg/m3), and generally expressed in g/cm3. 0.04 mol/L Zinc chloride TS Dissolve 5.452 g of zinc Apparatus chloride in water to make 1000 mL. The schematic diagram of particle density apparatus for gas displacement pycnometric measurement is shown in Figure 1. The apparatus consists of a test cell in which the (3) Standard Solutions for sample is placed, a reference cell and a manometer. Volumetric Analysis Generally, helium is used as the measurement gas. The apparatus has to be equipped with a system capable of Change the following: pressuring the test cell to the deˆned pressure through the manometer. Iodine, 0.05 mol/L Calibration of apparatus The volumes of the test cell 1000 mL of this solution contains 12.690 g of iodine (V ) and the reference cell (V )mustbeaccuratelydeter- (I: 126.90). c r minedtothenearest0.001cm3, and to assure accuracy of the Preparation—Dissolve 13 g of iodine in 100 mL of a solu- results of volume obtained, calibration of the apparatus is tion of potassium iodide (2 in 5), add 1 mL of dilute carried out as follows using a calibration ball of known hydrochloric acid and water to make 1000 mL, and stan- volume for particle density measurement. The ˆnal pressures dardize the solution as follows: (P ) are determined for the initial empty test cell followed by Standardization—Measure exactly 15 mL of the iodine f the test cell placed with the calibration ball for particle solution, and titrate with 0.1 mol/L sodium thiosulfate VS density measurement in accordance with the procedures, and (Indicator method: starch TS; or potentiometric titration: V and V are calculated using the equation described in the platinum electrode). In the indicator method, when the c r section of Procedure. Calculation can be made taking into solution assumes a pale yellow color as the end point is account that the sample volume (V ) is zero in the ˆrst run. approached, add 3 mL of starch TS, and continue the titra- s tion until the blue color disappears. Calculate the molarity factor. 1686 General Tests, Processes and Apparatus Supplement II, JPXIV

the system is stable, and then read the system reference

pressure (Pr). Secondly, close the valve that connects to the two cells, and introduce the measurement gas into the test cell to achieve positive pressure. Conˆrm with the manome- ter that the pressure inside the system is stable, and then read

the initial pressure (Pi). Open the valve to connect the test cell with the reference cell. After conˆrming that the indica-

torofthemanometerisstable,readtheˆnalpressure(Pf),

and calculate the sample volume (Vs) with the following equation.

3 Vr Vr: Reference cell volume (cm ) Vs＝Vc－ 3 Pi－Pr Vc: Test cell volume (cm ) －1 3 Pf－Pr Vs: Sample volume (cm ) 3 M: Manometer Vr: Reference cell volume (cm ) 3 Vc: Test cell volume (cm ) Figure 1. Schematic diagram of a gas displacement py- 3 Vs: Sample volume (cm ) cnometer Pi:Initialpressure(kPa)

Pf: Final pressure (kPa) Procedure Pr: Reference pressure (kPa) The measurement of the particle density is carried out Repeat the measurement sequence for the same powder between 15 and 309C, and temperature must not vary by sample until consecutive measurements of the sample more than 29C during the course of measurement. volume agree to within 0.5z, and calculate the mean of Firstly, weigh the mass of the test cell and record it. After sample volumes (Vs). Finally, unload the test cell, weigh the weighing out the amount of the sample as described in the mass of test cell, and calculate the ˆnal sample mass by individual monograph and placing it in the test cell, seal the deducting the empty cell mass from the test cell mass. The cell in the pycnometer. Secondly, introduce the measure- powder particle density r is calculated by the following ment gas (helium) into the test cell, and remove volatile equation. contaminants in the powder. If necessary, keep the sample m powder under reduced pressure to remove the volatile r＝ Vs contaminants in advance and use it as the test sample for r: Powder particle density (g/cm3) measurement. m: Final sample mass (g) Open the valve which connects the reference cell with the 3 Vs: Sample volume (cm ) test cell, conˆrm with the manometer that the pressure inside O‹cial Monographs for Part I

Detector temperature: A constant temperature of about 2109C. Acetohexamide Carrier gas: Helium Flow rate: Adjust the ‰ow rate so that the retention time アセトヘキサミド of cyclohexylamine is about 4 minutes. Split ratio: 1:1 Change the origin/limits of content to read: System suitability— System performance: When the procedure is run with 2 mL Acetohexamide, when dried, contains not less than of the standard solution under the above operating 98.0z and not more than 101.0z of C15H20N2O4S. conditions, the number of theoretical plates of the peak of cyclohexylamine is not less than 8000. Change the Description to read: System repeatability: When the test is repeated 6 times with 2 mL of the standard solution under the above operat- Description Acetohexamide occurs as a white to yellowish ing conditions, the relative standard deviation of the peak white powder. area of cyclohexylamine is not more than 5z. It is freely soluble in N,N-dimethylformamide, sparingly (ii) Dicyclohexylurea—Dissolve exactly 1.0 g of Aceto- soluble in acetone, slightly soluble in methanol and in hexamide in exactly 10 mL of 0.5 mol/L sodium hydroxide ethanol (99.5), and practically insoluble in water. TS, add exactly 20 mL of methanol, shake, then add exactly Melting point: about 1859C (with decomposition). 5 mL of diluted hydrochloric acid (1 in 10), shake vigorously for 15 minutes, and centrifuge. Filter 10 mL or more of the Change the Purity (5) to read: supernatant liquid through a membrane ˆlter with pore size Purity of not larger than 0.5 mm. Discard the ˆrst 5 mL of the (5) Related substances (i) Cyclohexylamine—Dissolve ˆltrate, and use the subsequent ˆltrate as the sample solu- exactly 1.0 g of Acetohexamide in exactly 30 mL of 0.5 tion. Separately, dissolve exactly 50 mg of dicyclohexylurea mol/L sodium hydroxide TS, add exactly 5 mL of hexane, in methanol to make exactly 100 mL. Pipet 2 mL of this so- shake vigorously for 60 minutes, allow to stand for 5 lution, and add methanol to make exactly 100 mL. Pipet 20 minutes, and use the upper layer as the sample solution. mL of this solution, add exactly 10 mL of 0.5 mol/L sodium Separately, dissolve exactly 50 mg of cyclohexylamine in 0.5 hydroxide TS, shake, then add exactly 5 mL of diluted mol/L sodium hydroxide TS to make exactly 50 mL. Pipet 2 hydrochloric acid (1 in 10), shake, and use this solution as mL of this solution, and add 0.5 mol/L sodium hydroxide the standard solution. Perform the test with exactly 50 mL TS to make exactly 300 mL. Pipet 30 mL of this solution, each of the sample solution and the standard solution as add exactly 5 mL of hexane, shake vigorously for 60 directed under the Liquid Chromatography according to the minutes, allow to stand for 5 minutes, and use the upper lay- following conditions, and determine the peak area of dicy- er as the standard solution. Perform the test with exactly 2 clohexylurea by the automatic integration method: the peak mL each of the sample solution and the standard solution as area of dicyclohexylurea is not more than that with the stan- directed under the Gas Chromatography according to the dard solution. following conditions, and determine the peak area of cyclo- Operating conditions— hexylamine by the automatic integration method: the peak Detector: An ultraviolet absorption photometer area of cyclohexylamine is not more than that with the stan- (wavelength: 210 nm). dard solution. Column: A stainless steel column 4.6 mm in inside Operating conditions— diameter and 25 cm in length, packed with octadecyl- Detector: A hydrogen ‰ame-ionization detector. silanized silica gel for liquid chromatography (5 mminparti- Column: A fused-silica column 0.53 mm in inside di- cle diameter). ameter and 30 m in length, coated the inner surface with Column temperature: A constant temperature of about methylsilicone polymer for gas chromatography 1.5 mmin 259C. thickness. Mobile phase: Dissolve 0.5 g of sodium hydroxide in 1000 Column temperature: A constant temperature of about mL of 0.05 mol/L sodium dihydrogen phosphate TS, and 909C. adjust the pH to 6.5 with 0.5 mol/L sodium hydroxide TS. Injection port temperature: A constant temperature of To 500 mL of this solution add 500 mL of acetonitrile. about 1509C. Flow rate: Adjust the ‰ow rate so that the retention time 1687 1688 O‹cial Monographs for Part I Supplement II, JPXIV of dicyclohexylurea is about 10 minutes. shows no absorption between 210 nm and 350 nm. Separate- System suitability— ly, to 10 mL of this solution add 1 mL of potassium hydrox- System performance: When the procedure is run with ide-ethanol TS, allow to stand for 15 minutes, and determine 50 mL of the standard solution under the above operating the absorption spectrum in the same way. Compare the conditions, the number of theoretical plates of the peak of spectrum so obtained with the Reference Spectrum or the dicyclohexylurea is not less than 10,000. spectrum of a solution of Alprostadil Reference Standard System repeatability: When the test is repeated 6 times prepared in the same manner as the sample solution: both with 50 mL of the standard solution under the above operat- spectra exhibit similar intensities of absorption at the same ing conditions, the relative standard deviation of the peak wavelengths. area of dicyclohexylurea is not more than 2.0z. (2) Determine the infrared absorption spectrum of (iii) Other related substances—Dissolve 0.10 g of Aceto- Alprostadil, previously dried, as directed in the potassium hexamide in 10 mL of acetone, and use this solution as the bromide disk method under the Infrared Spectrophotomet- sample solution. Pipet 1 mL of the sample solution, and add ry, and compare the spectrum with the Reference Spectrum acetone to make exactly 20 mL. Pipet two 1-mL portions of or the spectrum of previously dried Alprostadil Reference this solution, add acetone to make exactly 10 mL and Standard: both spectra exhibit similar intensities of absorp- 25 mL, respectively, and use these solutions as the standard tion at the same wave numbers. solution (1) and the standard solution (2). Perform the test Optical rotation [a]20: －53 – －619(after drying, 25 mg, with these solutions as directed under the Thin-layer D tetrahydrofuran, 5 mL, 100 mm). Chromatography. Spot 10 mL each of the sample solution, the standard solution (1) and the standard solution (2) on a Melting point 114–1189C plate of silica gel with ‰uorescent indicator for thin-layer Purity Related substances—Dissolve 4 mg of Alprostadil chromatography. Develop the plate with a mixture of ethyl in 2 mL of a mixture of acetonitrile for liquid chro- acetate, methanol, ammonia water (28) and cyclohexane matography and water (9:1), and use this solution as the (6:2:1:1) to a distance of about 10 cm, and air-dry the plate. sample solution. Pipet 0.5 mL of the sample solution, and Examine under ultraviolet light (main wavelength: 254 nm): add the mixture of acetonitrile for liquid chromatography the spots other than the principal spot from the sample and water (9:1) to make exactly 10 mL. Pipet 2 mL of this solution are not more intense than spot from the standard solution, add the mixture of acetonitrile for liquid chro- solution (1), and the number of them which are more intense matography and water (9:1) to make exactly 10 mL, and use than the spot from the standard solution (2) is not more this solution as the standard solution. Perform the test with than 4. exactly 5 mL each of the sample solution and the standard so- lution as directed under the Liquid Chromatography accord- ing to the following conditions, and determine each peak Add the following: area by the automatic integration method: the area of the peaks, having the relative retention time of about 0.70 and Alprostadil 1.26 with respect to alprostadil, is not larger than 1/2 times the peak area of alprostadil with the standard solution, the アルプロスタジル area of the peaks, having the relative retention time of about Prostaglandin E1 0.88 and 1.18 with respect to alprostadil, is not larger than the peak area of alprostadil with the standard solution, the area of the peaks other than alprostadil and the peaks men- tioned above is not larger than 1/10 times the peak area of alprostadil with the standard solution and the total area of

C20H34O5: 354.48 the peaks other than alprostadil is not larger than 2 times the 7-s(1R,2R,3R)-3-Hydroxy-2-[(1E,3S)-3-hydroxyoct-1-en- peak area of alprostadil with the standard solution. 1-yl]-5-oxocyclopentyltheptanoic acid [745-65-3] Operating conditions— Detector, column, column temperature, mobile phase, Alprostadil, when dried, contains not less than and ‰ow rate: Proceed as directed in the operating condi-

97.0z and not more than 103.0z of C20H34O5. tions in the Assay. Time span of measurement: About 5 times as long as the Description Alprostadil occurs as white crystals or crystal- retention time of alprostadil after the solvent peak. line powder. System suitability— It is freely soluble in ethanol (99.5) and in tetrahydrofu- Test for required detectability: Measure exactly 2 mL of ran, slightly soluble in acetonitrile, and practically insoluble the standard solution, add the mixture of acetonitrile for in water. liquid chromatography and water (9:1) to make exactly Identiˆcation (1) The absorption spectrum of a solution 20 mL. Conˆrm that the peak area of alprostadil obtained of Alprostadil in ethanol (99.5) (1 in 100,000) determined as with 5 mL of this solution is equivalent to 7 to 13z of that directed under the Ultraviolet-visible Spectrophotometry with 5 mL of the standard solution. Supplement II, JPXIV O‹cial Monographs for Part I 1689

System performance: Proceed as directed in the system exceeding 59C. suitability in the Assay. System repeatability: When the test is repeated 6 times with 5 mL of the standard solution under the above operat- Atropine Sulfate Injection ing conditions, the relative standard deviation of the peak area of alprostadil is not more than 1.5z. 硫酸アトロピン注射液

Loss on drying Not more than 1.0z (0.1 g, in vacuum, Change the origin/limits of content to read: phosphorus (V) oxide, 4 hours).

Assay Weigh accurately about 5 mg each of Alprostadil Atropine Sulfate Injection is an aqueous solution and Alprostadil Reference Standard, previously dried, for injection. dissolve in exactly 5 mL of the internal standard solution, It contains not less than 93.0z and not more than add a mixture of acetonitrile for liquid chromatography and 107.0z of the labeled amount of atropine sulfate water (9:1) to make 50 mL, and use these solutions as the [(C17H23NO3)2.H2SO4.H2O: 694.83]. sample solution and the standard solution, respectively. Perform the test with 5 mL each of the sample solution and Change the Identiˆcation (2) to read: the standard solution as directed under the Liquid Chro- Identiˆcation matography according to the following conditions, and (2) Evaporate an exactly measured volume of Atropine determine the ratios, Q and Q , of the peak area of T S Sulfate Injection, equivalent to 5 mg of Atropine Sulfate alprostadil to that of the internal standard. according to the labeled amount, on a water bath to dryness. Q Amount (mg) of C H O ＝W × T After cooling, dissolve the residue in 1 mL of ethanol (95), 20 34 5 S Q S and use this solution as the sample solution. If insoluble W : Amount (mg) of Alprostadil Reference Standard S substance remains, crush it, allow to stand, and use the Internal standard solution—A solution of dimethyl phtha- supernatant liquid as the sample solution. Separately, late in the mixture of acetonitrile for liquid chromatography dissolve 10 mg of Atropine Sulfate Reference Standard in and water (9:1) (1 in 10,000). 2 mL of ethanol (95), and use this solution as the standard Operating conditions— solution. Perform the test with these solutions as directed Detector: An ultraviolet absorption photometer under the Thin-layer Chromatography. Spot 5 mLeachof (wavelength: 196 nm). the sample solution and the standard solution on a plate of Column: A stainless steel column 4.6 mm in inside silica gel for thin-layer chromatography. Develop the plate diameter and 15 cm in length, packed with octadecyl- with a mixture of acetone, water and ammonia water (28) silanized silica gel for liquid chromatography (5 mmin (90:7:3) to a distance of about 10 cm, and dry the plate at particle diameter). 809C for 10 minutes. After cooling, spray evenly Dragen- Column temperature: A constant temperature of about dorŠ's TS for spraying on the plate: the spots obtained from 409C. the sample solution and the standard solution show an Mobile phase: Dissolve 9.07 g of potassium dihydrogen orange color and the same Rfvalue. phosphate in water to make 1000 mL. Adjust the pH to 6.3 with a solution prepared by dissolving 9.46 g of disodium Add the following next to Identiˆcation: hydrogen phosphate in water to make 1000 mL, and dilute Bacterial endotoxins Less than 75 EU/mg. to 10 times its volume with water. To 360 mL of this solution add 110 mL of acetonitrile for liquid chromatography and Actual volume It meets the requirements of Injections. 30 mL of methanol for liquid chromatography. Foreign insoluble matter Perform the test according to Flow rate: Adjust the ‰ow rate so that the retention time Method 1: it meets the requirement of the Foreign Insoluble of alprostadil is about 10 minutes. Matter Test for Injections. System suitability— System performance: When the procedure is run with 5 mL Insoluble particulate matter Perform the test according to of the standard solution under the above operating condi- Method 1: it meets the requirement of the Insoluble Particu- tions, alprostadil and the internal standard are eluted in this late Matter Test for Injections. order with the resolution between these peaks being not less Sterility Perform the test: it meets the requirement of the than 9. Sterility Test. System repeatability: When the test is repeated 6 times with 5 mL of the standard solution under the above operat- Change the Assay to read: ing conditions, the relative standard deviation of the ratio of the peak area of alprostadil to that of the internal standard is Assay To an exactly measured volume of Atropine Sulfate not more than 1.0z. Injection, equivalent to about 5 mg of atropine sulfate

[(C17H23NO3)2.H2SO4.H2O], add exactly 3 mL of the inter- Containers and storage Containers—Tight containers. nal standard solution and water to make 50 mL, and use this Storage—Light-resistant, and at a temperature not 1690 O‹cial Monographs for Part I Supplement II, JPXIV solution as the sample solution. Separately, weigh accurately Add the following: about 25 mg of Atropine Sulfate Reference Standard, separately determine its loss on drying in the same condi- Azithromycin Hydrate tions as for Atropine Sulfate, and dissolve in water to make exactly 50 mL. Pipet 10 mL of this solution, add exactly 3 アジスロマイシン水和物 mL of the internal standard solution and water to make 50 mL, and use this solution as the standard solution. Perform the test with 20 mL each of the sample solution and the stan- dard solution as directed under the Liquid Chromatography according to the following conditions, and determine the ratios, QT and QS, of the peak area of atropine sulfate to that of the internal standard.

Amount (mg) of atropine sulfate

[(C17H23NO3)2.H2SO4.H2O]

QT 1 ＝WS× × ×1.027 QS 5 C38H72N2O12.2H2O: 785.02

WS: Amount (mg) of Atropine Reference Standard, calcu- (2R,3S,4S,5R,6R,8R,11R,12R,13S,14R)-5-(3,4,6-Trideoxy- lated based on the dried basis 3-dimethylamino-b-D-xylo-hexopyranosyloxy)-3- (2,6-dideoxy-3-C-methyl-3-O-methyl-a-L-ribo- Internal standard solution—A solution of etilefrine hexopyranosyloxy)-10-aza-6,12,13-trihydroxy- hydrochloride (1 in 1000). 2,4,6,8,10,11,13-heptamethylhexadecan-14-olide dihydrate Operating conditions— [117772-70-0] Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Azithromycin Hydrate is the derivative of Column: A stainless steel column 4.6 mm in inside erythromycin. diameter and 15 cm in length, packed with octadecyl- It contains not less than 945 mg (potency) and not silanized silica gel for liquid chromatography (5 mminparti- more than 1030 mg (potency) per mg, calculated on the cle diameter). anhydrous basis. The potency of Azithromycin Hy- Column temperature: A constant temperature of about drate is expressed as mass (potency) of azithromycin 409C. (C38H72N2O12:748.98). Mobile phase: To 0.4 g of sodium lauryl sulfate add 500 mL of diluted phosphoric acid (1 in 1000) to dissolve, Description Azithromycin Hydrate occurs as a white and adjust the pH to 3.0 with sodium hydroxide TS. To crystalline powder. 240 mL of this solution add 70 mL of tetrahydrofuran, and It is freely soluble in methanol and in ethanol (99.5), and mix. practically insoluble in water. Flow rate: Adjust the ‰ow rate so that the retention time Identiˆcation Determine the infrared absorption spectrum of atropine sulfate is about 16 minutes. of Azithromycin Hydrate as directed in the potassium System suitability— bromide disk method under the Infrared Spectrophotomet- System performance: When the procedure is run with 20 ry, and compare the spectrum with the Reference Spectrum mL of the standard solution under the above operating or the spectrum of Azithromycin Reference Standard: both conditions, the internal standard and atropine sulfate are spectra exhibit similar intensities of absorption at the same eluted in this order with the resolution between these peaks wave numbers. being not less than 3. 20 System repeatability: When the test is repeated 6 times Optical rotation [a]D : －45 – －499(400 mg calculated on with 20 mL of the standard solution under the above operat- the anhydrous basis, ethanol (99.5), 20 mL, 100 mm). ing conditions, the relative standard deviation of the ratio of Purity (1) Heavy metals—Proceed with 1.0 g of the peak area of atropine sulfate to that of the internal Azithromycin Hydrate according to Method 2, and perform standard is not more than 1.5z. the test. Prepare the control solution with 1.0 mL of Standard Lead Solution (not more than 10 ppm). (2) Related substances—Being speciˆed separately. (3) Residual solvent—Being speciˆed separately.

Water Not less than 4.0z and not more than 5.0z (0.4 g, volumetric titration, direct titration).

Residue on ignition Not more than 0.1z (1 g).

Assay Weigh accurately an amount of Azithromycin Supplement II, JPXIV O‹cial Monographs for Part I 1691

Hydrate and Azithromycin Reference Standard, equivalent Add the following: to about 50 mg (potency), dissolve each in an adequate amount of a mixture of acetonitrile and water (3:2), add Benidipine Hydrochloride exactly 2 mL of the internal standard solution and the mixture of acetonitrile and water (3:2) to make 50 mL, and 塩酸ベニジピン use these solutions as the sample solution and the standard solution. Perform the test with 5 mLeachofthesample solution and the standard solution as directed under the Liquid Chromatography according to the following condi- tions, and determine the ratios, QT and QS, of the peak area of azithromycin to that of the internal standard.

Amount [mg (potency)] of azithromycin (C38H72N2O12)

QR C28H31N3O6.HCl: 542.02 ＝WS× ×1000 QS 3-[(3RS)-1-Benzylpiperidin-3-yl] 5-methyl (4RS)- 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5- W : Amount [mg (potency)] of Azithromycin Reference S dicarboxylate monohydrochloride [91599-74-5] Standard Internal standard solution—A solution of 4,4?- Benidipine Hydrochloride, when dried, contains not bis(diethylamino)benzophenone in acetonitrile (3 in 4000). less than 99.0z and not more than 101.0z of

Operating conditions— C28H31N3O6.HCl. Detector: An ultraviolet absorption photometer Description Benidipine Hydrochloride occurs as a yellow (wavelength: 215 nm). crystalline powder. Column: A stainless steel column 4.6 mm in inside It is very soluble in formic acid, soluble in methanol, diameter and 25 cm in length, packed with octadecyl- sparingly soluble in ethanol (99.5), and practically insoluble silanized polyvinyl alcohol gel polymer for liquid chro- in water. matography (5 mm in particle diameter). A solution of Benidipine Hydrochloride in methanol (1 in Column temperature: A constant temperature of about 100) shows no optical rotation. 409C. Melting point: about 2009C (with decomposition). Mobile phase: Dissolve 6.97 g of dipotassium hydrogen phosphate in about 750 mL of water, adjust the pH to 11.0 Identiˆcation (1) Determine the absorption spectrum of with potassium hydroxide TS, and add water to make 1000 a solution of Benidipine Hydrochloride in methanol (1 in mL. To 400 mL of this solution add 600 mL of acetonitrile 100,000) as directed under the Ultraviolet-visible Spec- for liquid chromatography. trophotometry, and compare the spectrum with the Flow rate: Adjust the ‰ow rate so that the retention time Reference Spectrum: both spectra exhibit similar intensities of azithromycin is about 10 minutes. of absorption at the same wavelengths. System suitability— (2) Determine the infrared absorption spectrum of System performance: When the procedure is run with 5 mL Benidipine Hydrochloride, previously dried, as directed in of the standard solution under the above operating the potassium chloride disk method under the Infrared Spec- conditions, azithromycin and the internal standard are trophotometry, and compare the spectrum with the Refer- eluted in this order with the resolution between these peaks ence Spectrum: both spectra exhibit similar intensities of ab- being not less than 2.0. sorption at the same wave numbers. System repeatability: When the test is repeated 6 times (3) To 5 mL of a solution of Benidipine Hydrochloride with 5 mL of the standard solution under the above operat- (1 in 10) add 5 mL of ammonia TS, heat on a water bath for ing conditions, the relative standard deviation of the ratios 5 minutes, cool, and ˆlter. The ˆltrate, which is acidiˆed ofthepeakareaofazithromycintothatoftheinternal with dilute nitric acid, responds to the Qualitative Tests (2) standard is not more than 1.0z. for chloride.

Containers and storage Containers—Tight containers. Purity (1) Heavy metals—Proceed with 1.0 g of Benidi- pine Hydrochloride according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of Standard Lead Solution (not more than 20 ppm). (2) Related substances—Dissolve 20 mg of Benidipine Hydrochloride in 100 mL of a mixture of water and methanol (1:1), and use this solution as the sample solution. Pipet 1 mL of the sample solution, add the mixture of water and methanol (1:1) to make exactly 500 mL, and use this solution as the standard solution. Perform the test with ex- actly 10 mL each of the sample solution and the standard so- 1692 O‹cial Monographs for Part I Supplement II, JPXIV lution as directed under the Liquid Chromatography accord- Each mL of 0.1 mol/L perchloric acid VS ing to the following conditions, and determine each peak ＝54.20 mg of C H N O .HCl area by the automatic integration method: the peak areas of 28 31 3 6 bisbenzylpiperidyl ester having the relative retention time of Containers and storage Containers—Tight containers. about 0.35 with respect to benidipine, dehydro derivative having the relative retention time of about 0.75 and other related substances are not larger than 1/2 times the peak Add the following: area of benidipine with the standard solution, and the total area of the peaks other than benidipine is not larger than the Benidipine Hydrochloride Tablets peak area of benidipine with the standard solution. For this calculation, use the peak areas of bisbenzylpiperidyl ester 塩酸ベニジピン錠 and dehydro derivative after multiplying by their response factors, 1.6, respectively. Benidipine Hydrochloride Tablets contain not Operating conditions— less than 95.0z and not more than 105.0z of the Detector: An ultraviolet absorption photometer labeled amount of benidipine hydrochloride (wavelength: 237 nm). (C28H31N3O6.HCl: 542.02). Column: A stainless steel column 4.6 mm in inside Method of preparation Prepare as directed under Tablets, diameter and 10 cm in length, packed with octadecyl- with Benidipine Hydrochloride. silanized silica gel for liquid chromatography (3 mminparti- cle diameter). Identiˆcation Shake well a quantity of powdered Benidi- Column temperature: A constant temperature of about pine Hydrochloride Tablets, equivalent to 10 mg of Benidi- 259C. pine Hydrochloride according to the labeled amount, with Mobile phase: A mixture of 0.05 mol/L potassium di- 100 mL of methanol, and centrifuge. To 10 mL of the super- hydrogen phosphate TS, pH 3.0, methanol and tetra- natant liquid add methanol to make 100 mL, and use this so- hydrofuran (65:27:8). lution as the sample solution. Determine the absorption Flow rate: Adjust the ‰ow rate so that the retention time spectrum of the sample solution as directed under the of benidipine is about 20 minutes. Ultraviolet-visible Spectrophotometry: it exhibits maxima Time span of measurement: About 2 times as long as the between 235 nm and 239 nm, and between 350 nm and 360 retention time of benidipine after the solvent peak. nm. System suitability— Purity Dehydro derivative—Weigh accurately the mass of Test for required detectability: Measure exactly 5 mL of not less than 20 Benidipine Hydrochloride Tablets, and the standard solution, and add the mixture of water and powder in an agate mortar. To an amount of the powder, methanol (1:1) to make exactly 20 mL. Conˆrm that the equivalent to 20 mg of Benidipine Hydrochloride, add about peak area of benidipine obtained with 10 mL of this solution 80 mL of a mixture of diluted phosphoric acid (1 in 500) and is equivalent to 18 to 32z of that with 10 mL of the standard methanol (1:1), shake well, and add the mixture of diluted solution. phosphoric acid (1 in 500) and methanol (1:1) to make System performance: Dissolve 6 mg of Benidipine exactly 100 mL. Filter through a membrane ˆlter with pore Hydrochloride and 5 mg of benzoin in 200 mL of the size of 0.45 mm, and use the ˆltrate as the sample solution. mixture of water and methanol (1:1). When the procedure is Separately, dissolve 20 mg of benidipine hydrochloride for run with 10 mL of this solution under the above operating assay in the mixture of diluted phosphoric acid (1 in 500) and conditions, benzoin and benidipine are eluted in this order methanol (1:1) to make exactly 100 mL. Pipet 1 mL of this with the resolution between these peaks being not less solution, add the mixture of diluted phosphoric acid (1 in than 8. 500) and methanol (1:1) to make exactly 100 mL, and use System repeatability: When the test is repeated 6 times this solution as the standard solution. Perform the test with with 10 mL of the standard solution under the above operat- exactly 10 mL each of the sample solution and the standard ing conditions, the relative standard deviation of the peak solution as directed under the Liquid Chromatography ac- area of benidipine is not more than 3.5z. cording to the following conditions, and determine each Loss on drying Not more than 0.5z (0.5 g, 1059C, peak area by the automatic integration method: the peak 2 hours). area of dehydro derivative having the relative retention time of about 0.75 with respect to benidipine is not larger than Residue on ignition Not more than 0.10z (1 g). 1/2 times the peak area of benidipine with the standard solu- Assay Weigh accurately about 0.7 g of Benidipine tion. For this calculation, use the peak area of dehydro Hydrochloride, previously dried, dissolve in 10 mL of derivative after multiplying by the relative response factor, formic acid, add 70 mL of acetic anhydride, and titrate with 1.6. 0.1 mol/L perchloric acid VS (potentiometric titration). Operating conditions— Perform a blank determination in the same manner, and Perform as directed in the operating conditions in the make any necessary correction. Assay. Supplement II, JPXIV O‹cial Monographs for Part I 1693

System suitability— (C28H31N3O6.HCl) in 1 tablet. Test for required detectability: Measure exactly 2 mL of Operating conditions— the standard solution, and add the mixture of diluted phos- Detector: An ultraviolet absorption photometer phoric acid (1 in 500) and methanol (1:1) to make exactly (wavelength: 237 nm). 20 mL. Conˆrm that the peak area of benidipine obtained Column: A stainless steel column 4.6 mm in inside with 10 mLofthissolutionisequivalentto7to13z of that diameter and 15 cm in length, packed with octadecyl- with 10 mL of the standard solution. silanized silica gel for liquid chromatography (5 mmin System performance: Dissolve 6 mg of benidipine particle diameter). hydrochloride and 5 mg of benzoin in 200 mL of a mixture Column temperature: A constant temperature of about of water and methanol (1:1). When the procedure is run with 259C. 10 mL of this solution under the above operating conditions, Mobile phase: A mixture of 0.05 mol/L potassium di- benzoin and benidipine are eluted in this order with the hydrogen phosphate TS, pH 3.0 and acetonitrile (11:9). resolution between these peaks being not less than 8. Flow rate: Adjust the ‰ow rate so that the retention time System repeatability: When the test is repeated 6 times of benidipine is about 5 minutes. with 10 mL of the standard solution under the above operat- System suitability— ing conditions, the relative standard deviation of the peak System performance: When the procedure is run with area of benidipine is not more than 2.0z. 50 mL of the standard solution under the above operating conditions, the number of theoretical plates and the symmet- Content uniformity Perform the test as directed in the ry factor of the peak of benidipine are not less than 3000 and Assay, and determine the content: it meets the requirements not more than 2.0, respectively. of the Content Uniformity Test. System repeatability: When the test is repeated 6 times Dissolution Perform the test with 1 tablet of Benidipine with 50 mL of the standard solution under the above Hydrochloride Tablets at 50 revolutions per minute operating conditions, the relative standard deviation of the according to Method 2 under the Dissolution Test using the peak area of benidipine is not more than 1.5z. sinker, using 900 mL of the 1st ‰uid under the Disintegra- Assay Shake 1 tablet of Benidipine Hydrochloride Tablets tion Test as the dissolution medium. Withdraw 20 mL or with 40 mL of a mixture of diluted phosphoric acid (1 in 500) more of the dissolution medium 30 minutes after starting the and methanol (1:1) to disintegrate, add the mixture of test for a 2-mg and a 4-mg tablet or 45 minutes after starting diluted phosphoric acid (1 in 500) and methanol (1:1) to the test for a 8-mg tablet, and ˆlter through a membrane make exactly V mL so that each mL contains 40 mgof ˆlter with pore size of not more than 0.45 mm. Discard the benidipine hydrochloride (C H N O .HCl), and cen- ˆrst 10 mL of the ˆltrate, pipet the subsequent V mL, and 28 31 3 6 trifuge. Pipet 20 mL of the supernatant liquid, add exactly add the 1st ‰uid to make exactly V? mL so that each mL 10 mL of the internal standard solution and the mixture of contains about 2.2 mg of benidipine hydrochloride diluted phosphoric acid (1 in 500) and methanol (1:1) to (C H N O .HCl) according to the labeled amount. Pipet 28 31 3 6 make 50 mL, and use this solution as the sample solution. 5 mL of this solution, add exactly 5 mL of the mobile phase, Separately, weigh accurately about 40 mg of benidipine and use this solution as the sample solution. Separately, hydrochloride for assay, previously dried at 1059Cfor2 weigh accurately about 22 mg of benidipine hydrochloride hours, and dissolve in the mixture of diluted phosphoric acid for assay, previously dried at 1059C for 2 hours, and (1 in 500) and methanol (1:1) to make exactly 100 mL. Pipet dissolve in the mobile phase to make exactly 100 mL. Pipet 2 mL of this solution, add exactly 10 mL of the internal stan- 2 mL of this solution, and add the mobile phase to make dard solution and the mixture of diluted phosphoric acid (1 exactly 50 mL. Pipet 5 mL of this solution, and add the in 500) and methanol (1:1) to make 50 mL, and use this solu- mobile phase to make exactly 20 mL. Pipet 5 mL of this tion as the standard solution. Perform the test with 10 mL solution, add exactly 5 mL of the 1st ‰uid, and use this solu- each of the sample solution and the standard solution as tion as the standard solution. Perform the test with exactly directed under the Liquid Chromatography according to the 50 mL each of the sample solution and the standard solution following conditions, and determine the ratios, Q and Q , as directed under the Liquid Chromatography according to T S of the peak area of benidipine to that of the internal stan- the following conditions, and determine the peak areas, A T dard. Perform the test described above with not less than 10 and A , of benidipine: the dissolution rates for 2-mg or 4-mg S tablets, and calculate the average of these results. tablet in 30 minutes and for 8-mg tablet in 45 minutes are not less than 80z and not less than 85z, respectively. Amount (mg) of benidipine hydrochloride (C H N O .HCl) Dissolution rate (z) with respect to the labeled amount of 28 31 3 6 QT V benidipine hydrochloride ＝WS× × QS 1000 (C28H31N3O6.HCl) A V? 1 W : Amount (mg) of benidipine hydrochloride for assay ＝W × T × × ×9 S S A V C S Internal standard solution—A solution of benzoin in a W : Amount (mg) of benidipine hydrochloride for assay S mixture of water and methanol (1:1) (13 in 200,000). C: Labeled amount (mg) of benidipine hydrochloride Operating conditions— 1694 O‹cial Monographs for Part I Supplement II, JPXIV

Detector: An ultraviolet absorption photometer solution, add the mobile phase A to make exactly 100 mL, (wavelength: 237 nm). and use this solution as the standard solution. Perform the Column: A stainless steel column 4.6 mm in inside test with exactly 20 mL each of the sample solution and the diameter and 10 cm in length, packed with octadecyl- standard solution as directed under the Liquid Chro- silanized silica gel for liquid chromatography (3 mminparti- matography according to the following conditions, and de- cle diameter). termine each peak area by the automatic integration Column temperature: A constant temperature of about method: the area of the peak having the relative retention 259C. time of about 2.4 with respect to benzylpenicillin is not more Mobile phase: A mixture of 0.05 mol/L potassium di- than 2 times the total area of the peaks of benzylpenicillin hydrogen phosphate TS, pH 3.0, methanol and tetra- and N,N?-dibenzylethylenediamine obtained from the stan- hydrofuran (65:27:8). dard solution, and the area of the peak other than benzyl- Flow rate: Adjust the ‰ow rate so that the retention time penicillin, N,N?-dibenzylethylenediamine and the peak hav- of benidipine is about 20 minutes. ing the relative retention time of about 2.4 is not more than System suitability— the total area of the peaks of benzylpenicillin and System performance: When the procedure is run with N,N?-dibenzylethylenediamine obtained from the standard 10 mL of the standard solution under the above operating solution. conditions, the internal standard and benidipine are eluted Operating conditions— in this order with the resolution between these peaks being Detector: An ultraviolet absorption photometer not less than 8. (wavelength: 220 nm). System repeatability: When the test is repeated 6 times Column: A stainless steel column 4.0 mm in inside with 10 mL of the standard solution under the above operat- diameter and 25 cm in length, packed with octadecyl- ing conditions, the relative standard deviation of the ratio of silanized silica gel for liquid chromatography (5 mminparti- the peak area of benidipine to that of the internal standard is cle diameter). not more than 1.0z. Column temperature: A constant temperature of about 409C. Containers and storage Containers—Well-closed contain- Mobile phase A: A mixture of water, methanol and ers. 0.25 mol/L potassium dihydrogen phosphate TS, pH 3.5 (6:3:1). Mobile phase B: A mixture of methanol, water and Benzylpenicillin Benzathine 0.25 mol/L potassium dihydrogen phosphate TS, pH 3.5 (6:3:1). ベンジルペニシリンベンザチン Flowing of the mobile phase: Control the gradient by mixing the mobile phases A and B as directed in the Change the origin/limits of content to read: following table.

Benzylpenicillin Benzathine is the N,N?-diben- Time after injection Mobile phase Mobile phase zylethylenediamine salt of a penicillin compound of sample (min) A(z) B(z) having antibacterial activity produced by the growth 0–10 75 25 of Penicillium species. 10–20 75ª 025ª 100 It contains not less than 1152 Units and not more 20–55 0 100 than 1272 Units per mg, calculated on the anhydrous basis. The potency of Benzylpenicillin Benzathine is Flow rate: 1.0 mL/min expressed as unit calculated from the amount of Time span of measurement: About 3 times as long as the benzylpenicillin sodium (C16H17N2NaO4S: 356.37). retention time of benzylpenicillin after the solvent peak. 1 Unit of Benzylpenicillin Benzathine is equivalent to System suitability—

0.6 mg of benzylpenicillin sodium (C16H17N2NaO4S). It Test for required detectability: To exactly 1 mL of the contains not less than 24.0z and not more than standard solution add the mobile phase A to make exactly

27.0z of N,N?-dibenzylethylenediamine (C16H20N2: 20 mL. Conˆrm that the peak area of benzylpenicillin 240.34), calculated on the anhydrous basis. obtained from 20 mL of this solution is equivalent to 3.5 to 6.5z of that from the standard solution. Change the Purity (3) to read: System performance: When the procedure is run with 20 mL of the standard solution under the above operating Purity conditions, N,N?-dibenzylethylenediamine and benzyl- (3) Related substances—Dissolve 70 mg of Benzyl- penicillin are eluted in this order with the resolution between penicillin Benzathine in 25 mL of methanol, add a solution these peaks being not less than 25. prepared by dissolving 1.02 g of disodium hydrogen phos- System repeatability: When the test is repeated 3 times phate and 6.80 g of potassium dihydrogen phosphate in with 20 mL of the standard solution under the above operat- water to make 1000 mL to make 50 mL, and use this ing conditions, the relative standard deviation of the peak solution as the sample solution. Pipet 1 mL of the sample Supplement II, JPXIV O‹cial Monographs for Part I 1695 areas of benzylpenicillin is not more than 2.0z. areas of N,N?-dibenzylethylenediamine and benzylpenicillin Change the Assay to read: are not more than 2.0z, respectively. (2) N,N?-Dibenzylethylenediamine—Determine the Assay (1) Benzylpenicillin—Weigh accurately an amount areas, A and A , of the peak corresponding to N,N?-diben- of Benzylpenicillin Benzathine, equivalent to about 85,000 T S zylethylenediamine on the chromatograms obtained in (1) Units, dissolve in 25 mL of methanol, and add a solution with the sample solution and the standard solution. containing 1.02 g of disodium hydrogen phosphate and

6.80 g of potassium dihydrogen phosphate in 1000 mL of Amount (z)ofN,N?-dibenzylethylenediamine (C16H20N2) water to make exactly 50 mL. Pipet 5 mL of this solution, W A ＝ S × T ×100×0.667 add a mixture of the solution containing 1.02 g of disodium WT AS hydrogen phosphate and 6.80 g of potassium dihydrogen W :Amount(mg)ofN,N?-dibenzylethylenediamine phosphate in 1000 mL of water and methanol (1:1) to make S diacetate exactly 20 mL, and use this solution as the sample solution. W : Amount (mg) of the sample Separately, weigh accurately an amount of Benzylpenicillin T 0.667: Conversion factor for the molecular mass Potassium Reference Standard, equivalent to about 85,000 of N,N?-dibenzylethylenediamine diacetate Units, and about 25 mg of N,N?-dibenzylethylenediamine (C H N ･2CH COOH)tothatofN,N?-diben- diacetate, dissolve in 25 mL of methanol, and add the 16 20 2 3 zylethylenediamine (benzathine, C H N ) solution containing 1.02 g of disodium hydrogen phosphate 16 20 2 and 6.80 g of potassium dihydrogen phosphate in 1000 mL of water to make exactly 50 mL. Pipet 5 mL of this solution, Add the following: add a mixture of the solution containing 1.02 g of disodium hydrogen phosphate and 6.80 g of potassium dihydrogen phosphate in 1000 mL of water and methanol (1:1) to make Cefepime Dihydrochloride for exactly 20 mL, and use this solution as the standard Injection solution. Perform the test with exactly 20 mLeachofthe sample solution and the standard solution as directed under 注射用塩酸セフェピム the Liquid Chromatography according to the following conditions, and determine the peak areas, AT ands AS,of Cefepime Dihydrochloride for Injection is a prepa- benzylpenicillin. ration for injection, which is dissolved before use. It contains not less than 95.0z and not more Amount (unit) of benzylpenicillin sodium (C16H17N2NaO4S) than 110.0z of the labeled amount of cefepime AT ＝WS× (C19H24N6O5S2: 480.56). AS Method of preparation Prepare as directed under Injec- WS: Amount (unit) of Benzylpenicillin Potassium Refer- tions, with Cefepime Dihydrochloride. ence Standard Operating conditions— Description Cefepime Dihydrochloride for Injection Detector: An ultraviolet absorption photometer occurs as a white to pale yellow powder. (wavelength: 220 nm). Identiˆcation (1) Dissolve 40 mg of Cefepime Di- Column: A stainless steel column 4.6 mm in inside hydrochloride in 2 mL of water, add 1 mL of a solution of diameter and 25 cm in length, packed with octadecyl- hydroxylammonium chloride (1 in 10) and 2 mL of sodium silanized silica gel for liquid chromatography (5 mminparti- hydroxide TS, allow to stand for 5 minutes, then add 3 mL cle diameter). of 1 mol/L hydrochloric acid TS and 3 drops of iron (III) Column temperature: A constant temperature of about chloride TS: a red-brown color develops. 409C. (2) Determine the absorption spectrum of a solution of Mobile phase: A mixture of water, methanol and 0.25 Cefepime Dihydrochloride for Injection (1 in 12,500) as mol/L potassium dihydrogen phosphate TS, pH 3.5 directed under the Ultraviolet-visible Spectrophotometry: it (11:7:2). exhibits maxima between 233 nm and 237 nm and between Flow rate: Adjust the ‰ow rate so that the retention time 255 nm and 259 nm. of benzylpenicillin is about 18 minutes. System suitability— pH The pH of a solution obtained by dissolving an amount System performance: When the procedure is run with of Cefepime Dihydrochloride for Injection, equivalent to 20 mL of the standard solution under the above operating 0.5 g (potency) of Cefepime Dihydrochloride according to conditions, N,N?-dibenzylethylenediamine and benzyl- the labeled amount, in 5 mL of water is between 4.0 and 6.0. penicillin are eluted in this order with the resolution between Purity (1) Clarity and color of solution—Dissolve an these peaks being not less than 20. amount of Cefepime Dihydrochloride for Injection, equiva- System repeatability: When the test is repeated 6 times lent to 0.5 g (potency) of Cefepime Dihydrochloride accord- with 20 mL of the standard solution under the above operat- ing to the labeled amount, in 5 mL of water: the solution is ing conditions, the relative standard deviations of the peak clear and colorless or light yellow. The color is not darker 1696 O‹cial Monographs for Part I Supplement II, JPXIV than Matching Fluid I. accurately an amount of the content, equivalent to about (2) N-Methylpyrrolidine—Weigh accurately an amount 60 mg (potency) of Cefepime Dihydrochloride according to of Cefepime Dihydrochloride for Injection, equivalent to the labeled amount, dissolve in the mobile phase to make about 0.2 g (potency) of Cefepime Dihydrochloride accord- exactly 50 mL, and use this solution as the sample solution. ingtothelabeledamount,dissolveindilutednitricacid(2in Separately, weigh accurately an amount of Cefepime 625) to make exactly 20 mL, and use this solution as the Dihydrochloride Reference Standard, equivalent to about sample solution. Separately, transfer 30 mL of water into a 60 mg (potency), dissolve in the mobile phase to make 100-mL volumetric ‰ask, weigh accurately the mass of the exactly 50 mL, and use this solution as the standard ‰ask, add about 0.125 g of N-methylpyrrolidine, then weigh solution. Proceed as directed in the Assay under Cefepime accurately the mass, and add water to make exactly 100 mL. Dihydrochloride. Pipet 4 mL of this solution, add diluted nitric acid (2 in Amount [mg (potency)] of cefepime (C H N O S ) 3125) to make exactly 100 mL, and use this solution as the 19 24 6 5 2 A standard solution. Perform the test with exactly 100 mLeach ＝W × T ×1000 S A of the sample solution and the standard solution as directed S under the Liquid Chromatography according to the follow- WS: Amount [mg (potency)] of Cefepime Dihydrochloride ing conditions, and determine the peak areas of N-methyl- Reference Standard pyrrolidine, A and A , by the automatic integration method T S Containers and storage Containers—Hermetic containers. within 20 minutes after the sample solution is prepared. Storage—Light-resistant. Calculate the amount of N-methylpyrrolidine per mg (poten- cy) of Cefepime Dihydrochloride for Injection by the following formula: not more than 1.0z. Cefuroxime Axetil Amount (z)ofN-methylpyrrolidine W ×f A 1 セフロキシムアキセチル ＝ S × T × WT AS 125 Change the graphic formula to read: WS: Amount (mg) of N-methylpyrrolidine

WT: Amount [mg (potency)] of cefepime in the sample taken f:Purity(z)ofN-methylpyrrolidine

Operating conditions— Proceed as directed in the operating conditions in the Purity (3) under Cefepime Dihydrochloride. System suitability— Proceed as directed in the system suitability in the Purity Change the chemical name to read: (3) under Cefepime Dihydrochloride. (1RS)-1-Acetoxyethyl (6R,7R)-3-carbamoyloxymethyl-7- Water Not more than 4.0z (Weigh accurately about [(Z)-2-furan-2-yl-2-methoxyiminoacetylamino]-8-oxo-5- 50 mg of Cefepime Dihydrochloride for Injection, dissolve thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate in exactly 2 mL of methanol for Karl Fischer method, and perform the test with exactly 0.5 mL of this solution. Coulo- metric titration). Cefuroxime Sodium

Bacterial endotoxins Less than 0.06 EU/mg (potency). セフロキシムナトリウム Mass variation It meets the requirements of the Mass Variation Test. Change the Purity (1) to read:

Foreign insoluble matter Perform the test according to Purity (1) Clarity and color of solution—Dissolve 1.0 g Method 2: it meets the requirements of the Foreign Insoluble of Cefuroxime Sodium in 10 mL of water: the solution is Matter Test for Injections. clear, and its absorbance at 450 nm is not more than 0.25.

Insoluble particulate matter Perform the test according to Method 1: it meets the requirements of the Insoluble Particulate Matter Test for Injections.

Sterility Perform the test according to the Membrane ˆltration method: it meets the requirements of the Sterility Test.

Assay Weigh accurately the mass of the contents of not less than 10 Cefepime Dihydrochloride for Injection. Weigh Supplement II, JPXIV O‹cial Monographs for Part I 1697

calculated on the dried basis

Ciclosporin Operating conditions— Detector: An ultraviolet absorption photometer シクロスポリン (wavelength: 210 nm). Column: A stainless steel column 4 mm in inside diameter Change the Purity (3) to read: and 25 cm in length, packed with octadecylsilanized silica gel Purity for liquid chromatography (5 mm in particle diameter). (3) Related substances—Use the sample solution ob- Connect the sample injection port and the column with a tained in the Assay as the sample solution. Pipet 2 mL of the stainless steel tube 0.3 mm in inside diameter and 1 m in sample solution, add a mixture of water and acetonitrile length. (1:1) to make exactly 200 mL, and use this solution as the Column temperature: A constant temperature of about standard solution. Perform the test with exactly 20 mLeach 809C (including the sample injection port and the connecting of the sample solution and the standard soluton as directed tube). under the Liquid Chromatography according to the follow- Mobile phase: A mixture of water, acetonitrile, tert-butyl ing conditions. Determine each peak area of both solutions methyl ether and phosphoric acid (520:430:50:1). by the automatic integration method: the area of the peak Flow rate: Adjust the ‰ow rate so that the retention time other than the ciclosporin is not more than 0.7 times of the of ciclosporin is about 27 minutes. peak area of ciclosporin from the standard solution, and the System suitability— total area of all peaks other than the ciclosporin is not more System performance: Dissolve 3 mg of Ciclosporin U in than 1.5 times of the peak area of ciclosporin from the stan- 2.5 mL of a mixture of water and acetonitrile (1:1), and add dard solution. 2.5 mL of the standard solution. When the procedure is run Operating conditions— with 20 mL of this solution under the above operating Detector, column, column temperature, mobile phase, conditions, ciclosporin U and ciclosporin are eluted in this and ‰ow rate: Proceed as directed in the operating condi- order with the resolution between these peaks being not less tions in the Assay. than 1.2. Time span of measurement: About 2 times as long as the System repeatability: When the test is repeated 6 times retention time of ciclosporin after the solvent peak. with 20 mL of the standard solution under the above operat- System suitability— ing conditions, the relative standard deviation of the peak Test for required detection: To exactly 2 mL of the stan- area of ciclosporin is not more than 1.0z. dard solution add a mixture of water and acetonitrile (1:1) to make exactly 20 mL. Conˆrm that the peak area of ciclospo- rin obtained from 20 mL of this solution is equivalent to 7 to Add the following: 13z of that of ciclosporin obtained from 20 mLofthe standard solution. Cisplatin System performance: Proceed as directed in the system suitability in the Assay. シスプラチン System repeatability: When the test is repeated 6 times with 20 mL of the standard solution under the above operating conditions, the relative standard deviation of the peak area of ciclosporin is not more than 3.0z. Cl2H6N2Pt: 300.05 (SP-4-2)-Diamminedichloroplatinum [15663-27-1] Change the Assay to read: Assay Weigh accurately about 30 mg each of Ciclosporin Cisplatin, when dried, contains not less than 98.0z and Ciclosporin Reference Standard, previously determined and not more than 102.0z of Cl2H6N2Pt. the loss on drying as the same manner as Ciclosporin, and Description Cisplatin occurs as a yellow crystalline pow- dissolve each in a mixture of water and acetonitrile (1:1) to der. make exactly 25 mL, and use these solutions as the sample It is sparingly soluble in N,N-dimethylformamide, slightly solution and the standard solution. Perform the test with ex- soluble in water, and practically insoluble in ethanol (99.5). actly 20 mL each of the sample solution and the standard so- lution as directed under the Liquid Chromatography accord- Identiˆcation (1) To 5 mL of a solution of Cisplatin (1 in ing to the following conditions, and determine the peak 2000) add 2 to 3 drops of a solution of tin (II) chloride dihydrate (1 in 100): a brown precipitate is formed. areas, AT and AS, of ciclosporin. (2) Determine the absorption spectrum of a solution of Amount (mg) of C62H111N11O12 Cisplatin in a solution of sodium chloride in 0.01 mol/L AS hydrochloric acid TS (9 in 1000) (1 in 2000) as directed under ＝WS× AT the Ultraviolet-visible Spectrophotometry, and compare the WS: Amount (mg) of Ciclosporin Reference Standard, spectrum with the Reference Spectrum or the spectrum of a 1698 O‹cial Monographs for Part I Supplement II, JPXIV solution of Cisplatin Reference Standard prepared in the N,N-dimethylformamide to make exactly 25 mL, and use same manner as the sample solution: both spectra exhibit these solutions as the sample solution and the standard similar intensities of absorption at the same wavelengths. solution. Perform the test with exactly 40 mLeachofthe (3) Determine the infrared absorption spectrum of sample solution and the standard solution as directed under Cisplatin as directed in the potassium bromide disk method the Liquid Chromatography according to the following under the Infrared Spectrophotometry, and compare the conditions, and determine the peak areas, AT and AS,of spectrum with the Reference Spectrum or the spectrum of cisplatin by the automatic integration method.

Cisplatin Reference Standard: both spectra exhibit similar AT Amount (mg) of Cl2H6N2Pt＝WS× intensities of absorption at the same wave numbers. AS (4) A solution of Cisplatin (1 in 2000) responds to the W : Amount (mg) of Cisplatin Reference Standard Qualitative Test (1) for chloride. S Operating conditions— Purity Ammonium amminetrichloroplatinate—Conduct Detector: An ultraviolet absorption photometer this procedure using light-resistant vessels. Dissolve 50 mg (wavelength: 310 nm). of Cisplatin in a solution of sodium chloride (9 in 1000) to Column: A stainless steel column 4.6 mm in inside make exactly 100 mL, and use this solution as the sample diameter and 25 cm in length, packed with aminopropyl- solution. Separately, dissolve 10 mg of ammonium am- silanized silica gel for liquid chromatography (5 mmin minetrichloroplatinate for liquid chromatography, previous- particle diameter). ly dried at 809C for 3 hours, in the solution of sodium Column temperature: A constant temperature of about chloride (9 in 1000) to make exactly 200 mL. Pipet 2 mL of 259C. this solution, add the solution of sodium chloride (9 in 1000) Mobile phase: A mixture of ethyl acetate, methanol, water to make exactly 20 mL, and use this solution as the standard and N,N-dimethylformamide (25:16:5:5). solution. Perform the test with exactly 40 mLeachofthe Flow rate: Adjust the ‰ow rate so that the retention time sample solution and the standard solution as directed under of cisplatin is about 4 minutes. the Liquid Chromatography according to the following System suitability— conditions, and determine the peak area of ammonium System performance: When the procedure is run with amminetrichloroplatinate by the automatic integration 40 mL of the standard solution under the above operating method: the peak area from the sample solution is not more conditions, the number of theoretical plates and the symmet- than that from the standard solution. ry factor of the peak of cisplatin are not less than 3000 and Operating conditions— not more than 2.0, respectively. Detector: An ultraviolet absorption photometer System repeatability: When the test is repeated 6 times (wavelength: 209 nm). with 40 mL of the standard solution under the above operat- Column: A stainless steel column 4.6 mm in inside ing conditions, the relative standard deviation of the peak diameter and 25 cm in length, packed with silica gel for area of cisplatin is not more than 1.0z. liquid chromatography having quaternary ammonium groups (10 mm in particle diameter). Containers and storage Containers—Tight containers. Column temperature: A constant temperature of about 259C. Mobile phase: A solution of ammonium sulfate (1 in 800). Anhydrous Citric Acid Flow rate: Adjust the ‰ow rate so that the retention time of ammonium amminetrichloroplatinate is about 8 minutes. 無水クエン酸 System suitability— System performance: When the procedure is run with Change the Identiˆcation to read: 40 mL of the standard solution under the above operating Identiˆcation Determine the infrared absorption spectrum conditions, the number of theoretical plates and the of Anhydrous Citric Acid, previously dried at 1059Cfor symmetry factor of the peak of ammonium am- 2 hours, as directed in the potassium bromide disk method minetrichloroplatinate are not less than 1500 and not more under the Infrared Spectrophotometry, and compare the than 2.0, respectively. spectrum with the Reference Spectrum: both spectra exhibit System repeatability: When the test is repeated 6 times similar intensities of absorption at the same wave numbers. with 40 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the peak area of ammonium amminetrichloroplatinate is not more than 3.0z.

Loss on drying Not more than 0.1z (1 g, 1059C, 4 hours).

Assay Conduct this procedure using light-resistant vessels. Weigh accurately about 25 mg each of Cisplatin and Cisplatin Reference Standard, previously dried, dissolve in Supplement II, JPXIV O‹cial Monographs for Part I 1699

and ‰ow rate: Proceed as directed in the operating condi- Clarithromycin tions in the Assay. Time span of measurement: About 5 times as long as the クラリスロマイシン retentiontimeofthemainpeakafter2minutesofsample injection. Change the origin/limits of content to read: System suitability— Test for required detection: To exactly 2 mL of the stan- Clarithromycin is a derivative of erythromycin. dard solution add the mobile phase to make exactly 10 mL, It contains not less than 950 mg (potency) and not and use this solution as the solution for system suitability more than 1050 mg (potency) per mg, calculated on the test. Conˆrm that when the procedure is run with 10 mLof anhydrous basis. The potency of Clarithromycin is the solution for system suitability test, the peak area of expressed as mass (potency) of clarithromycin clarithromycin is equivalent to 14 – 26z of that obtained (C38H69NO13). from the standard solution. System performance: Proceed as directed in the system Change the Melting point to read: suitability in the Assay. System repeatability: When the test is repeated 6 times Melting point 220–2279C with 10 mL of the solution for system suitability test under the above operating conditions, the relative standard Change the Purity (2) and (3) to read: deviation of the peak area of clarithromycin is not more Purity than 3.0z. (2) Arsenic—Prepare the test solution with 1.0 g of Clarithromycin according to Method 3, and perform the test (not more than 2 ppm). Colchicine (3) Related substances—Weigh accurately about 0.1 g of Clarithromycin, dissolve in the mobile phase to make exactly コルヒチン 20 mL, and use this solution as the sample solution. Separately, weigh accurately about 10 mg of Clarithromycin Change to read except the structural formula Reference Standard, dissolve in the mobile phase to make and chemical name: exactly 20 mL, and use this solution as the standard solu- tion. Perform the test with exactly 10 mLeachofthesample Colchicine contains not less than 97.0z and not solution and the standard solution as directed under the Liq- more than 102.0z of C22H25NO6,calculatedonthe uid Chromatography according to the following conditions, anhydrous basis and corrected by the amount of ethyl and determine the each peak area by the automatic integra- acetate. tion method: the amount of each related substance calculat- Description Colchicine occurs as a yellowish white ed on the anhydrous basis is not more than 2.0z,andthe powder. total of them is not more than 5.0z. Exclude any peak with It is very soluble in methanol, freely soluble in N,N- an area of less than 0.05z. dimethylformamide, in ethanol (95) and in acetic anhydride, Amount (z)ofeachrelatedsubstancecalculatedonthe and sparingly soluble in water. anhydrous basis It is colored by light. W A ＝ S × T ×100 Identiˆcation (1) Determine the absorption spectrum of W A T S a solution of Colchicine in ethanol (95) (1 in 100,000) as Total amount (z) of the related substances calculated on the directed under the Ultraviolet-visible Spectrophotometry, anhydrous basis and compare the spectrum with the Reference Spectrum: W SA both spectra exhibit similar intensities of absorption at the ＝ S × T ×100 WT AS same wavelengths. (2) To 1 g of potassium bromide for infrared absorption W : Amount (mg) of Clarithromycin Reference Standard S spectrum add 0.5 mL of a solution of Colchicine in W : Amount (mg) of the sample, calculated on the anhy- T methanol (1 in 50), grind thoroughly, and dry in vacuum at drous basis 809C for 1 hour. Determine the infrared absorption spec- A :Peakareaofclarithromycinobtainedwiththestan- S trum of this powder as directed in the potassium bromide dard solution disk method under the Infrared Spectrophotometry, and A :Peakareaofeachrelatedsubstanceobtainedwiththe T compare the spectrum with the Reference Spectrum: both sample solution spectra exhibit similar intensities of absorption at the same SA : Total area of the peaks other than clarithromycin T wave numbers. obtained with the sample solution 20 Operating conditions— Optical rotation [a]D : －235 – －2509(0.1 g calculated on Detector, column, column temperature, mobile phase, the anhydrous basis and corrected by the amount of ethyl 1700 O‹cial Monographs for Part I Supplement II, JPXIV acetate, ethanol (95), 10 mL, 100 mm). dimethylformamide to make exactly 50 mL. Conˆrm that the peak area of ethyl acetate obtained from 2 mLofthis Purity (1) Colchiceine—Dissolve 0.10 g of Colchicine in solution is equivalent to 0.11 to 0.21z of that obtained from 10 mL of water, and to 5 mL of this solution add 2 drops of 2 mL of the standard solution (2). iron (III) chloride TS: no deˆnite green color develops. System performance: To 1 mL of chloroform add N,N- (2) Chloroform and ethyl acetate—Weigh accurately dimethylformamide to make 10 mL. To 1 mL of this solu- about 0.60 g of Colchicine, dissolve in exactly 2 mL of the tion add 2 mL of ethyl acetate and N,N-dimethylformamide internal standard solution, add N,N-dimethylformamide to to make 100 mL. To 2 mL of this solution add 2 mL of the make 10 mL, and use this solution as the sample solution. internal standard solution and N,N-dimethylformamide to Separately, weigh 0.30 g of chloroform using a 100-mL make 10 mL. When the procedure is run with 2 mLofthis volumetric ‰ask containing about 20 mL of N,N-dimethyl- solution under the above operating conditions, ethyl acetate, formamide, and add N,N-dimethylformamide to make ex- chloroform and the internal standard are eluted in this order actly 100 mL. Pipet 2 mL of this solution, add N,N- with the resolution between the peaks of chloroform and the dimethylformamide to make exactly 200 mL, and use this internal standard being not less than 2.0. solution as the standard solution (1). Separately, weigh ac- System repeatability: When the test is repeated 3 times curately about 1.8 g of ethyl acetate using a 100-mL volu- with 2 mL of the standard solution (2) under the above metric ‰ask containing about 20 mL of N,N-dimethylfor- operating conditions, the relative standard deviation of the mamide, and add N,N-dimethylformamide to make exactly ratio of the peak area of ethyl acetate to that of the internal 100 mL. Pipet 2 mL of this solution, add exactly 2 mL of the standard is not more than 3.0z. internal standard solution and N,N-dimethylformamide to (3) Related substances Dissolve 60 mg of Colchicine in make 10 mL, and use this solution as the standard solution 100 mL of diluted methanol (1 in 2). Pipet 1 mL of this (2). Perform the test with 2 mL each of the sample solution solution, add diluted methanol (1 in 2) to make exactly 100 and the standard solutions (1) and (2) as directed under the mL, and use this solution as the sample solution. Perform Gas Chromatography according to the following conditions: the test with 20 mL of the sample solution as directed under the peak area of chloroform is not more than that from the the Liquid Chromatography according to the following standard solution (1). Determine the ratios of the peak area conditions, and determine each peak area by the automatic of ethyl acetate to that of the internal standard, Q and Q , T S integration method. Calculate the total amount of the peaks of the sample solution and the standard solution (2), and cal- other than colchicine by the area percentage method: not culate the amount of ethyl acetate by the following formula: more than 5.0z. the amount of ethyl acetate is not more than 6.0z. Operating conditions—

WS QT Detector: An ultraviolet absorption photometer Amount (z) of ethyl acetate (C4H8O2)＝ × ×2 WT QS (wavelength: 254 nm). Column: A stainless steel column 4.6 mm in inside di- W : Amount (g) of ethyl acetate S ameter and 25 cm in length, packed with octylsilanized silica W : Amount (g) of the sample T gel for liquid chromatography (5 mm in particle diameter). Internal standard solution—A solution of 1-propanol in Column temperature: A constant temperature of about N,N-dimethylformamide (3 in 200) 259C. Operating conditions— Mobile phase: To 450 mL of 0.05 mol/L potassium di- Detector: A hydrogen ‰ame-ionization detector hydrogen phosphate TS add methanol to make 1000 mL. Column: A fused silica column 0.53 mm in inside di- Adjust the pH to 5.5 with diluted phosphoric acid (7 in 200). ameter and 30 m in length, coated inside surface with Flow rate: Adjust the ‰ow rate so that the retention time polyethylene glycol 20 M for gas chromatography 1.0 mmin of colchicine is about 7 minutes. thickness. Time span of measurement: About 2 times as long as the Column temperature: 609C for 7 minutes, then up to retention time of colchicine after the solvent peak. 1009Catarateof409C per minute if necessary, and hold at System suitability— 1009Cfor10minutes. Test for required detectability: Pipet 1 mL of the sample Injection port temperature: A constant temperature of solution, and add diluted methanol (1 in 2) to make exactly about 1309C 50 mL. Conˆrm that the peak area of colchicine obtained Detector temperature: A constant temperature of about from 20 mL of this solution is equivalent to 1.4 to 2.6z of 2009C that obtained from 20 mL of the sample solution. Carrier gas: Helium System performance: When the procedure is run with Flow rate: Adjust the ‰ow rate so that the retention time 20 mL of the sample solution under the above operating of ethyl acetate is about 3 minutes. conditions, the number of theoretical plates and the symmet- Split ratio: 1:20 ry factor of the peak of colchicine are not less than 6000 and System suitability— not more than 1.5, respectively. Test for required detectability: Pipet 2 mL of the standard System repeatability: When the test is repeated 6 times solution (2), and add N,N-dimethylformamide to make with 20 mL of the sample solution under the above operating exactly 25 mL. Pipet 1 mL of this solution, and add N,N- conditions, the relative standard deviation of the peak area Supplement II, JPXIV O‹cial Monographs for Part I 1701 of colchicine is not more than 2.0z. water to make exactly 25 mL and use this solution as the standard solution (2). Perform the test with these solutions Water Not more than 2.0z (0.5 g, volumetric titration, as directed under the Thin-layer Chromatography. Spot back titration). 10 mL each of the sample solution and the standard solutions Assay Weigh accurately about 0.4 g of Colchicine, dissolve (1) and (2) on a plate of silica gel with ‰uorescent indicator in 25 mL of acetic anhydride, and titrate with 0.05 mol/L for thin-layer chromatography. Develop the plate with perchloric acid VS (potentiometric titration). Perform a 1-butanol saturated with water to a distance of about 12 cm, blank determination in the same manner, and make any and air-dry the plate. Examine under ultraviolet light (main necessary correction. wavelength: 254 nm): the spots other than the principal spot from the sample solution are not more intense than the spot Each mL of 0.05 mol/L perchloric acid VS from the standard solution (1), and the number of them ＝19.97 mg of C H NO 22 25 6 which are more intense than the spot from the standard solu- Containers and storage Containers—Tight containers. tion (2) is not more than two. Spray evenly acidic potassium Storage—Light-resistant. permanganate TS on the plate: any spot other than the prin- cipal spot does not appear. Cytarabine Deferoxamine Mesilate シタラビン メシル酸デフェロキサミン Change the origin/limits of content to read: Change the Purity (6) to read: Cytarabine, when dried, contains not less than Purity 98.5z and not more than 101.0z of C H N O . 9 13 3 5 (6) Related substances—Dissolve 50 mg of Deferoxa- mine Mesilata in 50 mL of the mobile phase, and use this Change the Description to read: solution as the sample solution. Pipet 3 mL of the sample so- Description Cytarabine occurs as white crystals or crystal- lution, add the mobile phase to make exactly 50 mL, and use line powder. this solution as the standard solution. Perform the test with It is freely soluble in water, soluble in acetic acid (100), exactly 20 mL each of the sample solution and the standard and very slightly soluble in ethanol (99.5). solution as directed under the Liquid Chromatography ac- It dissolves in 0.1 mol/L hydrochloric acid TS. cording to the following conditions. Determine each peak Melting point: about 2149C (with decomposition). area of both solutions by the automatic integration method: the total area of all peaks other than the peak of deferoxa- Change the Identiˆcation to read: mine is not larger than the peak area of deferoxamine from the standard solution. Identiˆcation (1) Determine the absorption spectrum of Operating conditions— a solution of Cytarabine in 0.1 mol/L hydrochloric acid TS Detector: An ultraviolet absorption photometer (1 in 100,000) as directed under the Ultraviolet-visible (wavelength: 230 nm). Spectrophotometry, and compare the spectrum with the Column: A stainless steel column 4 mm in inside diameter Reference Spectrum: both spectra exhibit similar intensities and 20 cm in length, packed with octadecylsilanized silica gel of absorption at the same wavelengths. for liquid chromatography (10 mm in particle diameter). (2) Determine the infrared absorption spectrum of Column temperature: A constant temperature of about Cytarabine as directed in the potassium bromide disk 409C. method under the Infrared Spectrophotometry, and Mobile phase: Dissolve 1.32 g of diammonium hydrogen compare the spectrum with the Reference Spectrum: both phosphate, 0.37 g of disodium dihydrogen ethylenediamine spectra exhibit similar intensities of absorption at the same tetraacetate dihydrate and 1.08 g of sodium 1-heptanesul- wave numbers. fonate in 950 mL of water, and adjust the pH of this solution to 2.8 with phosphoric acid. To 800 mL of this Delete the Absorbance: solution add 100 mL of 2-propanol. Flow rate: Adjust the ‰ow rate so that the retention time Change the Purity (4) and (5) to read: of deferoxamine is about 15 minutes. Purity Time span of measurement: About two times as long as (4) Related substances—Dissolve 0.10 g of Cytarabine in the retention time of deferoxamine after the solvent peak. 10 mL of water, and use this solution as the sample solution. System suitability— Pipet 1 mL of the sample solution, add water to make Test for required detection: To exactly 2 mL of the stan- exactly 200 mL, and use this solution as the standard dard solution add the mobile phase to make exactly 100 mL. solution (1). Pipet 10 mL of the standard solution (1), add Conˆrm that the peak area of deferoxamine obtained from 1702 O‹cial Monographs for Part I Supplement II, JPXIV

20 mLofthissolutionisequivalentto1.5to2.5z of that of than digitoxin and gitoxin, obtained by the area percentage deferoxamine obtained from 20 mL of the standard solution. method, is not more than 3z. System performance: Dissolve 16 mg of Deferoxamine Operating conditions— Mesilate and 4 mg of methyl parahydroxybenzoate in 50 mL Detector, column, column temperature, mobile phase, of the mobile phase. When the procedure is run with 20 mL and ‰ow rate: Proceed as directed in the operating condi- of this solution under the above operating conditions, tions in the Assay. deferoxamine and methyl parahydroxybenzoate are eluted in Time span of measurement: About 4 times as long as the this order with the resolution between these peaks being not retention time of digoxin after the solvent peak. less than 4. System suitability— System repeatability: When the test is repeated 6 times Test for required detectability: Pipet 1 mL of the sample with 20 mL of the standard solution under the above operat- solution, add the mobile phase to make exactly 100 mL, and ing conditions, the relative standard deviation of the peak use this solution as the solution for system suitability test. areas of deferoxamine is not more than 3.0z. Pipet 1 mL of the solution, and add the mobile phase to make exactly 10 mL. Conˆrm that the peak area of digoxin obtained from 10 mLofthissolutionisequivalentto7to Digoxin 13z of that from the solution for system suitability test. System performance: Dissolve 25 mg of digoxin in 50 mL ジゴキシン of warm ethanol (95), cool, and add ethanol (95) to make exactly 100 mL. Pipet 10 mL of this solution, add exactly Change the Description to read: 5 mL of a solution of propyl parahydroxybenzoate in ethanol (95) (1 in 4000), 10 mL of water and dilute ethanol to Description Digoxin occurs as colorless or white crystals or make 50 mL. When the procedure is run with 10 mLofthis a white crystalline powder. solution under the above operating conditions, digoxin and It is freely soluble in pyridine, slightly soluble in ethanol propyl parahydroxybenzoate are eluted in this order with the (95), very slightly soluble in acetic acid (100), and practically resolution between these peaks being not less than 5. insoluble in water. System repeatability: When the test is repeated 6 times Change the Identiˆcation (2) to read: with 10 mL of the solution for system suitability test under the above operating conditions, the relative standard devia- Identiˆcation tion of the peak area of digoxin is not more than 1.0z. (2) Determine the infrared absorption spectrum of Digoxin, previously dried, as directed in the potassium Change the Assay to read: bromide disk method under the Infrared Spectrophotomet- Assay Weigh accurately about 25 mg each of Digoxin and ry, and compare the spectrum with the Reference Spectrum: Digoxin Reference Standard, previously dried, dissolve in both spectra exhibit similar intensities of absorption at the 50 mL of warm ethanol (95), cool, and add ethanol (95) to same wave numbers. make exactly 100 mL. Pipet 10 mL of these solutions, add exactly 5 mL of the internal standard solution, 10 mL of Change the Optical rotation to read: water and dilute ethanol to make 50 mL, and use these 20 Optical rotation [a]D : ＋10.0 – ＋13.09(after drying, 0.20 g, solutions as the sample solution and the standard solution. dehydratead pyridine, 10 mL, 100 mm). Perform the test with 10 mL each of the sample solution and the standard solution as directed under the Liquid Chro- Change the Purity (2) to read: matography according to the following conditions, and determine the ratios, Q and Q , of the peak area of digoxin Purity T S to that of the internal standard. (2) Related substances—Dissolve exactly 25.0 mg of

Digoxin in 50 mL of warm ethanol (95), cool, and add QT Amount (mg) of C41H64O14＝WS× ethanol (95) to make exactly 100 mL. Pipet 10 mL of this QS solution, add 10 mL of water and dilute ethanol to make 50 W : Amount (mg) of Digoxin Reference Standard mL, and use this solution as the sample solution. Separately, S dissolve exactly 5.0 mg of Gitoxin Reference Standard, Internal standard solution—A solution of propyl para- previously dried under reduced pressure at 1059Cfor1hour, hydroxybenzoate in ethanol (95) (1 in 4000). in a mixture of acetonitrile and water (7:3) to make exactly Operating conditions— 200 mL. Pipet 2 mL of this solution, add dilute ethanol to Detector: An ultraviolet absorption photometer make 50 mL, and use this solution as the standard solution. (wavelength: 220 nm). Perform the test with exactly 10 mL each of the sample solu- Column: A stainless steel column 4.6 mm in inside tion and the standard solution as directed under the Liquid diameter and 25 cm in length, packed with octadecyl- Chromatography according to the following conditions, and silanized silica gel for liquid chromatography (5 mminparti- determine the peak areas, AT and AS,ofgitoxin:AT is not cle diameter). larger than AS, and the total of the areas of the peaks other Column temperature: A constant temperature of about Supplement II, JPXIV O‹cial Monographs for Part I 1703

309C. Matter Test for Injections. Mobile phase: A mixture of water and acetonitrile (7:3). Insoluble particulate matter Perform the test according to Flow rate: Adjust the ‰ow rate so that the retention time Method 1: it meets the requirement of the Insoluble Particu- of digoxin is about 10 minutes. late Matter Test for Injections. System suitability— System performance: When the procedure is run with Sterility Perform the test according to the Membrane 10 mL of the standard solution under the above operating ˆltration method: it meets the requirements of the Sterility conditions, digoxin and the internal standard are eluted in Test. this order with the resolution between these peaks being not Assay To an exact volume of Digoxin Injection, equivalent less than 5. to about 2.5 mg of digoxin (C H O ), add exactly 5 mL of System repeatability: When the test is repeated 6 times 41 64 14 the internal standard solution and dilute ethanol to make with 10 mL of the standard solution under the above operat- 50 mL, and use this solution as the sample solution. ing conditions, the relative standard deviation of the ratio of Separately, weigh accurately about 25 mg of Digoxin Refer- the peak area of digoxin to that of the internal standard is ence Standard, previously dried under reduced pressure at not more than 1.0z. 1059C for 1 hour, dissolve in 50 mL of warm ethanol (95), cool, and add ethanol (95) to make exactly 100 mL. Pipet 10 mL of this solution, add exactly 5 mL of the internal stan- Change to read: dard solution, 10 mL of water and dilute ethanol to make 50 mL, and use this solution as the standard solution. Perform Digoxin Injection the test with 10 mL each of the sample solution and the stan- dard solution as directed under the Liquid Chromatography ジゴキシン注射液 according to the following conditions, and determine the ratios, Q and Q , of the peak area of digoxin to that of the Digoxin Injection is an aqueous solution for T S injection. internal standard.

It contains not less than 90.0z and not more than QT 1 Amount (mg) of digoxin (C41H64O14)＝WS× × 105.0z of the labeled amount of digoxin (C41H64O14: QS 10 780.94). WS: Amount (mg) of Digoxin Reference Standard Method of preparation Prepare as directed under Injec- Internal standard solution—A solution of propyl para- tions, with a solution of Digoxin in 5 to 50 volz ethanol. hydroxybenzoate in ethanol (95) (1 in 4000). Description Digoxin Injection is a clear, colorless liquid. Operating conditions— Detector: An ultraviolet absorption photometer Identiˆcation Dilute Digoxin Injection, if necessary, with (wavelength: 220 nm). methanol so that each mL contains about 0.25 mg of Digox- Column: A stainless steel column 4.6 mm in inside inaccordingtothelabeledamount,andusethissolutionas diameter and 25 cm in length, packed with octadecyl- the sample solution. In case where ingredients are suspected silanized silica gel for liquid chromatography (5 mminparti- to aŠect the test, remove them by means of a solid-phase ex- cle diameter). traction. Separately, dissolve 0.5 mg of Digoxin Reference Column temperature: A constant temperature of about Standard in 2 mL of methanol, and use this solution as the 309C. standard solution. Perform the test with these solutions as Mobile phase: A mixture of water and acetonitrile (7:3). directed under the Thin-layer Chromatography. Spot 10 mL Flow rate: Adjust the ‰ow rate so that the retention time each of the sample solution and the standard solution on a of digoxin is about 10 minutes. plate of octadecylsilanized silica gel for thin-layer chro- System suitability— matography. Develop the plate with a mixture of methanol System performance: When the procedure is run with 10 and water (7:3) to a distance of about 10 cm, and air-dry the mL of the standard solution under the above operating con- plate. Spray evenly a mixture of a solution of trichloroacetic ditions, digoxin and the internal standard are eluted in this acid in ethanol (99.5) (1 in 4) and a freshly prepared solution order with the resolution between these peaks being not less of sodium toluenesulfonchloramide trihydrate (3 in 100) than 5. (4:1) on the plate, heat at 1109C for 10 minutes, and System repeatability: When the test is repeated 6 times examine under ultraviolet light (main wavelength: 366 nm): with 10 mL of the standard solution under the above the Rf values of the principal spots with the sample solution operating conditions, the relative standard deviation of the and the standard solution are not diŠerent each other. ratio of the peak area of digoxin to that of the internal Bacterial endotoxins Less than 200 EU/mg. standard is not more than 1.0z.

Actual volume It meets the requirements of Injections. Containers and storage Containers—Hermetic containers, and colored containers may be used. Foreign insoluble matter Perform the test according to Storage—Light-resistant. Method 1: it meets the requirements of the Foreign Insoluble 1704 O‹cial Monographs for Part I Supplement II, JPXIV

Change to read: Dissolution Perform the test with 1 tablet of Digoxin Tablets, using 500 mL of diluted hydrochloric acid (3 in 500) Digoxin Tablets at 100 revolutions per minute according to the Method 1 un- der the Dissolution Test as the dissolution medium. ジゴキシン錠 Withdraw 30 mL or more of the dissolved solution 60 minutes after starting the test, and ˆlter through a mem- Digoxin Tablets contain not less than 90.0z and not brane ˆlter (less than 0.8 mm in pore size). Discard the ˆrst more than 105.0z of the labeled amount of digoxin 10 mL of the ˆltrate, and use the subsequent ˆltrate as the (C41H64O14:780.94). sample solution. Separately, weigh accurately about 25 mg of Digoxin Reference Standard, previously dried in vacuum Method of preparation Prepare as directed under Tablets, at 1059C for 1 hour, dissolve in a small portion of ethanol with Digoxin. (95), and add a mixture of ethanol (95) and water (4:1) to Identiˆcation To an amount of pulverized Digoxin make exactly 500 mL. Pipet 5 mL of this solution, add the Tablets, equivalent to 0.5 mg of Digoxin according to the la- dissolution medium to make exactly 500 mL, and use this so- beled amount, add 2 mL of methanol, shake for 10 minutes, lution as the standard solution. Pipet 2 mL each of the sam- ˆlter, and use the ˆltrate as the sample solution. Separately, ple solution, the standard solution and the dissolution medi- dissolve 0.5 mg of Digoxin Reference Standard in 2 mL of um, and transfer to brown glass-stoppered test tubes. Add methanol, and use this as the standard solution. Perform the exactly 10 mL of 0.012 g/dL L-ascorbic acid-hydrochloric test with these solutions as directed under the Thin-layer acid TS to these tubes, and shake. Immediately add exactly 1 Chromatography. Spot 10 mL each of the sample solution mL of dilute hydrogen peroxide TS, shake well, and allow to and the standard solution on a plate of octadecylsilanized stand at a constant temperature between 259Cand309Cfor silica gel for thin-layer chromatography. Develop the plate 45 minutes. Determine the ‰uorescence intensities, FT, FS, with a mixture of methanol and water (7:3) to a distance of and FB, of these solutions at 360 nm of the excitation about 10 cm, and air-dry the plate. Spray evenly a mixture of wavelength and at 485 nm of the ‰uorescence wavelength as a solution of trichloroacetic acid in ethanol (99.5) (1 in 4) directed under the Fluorometry, respectively: the dissolution and a freshly prepared solution of sodium toluenesulfon- rate in 60 minutes is not less than 65z. No retest require- chloramide trihydrate (3 in 100) (4:1) on the plate, heat at ment is applied to Digoxin Tablets. 1109C for 10 minutes, and examine under ultraviolet light Dissolution rate (z) with respect to the labeled amount of (main wavelength: 366 nm): the Rf values of the principal digoxin (C H O ) spots with the sample solution and the standard solution are 41 64 14 FT－FB 1 not diŠerent each other. ＝WS× × FS－FB C Content uniformity Perform the test according to the fol- W : Amount (mg) of Digoxin Reference Standard lowing method: it meets the requirements of the Content S C: The labeled amount (mg) of digoxin (C H O )in1 Uniformity Test. 41 64 14 tablet To 1 tablet of Digoxin Tablets add 0.5 mL of water to disintegrate, then add exactly 0.5 mL of the internal stan- Assay Weigh accurately the mass of not less than 20 dard solution, and add V mL of dilute ethanol so that each Digoxin Tablets, and powder. Weigh accurately a portion of mL contains about 21 mg of digoxin (C41H64O14). Exposure the powder, equivalent to about 2.5 mg of digoxin (C41H64O this solution to ultrasonic waves for 20 minutes, shake for 5 14), add 30 mL of dilute ethanol, exposure to ultrasonic minutes, ˆlter, and use the ˆltrate as the sample solution. waves for 20 minutes, and shake for 5 minutes. After cool- Separately, weigh accurately about 25 mg of Digoxin Refer- ing, add exactly 5 mL of the internal standard solution and ence Standard, previously dried under reduced pressure at dilute ethanol to make 50 mL, centrifuge, and use the super- 1059C for 1 hour, dissolve in 50 mL of warm ethanol (95), natant liquid as the sample solution. Separately, weigh ac- cool, and add ethanol (95) to make exactly 100 mL. Pipet curately about 25 mg of Digoxin Reference Standard, previ- 10 mL of this solution, and add ethanol (95) to make exactly ously dried under reduced pressure at 1059C for 1 hour , dis- 20 mL. Pipet 1 mL of this solution, add exactly 0.5 mL of solve in 50 mL of warm ethanol (95), cool, and add ethanol the internal standard solution, then add 1.5 mL of water and (95) to make exactly 100 mL. Pipet 10 mL of this solution, (V – 2) mL of dilute ethanol, and use this as the standard add exactly 5 mL of the internal standard solution, 10 mL of solution. Proceed with the sample solution and the standard water and dilute ethanol to make 50 mL, and use this solu- solution as directed in the Assay. tion as the standard solution. Perform the test with 10 mL each of the sample solution and the standard solution as QT 1 Amount (mg) of digoxin (C41H64O14)＝WS× × directed under the Liquid Chromatography according to the QS 200 following conditions, and determine the ratios, QT and QS, WS: Amount (mg) of Digoxin Reference Standard of the peak area of digoxin to that of the internal standard.

Internal standard solution—A solution of propyl para- QT 1 Amount (mg) of digoxin (C41H64O14)＝WS× × hydroxybenzoate in ethanol (95) (1 in 40,000/V). QS 10 Supplement II, JPXIV O‹cial Monographs for Part I 1705

WS: Amount (mg) of Digoxin Reference Standard Dimorpholamine, previously dried, as directed in the potas- sium bromide disk method under the Infrared Spectrophoto- Internal standard solution—A solution of propyl para- metry, and compare the spectrum with the Reference hydroxybenzoate in ethanol (95) (1 in 4000). Spectrum: both spectra exhibit similar intensities of absorp- Operating conditions— tion at the same wave numbers. Detector: An ultraviolet absorption photometer (wavelength: 220 nm). AddthefollowingnexttoPurity(4): Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with octadecyl- (5) Related substances—Dissolve 0.20 g of Dimorphola- silanized silica gel for liquid chromatography (5 mminparti- mine in 10 mL of ethanol (99.5), and use this solution as the cle diameter). sample solution. Pipet 1 mL of the sample solution, add Column temperature: A constant temperature of about ethanol (99.5) to make exactly 100 mL, and use this solution 309C. as the standard solution. Perform the test with these solu- Mobile phase: A mixture of water and acetonitrile (7:3). tions as directed under the Thin-layer Chromatography. Flow rate: Adjust the ‰ow rate so that the retention time Spot 10 mL each of the sample solution and the standard of digoxin is about 10 minutes. solution on a plate of silica gel for thin-layer chro- System suitability— matography. Develop the plate with a mixture of ethanol System performance: When the procedure is run with (99.5) and water (4:1) to a distance of about 10 cm, and 10 mL of the standard solution under the above operating air-dry the plate. Allow the plate to stand in iodine vapor for conditions, digoxin and the internal standard are eluted in 10 minutes: the spot other than the principal spot from the this order with the resolution between these peaks being not sample solution is not more intense than the spot from the less than 5. standard solution. System repeatability: When the test is repeated 6 times with 10 mL of the standard solution under the above operat- Change the Assay to read: ing conditions, the relative standard deviation of the ratio of Assay Weigh accurately about 0.6 g of Dimorpholamine, the peak area of digoxin to that of the internal standard is previously dried, dissolve in 50 mL of acetic anhydride, and not more than 1.0z. titrate with 0.1 mol/L perchloric acid VS (potentiometric Containers and storage Containers—Tight containers. titration). Perform a blank determination, and make any Storage—Light-resistant. necessary correction.

Each mL of 0.1 mol/L perchloric acid VS Dimorpholamine ＝39.85 mg of C20H38N4O4

ジモルホラミン Dimorpholamine Injection Change the origin/limits of the content to read: ジモルホラミン注射液 Dimorpholamine, when dried, contains not less than Change the origin/limits of content to read: 98.0z and not more than 101.0z of C20H38N4O4. Dimorpholamine Injection is an aqueous solution Change the Description to read: for injection. Description Dimorpholamine is a white to light yellow, It contains not less than 95.0z and not more than crystalline powder, masses or syrupy liquid. 105.0z of the labeled amount of dimorpholamine It is very soluble in ethanol (99.5) and in acetic anhydride, (C20H38N4O4:398.54). and soluble in water. The pH of a solution prepared by dissolving 1.0 g of Change the Description to read: Dimorpholamine in 10 mL of water is between 6.0 and 7.0. Description Dimorpholamine Injection is a clear, colorless It is hygroscopic. liquid. pH:3.0–5.5 Change the Identiˆcation to read: Identiˆcation (1) Determine the absorption spectrum of Change the Identiˆcation (2) to read: a solution of Dimorpholamine (1 in 50,000) as directed Identiˆcation under the Ultraviolet-visible Spectrophotometry, and (2) To a volume of Dimorpholamine Injection, equiva- compare the spectrum with the Reference Spectrum: both lent to 50 mg of Dimorpholamine according to the labeled spectra exhibit similar intensities of absorption at the same amount, add 1 mL of dilute hydrochloric acid, and wavelengths. evaporate on a water bath to dryness. Dissolve the residue in (2) Determine the infrared absorption spectrum of 1706 O‹cial Monographs for Part I Supplement II, JPXIV

2 mL of hydrochloric acid, boil for 10 minutes under a re‰ux Column temperature: A constant temperature of about condenser, and evaporate to dryness on a water bath. 409C. Dissolve the residue with 1 mL of water, neurtralize with Mobile phase: A mixture of water and acetonitrile (1:1). sodium hydroxide TS, and add 0.2 mL of a solution of Flow rate: Adjust the ‰ow rate so that the retention time acetaldehyde (1 in 20), 0.1 mL of sodium pentacyanonitrosyl of dimorpholamine is about 4 minutes. ferrate (III) TS and 0.5 mL of sodium carbonate TS: a blue System suitability— color develops. System performance: When the procedure is run with 10 mL of the standard solution under the above operating Add the following next to Identiˆcation: conditions, dimorpholamine and the internal standard are eluted in this order with the resolution between these peaks Bacterial endotoxins Less than 5.0 EU/mg. Perform the being not less than 2.0. test with the sample diluted to 0.15 w/vz with water for System repeatability: When the test is repeated 6 times bacterial endotoxins test. with 10 mL of the standard solution under the above operat- Actual volume It meets the requirements of Injections. ing conditions, the relative standard deviation of the ratio of the peak area of dimorpholamine to that of the internal Foreign insoluble matter Perform the test according to standard is not more than 1.0z. Method 1: it meets the requirements of the Foreign Insoluble Matter Test for Injections. Insoluble particulate matter Perform the test according to Add the following: Method 1: it meets the requirements of the Insoluble Par- ticulate Matter Test for Injections. Eperisone Hydrochloride Sterility Perform the test according to the Membrane 塩酸エペリゾン ˆltration method: it meets the requirements of the Sterility Test.

Change the Assay to read:

Assay Measure exactly a volume of Dimorpholamine C17H25NO.HCl: 295.85 Injection, equivalent to about 30 mg of dimorpholamine (2RS)-1-(4-Ethylphenyl)-2-methyl-3-piperidin-1-ylpropan-1-

(C20H38N4O4), and add water to make exactly 200 mL. Pipet one monohydrochloride [56839-43-1] 1 mL of this solution, shake with exactly 4 mL of the inernal standard solution for 5 minutes, and use this solution as the Eperisone Hydrochloride contains not less than sample solution. Separately, weigh accurately about 0.15 g 98.5z and not more than 101.0z of C17H25NO.HCl, of dimorpholamine for assay, previously dried in a desicca- calculated on the anhydrous basis. tor (in vacuum, phosphorus (V) oxide) for 8 hours, and Description Eperisone Hydrochloride occurs as a white dissolve in water to make exactly 1000 mL. Pipet 1 mL of crystalline powder. this solutionn, shake with exactly 4 mL of the inernal It is freely soluble in water, in methanol and in acetic acid standard solution for 5 minutes, and use this solution as the (100), and soluble in ethanol (99.5). standard solution. Perform the test with 10 mLeachofthe Melting point: about 1679C (with decomposition). sample solution and the standard solution as directed under A solution of Eperisone Hydrochloride in methanol (1 in the Liquid Chromatography according to the following 100) shows no optical rotation. conditions, and calculate the ratios, QT and QS,ofthepeak area of dimorpholamine to that of the inernal standard. Identiˆcation (1) Determine the absorption spectrum of a solution of Eperisone Hydrochloride in methanol (1 in Amount (mg) of dimorpholamine (C20H38N4O4) 100,000) as directed under the Ultraviolet-visible Spec- QT 1 ＝WS× × trophotometry, and compare the spectrum with the Refer- QS 5 ence Spectrum: both spectra exhibit similar intensities of

WS: Amount (mg) of dimorpholamine for assay absorption at the same wavelengths. (2) Determine the infrared absorption spectrum of Internal standard solution—A solution of butyl parahydrox- Eperisone Hydrochloride as directed in the potassium chlo- ybenzoate in acetonitrile (1 in 25,000). ride disk method under the Infrared Spectrophotometry, Operating conditions— and compare the spectrum with the Reference Spectrum: Detector: An ultraviolet absorption photometer both spectra exhibit similar intensities of absorption at the (wavelength: 216 nm). same wave numbers. Column: A stainless steel column 4.6 mm in inside di- (3) A solution of Eperisone Hydrochloride (1 in 50) ameter and 15 cm in length, packed with octadecylsilanized responds to the Qualitative Tests for chloride. silica gel for liquid chromatography (5 mminparticledi- ameter). Purity (1) Heavy metals—Proceed with 1.0 g of Eperi- Supplement II, JPXIV O‹cial Monographs for Part I 1707 sone Hydrochloride according to Method 1, and perform the ing conditions, the relative standard deviation of the peak test. Prepare the control solution with 2.0 mL of Standard area of eperisone is not more than 3.0z. Lead Solution (not more than 20 ppm). Water Not more than 0.2z (0.1 g, coulometric titration). (2) Piperidine hydrochloride—Dissolve 1.0 g of Eperi- sone Hydrochloride in 20 mL of water, add 2.0 mL of Residue on ignition Not more than 0.2z (1 g). dilutedhydrochloricacid(1in2),2.0mLofasolutionof Assay Weigh accurately about 0.6 g of Eperisone copper (II) sulfate pentahydrate (1 in 20) and 1.5 mL of am- Hydrochloride, dissolve in 20 mL of acetic acid (100), add monia solution (28), and use this solution as the sample solu- 80 mL of acetic anhydride, and titrate with 0.1 mol/L per- tion. Separately, to 2.0 mL of a solution of piperidine chloric acid VS (potentiometric titration). Perform a blank hydrochloride (1 in 1000) add 18 mL of water, 2.0 mL of determination in the same manner, and make any necessary dilutedhydrochloricacid(1in2),2.0mLofasolutionof correction. copper (II) sulfate pentahydrate (1 in 20) and 1.5 mL of ammonia solution (28), and use this solution as the standard Each mL of 0.1 mol/L perchloric acid VS solution. To each of the sample solution and the standard ＝29.58 mg of C17H25NO.HCl solution add 10 mL of a mixture of isopropylether and Containers and storage Containers—Well-closed contain- carbon disulˆde (3:1), shake for 30 seconds, allow them to ers. stand for 2 minutes, and compare the color of the upper layer: the color obtained from the sample solution is not more darker than that from the standard solution. (3) Related substances—Dissolve 0.1 g of Eperisone Ethionamide Hydrochloride in 100 mL of the mobile phase, and use this エチオナミド solution as the sample solution. Pipet 1 mL of the sample solution, add the mobile phase to make exactly 100 mL, and Change to read except the structural formula use this solution as the standard solution. Perform the test and chemical name: with exactly 10 mL each of the sample solution and the stan- dard solution as directed under the Liquid Chromatography Ethionamide, when dried, contains not less than according to the following conditions, and determine each 98.5z and not more than 101.0z of C H N S. peak area by the automatic integration method: the total 8 10 2 area of the peaks other than the peak of eperisone is not Description Ethionamide occurs as yellow crystals or crys- more than 1/5 of the peak area of eperisone from the stan- talline powder, having a characteristic odor. dard solution. It is soluble in methanol and in acetic acid (100), sparingly Operating conditions— solubleinethanol(99.5)andinacetone,andpracticallyin- Detector: An ultraviolet absorption photometer soluble in water. (wavelength: 254 nm). Identiˆcation (1) Determine the absorption spectrum of Column: A stainless steel column 4.6 mm in inside diameter a solution of Ethionamide in methanol (3 in 160,000) as and 15 cm in length, packed with octadecylsilanized silica gel directed under the Ultraviolet-visible Spectrophotometry, for liquid chromatography (5 mminparticlediameter). and compare the spectrum with the Reference Spectrum: Column temperature: A constant temperature of about both spectra exhibit similar intensities of absorption at the 309C. same wavelengths. Mobile phase: A mixture of methanol, 0.0375 mol/L sodi- (2) Determine the infrared absorption spectrum of um 1-decanesulfonate TS and perchloric acid (600:400:1). Ethionamide as directed in the potassium bromide disk Flow rate: Adjust the ‰ow rate so that the retention time method under the Infrared Spectrophotometry, and com- of eperisone is about 17 minutes. pare the spectrum with the Reference Spectrum: both spec- Time span of measurement: About 2 times as long as the tra exhibit similar intensities of absorption at the same wave retention time of eperisone. numbers. System suitability— Test for required detectability: Pipet 1 mL of the standard Melting point 161–1659C solution, and add the mobile phase to make exactly 10 mL. Purity Conˆrm that the peak area of eperisone obtained from 10 (1) Acid—Dissolve 3.0 g of Ethionamide in 30 mL of mLofthissolutionisequivalentto7to13z of that obtained methanol by warming, add 90 mL of water, allow to stand in from 10 mL of the standard solution. ice water for 1 hour, and ˆlter. To 80 mL of the ˆltrate add System performance: When the procedure is run with 10 0.8mLofcresolredTSand0.20mLof0.1mol/Lsodium mL of the standard solution under the above operating hydroxide VS: a red color develops. conditions, the number of theoretical plates and the symmet- (2) Heavy metals—Proceed with 1.0 g of Ethionamide ry factor of the peak of eperisone are not less than 4000 steps according to Method 2, and perform the test. Prepare the and not more than 2.0, respectively. control solution with 2.0 mL of Standard Lead Solution (not System repeatability: When the test is repeated 6 times more than 20 ppm). with 10 mL of the standard solution under the above operat- 1708 O‹cial Monographs for Part I Supplement II, JPXIV

(3) Arsenic—Prepare the test solution with 1.0 g of Change the Identiˆcation to read: Ethionamide according to Method 3. Add 10 mL of a solu- Identiˆcation To a quantity of powdered Etilefrine tion of magnesium nitrate hexahydrate in ethanol (95) (1 in Hydrochloride Tablets, equivalent to 5 mg of Etilefrine 50), then add 1.5 mL of hydrogen peroxide (30), and ˆre to Hydrochloride according to the labeled amount, add 60 mL burn (not more than 2 ppm). of diluted hydrochloric acid (1 in 1000), shake well, add (4) Related substances—Conduct this procedure without 40 mL of diluted hydrochloric acid (1 in 1000), and ˆlter. exposure to light, using light-resistant vessels. Dissolve 0.20 Determine the absorption spectrum of the ˆltrate as directed g of Ethionamide in 10 mL of acetone, and use this solution under the Ultraviolet-visible Spectrophotometry, using as the sample solution. Pipet 0.5 mL of the sample solution, diluted hydrochloric acid (1 in 1000) as the blank: it exhibits add acetone to make exactly 100 mL, and use this solution as a maximum between 271 nm and 275 nm. the standard solution (1). Separately, pipet exactly 0.2 mL of the sample solution, add acetone to make exactly 100 mL, Add the following next to Identiˆcation: and use this solution as the standard solution (2). Perform the test with these solutions as directed under the Thin-layer Content uniformity Perform the test according to the fol- Chromatography. Spot 10 mL each of the sample solution lowing method: it meets the requirements of the Content and the standard solutions (1) and (2) on a plate of silica gel Uniformity Test. with ‰uorescent indicator for thin-layer chromatography, To 1 tablet of Etilefrine Hydrochloride Tablets add 60 mL develop with a mixture of ethyl acetate, hexane and of diluted hydrochloric acid (1 in 1000), and proceed as methanol (6:2:1) to a distance of about 15 cm, and air-dry directed in the Assay. the plate. Examine under ultraviolet light (main wavelength: Amount (mg) of etilefrine hydrochloride (C H NO .HCl) 254 nm): the spot other than the principal spot obtained with 10 15 2 A 1 the sample solution is not more intense than the spot with ＝W × T × S A 10 the standard solution (1), and number of the spot other than S the principal spot obtained with the sample solution which is WS: Amount (mg) of etilefrine hydrochloride for assay more intense than the spot with the standard solution (2) is not more than one. Change the Assay to read: Loss on drying Not more than 0.5z (1 g, 1059C, 3 hours). Assay Weigh accurately the mass of not less than 20 Etilefrine Hydrochloride Tablets, and powder. Weigh ac- Residue on ignition Not more than 0.10z (1 g). curately a portion of the powder, equivalent to about 5 mg

Assay Weigh accurately about 0.3 g of Ethionamide, of etilefrine hydrochloride (C10H15NO2.HCl), add 60 mL of previously dried, dissolve in 50 mL of acetic acid (100), and diluted hydrochloric acid (1 in 1000), shake for 10 minutes, titrate with 0.1 mol/L perchloric acid VS until the color of add diluted hydrochloric acid (1 in 1000) to make exactly 100 the solution changes from orange-red to dark orange-brown mL, and ˆlter. Discard the ˆrst 20 mL of the ˆltrate, and use (indicator: 2 mL of p-naphtholbenzein TS). Perform a blank the subsequent ˆltrate as the sample solution. Separately, determination in the same manner, and make any necessary weigh accurately about 50 mg of etilefrine hydrochloride for correction. assay, previously dried at 1059C for 4 hours, and dissolve in diluted hydrochloric acid (1 in 1000) to make exactly 100 Each mL of 0.1 mol/L perchloric acid VS mL. Pipet 10 mL of this solution, add diluted hydrochloric ＝16.62 mg of C H N S 8 10 2 acid (1 in 1000) to make exactly 100 mL, and use this solu- Containers and storage Containers—Well-closed contain- tion as the standard solution. Perform the test with exactly ers. 20 mL each of the sample solution and the standard solution as direct under the Liquid Chromatography according to the

following conditions, and determine the peak areas, AT and Etilefrine Hydrochloride Tablets AS, of etilefrine. Amount (mg) of etilefrine hydrochloride (C H NO .HCl) 塩酸エチレフリン錠 10 15 2 A 1 ＝W × T × S A 10 Change the origin/limits of content to read: S

WS: Amount (mg) of etilefrine hydrochloride for assay Etilefrine Hydrochloride Tablets contain not less Operating conditions— than 93.0z and not more than 107.0z of the labeled Detector: An ultraviolet absorption photometer amount of etilefrine hydrochloride (C H NO .HCl: 10 15 2 (wavelength: 220 nm). 217.69). Column: A stainless steel column 4.6 mm in inside di- ameter and 25 cm in length, packed with octylsilanized silica gel for liquid chromatography (5 mm in particle diameter). Column temperature: A constant temperature of about 409C. Supplement II, JPXIV O‹cial Monographs for Part I 1709

Mobile phase: Dissolve 5 g of sodium lauryl sulfate in Etoposide as directed in the potassium bromide disk method 940 mL of water and 500 mL of acetonitrile, and adjust the under the Infrared Spectrophotometry, and compare the pH to 2.3 with phosphoric acid. spectrum with the Reference Spectrum or the spectrum of Flow rate: Adjust the ‰ow rate so that the retention time Etoposide Reference Standard: both spectra exhibit similar of etilefrine is about 6 minutes. intensities of absorption at the same wave numbers. System suitability— Optical rotation [a]20: －100 – －1059(0.1 g calculated on System performance: Dissolve 4 mg of bamethan sulfate D the anhydrous basis, methanol, 20 mL, 100 mm). and 4 mg of etilefrine hydrochloride in the mobile phase to make 50 mL. When the procedure is run with 20 mLofthis Purity (1) Heavy metals—Proceed with 2.0 g of Etopo- solution under the above operating conditions, etilefrine and side according to Method 2, and perform the test. Prepare bamethan are eluted in this order with the resolution the control solution with 2.0 mL of Standard Lead Solution between these peaks being not less than 5. (not more than 10 ppm). System repeatability: When the test is repeated 6 times (2) Related substances—Dissolve 50 mg of Etoposide in with 20 mL of the standard solution under the above operat- 10 mL of methanol, add the mobile phase to make 50 mL, ing conditions, the relative standard deviation of the peak and use this solution as the sample solution. Pipet 2 mL of area of etilefrine is not more than 1.0z. the sample solution, add the mobile phase to make exactly 200 mL, and use this solution as the standard solution. Per- form the test with exactly 50 mL each of the sample solution Add the following: and the standard solution as directed under the Liquid Chro- matography according to the following conditions, and Etoposide determine each peak area by the automatic integration method: the area of the peak other than etoposide is not エトポシド larger than 1/5 times the peak area of etoposide with the standard solution, and the total area of the peaks other than the peak of etoposide with the sample solution is not larger than 1/2 times the peak area of etoposide with the standard solution. Operating conditions— Detector, column, column temperature, mobile phase, and ‰ow rate: Proceed as directed in the operating condi- tions in the Assay. Time span of measurement: About 3 times as long as the retention time of etoposide after the solvent peak. System suitability—

C29H32O13: 588.56 Test for required detectability: Measure exactly 1 mL of (5R,5aR,8aR,9S)-9-s[4,6-O-(1R)-Ethylidene- the standard solution, and add the mobile phase to make b-D-glucopyranosyl]oxyt-5,8,8a,9-tetrahydro-5-(4-hydroxy- exactly 10 mL. Conˆrm that the peak area of etoposide 3,5-dimethoxyphenyl)furo[3?,4?:6,7]naphtha[2,3-d]-1,3- obtained with 50 mLofthissolutionisequivalentto7to dioxol-6(5aH)-one [33419-42-0] 13z of that with 50 mL of the standard solution. System performance: Proceed as directed in the system Etoposide contains not less than 98.0z and not suitability in the Assay. more than 102.0z of C29H32O13,calculatedonthe System repeatability: When the test is repeated 6 times anhydrous basis. with 50 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the peak Description Etoposide occurs as white crystals or crystal- area of etoposide is not more than 2.0z. line powder. It is sparingly soluble in methanol, slightly soluble in Water Not more than 4.0z (0.5 g, volumetric titration, ethanol (99.5), and very slightly soluble in water. direct titration). Melting point: about 2609C (with decomposition). Residue on ignition Not more than 0.1z (1 g). Identiˆcation (1) Determine the absorption spectrum of Assay Weigh accurately about 25 mg each of Etoposide a solution of Etoposide in methanol (1 in 10,000) as directed and Etoposide Reference Standard, separately determined under the Ultraviolet-visible Spectrophotometry, and the water content, and dissolve separately in methanol to compare the spectrum with the Reference Spectrum or the make exactly 25 mL. Pipet 10 mL each of these solutions, spectrum of a solution of Etoposide Reference Standard add exactly 5 mL of the internal standard solution and the prepared in the same manner as the sample solution: both mobile phase to make 50 mL, and use these solutions as the spectra exhibit similar intensities of absorption at the same sample solution and the standard solution. Perform the test wavelengths. with 50 mL each of the sample solution and the standard (2) Determine the infrared absorption spectrum of 1710 O‹cial Monographs for Part I Supplement II, JPXIV solution as directed under the Liquid Chromatography with 20 mL of the sample solution as directed under the according to the following conditions, and determine the Liquid Chromatography according to the following condi- ratios, QT and QS, of the peak area of etoposide to that of tions. Determine the peak area, A, of ‰avin adenine the internal standard. dinucleotide and the total area, S, of peaks other than the peak of ‰avin adenine dinucleotide by the automatic integra- Q Amount (mg) of C H O ＝W × T tion method: S/(A＋S) is not more than 0.10. 29 32 13 S Q S Operating conditions—

WS: Amount (mg) of Etoposide Reference Standard, Column, column temperature, mobile phase, ‰ow rate, calculated on the anhydrous basis and time span of measurement: Proceed as directed in the Internal standard solution—A solution of 2,6-dichloro- operating conditions in the Procedure (ii) under Assay (1). phenol in methanol (3 in 2500). Detector: An ultraviolet absorption photometer Operating conditions— (wavelength: 260 nm). Detector: An ultraviolet absorption photometer System suitability— (wavelength: 290 nm). Test for required detection: To exactly 2 mL of the sample Column: A stainless steel column 3.9 mm in inside solution add the mobile phase to make exactly 20 mL, and diameter and 30 cm in length, packed with phenylsilanized use this solution as the solution for system suitability test. silica gel for liquid chromatography (10 mminparticle Conˆrm that the peak area of ‰avin adenine dinucleotide diameter). obtained from 20 mLofthissolutionisequivalentto8to Column temperature: A constant temperature of about 12z of that of ‰avin adenine dinucleotide obtained from 359C. 20 mL of the sample solution. Mobile phase: Dissolve 6.44 g of sodium sulfate decahy- System performance: Proceed as directed in the system drate in diluted acetic acid (100) (1 in 100) to make 1000 mL, suitability in the Procedure (ii) under Assay (1). and add 250 mL of acetonitrile. System repeatability: When the test is repeated 6 times Flow rate: Adjust the ‰ow rate so that the retention time with 20 mL of the solution for system suitability test under of etoposide is about 20 minutes. the above operating conditions, the relative standard devia- System suitability— tion of the peak area of ‰avin adenine dinucleotide is not System performance: Dissolve 10 mg of Etoposide in more than 1.0z. 2 mL of methanol, add 8 mL of the mobile phase, and mix well. Add 0.1 mL of diluted acetic acid (100) (1 in 25) and Change the Assay (1) to read: 0.1 mL of phenolphthalein TS, and add sodium hydroxide Assay (1) Procedure (i) Total ‰avin content—Conduct TS until the color of the solution changes to faintly red. Af- this procedure without exposure to daylight, using light- ter allowing to stand for 15 minutes, add 0.1 mL of diluted resistant vessels. Weigh accurately about 0.1 g of Flavin acetic acid (100) (1 in 25). When the procedure is run with 10 Adenine Dinucleotide Sodium, and dissolve in water to mL of this solution under the above operating conditions, the make exactly 200 mL. Pipet 5 mL of this solution, add 5 mL resolution between the peak of etoposide and the peak hav- of zinc chloride TS, and heat in a water bath for 30 minutes. ing the relative retention time of about 1.3 with respect to After cooling, add water to make exactly 100 mL, and use etoposide is not less than 3. this solution as the sample solution. Separately, weigh System repeatability: When the test is repeated 6 times accurately about 50 mg of Ribo‰avin Reference Standard, with 50 mL of the standard solution under the above operat- previously dried at 1059C for 2 hours, dissolve in 200 mL of ing conditions, the relative standard deviation of the ratio of diluted acetic acid (100) (1 in 100) by warming, cool, add the peak area of etoposide to that of the internal standard is water to make exactly 500 mL. Pipet 10 mL of this solution, not more than 1.0z. add water to make exactly 100 mL, and use this solution as

Containers and storage Containers—Tight containers. the standard solution. Determine the absorbances, AT and A

Storage—Light-resistant. S, of the sample solution and the standard solution at 450 nm as directed under the Ultraviolet-visible Spectrophotometry, using water as the blank.

Flavin Adenine Dinucleotide Total amout (mg) of ‰avin

Sodium AT 4 ＝WS× × AS 5 フラビンアデニンジヌクレオチドナトリウム WS: Amount (mg) of Ribo‰avin Reference Standard Change the Purity (5) to read: (ii) Peak area ratio of ‰avin adenine dinucleotide—Dis- Purity solve 0.1 g of Flavin Adenine Dinucleotide Sodium in 200 (5) Related substances—Dissolve 0.10 g of Flavin Ade- mL of water, and use this solution as the sample solution. nine Dinucleotide Sodium in 200 mL of the mobile phase, Perform the test with 5 mL of this solution as directed under and use this solution as the sample solution. Perform the test the Liquid Chromatography according to the following conditions. Determine the peak area, A of ‰avin adenine Supplement II, JPXIV O‹cial Monographs for Part I 1711 dinucleotide, and the total area, S,ofthepeaksotherthan Add the following: ‰avin adenine dinucleotide by the automatic integration method. Flomoxef Sodium for Injection Peak area ratio of ‰avin adenine dinucleotide 注射用フロモキセフナトリウム 1.08×A ＝ 1.08×A＋S Flomoxef Sodium for Injection is a preparation for Operating conditions— injection which is dissolved before use. Detector: A visible spectrophotometer (wavelength: 450 It contains not less than 90.0z and not more nm). than 110.0z of the labeled amount of ‰omoxef Column: A stainless steel column 4 mm in inside diameter (C15H18F2N6O7S2:496.47). and 15 cm in length, packed with octadecylsilanized silica gel Method of preparation Prepare as directed under Injec- for liquid chromatography (5 mminparticlediameter). tions, with Flomoxef Sodium. Column temperature: A constant temperature of about 359C. Description Flomoxef Sodium for Injection occurs as Mobile phase: A mixture of a solution of potassium di- white to light yellowish white, friable masses or powder. hydrogen phosphate (1 in 500) and methanol (4:1). Identiˆcation Proceed as directed in the Identiˆcation (3) Flow rate: Adjust the ‰ow rate so that the retention time under Flomoxef Sodium. of ‰avin adenine dinucleotide is about 10 minutes. Time span of measurement: About 4.5 times as long as the pH The pH of a solution obtained by dissolving an amount retention time of ‰avin adenine dinucleotide. of Flomoxef Sodium for Injection, equivalent to 0.5 g System suitability— (potency) of Flomoxef Sodium according to the labeled Test for required detection: To exactly 2 mL of the sample amount, in 5 mL of water is between 4.0 and 5.5. solution add water to make exactly 20 mL, and use this solu- Purity (1) Clarity and color of solution—Dissolve an tion as the solution for system suitability test. Pipet 2 mL of amount of Flomoxef Sodium for Injection, equivalent to 1.0 the solution, and add water to make exactly 20 mL. Conˆrm g (potency) of Flomoxef Sodium according to the labeled that the peak area of ‰avin adenine dinucleotide obtained amount, in 10 mL of water: the solution is clear and color- from 5 mLofthissolutionisequivalentto8to12z of that less or pale yellow. of ‰avin adenine dinucleotide obtained from 5 mLofthe (2) 1-(2-Hydroxyethyl)-1H-tetrazol-5-thiol—Use the solution for system suitability test. sample solution obtained in the Assay as the sample solu- System performance: Dissolve 20 mg each of Flavin tion. Weigh accurately about 20 mg of 1-(2-hydroxyethyl)- Adenine Dinucleotide Sodium and ribo‰avin sodium phos- 1H-tetrazol-5-thiol, and dissolve in water to make exactly phatein100mLofwater.Whentheprocedureisrunwith 100 mL. Pipet 5 mL of this solution, add exactly 25 mL of 5 mL of this solution under the above operating conditions, the internal standard solution and water to make 50 mL, and ‰avin adenine dinucleotide and ribo‰avin phosphate are use this solution as the standard solution. Perform the test eluted in this order with the resolution between these peaks with 5 mL each of the sample solution and the standard solu- being not less than 2.0. tion as directed under the Liquid Chromatography accord- System repeatability: When the test is repeated 6 times ing to the following conditions, and determine the ratios, QT with 5 mL of the solution for system suitability test under the and QS, of the peak area of 1-(2-hydroxyethyl)-1H-tetrazol- above operating conditions, the relative standard deviation 5-thiol to that of the internal standard. Calculate the amount of the peak area of ‰avin adenine dinucleotide is not more of 1-(2-hydroxyethyl)-1H-tetrazol-5-thiol per 1 g (potency) than 1.0z. of Flomoxef Sodium for Injection by the following formula: (2) Calculation not more than 10 mg. Amount (mg) of C H N Na O P 27 31 9 2 15 2 Amount (mg) of 1-(2-hydroxyethyl)-1H-tetrazol-5-thiol ＝fT×fR×2.2040 (C3H6N4OS)

fT: Total amount (mg) of ‰avin in Flavin Adenine QT 1 ＝WS× × Dinucleotide Sodium obtained from the procedure (i). QS 10

fR: Peak area ratio of ‰avin adenine dinucleotide in Flavin WS: Amount (mg) of 1-(2-hydroxyethyl)-1H-tetrazol-5-thiol Adenine Dinucleotide Sodium obtained from the procedure (ii). Operating conditions— Proceed as directed in the Assay. System suitability— Test for required detectability: Pipet 1 mL of the standard solution, and add water to make exactly 20 mL. Conˆrm that the peak area of 1-(2-hydroxyethyl)-1H-tetrazol-5-thiol obtained from 5 mL of this solution is equivalent to 3.5 – 6.5z of that obtained from 5 mL of the standard solution. 1712 O‹cial Monographs for Part I Supplement II, JPXIV

System performance: When the procedure is run with 5 mL injection may be used. of the standard solution under the above operating condi- tions, 1-(2-hydroxyethyl)-1H-tetrazol-5-thiol and the inter- nal standard are eluted in this order with the resolution Add the following: between these peaks being not less than 20. System repeatability: When the test is repeated 3 times Flopropione Capsules with 5 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the ratio of フロプロピオンカプセル the peak area of 1-(2-hydroxyethyl)-1H-tetrazol-5-thiol to that of the internal standard is not more than 1.0z. Flopropione Capsules contain not less than 93.0z and not more than 107.0z of the labeled amount of Water Not more than 1.5z (0.5 g, volumetric titration, ‰opropione (C H O : 182.17). back titration). 9 10 4 Method of preparation Prepare as directed under the Bacterial endotoxins Less than 0.025 EU/mg (potency). Capsules, with Flopropione. Mass variation It meets the requirements of the Mass Identiˆcation (1) Powder the contents of Flopropione Variation Test. Capsules. To a portion of the powder, equivalent to 60 mg Foreign insoluble matter Perform the test according to of Flopropione according to the labeled amount, add 40 mL Method 2: it meets the requirements of the Foreign Insoluble of water, shake well, and ˆlter. To 5 mL of the ˆltrate add Matter Test for Injections. 1 mL of iron (III) nitrate TS: a red-purple color appears. (2) Powder the contents of Flopropione Capsules. To a Insoluble particulate matter Perform the test according to portion of the powder, equivalent to 90 mg of Flopropione Method 1: it meets the requirements of the Insoluble Par- according to the labeled amount, add 100 mL of ethanol ticulate Matter Test for Injections. (99.5), shake well, and ˆlter. To 5 mL of the ˆltrate add Sterility Perform the test according to the Membrane ethanol (99.5) to make 50 mL. To 5 mL of this solution add ˆltration method: it meets the requirements of the Sterility ethanol (99.5) to make 100 mL, and use this solution as the Test. sample solution. Determine the absorption spectrum of the sample solution as directed under the Ultraviolet-visible Assay Weigh accurately the mass of the contents of not Spectrophotometry: it exhibits a maximum between 283 nm less than 10 Flomoxef Sodium for Injection, and calculate and 287 nm. the average mass of the content. Spread out thinly about 1 g of the content in a petri dish, allow the dish to stand in a Content uniformity Perform the test as directed in the desiccator containing a saturated solution of magnesium Assay, and determine the content: it meets the requirements bromide without light exposure to equilibrate the sample to of the Content Uniformity Test. constant water content. Determine the water content, Dissolution Being speciˆed separately. separately, with about 0.1 g of the sample according to the method described in Water. Weigh accurately an amount of Assay To 1 capsule of Flopropione Capsules add 43 mL of the sample, equivalent to about 50 mg (potency) of Flomox- a mixture of water and phosphoric acid (86:1), and disinte- ef Sodium according to the labeled amount, add exactly 50 grate the capsule at 509C in a water bath. After cooling, add mL of the internal standard solution to dissolve, add water acetonitrile to make exactly 100 mL, stir for 10 minutes, and to make 100 mL, and use this solution as the sample solu- centrifuge a part of this solution for 5 minutes at 3000 rpm. tion. Separately weigh accurately about 50 mg (potency) of Use the supernatant liquid as the sample solution. Separate- Flomoxef Triethylammonium Reference Standard, add ly, weigh accurately about 40 mg of ‰opropione for assay, exactly 50 mL of the internal standard solution to dissolve, separately determine the water content in the same manner add water to make 100 mL, and use this solution as the as Flopropione, add 70 mL of the mobile phase, and dis- standard solution. Proceed as directed in the Assay under solve by exposure for 10 minutes to ultrasonic vibration. Flomoxef Sodium. Add the mobile phase to make exactly 100 mL, and use this solution as the standard solution. Perform the test with Amount [mg (potency)] of ‰omoxef (C H F N O S ) 15 18 2 6 7 2 exactly 5 mL each of the sample solution and the standard Q ＝W × T ×1000 solution as directed under the Liquid chromatography ac- S Q S cording to the following conditions, and determine the peak

WS: Amount [mg (potency)] of Flomoxef Triethylammo- areas, AT and AS, of ‰opropione. Repeat the procedure nium Reference Standard above with totally not less than 10 capsules, and calculate the mean. Internal standard solution—A solution of m-cresol (3 in

1000). AT Amount (mg) of ‰opropione (C9H10O4)＝WS× AS Containers and storage Containers—Hermetic containers.

Polyethylene or polypropylene containers for aqueous WS: Amount (mg) of ‰opropione for assay, calculated on Supplement II, JPXIV O‹cial Monographs for Part I 1713

the anhydrous basis Calcium in heavy water for nuclear magnetic resonance spectroscopy (1 in 300), using sodium 3-trimethylsilyl- Operating conditions— propanesulfonate for nuclear magnetic resonance spec- Detector: An ultraviolet absorption photometer troscopy as an internal reference compound, as directed (wavelength: 267 nm). under the Nuclear Magnetic Resonance Spectroscopy: it Column: A stainless steel column 4.6 mm in inside exhibits a double signal at around d 1.5 ppm, a duple double diameter and 15 cm in length, packed with octadecyl- signal at around d 2.9 ppm, a multiple signal at around d 3.3 silanized silica gel for liquid chromatography (5 mminparti- ppm,andnosignalataroundd 1.4 ppm. cle diameter). Column temperature: A constant temperature of about Change the Purity (1) to read: 359C. Mobile phase: A mixture of acetonitrile, water and phos- Purity (1) Heavy metals—To 1.0 g of Fosfomycin phoric acid (114:86:1) Calcium add 40 mL of 0.25 mol/L acetic acid TS and water Flow rate: Adjust the ‰ow rate so that the retention time to make 50 mL. Proceed with this solution according to of ‰opropione is about 3 minutes. Method 1, and perform the test. Prepare the control solution System suitability— with 2.0 mL of Standard Lead Solution (not more than 20 System performance: Dissolve 50 mg of ‰opropione in ppm). 50 mL of the mobile phase. To 20 mL of the solution add 25mLofasolutionpreparedbydissolving25mgofethyl parahydroxybenzoate in 30 mL of acetonitrile and add water Fosfomycin Sodium to make 50 mL, and then add the mobile phase to make 50 mL. When the procedure is run with 5 mLofthissolution ホスホマイシンナトリウム under the above operating conditions, ‰opropione and ethyl parahydroxybenzoate are eluted in this order with the reso- Change the origin/limits of content to read: lution between these peaks being not less than 2.0. System repeatability: When the test is repeated 6 times Fosfomycin Sodium is the sodium salt of a sub- with 5 mL of the standard solution under the above operat- stance having antibacterial activity produced by the ing conditions, the relative standard deviation of the peak growth of Streptomyces fradiae or by the chemical area of ‰opropione is not more than 1.0z. synthesis. It contains not less than 725 mg (potency) and not Containers and storage Containers—Tight containers. more than 770 mg (potency) per mg, calculated on the anhydrous basis. The potency of Fosfomycin Sodium Fosfomycin Calcium is expressed as mass (potency) of fosfomycin (C3H7O4P: 138.06). ホスホマイシンカルシウム Change the Description to read: Change the origin/limits of content to read: Description Fosfomycin Sodium occurs as a white crystal- line powder. Fosfomycin Calcium is the calcium salt of a sub- It is very soluble in water, sparingly soluble in methanol, stance having antibacterial actively produced by the and practically insoluble in ethanol (99.5). growth of Streptomyces fradiae or by the chemical synthesis. Change the Identiˆcation (2) to read: It contains not less than 725 mg (potency) and not Identiˆcation more than 805 mg (potency) per mg, calculated on the (2) Determine the spectrum of a solution of Fosfomycin anhydrous basis. The potency of Fosfomycin Calcium Sodium in heavy water for nuclear magnetic resonance is expressed as mass (potency) of fosfomycin spectroscopy (1 in 300), using sodium 3-trimethylsilyl- (C H O P: 138.06). 3 7 4 propanesulfonate for nuclear magnetic resonance spec- troscopy as an internal reference compound, as directed Change the Description to read: under the Nuclear Magnetic Resonance Spectroscopy: it Description Fosfomycin Calcium occurs as a white crystal- exhibits a double signal at around d 1.5 ppm, a duple double line powder. signal at around d 2.8 ppm, a multiple signal at around d It is slightly soluble in water, and practically insoluble in 3.3 ppm, and no signal at around d 1.3 ppm. methanol and in ethanol (99.5).

Change the Identiˆcation (2) to read: Identiˆcation (2) Determine the spectrum of a solution of Fosfomycin 1714 O‹cial Monographs for Part I Supplement II, JPXIV

more than 0.020z). Furosemide (3) Sulfate—To 20 mL of the ˆltrate obtained in (2) add 1 mL of dilute hydrochloric acid and water to make 50 mL, フロセミド and perform the test using this solution as the test solution. Prepare the control solution as follows: To 0.35 mL of 0.005 Change the origin/limits of content to read: mol/L sulfuric acid VS add 1 mL of dilute hydrochloric acid and water to make 50 mL (not more than 0.030z). Furosemide, when dried, contains not less than (4) Heavymetals—Proceedwith2.0gofFurosemide 98.0z and not more than 101.0z of C12H11ClN2O5S. according to Method 2, and perform the test. Prepare the control solution with 2.0 mL of Standard Lead Solution (not Change the Description to read: more than 10 ppm). (5) Related substances—Dissolve 25 mg of Furosemide Description Furosemide occurs as white, crystals or crys- in 25 mL of the dissolving solution, and use this solution as talline powder. the sample solution. Pipet 1 mL of the sample solution, add It is freely soluble in N,N-dimethylformamide, soluble in the dissolving solution to make exactly 200 mL, and use this methanol, sparingly soluble in ethanol (99.5), slightly solu- solution as the standard solution. Perform the test with 20 ble in acetonitrile and in acetic acid (100), and practically mL each of the sample solution and the standard solution as insoluble in water. directed under the Liquid Chromatography according to the It dissolves in dilute sodium hydroxide TS. following conditions, and determine each peak area by the It is gradually colored by light. automatic integration method: the area of each peak Melting point: about 2059C (with decomposition). appeared ahead of the peak of furosemide is not more than Change the Identiˆcation to read: 2/5 times the peak area of furosemide from the standard so- lution, the area of each peak appeared behind the peak of Identiˆcation (1) Dissolve 25 mg of Furosemide in 10 mL furosemide is not more than 1/4 times the peak area of of methanol. To 1 mL of this solution add 10 mL of 2 furosemide from the standard solution, and the total area of mol/L hydrochloric acid TS. Heat the solution under a these peaks is not more than 2 times the peak area of re‰ux condenser on a water bath for 15 minutes, cool, and furosemide from the standard solution. add 18 mL of sodium hydroxide TS to make weakly acidic: Dissolving solution—To 22 mL of acetic acid (100) add a the solution responds to the Qualitative Tests for primary mixture of water and acetonitrile (1:1) to make 1000 mL. aromatic amines, producing a red to red-purple color. Operating conditions— (2) Determine the absorption spectrum of a solution of Detector: An ultraviolet absorption photometer Furosemide in dilute sodium hydroxide TS (1 in 125,000) as (wavelength: 272 nm). directed under the Ultraviolet-visible Spectrophotometry, Column: A stainless steel column 4.6 mm in inside di- and compare the spectrum with the Reference Spectrum or ameter and 25 cm in length, packed with octadecylsilanized the spectrum of a solution of Furosemide Reference silica gel for liquid chromatography (5 mminparticledi- Standard prepared in the same manner as the sample solu- ameter). tion: both spectra exhibit similar intensities of absorption at Column temperature: A constant temperature of about thesamewavelengths. 259C. (3) Determine the infrared absorption spectrum of Mobile phase: A mixture of water, tetrahydrofuran and Furosemide as directed in the potassium bromide disk acetic acid (100) (70:30:1). method under the Infrared Spectrophotometry, and com- Flow rate: Adjust the ‰ow rate so that the retention time pare the spectrum with the Reference Spectrum or the of furosemide is about 18 minutes. spectrum of Furosemide Reference Standard: both spectra Time span of measurement: About 2.5 times as long as the exhibit similar intensities of absorption at the same wave retention time of furosemide after the solvent peak. numbers. System suitability— Test for required detectability: Measure exactly 2 mL of Change the Purity to read: the standard solution, and add the dissolving solution to make exactly 50 mL. Conˆrm that the peak area of Purity (1) Clarity and color of solution—Dissolve 0.5 g furosemide obtained from 20 mLofthissolutionisequiva- of Furosemide in 10 mL of a solution of sodium hydroxide lent to 3.2 to 4.8z of that obtained from 20 mLofthe (1 in 50): the solution is clear and colorless. standard solution. (2) Chloride—Dissolve 2.6 g of Furosemide in 90 mL of System performance: When the procedure is run with 20 dilute sodium hydroxide TS, add 2 mL of nitric acid, and mL of the standard solution under the above operating ˆlter. To 25 mL of the ˆltrate add 6 mL of dilute nitric acid conditions, the number of theoretical plates and the symmet- and water to make 50 mL, and perform the test using this so- ry factor of the peak of furosemide is not less than 7000 and lution as the test solution. Prepare the control solution as not more than 1.5, respectively. follows: To 0.40 mL of 0.01 mol/L hydrochloric acid VS System repeatability: When the test is repeated 6 times add 6 mL of dilute nitric acid and water to make 50 mL (not with 20 mL of the standard solution under the above operat- Supplement II, JPXIV O‹cial Monographs for Part I 1715 ing conditions, the relative standard deviation of the peak Content uniformity Perform the test according to the fol- area of furosemide is not more than 2.0z. lowing method: it meets the requirements of the Content Uniformity Test. Change the Assay to read: To 1 tablet of Furosemide Tablets add a suitable amount of 0.05 mol/L sodium hydroxide TS, shake to disintegrate, Assay Weigh accurately about 0.5 g of Furosemide, previ- then add 0.05 mol/L sodium hydroxide TS to make exactly ously dried, dissolve in 50 mL of N,N-dimethylformamide, V mL so that each mL contains about 0.4 mg of furosemide and titrate with 0.1 mol/L sodium hydroxide VS until the (C H ClN O S). Filter the solution, discard the ˆrst 10 mL color of the solution changes from yellow to blue (indicator: 12 11 2 5 of the ˆltrate, pipet the subsequent 2 mL of the ˆltrate, add 3 drops of bromothymol blue TS). Perform a blank determi- 0.05 mol/L sodium hydroxide TS to make exactly 100 mL, nation with a mixture of 50 mL of N,N-dimethylformamide and use this solution as the sample solution. Proceed as and 15 mL of water, and make any necessary correction directed in the Assay. Each mL of 0.1 mol/L sodium hydroxide VS Amount (mg) of furosemide (C12H11ClN2O5S) ＝33.07 mg of C12H11ClN2O5S AT V ＝WS× × AS 100

Add the following: WS: Amount (mg) of Furosemide Reference Standard Dissolution Perform the test with 1 tablet of Furosemide Furosemide Tablets Tablets at 50 revolutions per minute according to Method 2 under the Dissolution Test, using 900 mL of diluted phos- フロセミド錠 phate buŠer solution, pH 6.8 (1 in 2) as the dissolution medi- um. Withdraw 20 mL or more of the dissolution medium 15 Furosemide Tablets contain not less than 95.0z and minutes after starting the test for a 20-mg tablet or 30 not more than 105.0z of the labeled amount of furosemide (C H ClN O S: 330.74). minutes after for a 40-mg tablet, and ˆlter through a mem- 12 11 2 5 brane ˆlter with pore size of not more than 0.45 mm. Discard Method of preparation Prepare as directed under Tablets, the ˆrst 10 mL of the ˆltrate, pipet V mL of the subsequent with Furosemide. ˆltrate, add diluted phosphate buŠer solution, pH 6.8 (1 in 2) to make exactly V? mL so that each mL contains about 10 Identiˆcation (1) Shake well a quantity of powdered mgoffurosemide(C H ClN O S) according to the labeled Furosemide Tablets, equivalent to 0.2 g of Furosemide 12 11 2 5 amount, and use this solution as the sample solution. according to the labeled amount, with 40 mL of acetone, Separately, weigh accurately about 20 mg of Furosemide and ˆlter. To 0.5mL of the ˆltrate add 10mL of 2mol/L Reference Standard, previously dried at 1059C for 4 hours, hydrochloric acid TS, and heat under a re‰ux condenser on a anddissolvein5mLofmethanol,andadddilutedphos- water bath for 15 minutes. After cooling, add 18 mL of phate buŠer solution, pH 6.8 (1 in 2) to make exactly 100 sodium hydroxide TS to make the solution slightly acetic: mL. Pipet 5 mL of this solution, add diluted phosphate the solution responds to the Qualitative Tests for primary buŠer solution, pH 6.8 (1 in 2) to make exactly 100 mL, and aromatic amines, producing a red to red-purple color. use this solution as the standard solution. Determine the (2) Determine the absorption spectrum of the sample absorbances, A and A , of the sample solution and the solution obtained in the Assay as directed under the T S standard solution at 277 nm as directed under the Ultrav- Ultraviolet-visible Spectrophotometry: it exhibits maxima iolet-visible Spectrophotometry: the dissolution rates for a between 227 nm and 231 nm, between 269 nm and 273 nm, 20-mg tablet in 15 minutes and for a 40-mg tablet in 30 and between 330 nm and 336 nm. minutes are not less than 80z, respectively. Purity To a quantity of powdered Furosemide Tablets, Dissolution rate (z) with respect to the labeled amount of equivalent to 40 mg of Furosemide according to the labeled furosemide (C H ClN O S) amount, add about 30 mL of acetone, shake well, and add 12 11 2 5 A V? 1 acetone to make exactly 50 mL. Centrifuge the solution, add ＝W × T × × ×45 S A V C 3.0 mL of water to 1.0 mL of the supernatant liquid, cool in S ice, add 3.0 mL of dilute hydrochloric acid and 0.15 mL of WS: Amount (mg) of Furosemide Reference Standard sodium nitrite TS, shake, and allow to stand for 1 minute. C: Labeled amount (mg) of furosemide (C12H11ClN2O5S) Add 1.0 mL of ammonium amidosulfate TS, shake well, in 1 tablet allow to stand for 3 minutes, add 1.0 mL of N,N-diethyl-N?- Assay Weigh accurately the mass of not less than 20 1-naphthylethylenediamine oxalate TS, shake well, and Furosemide Tablets, and powder. Weigh accurately a por- allow to stand for 5 minutes. Perform the test with this solu- tion of the powder, equivalent to about 40 mg of furosemide tion as directed under the Ultraviolet-visible Spectrophoto- (C H ClN O S), add about 70 mL of 0.05 mol/L sodium metry, using a solution prepared in the same manner with 12 11 2 5 hydroxide TS, shake well, and add 0.05 mol/L sodium 1.0 mL of acetone as the blank: the absorbance at 530 nm is hydroxide TS to make exactly 100 mL. Filter, discard the not more than 0.10. ˆrst 10 mL of the ˆltrate, pipet 2 mL of the subsequent 1716 O‹cial Monographs for Part I Supplement II, JPXIV

ˆltrate, add 0.05 mol/L sodium hydroxide TS to make ex- Glutathione according to Method 1, and perform the test actly 100 mL, and use this solution as the sample solution. (not more than 2 ppm). Separately, weigh accurately about 20 mg of Furosemide (4) Related substances—Dissolve 50 mg of Glutathione Reference Standard, previously dried at 1059C for 4 hours, in 100 mL of the mobile phase, and use this solution as the and dissolve in 0.05 mol/L sodium hydroxide TS to make sample solution. Pipet 2 mL of the sample solution, add the exactly 50 mL. Pipet 2 mL of this solution, add 0.05 mol/L mobile phase to make exactly 100 mL, and use this solution sodium hydroxide TS to make exactly 100 mL, and use this as the standard solution. Perform the test with exactly 10 mL solution as the standard solution. Determine the absor- each of the sample solution and the standard solution as bances, AT and AS, of the sample solution and the standard directed under the Liquid Chromatography according to the solution at 271 nm as directed under the Ultraviolet-visible following conditions, and determine each peak area by the Spectrophotometry. automatic integration method: the area of the peak having the relative retention time of about 4 with respect to A Amount (mg) of furosemide (C H ClN O S)＝W × T glutathione is not more than 3/4 times the peak area of 12 11 2 5 S A S glutathione from the standard solution, and the total area of

WS: Amount (mg) of Furosemide Reference Standard the peaks other than the peak of glutathione is not more than the peak area of glutathione from the standard solu- Containers and storage Containers—Tight containers. tion. Storage—Light-resistant. Operating conditions— Detector: An ultraviolet absorption photometer (wavelength: 210 nm). Add the following: Column: A stainless steel column 4.6 mm in inside di- ameter and 15 cm in length, packed with octadecylsilanized Glutathione silica gel for liquid chromatography (5 mminparticledi- ameter). グルタチオン Column temperature: A constant temperature of about 309C. Mobile phase: Dissolve 6.8 g of potassium dihydrogen phosphate and 2.02 g of sodium 1-heptane sulfonate in 1000 mL of water, and adjust the pH to 3.0 with phosphoric acid. C10H17N3O6S: 307.32 To 970 mL of this solution add 30 mL of methanol. (2S)-2-Amino-4-[1-(carboxymethyl)carbamoyl-(2R)-2- Flow rate: Adjust the ‰ow rate so that the retention time sulfanylethylcarbamoyl]butanoic acid [70-18-8] of glutathione is about 5 minutes. Time span of measurement: About 6 times as long as the Glutathione, when dried, contains not less than retention time of glutathione after the solvent peak. 98.0z and not more than 101.0z of C10H17N3O6S. System suitability— Description Glutathione occurs as a white crystalline Test for required detectability: Pipet 10 mL of the stan- powder. dard solution, and add the mobile phase to make exactly 100 It is freely soluble in water, and practically insoluble in mL. Conˆrm that the peak area of glutathione obtained ethanol (99.5). from 10 mL of this solution is equivalent to 8 to 12z of that Melting point: about 1859C (with decomposition). obtained from 10 mL of the standard solution. System performance: Dissolve 50 mg of glutathione, Identiˆcation Determine the infrared absorption spectrum 10 mg of D-phenylglycine and 50 mg of ascorbic acid in 100 of Glutathione, previously dried, as directed in the potassi- mL of water. When the procedure is run with 10 mLofthis um bromide disk method under the Infrared Spectrophoto- solution under the above operating conditions, ascorbic metry, and compare the spectrum with the Reference Spec- acid, glutathione and D-phenylglycine are eluted in this trum: both spectra exhibit similar intensities of absorption at order, and the resolutions between the peaks of ascorbic acid thesamewavenumbers. and glutathione and between the peaks of glutathione and

20 D-phenylglycine are not less than 5, respectively. Optical rotation [a]D : －15.5 – －17.59(after drying, 2 g, water, 50 mL, 100 mm). System repeatability: When the test is repeated 6 times with 10 mL of the standard solution under the above operat- Purity (1) Clarity and color of solution—Dissolve 1.0 g ing conditions, the relative standard deviation of the peak of Glutathione in 10 mL of water: the solution is clear and area of glutathione is not more than 1.5z. colorless. (2) Heavy metals—Proceed with 2.0 g of Glutathione Loss on drying Not more than 0.5z (1 g, 1059C, 3 hours). according to Method 2, and perform the test. Prepare the Residue on ignition Not more than 0.1z (1 g). control solution with 2.0 mL of Standard Lead Solution (not more than 10 ppm). Assay Weigh accurately about 0.5 g of Glutathione, (3) Arsenic—Prepare the test solution with 1.0 g of previously dried, dissolve in 50 mL of a solution of Supplement II, JPXIV O‹cial Monographs for Part I 1717 metaphosphoric acid (1 in 50), and titrate with 0.05 mol/L iodine VS (indicator: 1 mL of starch TS). Perform a blank Heparin standard solution Human BuŠer Antithrombin Standard determination in the same manner, and make any necessary normal solution III solution solution correction. Heparin plasma (mL) (mL) (mL) No. concentration (mL) Each mL of 0.05 mol/L iodine VS (Unit/mL) ＝30.73 mg of C H N O S 10 17 3 6 (1) 0 800 100 100 0 Containers and storage Containers—Tight containers. (2) 0.02 700 100 100 100

(3) 0.04 600 100 100 200

Heparin Sodium (4) 0.06 500 100 100 300

ヘパリンナトリウム (5) 0.08 400 100 100 400

Change the Assay to read: (viii) Sample solution: Weigh accurately an adequate Assay (i) Substrate solution: Dissolve 15 mg of N-ben- amount of Heparin Sodium, dissolve in isotonic sodium zoyl-L-isoleucyl-L-glutamyl(g-OR)-glycyl-L-arginyl-p- chloride solution so that each mL contains about 0.5 Units nitroanilide hydrochloride in 20 mL of water. according to the labeled amount. To 100 mLofthissolution (ii) Antithrombin III solution: Dissolve human an- add 100 mL of antithrombin III solution, 100 mLofhuman tithrombin III in water to make a solution containing 1 Unit normal plasma and 700 mL of the buŠer solution, and use per mL. this solution as the sample solution. (iii) Activated blood coagulation factor X solution: Dis- (ix) Procedure: Transfer 400 mL of the sample solution solve bovine activated blood coagulation factor X in water to a test tube, and warm at 379C for 4 minutes. Add 200 mL to make a solution containing 0.426 Units per mL. of the activated blood coagulation factor X solution, mix (iv) Human normal plasma: Dissolve an amount of well, warm at 379C for exactly 30 seconds, add 400 mLofthe dried human normal plasma powder, equivalent to 1 mL of substrate solution, previously warmed at 379C, and mix human normal plasma, in 1 mL of water. Store at 2 – 109C well. Allow the tube to stand at 379C for exactly 3 minutes, and use within a week. add 600 mL of the reaction stop solution, mix immediately, (v) BuŠer solution: Dissolve 6.06 g of 2-amino-2- and determine the absorbance at 405 nm, using the blank hydroxymethyl-1,3-propanediol in 750 mL of water, adjust solution prepared by addition of 600 mL of the reaction stop the pH to 8.4 with 1 mol/L hydrochloric acid TS, and add solution and 600 mL of water to 400 mLofthesample water to make 1000 mL. solution. Proceed the same way with the heparin standard (vi) Reaction stop solution: To 20 mL of acetic acid solution (1), the heparin standard solution (2), the heparin (100) add water to make 40 mL. standard solution (3), the heparin standard solution (4) and (vii) Heparin standard solution: Dissolve Heparin Sodi- the heparin standard solution (5), and determine their absor- um Reference Standard in isotonic sodium chloride solution bances. to make a solution containing 10 Units per mL, and use as (x) Calculation: Plot the absorbances of the standard so- the standard stock solution. To 100 mL of the standard stock lutions on the vertical axis and their heparin concentrations solution add the buŠer solution to make exactly 5 mL, and on the horizontal axis to prepare a calibration curve. Deter- use this solution as the standard solution. Prepare the mine the heparin concentration, C, of the sample solution heparin standard solutions (1), (2), (3), (4) and (5) by from its absorbance by using the curve, and calculate addition of antithrombin III solution, human normal heparin Units per mg of Heparin Sodium from the following plasma and the buŠer solution to the standard solution as formula. directed in the following table. b Units per mg of Heparin Sodium＝C×10× a

a: Amount of sample (mg) b: Total volume (mL) of isotonic sodium chloride solution used to dissolve the sample to make the solution containing about 0.5 Units per mL 1718 O‹cial Monographs for Part I Supplement II, JPXIV

bitor TS and 0.05 mol/L phosphate buŠer solution, pH 7.0 Heparin Sodium Injection to make exactly 10 mL, and use this solution as the sample solution. Pipet 2.5 mL of substrate TS for kallidinogenase ヘパリンナトリウム注射液 assay (1), previously warmed at 30±0.59Cfor5minutes, place in a 1-cm cell, add exactly 0.5 mL of the sample solu- Add the following next to Bacterial endotoxins: tion,warmedat30±0.59C for 5 minutes, and start simul- taneously a chronograph. Perform the test at 30±0.59Cas Actual volume It meets the requirements of Injections. directed under the Ultraviolet-visible Spectrophotometry Foreign insoluble matter Perform the test according to using water as the blank, and determine the absorbances at

Method 1: it meets the requirements of the Foreign Insoluble 405 nm, AT2 and AT6, of this solution after allowing to stand Matter Test for Injections. for exactly 2 and 6 minutes. Separately, dissolve Kal- lidinogenase Reference Standard in 0.05 mol/L phosphate Insoluble particulate matter Perform the test according to buŠer solution, pH 7.0 to make a solutin so that each mL Method 1: it meets the requirements of the Insoluble Par- contains exactly 10 Units, and use this solution as the stan- ticulate Matter Test for Injections. dard stock solution. Pipet 4 mL of the stock solution, add Sterility Perform the test according to the Membrane exactly 1 mL of trypsin inhibitor TS and 0.05 mol/L phos- ˆltration method: it meets the requirements of the Sterility phate buŠer solution, pH 7.0 to make exactly 10 mL, and Test. use this solution as the standard solution. Take exactly 0.5 mL of the standard solution, perform the test in the same Change the Assay to read: manner as described for the sample solution, and determine the absorbances, A and A , of the solution after allowing Assay Proceed as directed in the Assay under Heparin S2 S6 to stand for exactly 2 and 6 minutes. Separately, take exactly Sodium, replacing the sample solution indicated in (viii) and 1 mL of the trypsin inhibitor TS, and add 0.05 mol/L phos- the calculation in (x) with the following. phate buŠer solution, pH 7.0 to make exactly 10 mL. Pipet Sample solution: Measure exactly an adequate portion of 0.5 mL of this solution, perform the test in the same manner Heparin Sodium Injection according to the labeled Units, as described for the sample solution, and determine the ab- dilute it with isotonic sodium chloride solution so that each sorbances, A and A , of the solution after allowing to mL contains about 0.5 Units. To 100 mL of this solution add O2 O6 stand for exactly 2 and 6 minutes. 100 mL of antithrombin III solution, 100 mLofhumannor- mal plasma and 700 mL of the buŠer solution, and use this Units per 1 mg of Kallidinogenase solution as the sample solution. (A －A )－(A －A ) W 1 Calculation: Plot the absorbances of the standard solu- ＝ T6 T2 O6 O2 × S × (A －A )－(A －A ) a b tions on the vertical axis and their heparin concentrations on S6 S2 O6 O2 the horizontal axis to prepare a calibration curve. Determine WS: Amount (Units) of Kallidinogenase Reference Standard the heparin concentration, C, of the sample solution from its a: Volume (mL) of the standard stock solution absorbance by using the curve, and calculate heparin Units b: Amount (mg) of Kallidinogenase in 1 mL of the sample per mL of Heparin Sodium Injection from the following stock solution formula.

b Units per mL of Heparin Sodium Injection＝C×10× a D-Mannitol

a: Amount of sample (mL) D-マンニトール b: Total volume (mL) of isotonic sodium chloride solution used to dilute the sample to make the solution contain- Change the Identiˆcation (2) to read: ing about 0.5 Units per mL Identiˆcation (2) Determine the infrared absorption spectrum of D- Mannitol as directed in the potassium bromide disk method Kallidinogenase under the Infrared Spectrophotometry, and compare the spectrum with the Reference Spectrum: both spectra exhibit カリジノゲナーゼ similar intensities of absorption at the same wave numbers. If any diŠerence appears between the spectra, dissolve 1 g of Change the Assay to read: D-Mannitol in 3 mL of warm water, then allow to stand at Assay Weigh accurately an appropriate amount of Kal- 59C for 24 hours or until crystals appear, and ˆlter. Wash lidinogenase according to the labeled Units, dissolve in 0.05 thecrystalssoobtainedwithafewamountofcoldwater, mol/L phosphate buŠer solution, pH 7.0 to prepare a solu- dry at 1059C for 4 hours, and perform the test with the tion containing about 10 Kallidinogenase Units per mL, and crystals. use this solution as the sample stock solution. Pipet 4 mL of the sample stock solution, add exactly 1 mL of trypsin inhi- Supplement II, JPXIV O‹cial Monographs for Part I 1719

Reference Spectrum: both spectra exhibit similar intensities Mepivacaine Hydrochloride of absorption at the same wave numbers. (3) A solution of dl-Methylephedrine Hydrochloride 塩酸メピバカイン (1 in 10) responds to the Qualitative Tests for chloride.

Change the graphic formula to read: Add the following next to Identiˆcation: pH The pH of a solution prepared by dissolving 1.0 g of dl-Methylephedrine Hydrochloride in 20 mL of water is be- tween 4.5 and 6.0.

Change the Purity to read: Change the chemical name to read: Purity (1) Clarity and color of solution—Dissolve 1.0 g (2RS)-N-(2,6-Dimethylphenyl)-1-methylpiperidine-2- of dl-Methylephedrine Hydrochloride in 10 mL of water: the carboxamide monohydrochloride solution is clear and colorless. (2) Heavy metals—Proceed with 1.0 g of dl-Methylephe- drine Hydrochloride according to Method 4, and perform Meropenem Trihydrate the test. Prepare the control solution with 1.0 mL of Stan- dard Lead Solution (not more than 10 ppm). メロペネム 三水和物 (3) Related substances—Dissolve 50 mg of dl- Methylephedrine Hydrochloride in 20 mL of water, and use Change the water to read: this solution as the sample solution. Pipet 1 mL of the sample solution, add water to make exactly 100 mL, and use Water Not less than 11.4z and not more than 13.4z this solution as the standard solution. Perform the test with (0.35 g, volumetric titration, direct titration). exactly 20 mL each of the sample solution and the standard solution as directed under the Liquid Chromatography ac- cording to the following conditions, and determine each dl-Methylephedrine Hydrochloride peak area by the automatic integration method: the total area of the peaks other than the peak of methylephedrine is dl-塩酸メチルエフェドリン not more than the peak area of methylephedrine from the standard solution. Change the origin/limits of the content to read: Operating conditions— Detector: An ultraviolet absorption photometer dl-Methylephedrine Hydrochloride, when dried, (wavelength: 257 nm). contains not less than 99.0z and not more than Column: A stainless steel column 4.6 mm in inside 101.0z of C11H17NO.HCl. diameter and 15 cm in length, packed with octadecyl- silanized silica gel for liquid chromatography (5 mminparti- Change the Description to read: cle diameter). Description dl-Methylephedrine Hydrochloride occurs as Column temperature: A constant temperature of about colorless crystals or a white, crystalline powder. 409C. It is freely soluble in water, sparingly soluble in ethanol Mobile phase: Dissolve 13.6 g of potassium dihydrogen (99.5), slightly soluble in acetic acid (100), and practically phosphate and 3 g of sodium 1-heptane sulfonate in 1000 insoluble in acetic anhydride. mL of water, and adjust the pH to 2.5 with phosphoric acid. Asolutionofdl-Methylephedrine Hydrochloride (1 in 20) To 900 mL of this solution add 200 mL of acetonitrile. shows no optical rotation. Flow rate: Adjust the ‰ow rate so that the retention time of methylephedrine is about 10 minutes. Change the Identiˆcation to read: Time span of measurement: About 2 times as long as the retention time of methylephedrine after the solvent peak. Identiˆcation (1) Determine the absorption spectrum of System suitability— asolutionofdl-Methylephedrine Hydrochloride (1 in 2000) Test for required detectability: To exactly 2 mL of the as directed under the Ultraviolet-visible Spectrophotometry, standard solution add water to make exactly 20 mL. Con- and compare the spectrum with the Reference Spectrum: ˆrm that the peak area of methylephedrine obtained from 20 both spectra exhibit similar intensities of absorption at the mL of this solution is equivalent to 7 to 13z of that of same wavelengths. methylephedrine obtained from 20 mL of the standard solu- (2) Determine the infrared absorption spectrum of dl- tion. Methylephedrine Hydrochloride, previously dried, as direct- System performance: Dissolve 50 mg of dl-Methylephe- ed in the potassium chloride disk method under the Infrared drine Hydrochloride and 0.4 mg of methyl parahydroxyben- Spectrophotometry, and compare the spectrum with the zoate in 50 mL of water. When the procedure is run with 1720 O‹cial Monographs for Part I Supplement II, JPXIV

20 mL of this solution under the above operating conditions, silanized silica gel for liquid chromatography (5 mminparti- methylephedrine and methyl parahydroxybenzoate are cle diameter). eluted in this order with the resolution between these peaks Column temperature: A constant temperature of about being not less than 3. 409C. System repeatability: When the test is repeated 6 times Mobile phase: Dissolve 13.6 g of potassium dihydrogen with 20 mL of the standard solution under the above operat- phosphate and 3 g of sodium 1-heptane sulfonate in 1000 ing conditions, the relative standard deviation of the peak mL of water, and adjust the pH to 2.5 with phosphoric acid. area of methylephedrine is not more than 2.0z. To 900 mL of this solution add 200 mL of acetonitrile. Flow rate: Adjust the ‰ow rate so that the retention time of methylephedrine is about 10 minutes. 10z dl-Methylephedrine System suitability— System performance: When the procedure is run with Hydrochloride Powder 20 mL of the standard solution under the above operating dl-塩酸メチルエフェドリン散10z conditions, methylephedrine and the internal standard are eluted in this order with the resolution between these peaks Delete the Description: being not less than 3. System repeatability: When the test is repeated 6 times Change the Identiˆcation to read: with 20 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the ratio of Identiˆcation Determine the absorption spectrum of a the peak area of methylephedrine to that of the internal solution of 10z dl-Methylephedrine Hydrochloride Powder standard is not more than 1.0z. (1 in 200) as directed under the Ultraviolet-visible Spec- trophotometry: it exhibits maxima between 250 nm and 253 nm, between 255 nm and 259 nm, and between 261 nm Add the following: and 264 nm. Methylprednisolone Succinate Change the Assay to read: コハク酸メチルプレドニゾロン Assay Weigh accurately about 0.5 g of 10z dl- Methylephedrine Hydrochloride Powder, add exactly 4 mL C H O :474.54 of the internal standard solution and 25 mL of water, shake 26 34 8 11b,17,21-Trihydroxy-6a-methylpregna-1,4-diene-3,20- vigorously for 20 minutes to dissolve, add water to make dione 21-(hydrogen succinate) [2921-57-5] 50 mL, ˆlter through a membrane ˆlter with pore size of 0.45 mm, if necessary, discard the ˆrst 10 mL of the ˆltrate, and use the subsequent ˆltrate as the sample solution. Separately, weigh accurately about 50 mg of dl-methylephe- drine hydrochloride for assay, previously dried at 1059Cfor 3 hours, add exactly 4 mL of the internal standard solution and water to make 50 mL, and use this solution as the standard solution. Perform the test with 20 mLeachofthe sample solution and the standard solution as directed under Methylprednisolone Succinate, when dried, contains the Liquid Chromatography according to the following not less than 97.0z and not more than 103.0z of conditions, and determine the ratios of the peak area, QT C26H34O8. and QS, of methylephedrine to that of the internal standard. Description Methylprednisolone Succinate occurs as a Amount (mg) of dl-methylephedrine hydrochloride white, crystals or crystalline powder.

(C11H17NO.HCl) It is soluble in methanol, sparingly soluble in ethanol (95),

QT and practically insoluble in water. ＝WS× QS Melting point: about 2359C (with decomposition).

WS: Amount (mg) of dl-methylephedrine hydrochloride Identiˆcation (1) Determine the absorption spectrum of for assay a solution of Methylprednisolone Succinate in methanol (1 in 50,000) as directed under the Ultraviolet-visible Internal standard solution—A solution of methyl para- Spectrophotometry, and compare the spectrum with the hydroxybenzoate in acetonitrile (1 in 10,000). Reference Spectrum or the spectrum of a solution of Methyl- Operating conditions— prednisolone Succinate Reference Standard prepared in the Detector: An ultraviolet absorption photometer same manner as the sample solution: both spectra exhibit (wavelength: 257 nm). similar intensities of absorption at the same wavelengths. Column: A stainless steel column 4.6 mm in inside (2) Determine the infrared absorption spectrum of diameter and 15 cm in length, packed with octadecyl- Supplement II, JPXIV O‹cial Monographs for Part I 1721

Methylprednisolone Succinate, previously dried, as directed 1.0z. in the potassium bromide disk method under the Infrared Loss on drying Not more than 1.0z (1 g, 1059C, 3 hours). Spectrophotometry, and compare the spectrum with the Reference Spectrum or the spectrum of previously dried Residue on ignition Not more than 0.2z (0.5 g). Methylprednisolone Succinate Reference Standard: both Assay Weigh accurately about 15 mg each of Methylpred- spectra exhibit similar intensities of absorption at the same nisolone Succinate and Methylprednisolone Succinate wave numbers. In case when some diŠerences are found be- Reference Standard, previously dried, dissolve separately in tween the spectra, repeat the test with residues obtained by 5 mL of methanol, and add the mixture of 0.05 mol/L phos- dissolving these substances in ethanol (95), evaporating to phate buŠer solution, pH 3.5 and acetonitrile (1:1) to make dryness, and drying. exactly 50 mL. Pipet 5 mL each of these solutions, add 25 Optical rotation [a]D : ＋99 – ＋1039(after drying, 0.2 g, exactly 5 mL of the internal standard solution, and use these ethanol (95), 20 mL, 100 mm). solutions as the sample solution and the standard solution, respectively. Perform the test with 5 mLeachofthesample Purity (1) Heavy metals—Proceed with 1.0 g of Methyl- solution and the standard solution as directed under the prednisolone Succinate according to Method 4, and perform Liquid Chromatography according to the following condi- the test. Prepare the control solution with 1.0 mL of tions, and determine the ratios, Q and Q , of the peak area Standard Lead Solution (not more than 10 ppm). T S of methylprednisolone succinate to that of the internal (2) Arsenic—Prepare the test solution with 2.0 g of standard. Methylprednisolone Succinate according to Method 3, and Q perform the test (not more than 1 ppm). Amount (mg) of C H O ＝W × T 26 34 8 S Q (3) Related substances—Dissolve 15 mg of Methylpred- S nisolone Succinate in 5 mL of methanol, add a mixture of WS: Amount (mg) of Methylprednisolone Succinate 0.05 mol/L phosphate buŠer solution, pH 3.5 and acetoni- Reference Standard trile (1:1) to make 50 mL, and use this solution as the sample Internal standard solution—A solution of ethyl parahydrox- solution. Pipet 1 mL of the sample solution, add the mixture ybenzoate in a mixture of 0.05 mol/L phosphate buŠer solu- of 0.05 mol/L phosphate buŠer solution, pH 3.5 and tion, pH 3.5 and acetonitrile (1:1) (3 in 20,000). acetonitrile (1:1) to make exactly 100 mL, and use this solu- Operating conditions— tion as the standard solution. Perform the test with exactly 5 Detector: An ultraviolet absorption photometer mL each of the sample solution and the standard solution as (wavelength: 254 nm). directed under the Liquid Chromatography according to the Column: A stainless steel column 4.6 mm in inside following conditions, and determine each peak area by the diameter and 25 cm in length, packed with octadecyl- automatic integration method: the area of the peaks other silanized silica gel for liquid chromatography (5 mminparti- than the peak of methylprednisolone succinate is not more cle diameter). than 1/2 of the peak area of methylprednisolone succinate Column temperature: A constant temperature of about from the standard solution, and the total area of the peaks 259C. other than the peak of methylprednisolone succinate is not Mobile phase: To 1000 mL of 0.05 mol/L potassium di- more than the peak area of methylprednisolone succinate hydrogen phosphate TS add a suitable amount of 0.05 from the standard solution. mol/L disodium hydrogen phosphate TS to make a solution Operating conditions— having pH 5.5. To 640 mL of this solution add 360 mL of Detector, column, column temperature, mobile phase, acetonitrile. and ‰ow rate: Proceed as directed in the operating condi- Flow rate: Adjust the ‰ow rate so that the retention time tions in the Assay. of methylprednisolone succinate is about 6 minutes. Time span of measurement: About 3 times as long as the System suitability— retention time of methylprednisolone succinate. System performance: When the procedure is run with 5 mL System suitability— of the standard solution under the above operating condi- Test for required detectability: Pipet 1 mL of the standard tions, methylprednisolone succinate and the internal stan- solution, and add the mixture of 0.05 mol/L phosphate dard are eluted in this order with the resolution between buŠer solution, pH 3.5 and acetonitrile (1:1) to make exactly these peaks being not less than 6. 10 mL. Conˆrm that the peak area of methylprednisolone System repeatability: When the test is repeated 6 times succinate obtained from 5 mLofthissolutionisequivalentto with 5 mL of the standard solution under the above operat- 7to13z of that obtained from 5 mL of the standard solu- ing conditions, the relative standard deviation of the ratio of tion. the peak area of methylprednisolone succinate to that of the System performance: Proceed as directed in the System internal standard is not more than 1.0z. suitability in the Assay. System repeatability: When the test is repeated 6 times Containers and storage Containers—Tight containers. with 5 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the peak area of methylprednisolone succinate is not more than 1722 O‹cial Monographs for Part I Supplement II, JPXIV

Operating conditions— Methyltestosterone Detector: An ultraviolet absorption photometer (wavelength: 241 nm). メチルテストステロン Column: A stainless steel column 6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel Change the origin/limits of content to read: for liquid chromatography (5 mminparticlediameter). Column temperature: A constant temperature of about Methyltestosterone, when dried, contains not less 359C. than 98.0z and not more than 102.0z of C20H30O2. Mobile phase: A mixture of acetonitrile and water (11:9). Flow rate: Adjust the ‰ow rate so that the retention time Change the Description to read: of methyltestosterone is about 10 minutes. System suitability— Description Methyltestosterone occurs as white to pale System performance: When the procedure is run with 10 yellow, crystals or crystalline powder. mL of the standard solution under the above operating It is freely soluble in methanol and in ethanol (95), and conditions, the internal standard and methyltestosterone are practically insoluble in water. eluted in this order with the resolution between these peaks Change the Identiˆcation to read: being not less than 9. System repeatability: When the test is repeated 6 times Identiˆcation with 10 mL of the standard solution under the above (1) Determine the absorption spectrum of a solution of operating conditions, the relative standard deviation of the Methyltestosterone in ethanol (95) (1 in 100,000) as directed ratios of the peak area of methyltestosterone to that of the under the Ultraviolet-visible Spectrophotometry, and com- internal standard is not more than 1.0z. pare the spectrum with the Reference Spectrum or the spec- trum of a solution of Methyltestosterone Reference Stan- dard prepared in the same manner as the sample solution: Methyltestosterone Tablets both spectra exhibit similar intensities of absorption at the same wavelengths. メチルテストステロン錠 (2) Determine the infrared absorption spectrum of Methyltestosterone, previously dried, as directed in the Change the origin/limits of content to read: potassium bromide disk method under the Infrared Spec- trophotometry, and compare the spectrum with the Refer- Methyltestosterone Tablets contain not less than ence Spectrum or the spectrum of dried Methyltestosterone 90.0z and not more than 110.0z of the labeled

Reference Standard: both spectra exhibit similar intensities amount of methyltestosterone (C20H30O2:302.45). of absorption at the same wave numbers. Change the Identiˆcation to read: Change the Assay to read: Identiˆcation To a portion of powdered Methyltestoster- Assay Weigh accurately about 20 mg each of one Tablets, equivalent to 0.01 g of Methyltestosterone Methyltestosterone and Methyltestosterone Reference accordingtothelabeledamount,add50mLofacetone, Standard, previously dried in a desiccator (in vacuum, phos- shake for 30 minutes, and ˆlter. Evaporate the ˆltrate to phorus (V) oxide) for 10 hours, dissolve each in methanol to dryness, dissolve the residue in 10 mL of acetone, and use make exactly 200 mL. Pipet 5 mL each of these solutions, this solution as the sample solution. Separately, dissolve add exactly 5 mL of the internal standard solution, add 0.01 g of Methyltestosterone Reference Standard in 10 mL methanol to make 50 mL, and use these solutions as the of acetone, and use this solution as the standard solution. sample solution and the standard solution. Perform the test Perform the test with these solutions as directed under the with 10 mL each of the sample solution and the standard Thin-layer Chromatography. Spot 10 mLeachofthesample solution as directed under the Liquid Chromatography solution and the standard solution on a plate of silica gel for according to the following conditions, and determine the thin-layer chromatography. Develop the plate with a mix- ratios, QT and QS, of the peak area of methyltestosterone to ture of chloroform and ethanol (95) (9:1) to a distance of that of the internal standard. about 12 cm, and air-dry the plate. Spray evenly dilute sul- furicacidontheplate,andheatat1109C for 10 minutes: the Amount (mg) of C H O 20 30 2 spot from the sample solution and the standard solution QT ＝WS× show the same Rfvalue. QS

WS: Amount (mg) of Methyltestosterone Reference Change the Assay to read: Standard Assay Weigh accurately the mass of not less than 20 Internal standard solution—A solution of propyl para- Methyltestosterone Tablets, and powder. Weigh accurately a hydroxybenzoate in methanol (1 in 10,000). portion of the powder, equivalent to about 25 mg of Supplement II, JPXIV O‹cial Monographs for Part I 1723

methyltestosterone (C20H30O2), add about 70 mL of Add the following: methanol, shake for 30 minutes, and add methanol to make exactly 100 mL. Pipet 2 mL of this solution, add exactly 5 Metoclopramide Tablets mL of the internal standard solution and methanol to make 50 mL, ˆlter through a membrane ˆlter (not exceeding 0.45 メトクロプラミド錠 mm in pore size), and use the ˆltrate as the sample solution. Separately, weigh accurately about 20 mg of Metoclopramide Tablets contain not less than Methyltestosterone Reference Standard, previously dried in 95.0z and not more than 105.0z of the labeled a desiccator (in vacuum, phosphorus (V) oxide) for 10 amount of metoclopramide (C14H22ClN3O2: 299.80). hours, dissolve in methanol to make exactly 200 mL. Pipet 5 Method of preparation Prepare as directed under Tablets, mL of this solution, add exactly 5 mL of the internal stan- with Metoclopramide. dard solution, add methanol to make 50 mL, and use this so- lution as the standard solution. Perform the test with 10 mL Identiˆcation (1) To a quantity of powdered each of the sample solution and the standard solution as Metoclopramide Tablets, equivalent to 50 mg of directed under the Liquid Chromatography according to the Metoclopramide according to the labeled amount, add 15 following conditions, and determine the ratios, QT and QS, mL of 0.5 mol/L hydrochloric acid TS, and heat in a water of the peak area of methyltestosterone to that of the internal bath at 709C for 15 minutes while frequent shaking. After standard. cooling, centrifuge for 10 minutes, and to 5 mL of the super- natant liquid add 1 mL of 4-dimethylaminobenzaldehyde- Amount (mg) of methyltestosterone (C H O ) 20 30 2 hydrochloric acid TS: a yellow color develops. Q 5 ＝W × T × (2) Determine the absorption spectrum of the sample S Q 2 S solution obtained in the Assay as directed under the

WS: Amount (mg) of Methyltestosterone Reference Ultraviolet-visible Spectrophotometry: it exhibits maxima Standard between 270 nm and 274 nm, and between 306 nm and 310 nm. Internal standard solution—A solution of propyl para- hydroxybenzoate in methanol (1 in 10,000). Content uniformity Perform the test according to the Operating conditions— following method: it meets the requirements of the Content Detector: An ultraviolet absorption photometer Uniformity Test. (wavelength: 241 nm). To 1 tablet of Metoclopramide Tablets add 10 mL of 0.1 Column: A stainless steel column 6 mm in inside diameter mol/L hydrochloric acid TS, disperse the particles with the and 15 cm in length, packed with octadecylsilanized silica gel aid of ultrasonic waves, then add 0.1 mol/L hydrochloric for liquid chromatography (5 mminparticlediameter). acid TS to make exactly 25 mL, and centrifuge for 10 Column temperature: A constant temperature of about minutes. Pipet 4 mL of the supernatant liquid, add 359C. 0.1 mol/L hydrochloric acid TS to make exactly V mL of a Mobile phase: A mixture of acetonitrile and water (11:9). solution so that each mL contains about 12 mgof

Flow rate: Adjust the ‰ow rate so that the retention time metoclopramide (C14H22ClN3O2), and use this solution as the of methyltestosterone is about 10 minutes. sample solution. Separately, weigh accurately about 80 mg System suitability— of metoclopramide for assay, previously dried at 1059Cfor System performance: When the procedure is run with 3 hours, and dissolve in 0.1 mol/L hydrochloric acid TS to 10 mL of the standard solution under the above operating make exactly 500 mL. Pipet 4 mL of this solution, add 0.1 conditions, the internal standard and methyltestosterone are mol/LhydrochloricacidTStomakeexactly50mL,anduse eluted in this order with the resolution between these peaks this solution as the standard solution. Determine the absor- being not less than 9. bances, AT and AS, of the sample solution and the standard System repeatability: When the test is repeated 6 times solution at 308 nm as directed under the Ultraviolet-visible with 10 mL of the standard solution under the above operat- Spectrophotometry. ing conditions, the relative standard deviation of the ratio of Amount (mg) of metoclopramide (C14H22ClN3O2) the peak area of methyltestosterone to that of the internal AT V standard is not more than 1.0z. ＝WS× × AS 1000

WS: Amount (mg) of metoclopramide for assay Dissolution Perform the test with 1 tablet of Metoclopramide Tablets at 50 revolutions per minute ac- cording to Method 2 under the Dissolution Test, using 900 mL of diluted phosphate buŠer solution, pH 6.8 (1 in 2) as the dissolution medium. Withdraw 20 mL or more of the dissolution medium 45 minutes after starting the test for a 3.84-mg tablet or 15 minutes after starting the test for a 1724 O‹cial Monographs for Part I Supplement II, JPXIV

7.67-mg tablet, and ˆlter through a membrane ˆlter with 4 mL of the supernatant liquid, add 0.1 mol/hydrochloric pore size of not more than 0.5 mm. Discard the ˆrst 10 mL of acid TS to make exactly 50 mL, and use this solution as the the ˆltrate, pipet V mL of the subsequent ˆltrate, add dilut- sample solution. Separately, weigh accurately about 80 mg ed phosphate buŠer solution, pH 6.8 (1 in 2) to make exactly of metoclopramide for assay, previously dried at 1059Cfor V? mL so that each mL contains about 4 mgof 3 hours, and dissolve in 0.1 mol/L hydrochloric acid TS to metoclopramide (C14H22ClN3O2) according to the labeled make exactly 500 mL. Pipet 4 mL of this solution, add 0.1 amount, and use this solution as the sample solution. mol/hydrochloric acid TS to make exactly 50 mL, and use Separately, weigh accurately about 20 mg of this solution as the standard solution. Determine the absor- metoclopramide for assay, previously dried at 1059Cfor3 bances, AT and AS, of the sample solution and the standard hours, and dissolve in diluted phosphate buŠer solution, pH solution at 308 nm as directed under the Ultraviolet-visible 6.8(1in2)tomakeexactly100mL.Pipet2mLofthissolu- Spectrophotometry. tion, add diluted phosphate buŠer solution, pH 6.8 (1 in 2) Amount (mg) of metoclopramide (C H ClN O ) to make exactly 100 mL, and use this solution as the stan- 14 22 3 2 A dard solution. Perform the test with exactly 50 mLeachof ＝W × T S A the sample solution and the standard solution as directed un- S der the Liquid Chromatography according to the following WS: Amount (mg) of metoclopramide for assay conditions, and determine the peak areas, A and A ,of T S Containers and storage Containers—Tight containers. metoclopramide. The dissolution rates for a 3.84-mg tablet in 45 minutes and for a 7.67-mg tablet in 15 minutes are not less than 80z and not less than 85z, respectively. Add the following: Dissolution rate (z) with respect to the labeled amount of metoclopramide (C14H22ClN3O2) Nicorandil AT V? 1 ＝WS× × × ×18 AS V C ニコランジル

WS: Amount (mg) of metoclopramide for assay C: Labeled amount (mg) of metoclopramide

(C14H22ClN3O2)in1tablet

Operating conditions— C8H9N3O4: 211.17 Detector: An ultraviolet absorption photometer N-[2-(Nitrooxy)ethyl]pyridine-3-carboxamide [65141-46-0] (wavelength: 275 nm). Column: A stainless steel column 4 mm in inside diameter Nicorandil contains not less than 98.5z and not and 15 cm in length, packed with octadecylsilanized silica gel more than 101.0z of C8H9N3O4,calculatedonthe for liquid chromatography (5 mminparticlediameter). anhydrous basis. Column temperature: A constant temperature of about Description Nicorandil occurs as white crystals. 259C. It is freely soluble in methanol, in ethanol (99.5) and in Mobile phase: Dissolve 0.79 g of sodium lauryl sulfate in acetic acid (100), soluble in acetic anhydride, and sparingly 550 mL of water, and add 450 mL of acetonitrile and 0.3 mL soluble in water. of acetic acid (100). Melting point: about 929C (with decomposition). Flow rate: Adjust the ‰ow rate so that the retention time of metoclopramide is about 5 minutes. Identiˆcation Determine the infrared absorption spectrum System suitability— of Nicorandil as directed in the potassium bromide disk System performance: When the procedure is run with method under the Infrared Spectrophotometry, and com- 50 mL of the standard solution under the above operating pare the spectrum with the Reference Spectrum: both spec- conditions, the number of theoretical plates and the symmet- tra exhibit similar intensities of absorption at the same wave ry factor of the peak of metoclopramide are not less than numbers. 3000 and not more than 1.5, respectively. Purity (1) Sulfate—Dissolve 2.0 g of Nicorandil in 20 mL System repeatability: When the test is repeated 6 times of dilute ethanol, add 1 mL of dilute hydrochloric acid and with 50 mL of the standard solution under the above operat- water to make 50 mL, and perform the test using this solu- ing conditions, the relative standard deviation of the peak tion as the test solution. Prepare the control solution with area of metoclopramide is not more than 1.0z. 0.40 mL of 0.005 mol/L sulfuric acid VS, 20 mL of dilute Assay Weigh accurately not less than 20 Metoclopramide ethanol and 1 mL of dilute hydrochloric acid, and dilute Tablets, and powder. Weigh accurately a portion of the withwatertomake50mL(notmorethan0.010z). powder, equivalent to about 75 mg of metoclopramide (2) Heavy metals—Proceed with 2.0 g of Nicorandil

(C14H22ClN3O2), add 300 mL of 0.1 mol/L hydrochloric acid according to Method 2, and perform the test. Prepare the TS, shake for 1 hour, and add 0.1 mol/L hydrochloric acid control solution with 2.0 mL of Standard Lead Solution (not TS to make exactly 500 mL. Centrifuge for 10 minutes, pipet more than 10 ppm). Supplement II, JPXIV O‹cial Monographs for Part I 1725

(3) Related substances—Dissolve 20 mg of Nicorandil in tion. 10 mL of the mobile phase, and use this solution as the Each mL of 0.1 mol/L perchloric acid VS sample solution. Perform the test with 10 mLofthesample ＝21.12 mg of C H N O solution as directed under the Liquid Chromatography 8 9 3 4 according to the following conditions, and determine each Containers and storage Containers—Tight containers. peak area by the automatic integration method: the peak Storage—At a temperature between 29Cand89C. area of N-(2-hydroxyethyl)isonicotinamide nitric ester, having the relative retention time of about 0.86 with respect to nicorandil, is not more than 0.5z of the peak area of Add the following: nicorandil, the area of all other peaks is less than 0.1z,and the sum area of the peaks other than nicorandil and N-(2- Nilvadipine hydroxyethyl)isonicotinamide nitric ester is not more than 0.25z of the total peak area. ニルバジピン Operating conditions— Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Column: A stainless steel column 4 mm in inside diameter and 25 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 mminparticlediameter). Column temperature: A constant temperature of about C19H19N3O6:385.37 259C. 5-Isopropyl 3-methyl (4RS)-2-cyano-1,4-dihydro- Mobile phase: A mixture of water, tetrahydrofuran, 6-methyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate triethylamine and tri‰uoroacetic acid (982:10:5:3). [75530-68-6] Flow rate: Adjust the ‰ow rate so that the retention time of nicorandil is about 18 minutes. Nilvadipine contains not less than 98.0z and not Time span of measurement: About 3 times as long as the more than 102.0z of C19H19N3O6. retention time of nicorandil after the solvent peak. System suitability— Description Nilvadipine occurs as a yellow crystalline Test for required detectability: Measure exactly 1 mL of powder. the sample solution, add the mobile phase to make exactly It is freely soluble in acetonitrile, soluble in methanol, 500 mL, and use this solution as the solution for system sparingly soluble in ethanol (99.5), and practically insoluble suitability test. Pipet 1 mL of the solution for system in water. suitability test, and add the mobile phase to make exactly A solution of Nilvadipine in acetonitrile (1 in 20) shows no 20 mL. Conˆrm that the peak area of nicorandil obtained optical rotation. with 10 mL of this solution is equivalent to 2 to 8z of that Identiˆcation (1) Determine the absorption spectrum of with 10 mL of the solution for system suitability test. a solution of Nilvadipine in ethanol (99.5) (1 in 100,000) as System performance: Dissolve 10 mg of N-(2-hydrox- directed under the Ultraviolet-visible Spectrophotometry, yethyl)isonicotinamide nitric ester in the mobile phase to and compare the spectrum with the Reference Spectrum or make 100 mL. To 1 mL of this solution add 10 mL of the the spectrum of a solution of Nilvadipine Reference sample solution. When the procedure is run with this solu- Standard prepared in the same manner as the sample solu- tion under the above operating conditions, N-(2-hydrox- tion: both spectra exhibit similar intensities of absorption at yethyl)isonicotinamide nitric ester and nicorandil are eluted thesamewavelengths. in this order with the resolution between these peaks being (2) Determine the infrared absorption spectrum of not less than 3.0. Nilvadipine as directed in the potassium bromide disk System repeatability: When the test is repeated 6 times method under the Infrared Spectrophotometry, and com- with 10 mL of the solution for system suitability test under pare the spectrum with the Reference Spectrum or the the above operating conditions, the relative standard devia- spectrum of Nilvadipine Reference Standard: both spectra tion of the peak area of nicorandil is not more than 1.5z. exhibit similar intensities of absorption at the same wave Water Not more than 0.1z (2 g, volumetric titration, numbers. direct titration). Melting point 167–1719C

Residue on ignition Not more than 0.1z (1 g). Purity (1) Heavy metals—Proceed with 2.0 g of Nilvadi- Assay Weigh accurately about 0.3 g of Nicorandil, dis- pine according to Method 2, and perform the test. Prepare solve in 30 mL of a mixture of acetic anhydride and acetic the control solution with 2.0 mL of Standard Lead Solution acid (100) (7:3), and titrate with 0.1 mol/L perchloric acid (not more than 10 ppm). VS (potentiometric titration). Perform a blank determina- (2) Related substances—Dissolve 20 mg of Nilvadipine tion in the same manner, and make any necessary correc- in 20 mL of acetonitrile, and use this solution as the sample 1726 O‹cial Monographs for Part I Supplement II, JPXIV solution. Perform the test with 5 mL of the sample solution Q as directed under the Liquid Chromatography according to Amount (mg) of C H N O ＝W × T 19 19 3 6 S Q the following conditions. Determine each peak area by the S automatic integration method, and calculate the amount of WS: Amount (mg) of Nilvadipine Reference Standard them by the area percentage method: the amount of each Internal standard solution—A solution of acenaphthene in related substance is not more than 0.3z, and the total of methanol (1 in 200). them is not more than 0.5z. Operating conditions— Operating conditions— Detector: An ultraviolet absorption photometer Detector: An ultraviolet absorption photometer (wavelength: 254 nm). (wavelength: 240 nm). Column: A stainless steel column 4 mm in inside diameter Column: A stainless steel column 4.6 mm in inside and 15 cm in length, packed with octadecylsilanized silica gel diameter and 15 cm in length, packed with octadecyl- for liquid chromatography (5 mminparticlediameter). silanized silica gel for liquid chromatography (5 mminparti- Column temperature: A constant temperature of about cle diameter). 259C. Column temperature: A constant temperature of about Mobile phase: Dissolve 2.5 g of diammonium hydrogen 259C. phosphate in 1000 mL of water, add 10 mL of tetrabutylam- Mobile phase: A mixture of phosphate buŠer solution, monium hydoxide TS, adjust the pH to 7.0 with diluted pH 7.4, methanol and acetonitrile (32:27:18). phosphoric acid (1 in 10), and add 900 mL of acetonitrile. Flow rate: Adjust the ‰ow rate so that the retention time Flow rate: Adjust the ‰ow rate so that the retention time of nilvadipine is about 12 minutes. of nilvadipine is about 12 minutes. Time span of measurement: About 2.5 times as long as the System suitability— retention time of nilvadipine after the solvent peak. System performance: When the procedure is run with 5 mL System suitability— of the standard solution under the above operating condi- Test for required detectability: Pipet 1 mL of the sample tions, nilvadipine and the internal standard are eluted in this solution, add acetonitrile to make exactly 100 mL, and use order with the resolution between these peaks being not less this solution as the solution for system suitability test. Pipet than 8. 1 mL of the solution for system suitability test, and add System repeatability: When the test is repeated 6 times acetonitrile to make exactly 10 mL. Conˆrm that the peak with 5 mL of the standard solution under the above operat- area of nilvadipine obtained from 5 mL of this solution is ing conditions, the relative standard deviation of the ratio of equivalent to 7 to 13z of that obtained from 5 mLofthe the peak area of nilvadipine to that of the internal standard solution for system suitability test. is not more than 1.0z. System performance: When the procedure is run with 5 mL of the solution for system suitability test under the above Containers and storage Containers—Well-closed contain- operating conditions, the number of theoretical plates and ers. the symmetry factor of the peak of nilvadipine is not less than 3300 and not more than 1.3, respectively. System repeatability: Pipet 1 mL of the solution for Add the following: system suitability test, and add acetonitrile to make exactly 10 mL. When the test is repeated 6 times with 5 mLofthis Nilvadipine Tablets solution under the above operating conditions, the relative standard deviation of the peak area of nilvadipine is not ニルバジピン錠 more than 1.5z. Nilvadipine Tablets contain not less than 93.0z and Loss on drying Not more than 0.1 (1 g, 105 C, 2 hours). z 9 not more than 107.0z of the labeled amount of

Residue on ignition Not more than 0.1z (1 g). nilvadipine (C19H19N3O6: 385.37). Assay Weigh accurately about 25 mg each of Nilvadipine Method of preparation Prepare as directed under Tablets, and Nilvadipine Reference Standard, dissolve in methanol to with Nilvadipine. make exactly 25 mL. Pipet 10 mL each of these solutions, Identiˆcation To a quantity of powdered Nilvadipine add exactly 20 mL of the internal standard solution, 20 mL Tablets, equivalent to 1 mg of Nilvadipine according to the of water and methanol to make 100 mL, and use these solu- labeled amount, add 100 mL of ethanol (99.5), shake for 10 tions as the sample solution and the standard solution, re- minutes, centrifuge, and use the supernatant liquid as the spectively. Perform the test with 5 mL each of the sample so- sample solution. Determine the absorption spectrum of the lution and the standard solution as directed under the Liquid sample solution as directed under the Ultraviolet-visible Chromatography according to the following conditions, and Spectrophotometry: it exhibits a maximum between 239 nm determine the ratios, Q and Q , of the peak area of nilvadi- T S and 243 nm and a maximum having a broad-ranging absorp- pine to that of the internal standard. tion between 371 nm and 381 nm. Supplement II, JPXIV O‹cial Monographs for Part I 1727

Content uniformity Perform the test according to the fol- Column temperature: A constant temperature of about lowing method: it meets the requirements of the Content 259C. Uniformity Test. Mobile phase: A mixture of phosphate buŠer solution, pH To 1 tablet of Nilvadipine Tablets add V mL of a mixture 7.4, methanol and acetonitrile (7:7:6) of acetonitrile and water (7:3) so that each mL of the solu- Flow rate: Adjust the ‰ow rate so that the retention time tion contains about 200 mg of nilvadipine (C19H19N3O6) of nilvadipineis about 5 minutes. according to the labeled amount, add exactly V mL of the System suitability— internal standard solution obtained in the Assay, and dis- System performance: When the procedure is run with 20 perse the particles with the aid of ultrasonic waves. Cen- mL of the standard solution under the above operating con- trifuge for 10 minutes, and use the supernatant liquid as the ditions, the number of theoretical plates and the symmetry sample solution. Separately, weigh accurately about 20 mg factor of the peak of nilvadipine are not less than 2000 and of Nilvadipine Reference Standard, dissolve in the mixture not more than 1.5, respectively. of acetonitrile and water (7:3) to make exactly 20 mL. Pipet System repeatability: When the test is repeated 6 times 5 mL of this solution, add exactly 25 mL of the internal with 20 mL of the standard solution under the above operat- standard solution obtained in the Assay and the mixture of ing conditions, the relative standard deviation of the peak acetonitrile and water (7:3) to make 50 mL, and use this area of nilvadipine is not more than 1.5z. solution as the standard solution. Proceed as directed in the Assay Weigh accurately not less than 20 Nilvadipine Assay. Tablets, and powder. Weigh accurately an amount of the

Amount (mg) of nilvadipine (C19H19N3O6) powder, equivalent to about 5 mg of nilvadipine

QT V (C19H19N3O6), add 10 mL of a mixture of acetonitrile and ＝WS× × QS 100 water (7:3) and exactly 25 mL of the internal standard solu- tion, shake for 15 minutes, and add the mixture of acetoni- W : Amount (mg) of Nilvadipine Reference Standard S trile and water (7:3) to make 50 mL. Centrifuge, and use the Dissolution Perform the test with 1 tablet of Nilvadipine supernatant liquid as the sample solution. Separately, weigh Tablets at 50 revolutions per minute according to Method 2 accurately about 20 mg of Nilvadipine Reference Standard, under the Dissolution Test, using 900 mL of water as the dissolve in the mixture of acetonitrile and water (7:3) to dissolution medium. Withdraw 20 mL or more of the disso- make exactly 20 mL. Pipet 5 mL of this solution, add exactly lution medium 30 minutes after starting the test, and ˆlter 25 mL of the internal standard solution and the mixture of through a membrane ˆlter with pore size of not more than acetonitrile and water (7:3) to make 50 mL, and use this 0.5 mm. Discard the ˆrst 10 mL of the ˆltrate, pipet 10 mL of solution as the standard solution. Perform the test with 5 mL the subsequent ˆltrate, add exactly 1 mL of methanol, and each of the sample solution and the standard solution as use this solution as the sample solution. Separately, weigh directed under the Liquid Chromatography according to the accurately an amount of Nilvadipine Reference Standard, following conditions, and determine the ratios, QT and QS, equivalent to 10 times the labeled amount of Nilvadipine of the peak area of nilvadipine to that of the internal stan- Tablets, and dissolve in methanol to make exactly 50 mL. dard.

Pipet 5 mL of this solution, and add methanol to make QT 1 Amount (mg) of nilvadipine (C19H19N3O6)＝WS× × exactly 100 mL. Pipet 1 mL of this solution, add exactly QS 4 10 mL of water, and use this solution as the standard W : Amount (mg) of Nilvadipine Reference Standard solution. Perform the test with exactly 20 mLeachofthe S sample solution and the standard solution as directed under Internal standard solution—A solution of acenaphthene in the Liquid Chromatography according to the following con- acetonitrile (1 in 500). ditions, and determine the peak areas, AT and AS, of nilvadi- Operating conditions— pine: the dissolution rate in 30 minutes is not less than 85z. Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Dissolution rate (z) with respect to the labeled amount of Column: A stainless steel column 4 mm in inside diameter nilvadipine (C H N O ) 19 19 3 6 and 15 cm in length, packed with octadecylsilanized silica gel A 1 ＝W × T × ×9 for liquid chromatography (5 mminparticlediameter). S A C S Column temperature: A constant temperature of about

WS: Amount (mg) of Nilvadipine Reference Standard 259C.

C: Labeled amount (mg) of nilvadipine (C19H19N3O6)in1 Mobile phase: Dissolve 2.5 g of diammonium hydrogen tablet phosphate in 1000 mL of water, add 10 mL of tetrabutylam- monium hydoxide TS, adjust the pH to 7.0 with diluted Operating conditions— phosphoric acid (1 in 10), and add 900 mL of acetonitrile. Detector: An ultraviolet absorption photometer Flow rate: Adjust the ‰ow rate so that the retention time (wavelength: 242 nm). of nilvadipine is about 12 minutes. Column: A stainless steel column 4 mm in inside diameter System suitability— and 15 cm in length, packed with octadecylsilanized silica gel System performance: When the procedure is run with 5 mL for liquid chromatography (5 mminparticlediameter). 1728 O‹cial Monographs for Part I Supplement II, JPXIV of the standard solution under the above operating condi- 37 mg of L-lysine hydrochloride, about 42 mg of L-histidine tions, nilvadipine and the internal standard are eluted in this hydrochloride monohydrate and about 42 mg of L-arginine order with the resolution between these peaks being not less hydrochloride, dissolve them in 10 mL of 1 mol/L than 8. hydrochloric acid TS, and add water to make exactly System repeatability: When the test is repeated 6 times 100 mL. Pipet 5 mL of this solution, add water to make ex- with 5 mL of the standard solution under the above operat- actly 20 mL, and use this solution as the standard solution. ing conditions, the relative standard deviation of the ratio of Perform the test with exactly 20 mL each of the sample solu- the peak area of nilvadipine to that of the internal standard tion and the standard solution as directed under the Liquid is not more than 1.0z. Chromatography according to the following conditions, and calculate the respective molar ratios with respect to leucine: Containers and storage Containers—Well-closed contain- 0.95 – 1.05 for aspartic acid, 0.95 – 1.05 for glutamic acid, ers. 0.95–1.05forproline,0.95–1.05forglycine,0.80–1.10 for isoleucine, 0.80 – 1.05 for tyrosine and 0.80 – 1.05 for cystine, and not more than 0.01 each for others. Add the following: Operating conditions— Detector: A visible spectrophotometer (wavelength: 440 Oxytocin nm and 570 nm). Column: A stainless steel column 4.6 mm in inside diameter オキシトシン and 8 cm in length, packed with strongly acidic ion-exchange resin for liquid chromatography (sodium type) composed with a sulfonated polystyrene copolymer (3 mminparticle C43H66N12O12S2: 1007.19 diameter). [50-56-6] Column temperature: A constant temperature of about 579C. Oxytocin is a synthetic peptide having the property of causing the contraction of uterine smooth muscle. Chemical reaction bath temperature: A constant tempera- ture of about 130 C. It contains not less than 540 oxytocin Units and not 9 more than 600 oxytocin Units per mg, calculated on Color developing time: About 1 minute. Mobile phase: Prepare mobile phases A, B and C accord- the dehydrated and de-acetic acid basis. ing to the following table. Description Oxytocin occurs as a white powder. It is very soluble in water, and freely soluble in ethanol Mobile phase A B C (99.5). Citric acid mono- It dissolves in hydrochloric acid TS. 19.80 g 22.00 g 6.10 g hydrate The pH of a solution prepared by dissolving 0.10 g of Trisodium citrate 6.19 g 7.74 g 26.67 g Oxytocin in 10 mL of freshly boiled and cooled water is be- dihydrate tween 4.0 and 6.0. Sodium chloride 5.66 g 7.07 g 54.35 g It is hygroscopic. Ethanol (99.5) 260.0 mL 20.0 mL — Benzyl alcohol — — 5.0 mL Identiˆcation Determine the absorption spectrum of a Thiodiglycol 5.0 mL 5.0 mL — solution of Oxytocin (1 in 2000) as directed under the Lauromacrogol 4.0mL 4.0mL 4.0mL Ultraviolet-visible Spectrophotometry, and compare the solution (1 in 4) spectrum with the Reference Spectrum: both spectra exhibit Capryric acid 0.1 mL 0.1 mL 0.1 mL similar intensities of absorption at the same wavelengths. Water a su‹cient a su‹cient a su‹cient amount amount amount Constituent amino acids Put about 1 mg of Oxytocin in a test tube for hydrolysis, add 6 mol/L hydrochloric acid TS Total amount 2000 mL 1000 mL 1000 mL to dissolve, replace the air in the tube with Nitrogen, seal the pH 3.3 3.2 4.9 tube under reduced pressure, and heat at 110 to 1159Cfor 16 hours. After cooling, open the tube, evaporate the Flowing of the mobile phase: Control the gradient by mix- hydrolyzate to dryness under reduced pressure, add 2 mL of ing the mobile phases A, B and C as directed in the following 0.02 mol/L hydrochloric acid TS to dissolve the residue, and table. use this solution as the sample solution. Separately, weigh accurately about 27 mg of L-aspartic acid, about 24 mg of L-threonine, about 21 mg of L-serine, about 29 mg of L-glutamic acid, about 23 mg of L-proline, about 15 mg of glycine, about 18 mg of L-alanine, about 23 mg of L-valine, about 48 mg of L-cystine, about 30 mg of methionine, about 26 mg of L-isoleucine, about 26 mg of L-leucine, about 36 mg of L-tyrosine, about 33 mg of phenylalanine, about Supplement II, JPXIV O‹cial Monographs for Part I 1729

Operating conditions— Time after Mobile Mobile Mobile Detector: An ultraviolet absorption photometer injection of phase phase phase sample (min) A(z) B(z) C(z) (wavelength: 210 nm). Column: A stainless steel column 4.6 mm in inside di- 0–9 100 0 0 ameter and 15 cm in length, packed with octadecylsilanized 9–25 0 100 0 silica gel for liquid chromatography (5 mminparticledi- 25 – 61 0 100 ª 00ª 100 ameter). 61–80 0 0 100 Column temperature: A constant temperature of about 409C. Reaction reagent: Mix 407 g of lithium acetate dihydrate, Mobile phase: To 0.7 mL of phosphoric acid add 900 mL 245 mL of acetic acid (100) and 801 mL of 1-methoxy-2- of water, adjust the pH to 3.0 with 8 mol/L sodium propanol, add water to make 2000 mL, stir for more than 10 hydroxide TS, and add water to make 1000 mL. To 950 mL minutes while passing Nitrogen, and use this solution as So- of this solution add 50 mL of methanol. lution A. Separately, to 1957 mL of 1-methoxy-2-propanol Flow rate: Adjust the ‰ow rate so that the retention time add 77 g of ninhydrin and 0.134 g of sodium borohydride, of acetic acid is about 3 minutes. stir for more than 30 minutes while passing Nitrogen, and System suitability— use this solution as Solution B. Mix Solution A and Solution System performance: When the procedure is run with 10 Bbeforeuse. mL of the standard solution under the above operating Flow rate of mobile phase: About 0.26 mL per minute. conditions, acetic acid and propionic acid are eluted in this Flow rate of reaction reagent: About 0.3 mL per minute. order with the resolution between these peaks being not less System suitability— than 14. System performance: When the procedure is run with System repeatability: When the test is repeated 6 times 20 mL of the standard solution under the above operating with 10 mL of the standard solution under the above operat- conditions, aspartic acid, threonine, serine, glutamic acid, ing conditions, the relative standard deviation of the ratio of proline, glycine, alanine, valine, cystine, methionine, isoleu- the peak area of acetic acid to that of the internal standard is cine, leucine, tyrosine, phenylalanine, lysine, histidine and not more than 2.0z. arginine are eluted in this order with the resolutions between (2) Related substances—Dissolve 25 mg of Oxytocin in the peaks of threonine and serine, glycine and alanine, and 100 mL of the mobile phase A, and use this solution as the isoleucine and leucine being not less than 1.5, 1.4 and 1.2, sample solution. Perform the test with 50 mLofthesample respectively. solution as directed under the Liquid Chromatography System repeatability: When the test is repeated 3 times according to the following conditions, determine each peak with 20 mL of the standard solution under the above operat- area by the automatic integration method, and calculate the ing conditions, the relative standard deviations of the peak amount of them by the area percentage method: the amount area of aspartic acid, proline, valine and arginine are not of each peak other than Oxytocin is not more than 1.5z, more than 2.0z, respectively. and the total of them is not more than 5.0z. Operating conditions— Purity (1) Acetic acid—Weigh accurately about 15 mg of Detector, column, column temperature, mobile phase, Oxytocin, dissolve in the internal standard solution to make ‰owing of mobile phase, and ‰ow rate: Proceed as directed exactly 10 mL, and use this solution as the sample solution. in the operating conditions in the Assay. Separately, weigh accurately about 1 g of acetic acid (100), Time span of measurement: About 2.5 times as long as the add the internal standard solution to make exactly 100 mL. retention time of oxytocin. Pipet 2 mL of this solution, add the internal standard System suitability— solution to make exactly 200 mL, and use this solution as the Test for required detectability: Measure exactly 1 mL of standard solution. Perform the test with 10 mLeachofthe the sample solution, add the mobile phase A to make exactly sample solution and the standard solution as directed under 100 mL, and use this solution as the solution for system the Liquid Chromatography according to the following suitability test. Pipet 1 mL of the solution for system conditions, and calculate the ratios, Q and Q ,ofthepeak T S suitability test, and add the mobile phase A to make exactly area of acetic acid to that of the internal standard: the 10 mL. Conˆrm that the peak area of oxytocin obtained amount of acetic acid is not less than 6.0z and not more from 50 mL of this solution is equivalent to 5 to 15z of that than 10.0z. from 50 mL of the solution for system suitability test.

Amount (z) of acetic acid (C2H4O2) System performance: Dissolve an adequate amount of W Q 1 oxytocin and vasopressin in the mobile phase A, so that each ＝ S × T × WT QS 10 mL contains about 0.1 mg each of them. When the proce- dure is run with 50 mL of this solution under the above oper- W : Amount (mg) of acetic acid (100) S ating conditions, vasopressin and oxytocin are eluted in this W : Amount (mg) of the sample T order with the resolution between these peaks being not less Internal standard solution—A solution of propionic acid in than 14, and the symmetry factor of the peak of oxytocin is the mobile phase (1 in 10,000). not more than 1.5. 1730 O‹cial Monographs for Part I Supplement II, JPXIV

System repeatability: When the test is repeated 6 times not more than 1.5. with 50 mL of the solution for system suitability test under System repeatability: When the test is repeated 6 times the above operating conditions, the relative standard devia- with 25 mL of the standard solution under the above operat- tion of the peak area of oxytocin is not more than 2.0z. ing conditions, the relative standard deviation of the peak area of oxytocin is not more than 1.0z. Water Not more than 5.0z (50 mg, coulometric titration). Containers and storage Containers—Tight containers. Assay Weigh accurately an amount of Oxytocin, equiva- Storage—At 2 to 89C. lent to about 13,000 Units, dissolve in the mobile phase A to make exactly 100 mL, and use this solution as the sample solution. Separately, dissolve oxytocin in 1 bottle of the Change to read: Posterior Pituitary Reference Standard in the mobile phase Atomakeaknownconcentrationsolutioncontainingeach mL contains about 130 Units, and use this solution as the Oxytocin Injection standard solution. Perform the test with exactly 25 mLeach オキシトシン注射液 of the sample solution and the standard solution as directed under the Liquid Chromatography according to the follow- Oxytocin Injection is an aqueous solution for injec- ing conditions, and determine the peak areas, AT and AS,of tion. oxytocin. It contains not less than 90.0z and not more than Units per mg of Oxytocin, calculated on the dehydrated and 110.0z of the labeled oxytocin Units. de-acetic acid basis Method of preparation Prepare as directed under Injec- W A ＝ S × T ×100 tions, with Oxytocin. WT AS Description Oxytocin Injection is a colorless, clear liquid. WS: Units per mL of the standard solution

WT: Amount (mg) of the sample, calculated on the de- pH 2.5–4.5 hydrated and de-acetic acid basis Bacterial endotoxins Less than 10 EU/oxytocin Unit. Operating conditions— Actual volume It meets the requirements of the Injections. Detector: An ultraviolet absorption photometer (wavelength: 220 nm). Foreign insoluble matter Perform the test according to Column: A stainless steel column 4.6 mm in inside di- Method 1: it meets the requirements of the Foreign Insoluble ameter and 15 cm in length, packed with octadecylsilanized Matter Test for Injections. silica gel for liquid chromatography (5 mminparticledi- Insoluble particulate matter Perform the test according to ameter). Method 1: it meets the requirements of the Insoluble Par- Column temperature: A constant temperature of about ticulate Matter Test for Injections. 259C. Mobile phase A: Dissolve 15.6 g of sodium dihydrogen Sterility Perform the test according to the Membrane phosphate dihydrate in 1000 mL of water. ˆltration method: it meets the requirements of the Sterility Mobile phase B: A mixture of water and acetonitrile (1:1). Test. Flowing of the mobile phase: Control the gradient by Assay Measure exactly a portion of Oxytocin Injection mixing the mobile phases A and B as directed in the according to the labeled Units, dilute with the diluent so that following table. each mL contains about 1 Unit, and use this solution as the sample solution. Separately, dissolve oxytocin in 1 bottle of Time after injection Mobile phase Mobile phase Posterior Pituitary Reference Standard in the mobile phase of sample (min) A(z) B(z) A to make exactly 20 mL. Pipet a suitable volume of this so- 0–30 70ª 40 30 ª 60 lution, dilute with the diluent to make a known concentra- 30–30.1 40ª 70 60 ª 30 tion solution so that each mL contains about 1 Unit, and use 30.1 – 45 70 30 this solution as the standard solution. Perform the test with exactly 100 mL each of the sample solution and the standard Flow rate: About 1.0 mL per minute. solution as directed under the Liquid Chromatography ac- System suitability— cording to the following conditions, and determine the peak

System performance: Dissolve an adequate amount of areas, AT and AS, of oxytocin. oxytocin and vasopressin in the mobile phase A, so that each A b mL contains about 0.1 mg each of them. When the proce- Units per mL of Oxytocin Injection＝W × T × S A a dure is run with 25 mL of this solution under the above oper- S ating conditions, vasopressin and oxytocin are eluted in this WS: Units per mL of the standard solution order with the resolution between these peaks being not less a: Volume (mL) of the sample than 14, and the symmetry factor of the peak of oxytocin is b: Total volume of the sample solution prepared by dilut- Supplement II, JPXIV O‹cial Monographs for Part I 1731

ing with the diluent directed under the Liquid Chromatography according to the Diluent: Dissolve 5 g of chlorobutanol, 1.1 g of sodium following conditions. Determine each peak area of both so- acetate trihydrate, 5 g of acetic acid (100) and 6 lutions by the automatic integration method: the total area mL of ethanol (99.5) in water to make 1000 mL. of the peaks other than pirenoxine is not larger than the peak Operating conditions— area of pirenoxine from the standard solution. Detector: An ultraviolet absorption photometer Operating conditions— (wavelength: 220 nm). Detector: An ultraviolet absorption photometer Column: A stainless steel column 4.6 mm in inside di- (wavelength: 230 nm). ameter and 15 cm in length, packed with octadecylsilanized Column: A stainless steel column 4 mm in inside diameter silica gel for liquid chromatography (5 mminparticledi- and 15 cm in length, packed with octadecylsilanized silica gel ameter). for liquid chromatography (5 mminparticlediameter). Column temperature: A constant temperature of about Column temperature: A constant temperature of about 259C. 359C. Mobile phase A: Dissolve 15.6 g of sodium dihydrogen Mobile phase: Dissolve 1.39 g of tetra n-butylammonium phosphate dihydrate in 1000 mL of water. chloride and 4.5 g of disodium hydrogen phosphate 12- Mobile phase B: A mixture of water and acetonitrile (1:1). water in 1000 mL of water, and adjust the pH to 6.5 with Flowing of the mobile phase: Control the gradient by phosphoric acid. To 700 mL of this solution add 200 mL of mixing the mobile phases A and B as directed in the follow- acetonitrile and 30 mL of tetrahydrofuran, and mix. ing table. Flow rate: Adjust the ‰ow rate so that the retention time of pirenoxine is about 10 minutes. Time after injection Mobile phase Mobile phase Time span of measurement: About 3 times as long as the of sample (min) A(z) B(z) retention time of pirenoxine. System suitability— 0–30 70ª 40 30 ª 60 Test for required detectability: To exactly 2 mL of the 30–30.1 40ª 70 60 ª 30 30.1 – 45 70 30 standard solution add the mobile phase to make exactly 30 mL. Conˆrm that the peak area of pirenoxine obtained from Flow rate: About 1.0 mL per minute. 5 mL of this solution is equivalent to 5 to 8z of that of System suitability— pirenoxine obtained from 5 mL of the standard solution. System performance: Dissolve an adequate amount of System performance: Dissolve 3 mg of Pirenoxine and oxytocin and vasopressin in the mobile phase A, so that each 16 mg of methyl parahydroxybenzoate in 100 mL of the mL contains about 0.02 mg each of them. When the proce- mobile phase. When the procedure is run with 5 mLofthis dure is run with 100 mL of this solution under the above solution under the above operating conditions, pirenoxine operating conditions, vasopressin and oxytocin are eluted in and methyl parahydroxybenzoate are eluted in this order this order with the resolution between these peaks being not with the resolution between these peaks being not less than less than 14, and the symmetry factor of the peak of oxyto- 2.0. cin is not more than 1.5. System repeatability: When the test is repeated 6 times System repeatability: When the test is repeated 6 times with 5 mL of the standard solution under the above operat- with 100 mL of the standard solution under the above oper- ing conditions, the relative standard deviation of the peak ating conditions, the relative standard deviation of the peak area of pirenoxine is not more than 1.0z. area of oxytocin is not more than 2.0z.

Containers and storage Containers—Hermetic containers. Storage—In a cold place, and avoid freezing.

Pirenoxine

ピレノキシン

Change the Purity (2) to read: Purity (2) Related substances—Dissolve 10 mg of Pirenoxine in 50 mL of the mobile phase, and use this solution as the sam- ple solution. Pipet 3 mL of the sample solution, add the mo- bile phase to make exactly 200 mL, and use this solution as the standard solution. Perform the test with exactly 5 mL each of the sample solution and the standard solution as 1732 O‹cial Monographs for Part I Supplement II, JPXIV

Add the following: Standard Lead Solution (not more than 10 ppm). (3) Related substances—Dissolve 0.3 g of Pirenzepine Pirenzepine Hydrochloride Hydrate Hydrochloride Hydrate in 10 mL of water. To 1 mL of this solution add 5 mL of methanol and the mobile phase A to 塩酸ピレンゼピン水和物 make 10 mL, and use this solution as the sample solution. Pipet 1 mL of the sample solution, and add 5 mL of methanol and the mobile phase A to make exactly 10 mL. Pipet 1 mL of this solution, add 5 mL of methanol and the mobile phase A to make exactly 10 mL, and use this solution as the standard solution. Perform the test with exactly 10 mL each of the sample solution and the standard solution as directed under the Liquid Chromatography according to the following conditions, and determine each peak area by the automatic integration method: the area of the peak other

C19H21N5O2.2HCl.H2O: 442.34 than pirenzepine is not more than 3/10 times the peak area 5,11-Dihydro-11-[(4-methylpiperazin-1-yl)acetyl]-6H- of pirenzepine from the standard solution, and the total area pyrido[2,3-b][1,4]benzodiazepin-6-one dihydrochloride of the peaks other than pirenzepine is not more than 3/5 monohydrate [29868-97-1,anhydride] times the peak area of pirenzepine from the standard solu- tion. Pirenzepine Hydrochloride Hydrate contains not Operating conditions— less than 98.5z and not more than 101.0z of pirenze- Detector: An ultraviolet absorption photometer (wavelength: 283 nm). pine hydrochloride (C19H21N5O2.2HCl: 424.32), calcu- lated on the anhydrous basis. Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecyl- Description Pirenzepine Hydrochloride Hydrate occurs as silanized silica gel for liquid chromatography (5 mmin a white to pale yellow crystalline powder. particle diameter). It is freely soluble in water and in formic acid, slightly Column temperature: A constant temperature of about soluble in methanol, and very slightly soluble in ethanol 409C. (99.5). Mobile phase A: Dissolve 2 g of sodium lauryl sulfate in The pH of a solution by dissolving 1 g of Pirenzepine 900 mL of water, adjust the pH to 3.2 with acetic acid (100), Hydrochloride Hydrate in 10 mL of water is between 1.0 and and add water to make 1000 mL. 2.0. Mobile phase B: Methanol Melting point: about 2459C (with decomposition). Mobile phase C: Acetonitrile It is gradually colored by light. Flowing of the mobile phase: Control the gradient by Identiˆcation (1) Determine the absorption spectrum of mixing the mobile phases A, B and C as directed in the fol- a solution of Pirenzepine Hydrochloride Hydrate (1 in lowing table. 40,000) as directed under the Ultraviolet-visible Spec- trophotometry, and compare the spectrum with the Time after injection Mobile phase Mobile phase Mobile phase Reference Spectrum: both spectra exhibit similar intensities of sample (min) A(z) B(z) C(z) of absorption at the same wavelengths. 0–15 55ª 25 30 15 ª 45 (2) Determine the infrared absorption spectrum of 15–253045 Pirenzepine Hydrochloride Hydrate as directed in the potas- sium chloride disk method under the Infrared Spectrophoto- Flow rate: Adjust the ‰ow rate so that the retention time metry, and compare the spectrum with the Reference of pirenzepine is about 8 minutes. Spectrum: both spectra exhibit similar intensities of absorp- Time span of measurement: About 2 times as long as the tion at the same wave numbers. retention time of pirenzepine after the solvent peak. (3) A solution of Pirenzepine Hydrochloride Hydrate (1 System suitability— in 50) responds to the Qualitative Tests for chloride. Test for required detectability: Pipet 1 mL of the standard Purity (1) Clarity and color of solution—A solution solution, and add 5 mL of methanol and the mobile phase A obtained by dissolving 1.0 g of Pirenzepine Hydrochloride to make exactly 10 mL. Conˆrm that the peak area of piren- Hydrate in 10 mL of water is clear and not more color than zepine obtained from 10 mLofthissolutionisequivalentto7 that of the following control solution. to 13z of that from 10 mL of the standard solution. Control solution: To 1.2 mL of Matching ‰uid for color F System performance: Dissolve 0.1 g of phenylpiperazine add8.8mLofdilutedhydrochloricacid(1in40). hydrochloride in 10 mL of methanol. Mix 1 mL of this (2) Heavy metals—Proceed with 2.0 g of Pirenzepine solution and 1 mL of the sample solution, and add 5 mL of Hydrochloride Hydrate according to Method 2, and per- methanol and the mobile phase A to make 10 mL. When the form the test. Prepare the control solution with 2.0 mL of procedure is run with 10 mL of this solution under the above Supplement II, JPXIV O‹cial Monographs for Part I 1733 operating conditions, pirenzepine and phenylpiperazine are Reference Spectrum: both spectra exhibit similar intensities eluted in this order with the resolution between these peaks of absorption at the same wavelengths. being not less than 5. (2) Determine the infrared absorption spectrum of System repeatability: When the test is repeated 6 times Piroxicam as directed in the potassium bromide disk method with 10 mL of the standard solution under the above under the Infrared Spectrophotometry, and compare the operating conditions, the relative standard deviation of the spectrum with the Reference Spectrum: both spectra exhibit peak area of pirenzepine is not more than 2.0z. similar intensities of absorption at the same wave numbers. If any diŠerence appears between the spectra, dissolve the Water Not less than 3.5z and not more than 5.0z (0.3 g, sample with dichloromethane, evaporate the solvent, dry the volumetric titration, direct titration). residue on a water bath, and perform the test. Residue on ignition Not more than 0.1z (1 g). Purity (1) Heavy metals—Proceed with 1.0 g of Piroxic- Assay Weigh accurately about 0.2 g of Pirenzepine am according to Method 2, and perform the test. Prepare Hydrochloride Hydrate, dissolve in 2 mL of formic acid, the control solution with 2.0 mL of Standard Lead Solution add 60 mL of acetic anhydride, and titrate with 0.1 mol/L (not more than 20 ppm). perchloric acid VS (potentiometric titration). Perform a (2) Related substances—Dissolve 75 mg of Piroxicam in blank determination in the same manner, and make any 50 mL of acetonitrile for liquid chromatography, and use necessary correction. this solution as the sample solution. Pipet 1 mL of the sample solution, add acetonitrile for liquid chromatography Each mL of 0.1 mol/L perchloric acid VS to make exactly 10 mL. Pipet 1 mL of this solution, add ＝14.14 mg of C H N O .2HCl 19 21 5 2 acetonitrile for liquid chromatography to make exactly 50 Containers and storage Containers—Well-closed contain- mL, and use this solution as the standard solution. Perform ers. the test with exactly 20 mL each of the sample solution and Storage—Light-resistant. the standard solution as directed under the Liquid Chro- matography according to the following conditions, and determine each peak area by the automatic integration Add the following: method: the area of the peak other than piroxicam is not larger than the peak area of piroxicam with the standard so- Piroxicam lution, and the total area of the peaks other than piroxicam is not larger than 2 times the peak area of piroxicam with the ピロキシカム standard solution. Operating conditions— Detector: An ultraviolet absorption photometer (wavelength: 230 nm). Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with octadecyl-

C15H13N3O4S: 331.35 silanized silica gel for liquid chromatography (5 mmin 4-Hydroxy-2-methyl-N-(pyridin-2-yl)-2H-1,2-benzothiazine- particle diameter). 3-carboxamide 1,1-dioxide [36322-90-4] Column temperature: A constant temperature of about 409C. Piroxicam contains not less than 98.5z and not Mobile phase: A mixture of 0.05 mol/L potassium di- more than 101.0z of C15H13N3O4S, calculated on the hydrogen phosphate TS, pH 3.0 and acetonitrile for liquid dried basis. chromatography (3:2). Flow rate: Adjust the ‰ow rate so that the retention time Description Piroxicam occurs as a white to pale yellow of piroxicam is about 10 minutes. crystalline powder. Time span of measurement: About 5 times as long as the It is sparingly soluble in acetic anhydride, slightly soluble retention time of piroxicam after the solvent peak. in acetonitrile, in methanol and in ethanol (99.5), very slight- System suitability— ly soluble in acetic acid (100), and practically insoluble in Test for required detectability: To exactly 5 mL of the water. standard solution add acetonitrile for liquid chro- Melting point: about 2009C (with decomposition). matography to make exactly 20 mL. Conˆrm that the peak Identiˆcation (1) Dissolve 0.1 g of Piroxicam in a mix- area of piroxicam obtained with 20 mL of this solution is ture of methanol and 0.5 mol/L hydrochloric acid TS equivalent to 17.5 to 32.5z of that with 20 mLofthe (490:1) to make 200 mL. To 1 mL of this solution add the standard solution. mixture of methanol and 0.5 mol/L hydrochloric acid TS System performance: When the procedure is run with (490:1) to make 100 mL. Determine the absorption spectrum 20 mL of the standard solution under the above operating of this solution as directed under the Ultraviolet-visible conditions, the number of theoretical plates and the symmet- Spectrophotometry, and compare the spectrum with the ry factor of the peak of piroxicam are not less than 6000 and 1734 O‹cial Monographs for Part I Supplement II, JPXIV not more than 1.5, respectively. ner, and make any necessary correction. System repeatability: When the test is repeated 6 times Each mL of 0.1 mol/L perchloric acid VS with 20 mL of the standard solution under the above operat- ＝12.31 mg of C H N O ing conditions, the relative standard deviation of the peak 5 5 3 area of piroxicam is not more than 2.0z. Loss on drying Not more than 0.5z (1 g, 1059C, 3 hours). Pyridoxine Hydrochloride Residue on ignition Not more than 0.2z (1 g). 塩酸ピリドキシン Assay Weigh accurately about 0.25 g of Piroxicam, dis- solve in 60 mL of a mixture of acetic anhydride and acetic Change the origin/limits of content to read: acid (100) (1:1), and titrate with 0.1 mol/L perchloric acid VS (potentiometric titration). Perform a blank determina- Pyridoxine Hydrochloride, when dried, contains not tion in the same manner, and make any necessary correc- less than 98.0z and not more than 101.0z of tion. C8H11NO3.HCl. Each mL of 0.1 mol/L perchloric acid VS Change the Description to read: ＝33.14 mg of C15H13N3O4S Description Pyridoxine Hydrochloride occurs as a white to Containers and storage Containers—Tight containers. pale yellow, crystalline powder. It is freely soluble in water, slightly soluble in ethanol (99.5), and practically insoluble in acetic anhydride and in Pyrazinamide acetic acid (100). It is gradually aŠected by light. ピラジナミド Melting point: about 2069C (with decomposition). Change the Description to read: Change the Identiˆcation to read: Description Pyrazinamide occurs as white crystals or Identiˆcation crystalline powder. (1) Determine the absorption spectrum of a solution of It is sparingly soluble in water and in methanol, and Pyridoxine Hydrochloride in 0.1 mol/L hydrochloric acid slightly soluble in ethanol (99.5) and in acetic anhydride. TS (1 in 100,000) as directed under the Ultraviolet-visible Spectrophotometry, and compare the spectrum with the Change the Purity to read: Reference Spectrum or the spectrum of a solution of Purity Pyridoxine Hydrochloride Reference Standard prepared in (1) Heavy metals—Proceed with 1.0 g of Pyrazinamide the same manner as the sample solution: both spectra exhibit according to Method 2, and perform the test. Prepare the similar intensities of absorption at the same wavelengths. control solution with 2.0 mL of Standard Lead Solution (not (2) Determine the infrared absorption spectrum of more than 20 ppm). Pyridoxine Hydrochloride as directed in the potassium (2) Related substances—Dissolve 0.10 g of Pyrazin- chloride disk method under the Infrared Spectrophotomet- amide in 10 mL of methanol, and use this solution as the ry, and compare the spectrum with the Reference Spectrum sample solution. Pipet 1 mL of the sample solution, add or the spectrum of Pyridoxine Hydrochloride Reference methanol to make exactly 200 mL, and use this solution as Standard: both spectra exhibit similar intensities of absorp- the standard solution. Perform the test with these solutions tion at the same wave numbers. as directed under the Thin-layer Chromatography. Spot 20 (3) A solution of Pyridoxine Hydrochloride (1 in 10) mL each of the sample solution and the standard solution on responds to the Qualitative Tests for chloride. a plate of silica gel with ‰uorescent indicator for thin-layer chromatography. Develop the plate with a mixture of 1- Add the following next to Identiˆcation: butanol, water and acetic acid (100) (3:1:1) to a distance of pH The pH of a solution prepared by dissolving 1.0 g of about 10 cm, and air-dry the plate. Examine under ultravio- Pyridoxine Hydrochloride in 50 mL of water is between 2.5 let light (main wavelength: 254 nm): the spot other than the and 3.5. principal spot obtained from the sample solution is not more intense than the spot from the standard solution. AddthefollowingnexttoPurity(2): Change the Assay to read: (3) Related substances—Dissolve 1.0 g of Pyridoxine Hydrochloride in 10 mL of water, and use this solution as Assay Weigh accurately about 0.1 g of Pyrazinamide, the sample solution. Pipet 2.5 mL of the sample solution, previously dried, dissolve in 50 mL of acetic anhydride, and and add water to make exactly 100 mL. Pipet 1 mL of this titrate with 0.1 mol/L perchloric acid VS (potentiometric solution, add water to make exactly 10 mL, and use this titration). Perform a blank determination in the same man- Supplement II, JPXIV O‹cial Monographs for Part I 1735 solution as the standard solution. Perform the test with these Add the following next to Identiˆcation: solutions as directed under the Thin-layer Chromatography. Endotoxins Less than 3.0 EU/mg. Spot 2 mL each of the sample solution and the standard solution on a plate of silica gel for thin-layer chro- matography, and air-dry the plate. Develop the plate with a Delete the following Monograph: mixture of acetone, tetrahydrofuran, hexane and ammonia solution (28) (65:13:13:9) to a distance of about 10 cm, and air-dry the plate. Spray evenly a solution of sodium Santonin Tablets carbonate in diluted ethanol (99.5) (3 in 10) (1 in 20) on the plate. After air-drying, spray evenly a solution of 2,6- dibromo-N-chloro-1,4-benzoquinone monoimine in ethanol Add the following: (99.5) (1 in 1000) on the plate, and air-dry: the spot other than the principal spot obtained from the sample solution is Serrapeptase not more intense than the spot from the standard solution. セラペプターゼ

Pyridoxine Hydrochloride Injection [95077-02-4]

塩酸ピリドキシン注射液 Serrapeptase is the enzyme preparation having pro- teolytic activity, produced by the growth of Serratia Change the origin/limits of content to read: species. Usually, it is diluted with Lactose. It contains not less than 2000 serrapeptase Units and Pyridoxine Hydrochloride Injection is an aqueous not more than 2600 serrapeptase Units per mg. solution for injection. It is hygroscopic. It contains not less than 95.0z and not more than Description Serrapeptase occurs as a grayish white to light 105.0 of the labeled amount of pyridoxine z brown powder, having a slight characteristic odor. hydrochloride (C8H11NO3.HCl: 205.64). Identiˆcation Dissolve 0.4 g of Serrapeptase in 100 mL of Change the Identiˆcation to read: acetic acid-sodium acetate buŠer solution, pH 5.0, transfer exactly 1 mL each of this solution into three tubes, and refer Identiˆcation tothem asA,B andC.TotubeA addexactly1 mL ofwater, (1) To a volume of Pyridoxine Hydrochloride Injection, to tubes B and C add exactly 1 mL of 0.04 mol/L disodium equivalent to 50 mg of Pyridoxine Hydrochloride according dihydrogen ethylenediamine tetraacetate TS, mix gently, to the labeled amount, add 0.1 mol/L hydrochloric acid TS and allow them to stand in a water bath at 4±19C for about to make 100 mL. To 2 mL of this solution add 0.1 mol/L 1 hour. Then, to the tube B add exactly 2 mL of 0.04 mol/L hydrochloric acid TS to make 100 mL. Determine the zinc chloride TS, to the tubes A and C add exactly 2 mL of absorption spectrum of this solution as directed under the water, mix gently, and allow them to stand in a water bath at Ultraviolet-visible spectrophotometry: it exhibits a maxi- 4±19C for about 1 hour. Pipet 1 mL each of these solu- mum between 288 nm and 292 nm. tions, add borate-hydrochloric acid buŠer solution, pH 9.0 (2) To a volume of Pyridoxine Hydrochloride Injection, to the solutions A and B to make exactly 200 mL, to the so- equivalent to 10 mg of Pyridoxine Hydrochloride according lution C to make exactly 50 mL, and use these solutions as to the labeled amount, add water to make 10 mL, and use the sample solutions. Proceed with these sample solutions as this solution as the sample solution. Separately, dissolve 10 directed in the Assay: the activities of the solutions A and B mg of Pyridoxine Hydrochloride Reference Standard in 10 are almost the same, and the activity of the solution C is not mL of water, and use this solution as the standard solution. more than 5z of that of the solution A. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 2 mLeachofthesample A 1 Activity of solutions A, B or C＝ T × ×D×176 solution and the standard solution on a plate of silica gel for AS 20 thin-layer chromatography, and air-dry the plate. Develop AS: Absorbance of the standard solution the plate with a mixture of acetone, tetrahydrofuran, hexane AT: Absorbance of the sample solution and ammonia solution (28) (65:13:13:9) to a distance of 20: Reaction time (minute) about 10 cm, and air-dry the plate. Spray evenly a solution D: Dilution rate (200 for solution A and B, 50 for solution of sodium carbonate in diluted ethanol (99.5) (3 in 10) (1 in C) 20) on the plate. After air-drying, spray evenly a solution of 176: Conversion factor (Total volume of enzyme reaction 2,6-dibromo-N-chloro-1,4-benzoquinone monoimine in solution/volume of ˆltrate taken×amount of tyrosine in 2 ethanol (99.5) (1 in 1000) on the plate: the spots obtained mL of tyrosine standard solution) from the sample solution and the standard solution are blue in color and have the same Rfvalue. Purity (1) Heavy metals—Put 1.0 g of Serrapeptase in a 1736 O‹cial Monographs for Part I Supplement II, JPXIV porcelain crucible, add 2 drops each of sulfuric acid and at 37±0.59C for 30 minutes, and ˆlter through a dried ˆlter nitric acid, and incinerate by ignition. After cooling, to the paper. Pipet 2 mL of the ˆltrate, add exactly 5 mL of a residue add 2 mL of hydrochloric acid, evaporate to dryness solution of anhydrous sodium carbonate (3 in 50), mix, add on a water bath, add 10 mL of a solution of hydroxylamine exactly 1 mL of diluted Folin's TS (1 in 3), mix well, and hydrochloride (3 in 100) and 2 mL of dilute acetic acid, and allow to stand at 37±0.59C for 30 minutes. Determine the heat on a water bath for 5 minutes. After cooling, ˆlter if absorbance of this solution at 660 nm, A1, as directed under necessary, wash the ˆlter paper with 10 mL of water, put the the Ultraviolet-visible Spectrophotometry using water as the ˆltrate and washing in a Nessler tube, add water to make 50 blank. Separately, pipet 1 mL of the sample solution, add mL, and use this solution as the test solution. Prepare the exactly 5 mL of trichloroacetic acid TS for serrapeptase, control solution as follows: Evaporate to dryness 2 drops mix, add exactly 5 mL of the substrate solution, allow to each of sulfuric acid and nitric acid on a sand bath, add 2 stand at 37±0.59C for 30 minutes, and proceed in the same mL of hydrochloric acid to the residue, evaporate to dryness manner as directed above to determine the absorbance A2. on a water bath, add 2.0 mL of Standard Lead Solution, 10 Separately, pipet 2 mL of the tyrosine standard solution, mL of a solution of hydroxylamine hydrochloride (3 in 100) add exactly 5 mL of a solution of anhydrous sodium and 2 mL of dilute acetic acid, and heat on a water bath for carbonate (3 in 50), mix, add exactly 1 mL of diluted Folin's 5 minutes. Proceed in the same manner as directed for the TS (1 in 3), mix well, and proceed in the same manner as preparation of the test solution, and add water to make 50 directed above to determine the absorbance A3. Separately, mL (not more than 20 ppm). pipet 2 mL of 0.2 mol/L hydrochloric acid TS, and proceed (2) Arsenic—Prepare the test solution with 0.40 g of in the same manner as directed above to determine the

Serrapeptase according to Method 3, excepting addition of 5 absorbance A4. mL of a solution of magnesium nitrate hexahydrate in Serrapeptase Unit per mg of Serrapeptase ethanol (95) (3 in 10) instead of a solution of magnesium ni- A －A 1 trate hexahydrate in ethanol (95) (1 in 50), evaporating to ＝ 1 2 × ×200×176 A －A 20 dryness on a water bath, then incinerating with a small 3 4 ‰ame, and perform the test (not more than 5 ppm). 20: Reaction time (minute) 200: Dilution rate Loss on drying Not more than 7.0z (1 g, 1059C, 4 hours). 176: Conversion factor (Total volume of enzyme reaction Residue on ignition Not more than 1.5z (1 g). solution/volume of ˆltrate taken×amount of tyro- sine in 2 mL of tyrosine standard solution) Assay (i) Sample solution: Dissolve exactly 0.100 g of One serrapeptase Unit corresponds to the amount of Serrapeptase in a solution of ammonium sulfate (1 in 20) to serrapeptase which produces 5 mgoftyrosineperminute make exactly 100 mL. Pipet 1 mL of this solution, add from 5 mL of the substrate solution under the above condi- borate-hydrochloric acid buŠer solution, pH 9.0 to make tions. exactly 200 mL, and use this solution as the sample solution. (ii) Tyrosine standard solution: Dissolve exactly 0.160 g Containers and storage Containers—Tight containers. of Tyrosine Reference Standard, previously dried at 1059C for 3 hours, in 0.2 mol/L hydrochloric acid TS to make exactly 1000 mL. Pipet 10 mL of this solution, and add Sodium Aurothiomalate 0.2 mol/L hydrochloric acid TS to make exactly 100 mL. Prepare before use. 金チオリンゴ酸ナトリウム (iii) Substrate solution: Previously determine the loss on drying (609C, reduced pressure not exceeding 0.67 kPa, 3 Change the origin/limits of content to read: hours) of milk casein, previously dried. To exactly 1.20 g of the milk casein, calculated based on the loss on drying, add Sodium Aurothiomalate contains not less than 160 mL of a solution of sodium borate (19 in 1000), and heat 49.0z and not more than 52.5z of gold (Au: 196.97), in a water bath to dissolve. After cooling, adjust the pH to calculated on the anhydrous basis and corrected by the exactly 9.0 with 1 mol/L hydrochloric acid TS, and add amount of ethanol. borate-hydrochloric acid buŠer solution, pH 9.0 to make ex- actly 200 mL. Use after warming to 37±0.59C. Prepare be- Change the Description to read: fore use. Description Sodium Aurothiomalate occurs as white to (iv) Precipitation reagent: Trichloroacetic acid TS for light yellow, powder or granules. serrapeptase. Use after warming to 37±0.59C. It is very soluble in water, and practically insoluble in (v) Procedure: Pipet 1 mL of the sample solution, put in ethanol (99.5). a glass-stoppered tube (15×130 mm), allow to stand at 37± It is hygroscopic. 0.59C for 5 minutes, add exactly 5 mL of the substrate It changes in color by light to greenish pale yellow. solution, and mix well immediately. Allow to stand at 37± 0.5 C for exactly 20 minutes, add exactly 5 mL of 9 Change the Identiˆcation (3) to read: trichloroacetic acid TS for serrapeptase, mix, allow to stand Identiˆcation Supplement II, JPXIV O‹cial Monographs for Part I 1737

(3) Place 2 mL of a solution of Sodium Aurothiomalate not more than 2.0z. (1 in 10) in a porcelain crucible, add 1 mL of ammonia TS and 1 mL of hydrogen peroxide (30), evaporate to dryness, AddthefollowingnexttoPurity: and ignite. Add 20 mL of water to the residue, and ˆlter: the Water Not more than 5.0z (0.1 g, coulometric titration). residue on the ˆlter paper occurs as a yellow or dark yellow, Use a water vaporizer (heating temperature: 1059C; heating powder or granules. time: 30 minutes). AddthefollowingnexttoIdentiˆcation(3): Delete the Loss on drying: Identiˆcation (4) The ˆltrate obtained in (3) responds to the Qualita- Change the Assay to read: tive Tests for sodium salt. Assay Weigh accurately about 25 mg of Sodium (5) The ˆltrate obtained in (3) responds to the Qualita- Aurothiomalate, and dissolve in 2 mL of aqua regia by heat- tive Tests for sulfate. ing. After cooling, add water to make exactly 100 mL. Pipet 2 mL of the solution, add water to make exactly 25 mL, and AddthefollowingnexttoPurity(3): use this solution as the sample solution. Separately, pipet 5 Purity mL, 10 mL and 15 mL of Standard Gold Solution for atom- (4) Ethanol—Weigh accurately about 0.2 g of Sodium ic absorption spectrophotometry, add water to make exactly Aurothiomalate, add exactly 3 mL of the internal standard 25 mL, and use these solutions as the standard solutions (1), solution and 2 mL of water to dissolve, and use this solution (2) and (3), respectively. Perform the test with the sample so- as the sample solution. Separately, pipet 3 mL of ethanol lution and the standard solutions (1), (2) and (3) as directed (99.5), and add water to make exactly 1000 mL. Pipet 2 mL under the Atomic Absorption Spectrophotometry under the of this solution, add exactly 3 mL of the internal standard following conditions. Determine the amount of gold in the solution, and use this solution as the standard solution. sample solution using the calibration curve obtained from Perform the test with 2 mL each of the sample solution and the absorbances of the standard solutions. the standard solution as directed under the Gas Chro- Gas: Combustible gas—Acetylene matography according to the following conditions, and Supporting gas—Air determine the ratios of the peak area of ethanol to that of Lamp: Gold hollow-cathode lamp the internal standard, QT and QS: the amount of ethanol is Wavelength: 242.8 nm not more than 3.0z.

Q Amount (mg) of ethanol＝ T ×6×0.793 Sodium Polystyrene Sulfonate QS 0.793: Density (g/mL) of ethanol (99.5) at 209C ポリスチレンスルホン酸ナトリウム

Internal standard solution—A solution of 2-propanol (1 in Change the Purity (4) to read: 500). Operating conditions— (4) Styrene—To 10.0 g of Sodium Polystyrene Sulfonate Detector: Hydrogen ‰ame-ionization detector. add 10 mL of acetone, shake for 30 minutes, centrifuge, and Column: A column 3 mm in inside diameter and 3 m in use the supernatant liquid as the sample solution. Separate- length, packed with porous styrene-divinylbenzene ly, dissolve 10 mg of styrene in acetone to make exactly 100 copolymer for gas chromatography (particle diameter: 150 – mL. Pipet 1 mL of this solution, add acetone to make 180 mm) (average pore size: 0.0085 mm; 300 – 400 m2/g). exactly 100 mL, and use this solution as the standard Column temperature: A constant temperature of about solution. Perform the test with 20 mL each of the sample so- 1809C. lution and the standard solution as directed under the Liquid Carrier gas: Nitrogen Chromatography according to the following conditions, and Flow rate: Adjust the ‰ow rate so that the retention time determine peak areas, AT and AS, of styrene in each solu- of the internal standard is about 7 minutes. tion: AT is not larger than AS. System suitability— Operating conditions— System performance: When the procedure is run with 2 mL Detector: An ultraviolet absorption photometer of the standard solution under the above operating condi- (wavelength: 254 nm). tions, ethanol and the internal standard are eluted in this Column: A stainless steel column 4 mm in inside diameter order with the resolution between these peaks being not less and 15 cm in length, packed with octadecylsilanized silica gel than 4. for liquid chromatography (5 mminparticlediameter). System repeatability: When the test is repeated 6 times Column temperature: A constant temperature of about with 2 mL of the standard solution under the above operat- 259C. ing conditions, the relative standard deviation of the ratio of Mobile phase: A mixture of water and acetonitrile (1:1). the peak area of ethanol to that of the internal standard is Flow rate: Adjust the ‰ow rate so that the retention time 1738 O‹cial Monographs for Part I Supplement II, JPXIV of styrene is about 8 minutes. System suitability— Tegafur System performance: Dissolve 20 mg each of styrene and butyl parahydroxybenzoate in 100 mL of acetone. To 5 mL テガフール ofthissolutionaddacetonetomake100mL.Whenthe procedure is run with 20 mL of this solution under the above Add the following next to Identiˆcation: operating conditions, butyl parahydroxybenzoate and sty- pH Dissolve 0.5 g of Tegafur in 50 mL of water: the pH of rene are eluted in this order with the resolution between this solution is between 4.2 and 5.2. these peaks being not less than 5. System repeatability: When the test is repeated 6 times with 20 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the peak Testosterone Propionate area of styrene is not more than 2.0z. プロピオン酸テストステロン

Change the Description to read: Sodium Thiosulfate Description Testosterone Propionate occurs as white to チオ硫酸ナトリウム pale yellow crystals or crystalline powder. It is freely soluble in methanol and in ethanol (95), and Change the origin/limits of content to read: practically insoluble in water.

Sodium Thiosulfate, when dried, contains not less Change the Identiˆcation to read: than 99.0z and not more than 101.0z of sodium Identiˆcation thiosulfate (Na S O :158.11). 2 2 3 (1) Determine the absorption spectrum of a solution of Testosterone Propionate in ethanol (95) (1 in 100,000) as Change the Description to read: directed under the Ultraviolet-visible Spectrophotometry, Description Sodium Thiosulfate occurs as colorless, crys- and compare the spectrum with the Reference Spectrum or tals or crystalline powder. It is odorless. the spectrum of a solution of Testosterone Propionate It is very soluble in water, and practically insoluble in Reference Standard prepared in the same manner as the ethanol (99.5). sample solution: both spectra exhibit similar intensities of It eŒoresces in dry air, and is deliquescent in moist air. absorption at the same wavelengths. (2) Determine the infrared absorption spectrum of Change the Identiˆcation to read: Testosterone Propionate, previously dried, as directed in the potassium bromide disk method under the Infrared Identiˆcation Spectrophotometry, and compare the spectrum with the (1) A solution of Sodium Thiosulfate (1 in 10) responds Reference Spectrum or the spectrum of Testosterone to the Qualitative Tests for thiosulfate. Propionate Reference Standard: both spectra exhibit similar (2) A solution of Sodium Thiosulfate (1 in 10) responds intensities of absorption at the same wave numbers. to the Qualitative Tests for sodium salt. Change the Optical rotation to read: Change the Loss on drying to read: 20 Optical rotation [a]D : ＋83 – ＋909(after drying, 0.1 g, Loss on drying 32.0 – 37.0z (1 g, in vacuum, 40 – 459C, ethanol (95), 10 mL, 100 mm). 16 hours). Change the Purity to read: Change the Assay to read: Purity Other steroids—Dissolve 40 mg of Testosterone Assay Weigh accurately about 0.4 g of Sodium Thiosul- Propionate in 2 mL of ethanol (95), and use this solution as fate, previously dried, dissolve in 30 mL of water, and titrate the sample solution. Pipet 1 mL of this solution, add ethanol with 0.05 mol/L iodine VS (indicator: 1 mL of starch TS). (95) to make exactly 100 mL, and use this solution as the Each mL of 0.05 mol/L iodine VS standard solution. Perform the test with these solutions as

＝15.81 mg of Na2S2O3 directed under the Thin-layer Chromatography. Spot 10 mL each of the sample solution and the standard solution on a plate of silica gel with ‰uorescent indicator for thin-layer chromatography. Develop the plate with a mixture of chlo- roform and diethylamine (19:1) to a distance of about 15 cm, and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 nm): the spot other than the principal Supplement II, JPXIV O‹cial Monographs for Part I 1739 spot from the sample solution is not more intense than the It contains not less than 92.5z and not more than spot from the standard solution. 107.5z of the labeled amount of testosterone

propionate (C22H32O3:344.49). Change the Assay to read: Change the Identiˆcation to read: Assay Weigh accurately each about 10 mg of Testosterone Propionate and Testosterone Propionate Reference Identiˆcation Dissolve the residue obtained as directed in Standard, previously dried, and dissolve in methanol to the procedure in the Assay in exactly 20 mL of methanol, make exactly 100 mL. To exactly 5 mL of these solutions and use this solution as the sample solution. Separately, add exactly 5 mL of the internal standard solution and dissolve 1 mg of Testosterone Propionate Reference methanol to make 20 mL, and use these solutions as the Standard in 10 mL of methanol, and use this solution as the sample solution and the standard solution. Perform the test standard solution. Perform the test with these solutions as with 5 mL each of the sample solution and the standard directed under the Thin-layer Chromatography. Spot 10 mL solution as directed under the Liquid Chromatography each of the sample solution and the standard solution on a according to the following conditions, and determine the plate of silica gel with ‰uorescent indicator for thin-layer ratios, QT and QS, of the peak area of testosterone chromatography. Develop the plate with a mixture of propionate to that of the internal standard. chloroform and diethylamine (19:1) to a distance of about 15 cm, and air-dry the plate. Examine under ultraviolet light Amount (mg) of C H O 22 32 3 (main wavelength: 254 nm): the Rfvaluesoftheprincipal Q ＝W × T spot with the sample solution and of the spot with the stan- S Q S dard solution are not diŠerent each other.

WS: Amount (mg) of Testosterone Propionate Reference Standard Add the following next to Identiˆcation: Internal standard solution—A solution of Progesterone in Actual volume It meets the requirements of Injections. methanol (9 in 100,000). Foreign insoluble matter Perform the test according to Operating conditions— Method 1: it meets the requirements of the Foreign Insoluble Detector: An ultraviolet absorption photometer Matter Test for Injections. (wavelength: 241 nm). Column: A stainless steel column 4.6 mm in inside di- Sterility Perform the test according to the Membrane ameter and 15 cm in length, packed with octadecylsilanized ˆltration method: it meets the requirements of the Sterility silica gel for liquid chromatography (5 mminparticledi- Test. ameter). Column temperature: A constant temperature of about Change the Assay to read: 359C. Assay Mobile phase: A mixture of acetonitrile and water (7:3). (i) Chromatographic tube A glass tube about 1 cm in Flow rate: Adjust the ‰ow rate so that the retention time inside diameter and about 18 cm in length, with a glass ˆlter of testosterone propionate is about 10 minutes. (G3) at the lower end. System suitability— (ii) Chromatographic column To about 2 g of silica gel System performance: When the procedure is run with 5 mL for liquid chromatography add 5 mL of dichloromethane, of the standard solution under the above operating condi- and mix gently. Transfer and wash into the chromatographic tions, the internal standard and testosterone propionate are tube with the aid of dichloromethane, allow to elute the eluted in this order with the resolution between these peaks dichloromethane through the column, and put a ˆlter paper being not less than 9. on the upper end of the silica gel. System repeatability: When the test is repeated 6 times (iii) Standard solution Weigh accurately about 10 mg with 5 mL of the standard solution under the above operat- of Testosterone Propionate Reference Standard, previously ing conditions, the relative standard deviation of the ratio of dried at 1059C for 4 hours, and dissolve in methanol to the peak area of testosterone propionate to that of the make exactly 100 mL. Pipet 5 mL of this solution, add internal standard is not more than 1.0z. exactly 5 mL of the internal standard solution and methanol to make exactly 20 mL. (iv) Sample stock solution To exactly a volume of Testosterone Propionate Injection Testosterone Propionate Injection, equivalent to about 20 mg of testosterone propionate (C H O ), add プロピオン酸テストステロン注射液 22 32 3 dichloromethane to make exactly 20 mL. (v) Procedure Transfer exactly 2 mL of the sample Change the origin/limits of content to read: stock solution into the chromatographic column, and elute to the upper surface of the silica gel. Wash the inner Testosterone Propionate Injection is an oily solution for injection. surface of the chromatographic tube with 15 mL of 1740 O‹cial Monographs for Part I Supplement II, JPXIV dichloromethane, elute to the upper surface of the silica gel, area of thiamine is not more than 1.0z. and discard the eŒuent. Elute 15 mL of a mixture of dichloromethane and methanol (39:1), discard the ˆrst Change the Assay to read: 5 mL of the eŒuent, and collect the subsequent eŒuent. Assay Weigh accurately about 0.1 g each of Thiamine Wash the lower part of the column with a few amount of Hydrochloride and Thiamine Hydrochloride Reference dichloromethane, combine the washings and the eŒuent, Standard (previously determine the water content), and and evaporate the solvent under reduced pressure. Dissolve dissolve them in the mobile phase to make exactly 50 mL. To the residue so obtained with methanol to make exactly 20 10 mL each of the solutions, accurately measured, add mL. Pipet 5 mL of this solution, add exactly 5 mL of the in- exactly 5 mL each of the internal standard solution, add the ternal standard solution and methanol to make exactly 20 mobile phase to make 50 mL, and use these solutions as the mL, and use this solution as the sample solution. Perform sample solution and the standard solution. Perform the test the test with 5 mL each of the sample solution and the stan- with 10 mL each of the sample solution and the standard dard solution as directed in the Assay under Testosterone solution as directed under the Liquid Chromatography ac- Propionate. cording to the following conditions, and calculate the ratios,

Amount (mg) of testosterone propionate (C22H32O3) QT and QS, of the peak area of thiamine to that of the inter-

QT nal standard. ＝WS× ×2 QS Amount (mg) of C12H17ClN4OS.HCl

WS: Amount (mg) of Testosterone Propionate Reference QT ＝WS× Standard QS

WS: Amount (mg) of Thiamine Hydrochloride Reference Thiamine Hydrochloride Standard, calculated on the anhydrous basis Internal standard solution—A solution methyl benzoate in 塩酸チアミン methanol (1 in 50). Operating conditions— Change the Purity (5) to read: Detector: An ultraviolet absorption photometer (wavelength: 254 nm). Purity Column: A stainless steel column 4.6 mm in inside (5) Related substances—Dissolve 0.10 g of Thiamine diameter and 15 cm in length, packed with octadecyl- Hydrochloride in 100 mL of the mobile phase, and use this silanized silica gel for liquid chromatography (5 mmin solution as the sample solution. Pipet 1 mL of the sample particle diameter). solution, add the mobile phase to make exactly 100 mL, and Column temperature: A constant temperature of about use this solution, as the standard solution. Perform the test 259C. with exactly 10 mL each of the sample solution and the the Mobile phase: Dissolve 1.1 g of sodium 1-octanesulfonate standard solution as directed under the Liquid Chro- in 1000 mL of diluted acetic acid (100) (1 in 100). To 600 mL matography according to the following conditions, and de- of this solution add 400 mL of a mixture of methanol and termine the area of each peak by the automatic integration acetonitrile (3:2). method: the total area of the peaks other than thiamine is Flow rate: Adjust the ‰ow rate so that the retention time not larger than the peak area of thiamine from the standard of thiamine is about 12 minutes. solution. System suitability— Operating conditions— System performance: When the procedure is run with Detector, column, column temperature, mobile phase, 10 mL of the standard solution under the above operating and ‰ow rate: Proceed as directed in the operating condi- conditions, thiamine and the internal standard are eluted in tions in the Assay. this order with the resolution between these peaks being not Time span of measurement: About 3 times as long as the less than 6. retention time of thiamine. System repeatability: When the test is repeated 6 times System suitability— with 10 mL of the standard solution under the above operat- Test for required detectability: To exactly 5 mL of the ing conditions, the relative standard deviation of the ratio of standard solution add water to make exactly 50 mL. Con- the peak area of thiamine to that of the internal standard is ˆrm that the peak area of thiamine obtained from 10 mLof not more than 1.0z. this solution is equivalent to 7 to 13z of that of thiamine obtained from 10 mL of the standard solution. System performance: Proceed as directed in the system suitability in the Assay. System repeatability: When the test is repeated 6 times with 10 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the peak Supplement II, JPXIV O‹cial Monographs for Part I 1741

the spot with the standard solution (2), and the spot other Thiamylal Sodium than the principal spot, the spot at origin and the spot men- tioned above obtained with the sample solution is not more チアミラールナトリウム intense than the spot with the standard solution (1).

Change the origin/limits of content to read: Change the Assay to read: Assay Weigh accurately about 0.25 g of Thiamylal Sodi- Thiamylal Sodium contains not less than 97.5z and um, dissolve in 50 mL of methanol and 5 mL of dilute not more than 101.0z of C12H17N2NaO2S, calculated on the dried basis. hydrochloric acid, and add methanol to make exactly 100 mL. Pipet 10 mL of this solution, and add methanol to make exactly 100 mL. Pipet 5 mL of this solution, add Change the Description to read: exactly 10 mL of the internal standard solution and the Description Thiamylal Sodium occurs as light yellow crys- mobile phase to make 200 mL, and use this solution as the tals or powder. sample solution. Separately, weigh accurately about 23 mg It is very soluble in water, and freely soluble in ethanol of Thiamylal Reference Standard, previously dried at 1059C (95). for 1 hour, dissolve in 50 mL of methanol and 0.5 mL of The pH of a solution of Thiamylal Sodium (1 in 10) is dilute hydrochloric acid, and add methanol to make exactly between 10.0 and 11.0. 100 mL. Pipet 5 mL of this solution, add exactly 10 mL of It is hygroscopic. the internal standard solution and the mobile phase to make It is gradually decomposed by light. 200 mL, and use this solution as the standard solution. Its solution in ethanol (95) (1 in 10) shows no optical Perform the test with 20 mL each of the sample solution and rotation. the standard solution as directed under the Liquid Chro- matography according to the following conditions, and

Change the Identiˆcation to read: determine the ratios, QT and QS, of the peak area of thiamylal to that of the internal standard. Identiˆcation

(1) Determine the absorption spectrum of a solution of QT Amount (mg) of C12H17N2NaO2S＝WS× ×10×1.0864 Thiamylal Sodium in ethanol (95) (7 in 1,000,000) as direct- QS ed under the Ultraviolet-visible Spectrophotometry, and W : Amount (mg) of Thiamylal Reference Standard compare the spectrum with the Reference Spectrum: both S spectra exhibit similar intensities of absorption at the same Internal standard solution—A solution of phenyl benzoate wavelengths. in methanol (3 in 500). (2) Determine the infrared absorption spectrum of Operating conditions— Thiamylal Sodium, previously dried, as directed in the Detector: An ultraviolet absorption photometer potassium bromide disk method under the Infrared (wavelength: 289 nm). Spectrophotometry, and compare the spectrum with the Column: A stainless steel column about 4 mm in inside Reference Spectrum: both spectra exhibit similar intensities diameter and about 15 cm in length, packed with octadecyl- of absorption at the same wave numbers. silanized silica gel for liquid chromatography (5 mmin (3) A solution of Thiamylal Sodium (1 in 10) responds to particle diameter). the Qualitative Tests for sodium salt. Column temperature: A constant temperature of about 259C. Change the Purity (3) to read: Mobile phase: A mixture of methanol and 0.05 mol/L acetic acid-sodium acetate buŠer solution, pH 4.6 (13:7). Purity Flow rate: Adjust the ‰ow rate so that the retention time (3) Related substances—Dissolve 0.10 g of Thiamylal of thiamylal is about 6 minutes. Sodium in 10 mL of ethanol (95), and use this solution as the System suitability— sample solution. Pipet 1 mL and 3 mL of the sample solu- System performance: When the procedure is run with tion, add ethanol (95) to make exactly 200 mL, and use these 20 mL of the standard solution under the above operating solutions as the standard solution (1) and the standard solu- conditions, thiamylal and the internal standard are eluted in tion (2). Perform the test with these solutions as directed this order with the resolution between these peaks being not under the Thin-layer Chromatography. Spot 10 mLeachof less than 12. the sample solution, the standard solution (1) and the System repeatability: When the test is repeated 6 times standard solution (2) on a plate of silica gel for thin-layer with 20 mL of the standard solution under the above operat- chromatography, develop with a mixture of toluene, ing conditions, the relative standard deviation of the ratio of methanol and ethyl acetate (40:7:3) to a distance of about the peak area of thiamylal to that of the internal standard is 12 cm, and air-dry the plate. Allow the plate to stand in not more than 1.0z. iodine vapor for a night: the spot appeared around Rf0.1 obtained with the sample solution is not more intense than 1742 O‹cial Monographs for Part I Supplement II, JPXIV

Change to read: Assay Open carefully 10 containers of Thiamylal Sodium for Injection, dissolve the contents with water, wash out the inside of each container with water, combine them, and add Thiamylal Sodium for Injection water to make exactly V mL so that each mL contains about 5 mg of thiamylal sodium (C12H17N2NaO2S). Pipet 5 mL of 注射用チアミラールナトリウム this solution, and add 0.5 mL of dilute hydrochloric acid and methanol to make exactly 100 mL. Pipet 5 mL of this Thiamylal Sodium for Injection is a preparation for solution, add exactly 10 mL of the internal standard solution injection which is dissolved before use. and the mobile phase to make 200 mL, and use this solution It contains not less than 93.0z and not more than as the sample solution. Proceed the test with the sample so- 107.0z of the labeled amount of thiamylal sodium lution as directed in the Assay under Thiamylal Sodium. (C12H17N2NaO2S: 276.33). Amount (mg) of thiamylal sodium (C12H17N2NaO2S) in 1 Method of preparation Prepare as directed under Injec- container tions, with 100 parts of Thiamylal Sodium and 7 parts of QT V ＝WS× × ×1.0864 Dried Sodium Carbonate in mass. QS 50

Description Thiamylal Sodium for Injection occurs as light WS: Amount (mg) of Thiamylal Reference Standard yellow crystals, powder or masses. Internal standard solution—A solution of phenyl benzoate It is hygroscopic. in methanol (3 in 500). It is gradually decomposed by light. Containers and storage Containers—Hermetic containers. Identiˆcation (1) To 1.0 g of Thiamylal Sodium for Storage—Light-resistant. Injection add 20 mL of ethanol (95), shake vigorously, and ˆlter. Dissolve the precipitate so obtained in 1 mL of water, and add 1 mL of barium chloride TS: a white precipitate is produced. Centrifuge this solution, take oŠ the supernatant Add the following: liquid, and to the precipitate add dilute hydrochloric acid dropwise: the precipitate dissolves with eŠervescence. Tiaramide Hydrochloride Tablets (2) To 50 mg of Thiamylal Sodium for Injection add 塩酸チアラミド錠 100 mL of ethanol (95), shake vigorously, and ˆlter. To 3 mL of the ˆltrate add ethanol (95) to make 200 mL. Tiaramide Hydrochloride Tablets contain not less Determine the absorption spectrum of this solution as direct- than 95.0z and not more than 105.0z of the labeled ed under the Ultraviolet-visible Spectrophotometry: it amount of tiaramide (C H ClN O S: 355.84). exhibits maxima between 236 nm and 240 nm, and between 15 18 3 3 287 nm and 291 nm. Method of preparation Prepare as directed under Tablets, with Tiaramide Hydrochloride. pH The pH of a solution obtained by dissolving 1.0 g of Thiamylal Sodium for Injection in 40 mL of water is be- Identiˆcation (1) Determine the absorption spectrum of tween 10.5 and 11.5. the sample solution obtained in the Assay as directed under the Ultraviolet-visible Spectrophotometry: it exhibits Purity Related substances—To 0.10 g of Thiamylal Sodi- maxima between 285 nm and 289 nm, and between 292 nm um for Injection add 10 mL of ethanol (95), shake vigorous- and 296 nm. ly, ˆlter, and use the ˆltrate as the sample solution. Proceed (2) To a quantity of powdered Tiaramide Hydrochloride as diected in the Purity (3) under Thiamylal Sodium. Tablets, equivalent to 0.1 g of tiaramide according to the Bacterial endotoxins Less than 1.0 EU/mg. labeled amount, add 10 mL of diluted ethanol (99.5) (7 in 10), shake well, ˆlter, and use the ˆltrate as the sample Mass variation It meets the requirements of the Mass solution. Separately, dissolve 0.11 g of tiaramide hydrochlo- Variation Test. ride for assay in 10 mL of diluted ethanol (99.5) (7 in 10), Foreign insoluble matter Perform the test according to and use this solution as the standard solution. Perform the Method 2: it meets the requirements of the Foreign Insoluble test with these solutions as directed under the Thin-layer Matter Test for Injections. Chromatography. Spot 20 mL each of the sample solution and the standard solution on a plate of silica gel for thin-lay- Insoluble particulate matter Perform the test according to er chromatography, develop with a mixture of 1-butamol, Method 1: it meets the requirements of the Insoluble Par- water and acetic acid (100) (4:2:1) to a distance of about 10 ticulate Matter Test for Injections. cm, and dry the plate at 1009C for 30 minutes. Spray evenly Sterility Perform the test according to the Membrane DragendorŠ's TS for spraying followed by diluted nitric acid ˆltration method: it meets the requirements of the Sterility (1 in 50) on the plate: the principal spot obtained with the Test. sample solution and the spot with the standard solution are yellow-redincolorandhavethesameRfvalue. Supplement II, JPXIV O‹cial Monographs for Part I 1743

Content uniformity Perform the test according to the curately an amount of the powder, equivalent to about 0.1 g following method: it meets the requirements of the Content of tiaramide (C15H18ClN3O3S), add 60 mL of 0.1 mol/L uniformity Test. hydrochloric acid TS, shake for 30 minutes, add 0.1 mol/L To 1 tablet of Tiaramide Hydrochloride Tablets add a hydrochloric acid TS to make exactly 100 mL, and ˆlter. volume of 0.1 mol/L hydrochloric acid TS, equivalent to Discard the ˆrst 20 mL of the ˆltrate, pipet 5 mL of the sub- 3/5 volume of V mLwhichmakesasolutionsothateachmL sequent ˆltrate, add water to make exactly 100 mL, and use contains about 1 mg of tiaramide (C15H18ClN3O3S) accord- this solution as the sample solution. Separately, weigh ac- ing to the labeled amount, shake for 60 minutes, then add curately about 0.11 g of tiaramide hydrochloride for assay, 0.1 mol/L hydrochloric acid TS to make exactly V mL, and previously dried at 1059C for 3 hours, and dissolve in 0.1 ˆlter. Discard the ˆrst 20 mL of the ˆltrate, pipet 5 mL of mol/L hydrochloric acid TS to make exactly 100 mL. Pipet the subsequent ˆltrate, add water to make exactly 100 mL, 5 mL of this solution, add water to make exactly 100 mL, and use this solution as the sample solution. Separately, and use this solution as the standard solution. Determine the weigh accurately about 55 mg of tiaramide hydrochloride absorbances, AT and AS, of the sample solution and the for assay, previously dried at 1059C for 3 hours, and dis- standard solution at 294 nm as directed under the Ultrav- solve in 0.1 mol/L hydrochloric acid TS to make exactly 50 iolet-visible Spectrophotometry. mL. Pipet 5 mL of this solution, add water to make exactly Amount (mg) of tiaramide (C H ClN O S) 100 mL, and use this solution as the standard solution. 15 18 3 3 A Determine the absorbances, A and A , of the sample solu- ＝W × T ×0.907 T S S A tion and the standard solution at 294 nm as directed under S the Ultraviolet-visible Spectrophotometry. WS: Amount (mg) of tiaramide hydrochloride for assay

Amount (mg) of tiaramide (C15H18ClN3O3S) Containers and storage Containers—Tight containers.

AT V ＝WS× × ×0.907 AS 50 Tinidazole WS: Amount (mg) of tiaramide hydrochloride for assay Dissolution Perform the test with 1 tablet of Tiaramide チニダゾール Hydrochloride Tablets at 50 revolutions per minute accord- ing to Method 2 under the Dissolution Test, using 900 mL of Change the origin/limits of content to read: water as the dissolution medium. Withdraw 20 mL or more of the dissolution medium 15 minutes after starting the test Tinidazole, when dried, contains not less than for a 50-mg tablet or 30 minutes after starting the test for a 98.5z and not more than 101.0z of C8H13N3O4S. 100-mg tablet, and ˆlter through a membrane ˆlter with pore size of not more than 0.5 mm. Discard the ˆrst 10 mL of the Change the Description to read: ˆltrate, pipet V mL of the subsequent ˆltrate, add water to Description Tinidazole occurs as a light yellow, crystalline make exactly V? mL so that each mL contains about 56 mgof powder. tiaramide (C H ClN O S) according to the labeled amount, 15 18 3 3 It is soluble in acetic anhydride and in acetone, sparingly and use this solution as the sample solution. Separately, soluble in methanol, slightly soluble in ethanol (99.5), and weigh accurately about 15 mg of tiaramide hydrochloride very slightly soluble in water. for assay, previously dried at 1059C for 3 hours, and dis- solve in water to make exactly 50 mL. Pipet 5 mL of this Change the Identiˆcation to read: solution, add water to make exactly 25 mL, and use this so- lution as the standard solution. Determine the absorbances, Identiˆcation

AT and AS, at 294 nm of the sample solution and the stan- (1) Determine the absorption spectrum of a solution of dard solution as directed under the Ultraviolet-visible Tinidazole in methanol (1 in 50,000) as directed under the Spectrophotometry: the dissolution rates for a 50-mg tablet Ultraviolet-visible Spectrophotometry, and compare the in15minutesanda100-mgtabletin30minutesarenotless spectrum with the Reference Spectrum: both spectra exhibit than 80z, respectively. similar intensities of absorption at the same wavelengths. (2) Determine the infrared absorption spectrum of Dissolution rate (z) with respect to the labeled amount of Tinidazole as directed in the potassium bromide disk method tiaramide (C H ClN O S) 15 18 3 3 under the Infrared Spectrophotometry, and compare the A V? 1 ＝W × T × × ×360×0.907 spectrum with the Reference Spectrum: both spectra exhibit S A V C S similar intensities of absorption at the same wave numbers.

WS: Amount (mg) of tiaramide hydrochloride for assay

C: Labeled amount (mg) of tiaramide (C15H18ClN3O3S) in 1tablet

Assay Weigh accurately the mass of more than 20 Tiaramide Hydrochloride Tablets, and powder. Weigh ac- 1744 O‹cial Monographs for Part I Supplement II, JPXIV

Add the following: (wavelength: 230 nm for about 3 minutes after sample injec- tion and 318 nm subsequently). Tizanidine Hydrochloride Column: A stainless steel column 4.6 mm in inside diameter and 12.5 cm in length, packed with octadecyl- 塩酸チザニジン silanized silica gel for liquid chromatography (5 mmin particle diameter). Column temperature: A constant temperature of about 259C. Mobile phase A: A mixture of water and formic acid (200:1), adjusted to pH 8.5 with ammonia water (28). Mobile phase B: A mixture of acetonitrile and the mobile C9H8ClN5S.HCl: 290.17 5-Chloro-4-[(4,5-dihydro-1H-imidazol-2- phase A (4:1). yl)amino]benzo[c][1,2,5]thiadiazole monohydrochloride Flowing of the mobile phase: Control the gradient by [64461-82-1] mixing the mobile phases A and B as directed in the follow- ing table. Tizanidine Hydrochloride, when dried, contains not less than 99.0z and not more than 101.0z of Time after injection Mobile phase Mobile phase of sample (min) A(z) B(z) C9H8ClN5S.HCl. Description Tizanidine Hydrochloride occurs as a white to 0–10 81ª 68 19 ª 32 10–13 68 32 light yellowish white crystalline powder. 13–26 68ª 10 32 ª 90 It is soluble in water, slightly soluble in ethanol (99.5), and 26–28 10 90 practically insoluble in acetic anhydride and in acetic acid (100). Flow rate: Adjust the ‰ow rate so that the retention time Melting point: about 2909C (with decomposition). of tizanidine is about 7 minute. Identiˆcation (1) Determine the absorption spectrum of Time span of measurement: About 4 times as long as the a solution of Tizanidine Hydrochloride in diluted 1 mol/L retention time of tizanidine after the solvent peak. ammonia TS (1 in 10) (1 in 125,000) as directed under the System suitability— Ultraviolet-visible Spectrophotometry, and compare the Test for required detectability: Measure exactly 2 mL of spectrum with the Reference Spectrum: both spectra exhibit the standard solution, and add the mixture of water and similar intensities of absorption at the same wavelengths. acetonitrile (17:3) to make exactly 10 mL. Conˆrm that the (2) Determine the infrared absorption spectrum of peak area of tizanidine obtained with 10 mL of this solution Tizanidine Hydrochloride as directed in the potassium chlo- is equivalent to 14 to 26z of that with 10 mL of the standard ride disk method under the Infrared Spectrophotometry, solution. and compare the spectrum with the Reference Spectrum: System performance: Dissolve 2 mg each of Tizanidine both spectra exhibit similar intensities of absorption at the Hydrochloride and p-toluenesulfonic acid monohydrate in same wave numbers. 100 mL of the mixture of water and acetonitrile (17:3). (3) A solution of Tizanidine Hydrochloride (1 in 50) When the procedure is run with 10 mLofthissolutionunder responds to the Qualitative Tests for chloride. the above operating conditions, p-toluenesulfonic acid and tizanidine are eluted in this order with the resolution Purity (1) Heavy metals—Proceed with 1.0 g of Tizani- between these peaks being not less than 10. dine Hydrochloride according to Method 3, and perform the System repeatability: When the test is repeated 6 times test. Prepare the control solution with 2.0 mL of Standard with 10 mL of the standard solution under the above operat- Lead Solution (not more than 20 ppm). ing conditions, the relative standard deviation of the peak (2) Related substances—Dissolve 60 mg of Tizanidine area of tizanidine is not more than 2.0z. Hydrochloride in 10 mL of a mixture of water and acetoni- trile (17:3), and use this solution as the sample solution. Loss on drying Not more than 0.2z (1 g, 1059C, 3 hours). Pipet 1 mL of the sample solution, add the mixture of water Residue on ignition Not more than 0.1z (1 g). and acetonitrile (17:3) to make exactly 200 mL, and use this solution as the standard solution. Perform the test with ex- Assay Weigh accurately about 0.2 g of Tizanidine actly 10 mL of the sample solution and the standard solution Hydrochloride, previously dried, dissolve in 60 mL of a as directed under the Liquid Chromatography according to mixture of acetic anhydride and acetic acid (100) (7:3) with the following conditions, and determine each peak area by the aid of warming. After cooling, titrate with 0.1 mol/L the automatic integration method: the area of the peak other perchloric acid VS (potentiometric titration). Perform a than tizanidine is not larger than 1/5 times the peak area of blank determination in the same manner, and make any tizanidine with the standard solution. necessary correction. Operating conditions— Detector: An ultraviolet absorption photometer Supplement II, JPXIV O‹cial Monographs for Part I 1745

control solution. Compare the sample solution and the stan- Each mL of 0.1 mol/L perchloric acid VS dard solution 2 minutes after they are prepared: the color of ＝29.02 mg of C H ClN S.HCl 9 8 5 the sample solution is not more intense than that of the stan- Containers and storage Containers—Well-closed contain- dard solution (not more than 10 ppm). ers. (4) Arsenic—Prepare the test solution by dissolving 1.0 g of Tranexamic Acid in 10 mL of water, and perform the test (not more than 2 ppm). Tranexamic Acid (5) Related substances—Dissolve 0.20 g of Tranexamic Acid in water to make exactly 20 mL, and use this solution トラネキサム酸 as the sample solution. Pipet 5 mL of the sample solution, and add water to make exactly 100 mL. Pipet 1 mL of this Change the origin/limits of content to read: solution, add water to make exactly 10 mL, and use this solution as the standard solution. Perform the test with ex- Tranexamic Acid, when dried, contains not less than actly 20 mL each of the sample solution and the standard so- 98.0z and not more than 101.0z of C8H15NO2. lution as directed under the Liquid Chromatography accord- ing to the following conditions, and determine each peak Change the Description to read: area by the automatic integration method: the area multi- plied by relative response factor 1.2 of the peak, having the Description Tranexamic Acid occurs as white crystals or relative retention time of about 1.5 with respect to tranexam- crystalline powder. ic acid, is not more than 2/5 of the peak area of tranexamic It is freely soluble in water, and practically insoluble in acid from the standard solution, and the area of the peak, ethanol (99.5). having the relative retention time of about 2.1 with respect to tranexamic acid, is not more than 1/5 of the peak area of Change the Identiˆcation to read: tranexamic acid from the standard solution. The area of Identiˆcation Determine the infrared absorption spectrum each peak other than tranexamic acid and other than the of Tranexamic Acid as directed in the potassium bromide peaks mentioned above is not more than 1/5 of the peak disk method under the Infrared Spectrophotometry, and area of tranexamic acid from the standard solution. For this compare the spectrum with the Reference Spectrum or the comparison, use the area of the peaks, having the relative spectrum of Tranexamic Acid Reference Standard: both retention time of about 1.1 and about 1.3, after multiplying spectra exhibit similar intensities of absorption at the same by their relative response factors 0.005 and 0.006, respec- wave numbers. tively. The total area of the peaks other than tranexamic acid is not more than the peak area of tranexamic acid from the Add the following next to Identiˆcation: standard solution. Operating conditions— pH The pH of a solution prepared by dissolving 1.0 g of Detector, column, column temperature, mobile phase, Tranexamic Acid in 20 mL of water is between 7.0 and 8.0. and ‰ow rate: Proceed as directed in the operating condi- Change the Purity to read: tions in the Assay. Time span of measurement: About 3 times as long as the Purity (1) Clarity and color of solution—Dissolve 1.0 g retention time of tranexamic acid after the solvent peak. of Tranexamic Acid in 10 mL of water: the solution is clear System suitability— and colorless. Test for required detectability: To exactly 5 mL of the (2) Chloride—Perform the test with 1.0 g of Tranexamic standard solution add water to make exactly 25 mL. Con- Acid. Prepare the control solution with 0.40 mL of 0.01 ˆrm that the peak area of tranexamic acid obtained from mol/L hydrochloric acid VS (not more than 0.014z). 20 mL of this solution is equivalent to 14 to 26z of that from (3) Heavy metals—Dissolve 2.0 g of Tranexamic Acid in 20 mL of the standard solution. water to make 20 mL, and use this solution as the sample System performance: Proceed as directed in the system stock solution. To 12 mL of the sample stock solution add suitability in the Assay. 2 mL of hydrochloric acid-ammonium acetate buŠer solu- System repeatability: When the test is repeated 6 times tion,pH3.5,mix,add1.2mLofthioacetamideTS,mix with 20 mL of the standard solution under the above operat- immediately, and use this solution as the sample solution. ing conditions, the relative standard deviation of the peak Separately, proceed in the same manner as above with a area of tranexamic acid is not more than 7z. mixture of 1 mL of Standard Lead Solution, 2 mL of the sample stock solution and 9 mL of water, and use the solu- Change the Assay to read: tion so obtained as the standard solution. Separately, pro- Assay Weigh accurately about 50 mg each of Tranexamic ceed in the same manner with a mixture of 10 mL of water Acid and Tranexamic Acid Reference Standard, previously and 2 mL of the sample stock solution, and use the solution dried, dissolve in water to make exactly 25 mL, and use these so obtained as the control solution. Conform that the color solutions as the sample solution and the standard solution. of the standard solution is slightly darker than that of the 1746 O‹cial Monographs for Part I Supplement II, JPXIV

Perform the test with exactly 20 mLeachofthesample color develops. solution and the standard solution as directed under the Mass variation It meets the requirements of the Mass Liquid Chromatography according to the following condi- Variation Test. tions, and determine the peak areas, AT and AS,oftranex- amic acid. Dissolution Being speciˆed separately.

AT Assay Weigh accurately the mass of the contents of not Amount (mg) of C8H15NO2＝WS× AS less than 20 Tranexamic Acid Capsules, and powder. Weigh accurately an amount of the powder, equivalent to about 0.1 W : Amount (mg) of Tranexamic Acid Reference S goftranexamicacid(CH NO ), add 30 mL of water, shake Standard 8 15 2 well, and add water to make exactly 50 mL. Centrifuge, ˆlter Operating conditions— the supernatant liquid through a membrane ˆlter with pore Detector: An ultraviolet absorption photometer size of not more than 0.45 mm, discard the ˆrst 10 mL of the (wavelength: 220 nm). ˆltrate, and use the subsequent ˆltrate as the sample solu- Column: A stainless steel column 6.0 mm in inside tion. Separately, weigh accurately about 50 mg of Tranex- diameter and 25 cm in length, packed with octadecyl- amic Acid Reference Standard, previously dried at 1059C silanized silica gel for liquid chromatography (5 mminparti- for 2 hours, dissolve in water to make exactly 25 mL, and cle diameter). use this solution as the standard solution. Perform the test Column temperature: A constant temperature of about with exactly 30 mL each of the sample solution and the stan- 259C. dard solution as directed under the Liquid Chromatography Mobile phase: Dissolve 11.0 g of sodium dihydrogen according to the following conditions, and determine the phosphate in 500 mL of water, and add 5 mL of triethyla- peak areas, A and A ,oftranexamicacid. mine and 1.4 g of sodium lauryl sulfate. Adjust the pH to T S

2.5 with phosphoric acid or diluted phosphoric acid (1 in Amount (mg) of tranexamic acid (C8H15NO2)

10), add water to make 600 mL, and add 400 mL of AT ＝WS× ×2 methanol. AS Flow rate: Adjust the ‰ow rate so that the retention time W : Amount (mg) of Tranexamic Acid Reference Stan- of tranexamic acid is about 20 minutes. S dard System suitability— System performance: To 5 mL of the standard solution Operating conditions— add 1 mL of a solution of 4-(aminomethyl)benzoic acid (1 in Detector, column, and mobile phase: Proceed as directed 10,000) and water to make 50 mL. When the procedure is in the operating conditions in the Assay under Tranexamic run with 20 mL of this solution under the above operating Acid. conditions, tranexamic acid and 4-(aminomethyl)benzoic Column temperature: A constant temperature of about acid are eluted in this order with the resolution between these 359C. peaks being not less than 5. Flow rate: Adjust the ‰ow rate so that the retention time System repeatability: When the test is repeated 6 times of tranexamic acid is about 16 minutes. with 20 mL of the standard solution under the above operat- System suitability— ing conditions, the relative standard deviation of the peak System performance: To 5 mL of the standard solution area of tranexamic acid is not more than 0.6z. add 1 mL of a solution of 4-(aminomethyl)benzoic acid (1 in 10,000) and water to make 50 mL. When the procedure is runwith30mL of this solution under the above operating Add the following: conditions, tranexamic acid and 4-(aminomethyl)benzoic acid are eluted in this order with the resolution between these Tranexamic Acid Capsules peaks being not less than 3. System repeatability: When the test is repeated 6 times トラネキサム酸カプセル with 30 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the peak Tranexamic Acid Capsules contain not less than area of tranexamic acid is not more than 1.0z. 95.0z and not more than 105.0z of the labeled Containers and storage Containers—Tight containers. amount of tranexamic acid (C8H15NO2: 157.21). Method of preparation Prepare as directed under Capsules, with Tranexamic Acid.

Identiˆcation Take an amount of powdered contents of Tranexamic Acid Capsules, equivalent to 0.5 g of Tranex- amic Acid according to the labeled amount, add 50 mL of water, shake well, and ˆlter. To 5 mL of the ˆltrate add 1 mL of ninhydrin TS, and heat for 3 minutes: a dark purple Supplement II, JPXIV O‹cial Monographs for Part I 1747

Add the following: 359C. Flow rate: Adjust the ‰ow rate so that the retention time Tranexamic Acid Injection of tranexamic acid is about 16 minutes. System suitability— トラネキサム酸注射液 System performance: To 5 mL of the standard solution add 1 mL of a solution of 4-(aminomethyl)benzoic acid (1 in Tranexamic Acid Injection is an aqueous injection. 10,000) and water to make 50 mL. When the procedure is It contains not less than 95.0z and not more than runwith30mL of this solution under the above operating 105.0z of the labeled amount of tranexamic acid conditions, tranexamic acid and 4-(aminomethyl)benzoic (C8H15NO2: 157.21). acid are eluted in this order with the resolution between these peaks being not less than 3. Method of preparation Prepare as directed under System repeatability: When the test is repeated 6 times Injections, with Tranexamic Acid. with 30 mL of the standard solution under the above operat- Description Tranexamic Acid Injection is a clear and ing conditions, the relative standard deviation of the peak colorless liquid. area of tranexamic acid is not more than 1.0z.

Identiˆcation To a volume of Tranexamic Acid Injection, Containers and storage Containers—Hermetic containers. equivalent to 50 mg of Tranexamic Acid according to the labeled amount, add water to make 5 mL, add 1 mL of ninhydrin TS, and heat: a dark purple color develops. Add the following: pH 7.0–8.0 Tranexamic Acid Tablets Bacterial endotoxins Not more than 0.12 EU/mg. トラネキサム酸錠 Actual volume It meets the requirements of Injections. Foreign insoluble matter Perform the test according to Tranexamic Acid Tablets contain not less than Method 1: it meets the requirements of the Foreign Insoluble 95.0z and not more than 105.0z of the labeled

Matter Test for Injections. amount of tranexamic acid (C8H15NO2: 157.21). Insoluble particulate matter Perform the test according to Method of preparation Prepare as directed under Tablets, Method 1: it meets the requirements of the Insoluble Par- with Tranexamic Acid. ticulate Matter Test for Injections. Identiˆcation To an amount of powdered Tranexamic Sterility Perform the test according to the Membrane Acid Tablets, equivalent to 0.5 g of Tranexamic Acid ˆltration method: it meets the requirements of the Sterility according to the labeled amount, add 50 mL of water, shake Test. well, and ˆlter. To 5 mL of the ˆltrate add 1 mL of ninhydrin TS, and heat for 3 minutes: a dark purple color Assay Take accurately a volume of Tranexamic Acid develops. Injection, equivalent to about 0.1 g of tranexamic acid

(C8H15NO2), add water to make exactly 50 mL, and use this Mass variation It meets the requirements of the Mass solution as the sample solution. Separately, weigh accurately Variation Test. about 50 mg of Tranexamic Acid Reference Standard, Dissolution Being speciˆed separately. previously dried at 1059C for 2 hours, dissolve in water to make exactly 25 mL, and use this solution as the standard Assay Weigh accurately the mass of not less than 20 solution. Perform the test with exactly 30 mLeachofthe Tranexamic Acid Tablets, and powder. Weigh accurately a sample solution and the standard solution as directed under quantity of the powder, equivalent to about 5 g of tranexam- the Liquid Chromatography according to the following ic acid (C8H15NO2), add 150 mL of water, disintegrate the conditions, and determine the peak areas, AT and AS,of tablets completely with the aid of ultrasonic waves, and add tranexamic acid. water to make exactly 200 mL. Centrifuge, pipet 4 mL of the supernatant liquid, and add water to make exactly 50 mL. A Amount (mg) of tranexamic acid (C H NO )＝W × T Filter through a membrane ˆlter with pore size of not more 8 15 2 S A S than 0.45 mm, discard the ˆrst 10 mL of the ˆltrate, and use

WS: Amount (mg) of Tranexamic Acid Reference the subsequent ˆltrate as the sample solution. Separately, Standard weigh accurately about 50 mg of Tranexamic Acid Reference Standard, previously dried at 1059C for 2 hours, dissolve in Operating conditions— water to make exactly 25 mL, and use this solution as the Detector, column, and mobile phase: Proceed as directed standard solution. Perform the test with exactly 30 mLeach in the operating conditions in the Assay under Tranexamic of the sample solution and the standard solution as directed Acid. under the Liquid Chromatography according to the follow- Column temperature: A constant temperature of about ing conditions, and determine the peak areas, AT and AS,of 1748 O‹cial Monographs for Part I Supplement II, JPXIV tranexamic acid. Standard prepared in the same manner as the sample solution: both spectra exhibit similar intensities of absorp- Amount (mg) of tranexamic acid (C H NO ) 8 15 2 tion at the same wavelengths. A ＝W × T ×100 (2) Determine the infrared absorption spectrum of S A S Trichlormethiazide as directed in the potassium bromide

WS: Amount (mg) of Tranexamic Acid Reference disk method under the Infrared Spectrophotometry, and Standard compare the spectrum with the Reference Spectrum or the spectrum of Trichlormethiazide Reference Standard: both Operating conditions— spectra exhibit similar intensities of absorption at the same Detector, column, and mobile phase: Proceed as directed wave numbers. in the operating conditions in the Assay under Tranexamic (3) Perform the test with Trichlormethiazide as directed Acid. under the Flame Coloration Test (2): a green color appears. Column temperature: A constant temperature of about 359C. Change the Purity (4) and (5) to read: Flow rate: Adjust the ‰ow rate so that the retention time of tranexamic acid is about 16 minutes. (4) Arsenic—Prepare the test solution with 0.6 g of System suitability— Trichlormethiazide according to Method 5, using 20 mL of System performance: To 5 mL of the standard solution N,N-dimethylformamide, and perform the test (not more add 1 mL of a solution of 4-(aminomethyl)benzoic acid (1 in than 3.3 ppm). 10,000) and water to make 50 mL. When the procedure is (5) Related substances―Dissolve 25 mg of Trichlor- run with 30 mL of this solution under the above operating methiazide in 50 mL of acetonitrile, and use the solution as conditions, tranexamic acid and 4-(aminomethyl)benzoic thesamplesolution.Performthetestwith10mLofthesam- acid are eluted in this order with the resolution between these ple solution as directed under the Liquid Chromatography peaks being not less than 3. according to the following conditions, determine each peak System repeatability: When the test is repeated 6 times area by the automatic integration method, and calculate the with 30 mL of the standard solution under the above operat- amount of related substances by the area percentage ing conditions, the relative standard deviation of the peak method: the amount of 4-amino-6-chlorobenzene-1,3-disul- area of tranexamic acid is not more than 1.0z. fonamide, having the relative retention time of about 0.3 with respect to trichlormethiazide, is not more than 2.0z, Containers and storage Containers—Tight containers. and the total amount of the related substances is not more than 2.5z. Operating conditions— Trichlormethiazide Detector: An ultraviolet absorption photometer (wavelength: 268 nm). トリクロルメチアジド Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with phenylsilanized Change the origin/limits of the content to read: silica gel for liquid chromatography (5 mminparticle Trichlormethiazide, when dried, contains not less diameter). Column temperature: A constant temperature of about than 97.5z and not more than 102.0z of C H Cl N O S . 259C. 8 8 3 3 4 2 Mobile phase A: A mixture of diluted phosphoric acid (1 in 1000) and acetonitrile (3:1). Change the Description to read: Mobile phase B: A mixture of acetonitrile and diluted Description Trichlormethiazide occurs as a white powder. phosphoric acid (1 in 1000) (3:1). It is freely soluble in N,N-dimethylformamide and in Flowing of the mobile phase: Control the gradient by acetone, slightly soluble in acetonitrile and in ethanol (95), mixing the mobile phases A and B as directed in the follow- and practically insoluble in water. ing table. A solution of Trichlormethiazide in acetone (1 in 50) shows no optical rotation. Time after injection Mobile phase Mobile phase Melting point: about 2709C (with decomposition). of sample (min) A(volz) B(volz)

0–10 100 0 Change the Identiˆcation to read: 10 – 20 100 ª 00ª 100 Identiˆcation (1) Determine the absorption spectrum of a solution of Flow rate: 1.5 mL per minute Trichlormethiazide in ethanol (95) (3 in 250,000) as directed Time span of measurement: About 2.5 times as long as the under the Ultraviolet-visible Spectrophotometry, and retention time of trichlormethiazide after the solvent peak. compare the spectrum with the Reference Spectrum or the System suitability— spectrum of a solution of Trichlormethiazide Reference Test for required detectability: To exactly 1 mL of the Supplement II, JPXIV O‹cial Monographs for Part I 1749 sample solution add acetonitrile to make exactly 50 mL, and 10 mL of the standard solution under the above operating use this solution as the solution for system suitability test. conditions, the internal standard and trichlormethiazide are Pipet 1 mL of the solution, and add acetonitrile to make ex- eluted in this order with the resolution between these peaks actly 20 mL. Conˆrm that the peak area of trichlor- being not less than 2.0. methiazide obtained from 10 mLofthissolutionisequiva- System repeatability: When the test is repeated 6 times lent to 3.5 to 6.5z of that of trichlormethiazide obtained with 10 mL of the standard solution under the above operat- from 10 mL of the solution for system suitability test. ing conditions, the relative standard deviation of the ratio of System performance: To 5 mL of the solution for system suitability test add 5 mL of water, and warm in a water bath the peak area of trichlormethiazide to that of the internal at 609C for 30 minutes. When the procedure is run with standard is not more than 1.0z. 10 mL of this solution, after cooling, under the above operat- ing conditions, 4-amino-6-chlorobenzene-1,3-disulfonamide and trichlormethiazide are eluted in this order, the relative Add the following: retention time of 4-amino-6-chlorobenzene-1,3-disul- fonamide with respect to trichlormethiazide is about 0.3, Trichlormethiazide Tablets and the number of theoretical plates and the symmetry fac- tor of the peak of trichlormethiazide are not less than 5000 トリクロルメチアジド錠 and not more than 1.2, respectively. System repeatability: When the test is repeated 3 times Trichlormethiazide Tablets contain not less than with 10 mL of the solution for system suitability test under 93.0z and not more than 107.0z of the labeled the above operating conditions, the relative standard devia- tion of the peak area of trichlormethiazide is not more than amount of trichlormethiazide (C8H8Cl3N3O4S2: 2.0z. 380.66). Method of preparation Prepare as directed under Tablets, Change the Assay to read: with Trichlormethiazide.

Assay Weigh accurately about 25 mg of Trichlor- Identiˆcation To an amount of pulverized Trichlor- methiazide and Trichlormethiazide Reference Standard, methiazide Tablets, equivalent to 4 mg of Trichlor- previously dried, and dissolve separately in exactly 20 mL of methiazide according to the labeled amount, add 10 mL of the internal standard solution. To 1 mL of these solutions acetone, shake vigorously for 5 minutes, centrifuge, and use add acetonitrile to make 20 mL, and use these solutions as the supernatant liquid as the sample solution. Separately, the sample solution and the standard solution. Perform the dissolve 4 mg of Trichlormethiazide Reference Standard in test with 10 mL each of the sample solution and the standard 10 mL of acetone, and use this solution as the standard solution as directed under the Liquid Chromatography solution. Perform the test with these solutions as directed according to the following conditions, and determine the under the Thin-layer Chromatography. Spot 5 mLeachof ratios, QT and QS, of the peak area of trichlormethiazide to the sample solution and the standard solution on a plate of that of the internal standard. silica gel with ‰uorescent indicator for thin-layer chro- matography. Develop the plate with a mixture of ethyl QT Amount (mg) of C8H8Cl3N3O4S2＝WS× QS acetate, hexane and methanol (10:4:1) to a distance of about 10 cm, and air-dry the plate. Examine under ultraviolet light WS: Amount (mg) of Trichlormethiazide Reference Stan- (main wavelength: 254 nm): the principal spots from the dard sample solution and the standard solution show the same Rf Internal standard solution—A solution of 3-nitrophenol in value. acetonitrile (1 in 800). Purity Related substances—Pulverize a suitable amount of Operating conditions— Trichlormethiazide Tablets in an agate mortar. Take an Detector: An ultraviolet absorption photometer amount of the powder, equivalent to 10 mg of Trichlor- (wavelength: 268 nm). methiazide according to the labeled amount, add 20 mL of Column: A stainless steel column 4.6 mm in inside acetonitrile, shake vigorously for 15 minutes, centrifuge, diameter and 15 cm in length, packed with phenylsilanized and use the supernatant liquid as the sample solution. Per- silica gel for liquid chromatography (5 mminparticle form the test with 10 mL of the sample solution as directed diameter). under the Liquid Chromatography according to the follow- Column temperature: A constant temperature of about ing conditions, determine each peak area by the automatic 259C. integration method, and calculate the amount of each Mobile phase: A mixture of diluted phosphoric acid (1 in related substance by the area percentage method: the 1000) and acetonitrile (3:1). amount of 4-amino-6-chlorobenzene-1,3-disulfoneamide, Flow rate: Adjust the ‰ow rate so that the retention time having the relative retention time of about 0.3 with respect of trichlormethiazide is about 8 minutes. to trichlormethiazide, is not more than 4.0z,andthetotal System suitability— amount of the peaks other than trichlormethiazide is not System performance: When the procedure is run with more than 5.0z. 1750 O‹cial Monographs for Part I Supplement II, JPXIV

Operating conditions— (C8H8Cl3N3O4S2) according to the labeled amount, add Detector: An ultraviolet absorption photometer acetonitrile to make 25 mL, shake vigorously for 15 (wavelength: 268 nm). minutes, and centrifuge. To an amount of the supernatant Column: A stainless steel column 4.6 mm in inside liquid add the mobile phase to make a solution so that it con- diameter and 15 cm in length, packed with phenylsilanized tains about 40 mg of trichlormethiazide (C8H8Cl3N3O4S2)in silica gel for liquid chromatography (5 mminparticle each mL, and use this solution as the sample solution. diameter). Separately, weigh accurately about 20 mg of Trichlor- Column temperature: A constant temperature of about methiazide Reference Standard, previously dried at 1059C 259C. for 3 hours, and dissolve in acetonitrile to make exactly 100 Mobile phase A: A mixture of diluted phosphoric acid mL. Pipet 5 mL of this solution, add exactly 5 mL of the in- (1 in 1000) and acetonitrile (3:1). ternal standard solution, add 10 mL of acetonitrile and 5 mL Mobile phase B: A mixture of acetonitrile and diluted of diluted phosphoric acid (1 in 50), and use this solution as phosphoric acid (1 in 1000) (3:1). the standard solution. Perform the test with 20 mLeachof Flowing of the mobile phase: Control the gradient by the sample solution and the standard solution as directed un- mixing the mobile phases A and B as directed in the follow- der the Liquid Chromatography according to the conditions ing table. described in Assay, and determine the ratios, QT and QS,of the peak area of trichlormethiazide to that of the internal Time after injection Mobile phase Mobile phase standard. of sample (min) A(z) B(z) Amount (mg) of trichlormethiazide (C8H8Cl3N3O4S2)

0–10 100 0 QT 1 ＝WS× ×C× 10–20 100ª 00ª 100 QS 20 W : Amount (mg) of Trichlormethiazide Reference Flow rate: 1.5 mL/minute S Standard Time span of measurement: About 2.5 times as long as the C: Labeled amount (mg) of trichlormethiazide retention time of trichlormethiazide after the solvent peak. (C H Cl N O S ) per tablet System suitability— 8 8 3 3 4 2 Test for required detectability: Dissolve 25 mg of Internal standard solution—A solution of 3-nitrophenol in Trichlormethiazide in 50 mL of acetonitrile. Pipet 1 mL of acetonitrile (1 in 5000). this solution, add acetonitrile to make exactly 50 mL, and Dissolution Perform the test with 1 tablet of Trichlor- use this solution as the solution for system suitability test. methiazide Tablets at 50 revolutions per minute according to Pipet 1 mL of the solution for system suitability test, and Method 2 under the Dissolution Test, using 900 mL of water add acetonitrile to make exactly 20 mL. Conˆrm that the as the dissolution medium. Withdraw 20 mL or more of the peak area of trichlormethiazide obtained from 10 mLofthis dissolution medium 15 minutes after starting the test, and solution is equivalent to 3.5 to 6.5z of that obtained from ˆlter through a membrane ˆlter with pore size of not more 10 mL of the solution for system suitability test. than 0.45 mm. Discard the ˆrst 10 mL of the ˆltrate, pipet System performance: To 5 mL of the solution for system the subsequent V mL of the ˆltrate, add diluted phosphoric suitability test add 5 mL of water, and warm in a water bath acid (1 in 50) to make exactly V? mL so that each mL con- of 609C for 30 minutes. When the procedure is run with 10 tains about 1.1 mg of trichlormethiazide (C H Cl N O S ) mL of this solution, after cooling, under the above operating 8 8 3 3 4 2 accordingtothelabeledamount,andusethissolutionasthe conditions, 4-amino-6-chlorobenzene-1,3-disulfonamide sample solution. Separately, weigh accurately about 22 mg and trichlormethiazide are eluted in this order, the relative of Trichlormethiazide Reference Standard, previously dried retention time of 4-amino-6-chlorobenzene-1,3-disul- at 1059C for 3 hours, and dissolve in acetonitrile to make ex- fonamide with respect to trichlormethiazide is about 0.3, actly 200 mL. Pipet 2 mL of this solution, add diluted phos- and the number of theoretical plates and the symmetry fac- phoric acid (1 in 50) to make exactly 200 mL, and use this so- tor of the peak of trichlormethiazide are not less than 5000 lution as the standard solution. Perform the test with exactly and not more than 1.2, respectively. 40 mL each of the sample solution and the standard solution System repeatability: When the test is repeated 3 times as directed under the Liquid Chromatography according to with 10 mL of the solution for system suitability test under the following conditions, and determine the peak areas, A the above operating conditions, the relative standard devia- Ta and A , of trichlormethiazide obtained with the sample so- tion of the peak area of trichlormethiazide is not more than Sa lution and the standard solution, and the area, A ,ofthe 2.0z. Tb peak, having the relative retention time of about 0.3 with Content uniformity Perform the test according to the fol- respect to trichlormethiazide, obtained with the sample solu- lowing method: it meets the requirements of the Content tion. The dissolution rate in 15 minutes is not less than 75z. Uniformity Test. To one tablet of Trichlormethiazide Dissolution rate (z) with respect to the labeled amount of Tablets add 5 mL of diluted phosphoric acid (1 in 50) to dis- trichlormethiazide (C H Cl N O S ) integrate. Add exactly an amount of the internal standard 8 8 3 3 4 2 solution, equivalent to 10 mL per 2 mg of trichlormethiazide Supplement II, JPXIV O‹cial Monographs for Part I 1751

ATa＋ATb×0.95 V? 1 9 Operating conditions— ＝WS× × × × ASa V C 2 Detector: An ultraviolet absorption photometer (wavelength: 268 nm). W : Amount (mg) of Trichlormethiazide Reference Stan- S Column: A stainless steel column 4.6 mm in inside dard diameter and 15 cm in length, packed with phenylsilanized C: Labeled amount (mg) of trichlormethiazide silica gel for liquid chromatography (5 mminparticle (C H Cl N O S ) per tablet 8 8 3 3 4 2 diameter). Operating conditions— Column temperature: A constant temperature of about Proceed as directed in the operating conditions in the 259C. Assay. Mobile phase: A mixture of diluted phosphoric acid (1 in System suitability— 1000) and acetonitrile (3:1). System performance: Dissolve 25 mg of Trichlor- Flow rate: Adjust the ‰ow rate so that the retention time methiazide in 50 mL of acetonitrile. To 1 mL of this solution of trichlormethiazide is about 8 minutes. add acetonitrile to make 50 mL. To 5 mL of this solution System suitability— add 5 mL of water, and heat at 609C in a water bath for 30 System performance: When the procedure is run with minutes. After cooling, when the procedure is run with 20 mL of the standard solution under the above operating 10 mL of this solution under the above operating conditions, conditions, the internal standard and trichlormethiazide are 4-amino-6-chlorobenzene-1,3-disulfonamide and trichlor- eluted in this order with the resolution between these peaks methiazide are eluted in this order, the relative retention being not less than 2.0. time of 4-amino-6-chlorobenzene-1,3-disulfonamide with System repeatability: When the test is repeated 6 times respect to trichlormethiazide is about 0.3, and the number of with 20 mL of the standard solution under the above operat- theoretical plates and the symmetry factor of the peak of ing conditions, the relative standard deviation of the ratio of trichlormethiazide are not less than 5000 and not more than the peak area of trichlormethiazide to that of the internal 1.2, respectively. standard is not more than 1.0z. System repeatability: When the test is repeated 6 times Containers and storage Containers—Tight containers. with 40 mL of the standard solution under the above operat- ing conditions, the relative standard deviation of the peak area of trichlormethiazide is not more than 2.0z. Vasopressin Injection Assay Weigh accurately the mass of not less than 20 Trichlormethiazide Tablets, and pulverize the tablets in an バソプレシン注射液 agate mortar. Weigh accurately an amount of the powder, equivalent to about 2 mg of trichlormethiazide Change the paragraph (i) under Purity to read: (C H Cl N O S ) according to the labeled amount, add 5 mL 8 8 3 3 4 2 Purity ofdilutedphosphoricacid(1in50)andexactly10mLofthe (i) Standard stock solution: Dissolve 200 oxytocin Units internal standard solution, add 10 mL of acetonitrile, shake of Posterior Pituitary Reference Standard, according to the vigorously for 15 minutes, and centrifuge. To 2 mL of the labeled Units, in exactly 10 mL of diluted acetic acid (100) supernatant liquid add 2 mL of the mobile phase, and use (1 in 400). Pipet 1 mL of this solution, and add diluted acetic this solution as the sample solution. Separately, weigh ac- acid (100) (1 in 400) to make exactly 10 mL. Store in a cold curately about 40 mg of Trichlormethiazide Reference Stan- place, avoiding freezing. Use within 6 months from the date dard, previously dried at 1059C for 3 hours, and dissolve in of preparation. acetonitrile to make exactly 200 mL. Pipet 5 mL of this solu- tion, add exactly 5 mL of the internal standard solution, add Change the paragraph (ii) under Assay to read: 10 mL of acetonitrile and 5 mL of diluted phosphoric acid (1 in 50), and use this solution as the standard solution. Per- Assay form the test with 20 mL each of the sample solution and the (ii) Standard stock solution: Dissolve 2000 vasopressin standard solution as directed under the Liquid Chro- Units of Posterior Pituitary Reference Standard, according matography according to the following conditions, and de- to the labeled Units, in exactly 100 mL of diluted acetic acid termine the ratios, QT and QS, of the peak area of trichlor- (100) (1 in 400). Pipet 1 mL of this solution, and add diluted methiazide to that of the internal standard. acetic acid (100) (1 in 400) to make exactly 10 mL. Store in a cold place, avoiding freezing. Use within 6 months from the Amount (mg) of trichlormethiazide (C H Cl N O S ) 8 8 3 3 4 2 date of preparation. QT 1 ＝WS× × QS 20

WS: Amount (mg) of Trichlormethiazide Reference Standard

Internal standard solution—A solution of 3-nitrophenol in acetonitrile (1 in 5000). 1752 O‹cial Monographs for Part I Supplement II, JPXIV

System suitability— Vinblastine Sulfate Test for required detectability: To exactly 2.5 mL of the standard solution add water to make exactly 100 mL. 硫酸ビンブラスチン Conˆrm that the peak area of vinblastine obtained from 200 mL of this solution is equivalent to 1.7 to 3.3z of that Change to read except the structural formula, from 200 mL of the standard solution. chemical name and origin/limits of content: System performance: Proceed as directed in the system suitability in the Assay. Description Vinblastine Sulfate occurs as a white to pale System repeatability: When the test is repeated 5 times yellow powder. with 200 mL of the standard solution under the above It is soluble in water, sparingly soluble in methanol, and operating conditions, the relative standard deviation of the practically insoluble in ethanol (99.5). peak area of vinblastine is not more than 1.5z. It is hygroscopic. 20 Optical rotation [a]D : －28 – －359(0.20 g calculated on Loss on drying Perform the test with about 10 mg of the dried basis, methanol, 10 mL, 100 mm). Vinblastine Sulfate as directed in Method 2 under the Ther- mal Analysis according to the following conditions: not Identiˆcation (1) Determine the absorption spectrum of more than 15.0z. a solution of Vinblastine Sulfate (1 in 50,000) as directed Operating conditions— under the Ultraviolet-visible Spectrophotometry, and com- Heating rate: 59C/minute pare the spectrum with the Reference Spectrum or the Temperature range: room temperature to 2009C spectrum of a solution of Vinblastine Sulfate Reference Atmospheric gas: dried Nitrogen Standard prepared in the same manner as the sample solu- Flow rate of atmospheric gas: 40 mL/minute tion: both spectra exhibit similar intensities of absorption at thesamewavelengths. Assay Weigh accurately about 10 mg each of Vinblastine (2) Determine the infrared absorption spectrum of Sulfate and Vinblastine Sulfate Reference Standard Vinblastine Sulfate as directed in the potassium bromide (separately determine its loss on drying), dissolve in water to disk method under the Infrared Spectrophotometry, and make exactly 25 mL, and use these solutions as the sample compare the spectrum with the Reference Spectrum or the solution and the standard solution, respectively. Perform spectrum of Vinblastine Sulfate Reference Standard: both the test with exactly 20 mL each of the sample solution and spectra exhibit similar intensities of absorption at the same the standard solution as directed under the Liquid Chro- wave numbers. matography according to the following conditions, and de-

(3) A solution of Vinblastine Sulfate (1 in 100) responds termine the peak areas, AT and AS,ofvinblastine. to the Qualitative Tests for sulfate. AT Amount (mg) of C46H58N4O9.H2SO4＝WS× pH Dissolve 15 mg of Vinblastine Sulfate in 10 mL of AS water: the pH of this solution is between 3.5 and 5.0. WS: Amount (mg) of Vinblastine Sulfate Reference Purity (1) Clarity and color of solution—Dissolve 50 mg Standard,calculatedonthedriedbasis of Vinblastine Sulfate in 10 mL of water: the solution is clear Operating conditions— and colorless. Detector: An ultraviolet absorption photometer (2) Related substances—Dissolve about 4 mg of Vin- (wavelength: 262 nm). blastine Sulfate in 10 mL of water, and use this solution as Column: A stainless steel column 4.6 mm in inside the sample solution. Pipet 1 mL of the sample solution, add diameter and 15 cm in length, packed with octadecyl- water to make exactly 25 mL, and use this solution as the silanized silica gel for liquid chromatography (5 mminparti- standard solution. Perform the test with exactly 200 mLeach cle diameter). of the sample solution and the standard solution as directed Column temperature: A constant temperature of about under the Liquid Chromatography according to the follow- 259C. ing conditions, and determine each peak area of these solu- Mobile phase: To 7 mL of diethylamine add water to tions by the automatic integration method: the area of peak make 500 mL, and adjust the pH to 7.5 with phosphoric other than the main peak is not larger than 1/4 of the peak acid. To 380 mL of this solution add 620 mL of a mixture of area of vinblastine from the standard solution, and the total methanol and acetonitrile (4:1). area of the peaks other than the main peak is not larger than Flow rate: Adjust the ‰ow rate so that the retention time 3/4 of the peak area of vinblastine from the standard of vinblastine is about 8 minutes. solution. System suitability— Operating conditions— System performance: Dissolve 10 mg each of Vinblastine Detector, column, column temperature, mobile phase, Sulfate and vincristine sulfate in 25 mL of water. When the and ‰ow rate: Proceed as directed in the operating condi- procedure is run with 20 mL of this solution under the above tions in the Assay. operating conditions, vincristine and vinblastine are eluted Time span of measurement: About 4 times as long as the in this order with the resolution between these peaks being retention time of vinblastine after the solvent peak. Supplement II, JPXIV O‹cial Monographs for Part I 1753 not less than 4. lowing method: it meets the requirements of the Content System repeatability: When the test is repeated 5 times Uniformity Test. Open a container of Vinblastine Sulfate with 20 mL of the standard solution under the above operat- for Injection, add an exact amount of water to make a solu- ing conditions, the relative standard deviation of the peak tion so that each mL contains about 0.4 mg of vinblastine area of vinblastine is not more than 1.0z. sulfate (C46H58N4O9.H2SO4) according to the labeled amount, and use this solution as the sample solution. Containers and storage Containers—Tight containers. Separately, weigh accurately about 10 mg of Vinblastine Storage—Light-resistant, at not exceeding －209C. Sulfate Reference Standard (separately determine its loss on drying in the same manner as for Vinblastine Sulfate), dis- solve in water to make exactly 25 mL, and use this solution Change to read: as the standard solution. Proceed as directed in the Assay Vinblastine Sulfate for Injection under Vinblastine Sulfate. Amount (mg) of vinblastine sulfate (C46H58N4O9.H2SO4)

注射用硫酸ビンブラスチン AT ＝WS× AS Vinblastine Sulfate for Injection is a preparation for injection, which is dissolved before use. It contains WS: Amount (mg) of Vinblastine Sulfate Reference Standard,calculatedonthedriedbasis not less than 90.0z and not more than 110.0z of the labeled amount of vinblastine sulfate Foreign insoluble matter Perform the test according to (C46H58N4O9.H2SO4: 909.05). Method 2: it meets the requirements of the Foreign Insoluble Matter Test for Injections. Method of preparation Prepare as directed under Injec- tions, with Vinblastine Sulfate. Insoluble particulate matter Perform the test according to Method 1: it meets the requirements of the Insoluble Par- Description Vinblastine Sulfate for Injection occurs as ticulate Matter Test for Injections. white to pale yellow, light masses or powder. It is freely soluble in water. Sterility Perform the test according to the Membrane The pH of a solution (1 in 1000) is 3.5 – 5.0. ˆltration method: it meets the requirements of the Sterility Test. Identiˆcation Proceed as directed in the Identiˆcation (1) under Vinblastine Sulfate. Assay Take an amount of Vinblastine Sulfate for Injec- tion, equivalent to 0.10 g of vinblastine sulfate Purity Related substances—Dissolve 4 mg of Vinblastine (C H N O .H SO ), dissolve each content with a suitable Sulfate for Injection in 10 mL of water, and use this solution 46 58 4 9 2 4 amount of water, transfer into a 100-mL volumetric ‰ask, as the sample solution. Pipet 1 mL of the sample solution, wash each container with water, transfer the washings into add water to make exactly 25 mL, and use this solution as the volumetric ‰ask, and add water to make exactly 100 mL. the standard solution. Perform the test with exactly 200 mL Pipet 10 mL of this solution, add water to make exactly 25 each of the sample solution and the standard solution as mL, and use this solution as the sample solution. Separately, directed under the Liquid Chromatography according to the weigh accurately about 10 mg of Vinblastine Sulfate Refer- following conditions, and determine each peak area by the ence Standard (separately determine its loss on drying in the automatic integration method: the area of the peak other same manner as for Vinblastine Sulfate), dissolve in water to than the main peak from the sample solution is not larger make exactly 25 mL, and use this solution as the standard than 1/2 of the peak area of vinblastine from the standard solution. Proceed as directed in the Assay under Vinblastine solution, and the total area of the peaks other than the main Sulfate. peak is not larger than 2 times the peak area of vinblastine from the standard solution. Amount (mg) of vinblastine sulfate (C46H58N4O9.H2SO4)

Operating conditions— AT ＝WS× Perform as directed in the operating conditions in Purity AS (2) under Vinblastine Sulfate. W : Amount (mg) of Vinblastine Sulfate Reference System suitability— S Standard,calculatedonthedriedbasis Perform as directed in the system suitability in Purity (2) under Vinblastine Sulfate. Containers and storage Containers—Hermetic containers, and colored containers may be used. Bacterial endotoxins Less than 10 EU/mg. Storage—Light-resistant, at 2 to 89C. Content uniformity Perform the test according to the fol- General Rules for Crude Drugs

Change the paragraph 1 to read: dron Bark, Picrasma Wood, Pinellia Tuber, Plantago Herb, Plantago Seed, Platycodon Root, Polygala 1. Crude drugs in the monographs include medici- Root, Polygonum Root, Powdered Acacia, Powdered nal parts obtained from plants or animals, cell inclu- Alisma Rhizome, Powdered Aloe, Powdered sions and secretes separated from the origins, their Amomum Seed, Powdered Atractylodes Lancea extracts, and minerals. General Rules for Crude Drugs Rhizome, Powdered Atractylodes Rhizome, Pow- and the Crude Drugs in General Tests, Processes and dered Calumba, Powdered Capsicum, Powdered Apparatus are applicable to the followings: Cinnamon Bark, Powdered Clove, Powdered Cnidi- Acacia, Achyranthes Root, Agar, Agar Powder, um Rhizome, Powdered Coix Seed, Powdered Coptis Akebia Stem, Alisma Rhizome, Aloe, Alpinia Rhizome, Powdered Cyperus Rhizome, Powdered O‹cinarum Rhizome, Amomum Seed, Anemarrhena Dioscorea Rhizome, Powdered Fennel, Powdered Rhizome, Angelica Dahurica Root, Apricot Kernel, Gambir, Powdered Gardenia Fruit, Powdered Genti- Areca, Artemisia Capillaris Flower, Asiasarum Root, an, Powdered Geranium Herb, Powdered Ginger, Asparagus Tuber, Astragalus Root, Atractylodes Powdered Ginseng, Powdered Glycyrrhiza, Powdered Lancea Rhizome, Atractylodes Rhizome, Bear Bile, Hoelen, Powdered Ipecac, Powdered Japanese Angel- Bearberry Leaf, Belladonna Root, Benzoin, Bitter ica Root, Powdered Japanese Gentian, Powdered Cardamon, Bitter Orange Peel, Bupleurum Root, Japanese Valerian, Powdered Magnolia Bark, Pow- Burdock Fruit, Calumba, Capsicum, Cardamon, dered Moutan Bark, Powdered Oyster Shell, Pow- Cassia Seed, Catalpa Fruit, Chrysanthemum Flower, dered Panax Rhizome, Powdered Peach Kernel, Pow- Chuling, Cimicifuga Rhizome, Cinnamon Bark, dered Peony Root, Powdered Phellodendron Bark, Citrus Unshiu Peel, Clematis Root, Clove, Cnidium Powdered Picrasma Wood, Powdered Platycodon Monnieri Fruit, Cnidium Rhizome, Coix Seed, Con- Root, Powdered Polygala Root, Powdered Polypou- durango, Coptis Rhizome, Cornus Fruit, Corydalis rus Sclerotium, Powdered Processed Aconite Root, Tuber, Cyperus Rhizome, Digenea, Dioscorea Rhi- Powdered Rhubarb, Powdered Rose Fruit, Powdered zome, Ephedra Herb, Epimedium Herb, Eucommia Scutellaria Root, Powdered Senega, Powdered Senna Bark, Evodia Fruit, Fennel, Forsythia Fruit, Fritillaria Leaf, Powdered Smilax Rhizome, Powdered Sophora Bulb, Gambir, Gardenia Fruit, Gastrodia Tuber, Root, Powdered Sweet Hydrangea Leaf, Powdered Gentian, Geranium Herb, Ginger, Ginseng, Glehnia Swertia Herb, Powdered Tragacanth, Powdered Root, Glycyrrhiza, Gypsum, Hemp Fruit, Hoelen, Zanthoxylum Fruit, Processed Aconite Root, Proc- Honey, Houttuynia Herb, Immature Orange, essed Ginger, Prunella Spike, Pueraria Root, Red Imperata Rhizome, Ipecac, Japanese Angelica Root, Ginseng, Rehmannia Root, Rhubarb, Rice Starch, Japanese Gentian, Japanese Valerian, Jujube, Jujube Rose Fruit, Rosin, SaŒower, SaŠron, Saposhnikovia Seed, Lindera Root, Lithospermum Root, Longgu, Root, Sappan Wood, Saussurea Root, Schisandra Lonicera Leaf and Stem, Loquat Leaf, Lycium Bark, Fruit, Schizonepeta Spike, Scopolia Rhizome, Scutel- Lycium Fruit, Magnolia Bark, Magnolia Flower, laria Root, Senega, Senna Leaf, Sinomenium Stem, Mallotus Bark, Mentha Herb, Moutan Bark, Smilax Rhizome, Sophora Root, Sweet Hydrangea Mulberry Bark, Notopterygium Rhizome, Nuphar Leaf, Swertia Herb, Termeric, Toad Venom, Rhizome, Nux Vomica, Ophiopogon Tuber, Oriental Tragacanth, Tribulus Fruit, Trichosanthes Root, Bezoar, Oyster Shell, Panax Rhizome, Peach Kernel, Uncaria Thorn, Zanthoxylum Fruit, Zedoary. Peony Root, Perilla Herb, Pharbitis Seed, Phelloden-

1755 O‹cial Monographs for Part II

solution as directed under the Liquid Chromatography Delete the following Monographs: according to the following conditions, and determine the

peak areas of barbaloin, AT and AS, of both solutions. Absorbent Cotton A Amount (mg) of barbaloin＝W × T ×2 S A Puriˆed Absorbent Cotton S WS: Amount (mg) of barbaloin for component determina- Sterile Absorbent Cotton tion Operating conditions— Sterile Puriˆed Absorbent Cotton Detector: An ultraviolet absorption photometer (wavelength: 360 nm). Column: A stainless steel column about 6 mm in inside Absorbent Gauze diameter and about 15 cm in length, packed with octadecyl- silanized silica gel for liquid chromatography (5 mminparti- Sterile Absorbent Gauze cle diameter). Column temperature: A constant temperature of about Adhesive Plaster 309C. Mobile phase: A mixture of water, acetonitrile and acetic acid (100) (74:26:1). Flow rate: Adjust the ‰ow rate so that the retention time Powdered Aloe of barbaloin is about 12 minutes. System suitability— アロエ末 System performance: To about 10 mg of barbaloin for component determination add 40 mg of oxalic acid dihy- Change the origin/limits of content to read: drate, and dissolve in methanol to make exactly 100 mL. Pipet 5 mL of the solution, add 1 mL of a solution of ethen- Powdered Aloe is the powder of Aloe. zamide in methanol (1 in 2000) and methanol to make It contains not less than 4.0z of barbaloin, calcu- exactly 10 mL. When the procedure is run with 5 mLofthis lated on the basis of dried material. solution under the above operating conditions except the wavelength of 300 nm, barbaloin and ethenzamide are Change the Loss on drying to read: eluted in this order with the resolution between these peaks being not less than 2.0. Loss on drying Not more than 12.0z. System repeatability: When the test is repeated 6 times with 5 mL of the standard solution under the above operat- Add the following next to Extract content: ing conditions, the relative standard deviation of the peak Component determination Weigh accurately about 0.1 g area of barbaloin is not more than 1.5z. of Powdered Aloe, add 40 mL of methanol, and heat under a re‰ex condenser on a water bath for 30 minutes. After cooling, ˆlter, and add methanol to the ˆltrate to make Benzyl Alcohol exactly 50 mL. Pipet 5 mL of the solution, add methanol to make exactly 10 mL, and use this solution as the sample ベンジルアルコール solution. Separately, weigh accurately about 10 mg of barbaloin for component determination, previously dried in Change to read except the structural formula a desiccator (in vacuum, phosphorus (V) oxide) for 24 and chemical name: hours, add 40 mg of oxalic acid dihydrate, and dissolve in methanol to make exactly 100 mL. Pipet 5 mL of the Benzyl Alcohol contains not less than 98.0z and solution, add methanol to make exactly 10 mL, and use this not more than 100.5z of C7H8O. solution as the standard solution. Perform the test with The label states, where applicable, that it is suitable exactly 5 mL each of the sample solution and the standard for use in the manufacture of injection forms.

1757 1758 O‹cial Monographs for Part II Supplement II, JPXIV

Description Benzyl Alcohol is a clear, colorless oily liquid. weigh exactly 0.250 g of benzaldehyde and 0.500 g of cyclo- It is miscible with ethanol (95), with fatty oils and with hexylmethanol, and add Benzyl Alcohol to make exactly volatile oils. 25 mL. Pipet 1 mL of this solution, add exactly 2 mL of the It is soluble in water. ethylbenzene internal standard solution and exactly 2 mL of 20 Speciˆc gravity d 20: 1.043 – 1.049 the dicyclohexyl internal standard solution, then add Benzyl Alcohol to make exactly 20 mL, and use this solution as the Identiˆcation Determine the infrared absorption spectrum standard solution (2). Perform the test with 0.1 mLeachof of Benzyl Alcohol as directed in the liquid ˆlm method the sample solution and the standard solution (2) as directed under the Infrared Spectrophotometry, and compare the under the Gas Chromatography according to the following spectrum with the Reference Spectrum: both spectra exhibit conditions: no peaks of ethylbenzene and dicyclohexyl similar intensities of absorption at the same wave numbers. appear on the chromatogram obtained with the sample 20 Refractive index nD : 1.538 – 1.541 solution. When 0.1 mL of the standard solution (2) is inject- ed, adjust the sensitivity of the detector so that the peak Purity (1) Clarity and color of solution—Dissolve 2.0 height of ethylbenzene is not more than 30z of the full scale mL of Benzyl Alcohol in 60 mL of water: the solution is of the recorder. The peak area of benzaldehyde of obtained clear and colorless. with the sample solution is not more than the deference (2) Acid—To 10 mL of Benzyl Alcohol add 10 mL of between the peak areas of benzaldehyde of the sample neutralized ethanol, 2 drops of phenolphthalein TS and 1.0 solution and the standard solution (2) (0.05z), and the peak mL of 0.1 mol/L sodium hydroxide VS: the color of the area of cyclohexylmethanol with the sample solution is not solution is red. more than the deference between the peak areas of cyclohex- (3) Benzaldehyde and other related substances—Use ylmethanol of the sample solution and the standard solution Benzyl Alcohol as the sample solution. Separately, weigh (2) (0.10z). The total area of the peaks having smaller exactly 0.750 g of benzaldehyde and 0.500 g of cyclohex- retention time than benzyl alcohol and other than benzalde- ylmethanol, and add Benzyl Alcohol to make exactly 25 mL. hyde and cyclohexylmethanol obtained with the sample Pipet 1 mL of this solution, add exactly 2 mL of ethylben- solution is not more than 2 times the peak area of ethylben- zene internal standard solution and exactly 3 mL of dicyclo- zene with the standard solution (2) (0.02z). The total area hexyl internal standard solution, then add Benzyl Alcohol to of the peaks having larger retention time than benzaldehyde make exactly 20 mL, and use this solution as the standard obtained with the sample solution is not more than the peak solution (1). Perform the test with 0.1 mLeachofthesample area of cyclohexylmethanol with the standard solution (2) solution and the standard solution (1) as directed under the (0.2z). For these calculation the peak areas less than 1/100 Gas Chromatography according to the following conditions: times the peak area of ethylbenzene with the standard no peaks of ethylbenzene and dicyclohexyl appear on the solution (2) are excluded. chromatogram obtained with the sample solution. When Ethylbenzene internal standard solution: Dissolve exactly 0.1 mL of the standard solution (1) is injected, adjust the 0.100 g of ethylbenzene in Benzyl Alcohol to make exactly sensitivity of the detector so that the peak height of ethyl- 10 mL. Pipet 2 mL of this solution, and add Benzyl Alcohol benzene is not more than 30z of the full scale of the record- to make exactly 20 mL. er. The peak area of benzaldehyde obtained with the sample Dicyclohexyl internal standard solution: Dissolve exactly solution is not more than the deference between the peak 2.000 g of dicyclohexyl in Benzyl Alcohol to make exactly areas of benzaldehyde of the sample solution and the 10 mL. Pipet 2 mL of this solution, and add Benzyl Alcohol standard solution (1) (0.15z), and the peak area of cyclo- to make exactly 20 mL. hexylmethanol with the sample solution is not more than the Operating conditions— deference between the peak areas of cyclohexylmethanol of Detector: A hydrogen ‰ame-ionization detector the sample solution and the standard solution (1) (0.10z). Column: A fused silica column 0.33 mm in inside The total area of the peaks having smaller retention time diameter and 30 m in length, coated inside with polyethylene than benzyl alcohol and other than benzaldehyde and glycol 20M for gas chromatography in 0.5 mm thickness. cyclohexylmethanol obtained with the sample solution is not Column temperature: Raise the temperature at a rate of more than 4 times the peak area of ethylbenzene with the 59C per minutes from 509Cto2209C, and maintain at standard solution (1) (0.04z).Thetotalareaofthepeaks 2209Cfor35minutes. having larger retention time than benzaldehyde obtained Temperature of injection port: A constant temperature of with the sample solution is not more than the peak area of about 2009C. cyclohexylmethanol with the standard solution (1) (0.3z). Temperature of detector: A constant temperature of For these calculations the peak areas less than 1/100 times about 3109C. the peak area of ethylbenzene with the standard solution (1) Carrier gas: Helium are excluded. Flow rate: Adjust the ‰ow rate so that the retention time Benzyl Alcohol labeled that it is suitable for use in the of benzyl alcohol is between 24 and 28 minutes. manufacture of injection forms meets the following require- Split ratio: Splitless ments. System suitability— Use Benzyl Alcohol as the sample solution. Separately, System performance: When the procedure is run with the Supplement II, JPXIV O‹cial Monographs for Part II 1759 standard solution (1) under the above operating conditions, It swells with water to form a suspension. the relative retention times of ethylbenzene, dicyclohexyl, The pH of a suspension, obtained by shaking 1.0 g of benzaldehyde and cyclohexylmethanol with respect to benzyl Carmellose Calcium with 100 mL of water, is between 4.5 alcohol are about 0.28, about 0.59, about 0.68 and about and 6.0. 0.71, respectively, and the resolution between the peaks of It is hygroscopic. benzaldehyde and cyclohexylmethanol is not less than 3.0. In the case of Benzyl Alcohol labeled to use for injection, Change the Identiˆcation (1) and (4) to read: proceed with the standard solution (2) instead of the Identiˆcation (1) Shake thoroughly 0.1 g of Carmellose standard solution (1). Calcium with 10 mL of water, followed by 2 mL of sodium (4) Peroxide value—Dissolve 5 g of Benzyl Alcohol in hydroxide TS, allow to stand for 10 minutes, and use this 30 mL of a mixture of acetic acid (100) and chloroform (3:2) solution as the sample solution. To 1 mL of the sample in a 250-mL glass-stoppered conical ‰ask. Add 0.5 mL of solution add water to make 5 mL. To 1 drop of this solution potassium iodide saturated solution, shake exactly for 1 add 0.5 mL of chromotropic acid TS, and heat in a water minute, add 30 mL of water, and titrate with 0.01 mol/L bath for 10 minutes: a red-purple color develops. sodium thiosulfate VS until the blue color of the solution (4) Ignite 1 g of Carmellose Calcium to ash, dissolve the disappears after addition of 10 mL of starch TS near the end residue in 10 mL of water and 6 mL of acetic acid (31), and point where the solution is a pale yellow color. Perform a ˆlter, if necessary. Boil the ˆltrate, cool, and neutralize with blank determination in the same manner. Calculate the ammonia TS: the solution responds to the Qualitative Tests amount of peroxide by the following formula: not more (1)and(3)forcalciumsalt. than 5.

(V －V ) Change the Purity to read: Amount (mEq/kg) of peroxide＝ 1 0 ×10 W Purity (1) Alkali—Shake thoroughly 1.0 g of Carmellose

V1: Volume (mL) of 0.01 mol/L sodium thiosulfate VS Calcium with 50 mL of freshly boiled and cooled water, and consumed in the test add 2 drops of phenolphthalein TS: no red color develops.

V0: Volume (mL) of 0.01 mol/L sodium thiosulfate VS (2) Chloride—Shake thoroughly 0.80 g of Carmellose consumed in the blank Calcium with 50 mL of water, add 10 mL of sodium W: Amount (g) of the sample hydroxide TS to dissolved, add water to make 100 mL, and use this solution as the sample solution. Heat 20 mL of the (5) Residue on evaporation—Perform the test after sample solution with 10 mL of 2 mol/L nitric acid on a conformation that the sample meets the requirement of the water bath until a ‰occulent precipitate is produced. After peroxide value. Evaporate to dryness 10.0 g of Benzyl cooling, centrifuge, and take out the supernatant liquid. Alcohol on a water bath, dry the residue at 1059Cfor1 Wash the precipitate with three 10-mL portions of water by hour, and allow to cool in a desiccator: not more than 5 mg. centrifuging each time, combine the supernatant and the Assay Weigh accurately about 0.9 g of Benzyl Alcohol, washings, and add water to make 100 mL. Take 25 mL of add exactly 15.0 mL of a mixture of pyridine and acetic this solution, and add 1 mL of nitric acid and water to make anhydride (7:1), and heat on a water bath under a re‰ux 50 mL. Perform the test using this solution as the test condenser for 30 minutes. Cool, add 25 mL of water, and solution. Prepare the control solution with 0.40 mL of 0.01 titrate the excess acetic acid with 1 mol/L sodium hydroxide mol/L hydrochloric acid VS (not more than 0.36z). VS (indicator: 2 drops of phenolphthalein TS). Perform a (3) Sulfate—Heat 10 mL of the sample solution ob- blank determination. tained in (2) with 1 mL of hydrochloric acid in a water bath until a ‰occulent precipitate is produced. Cool, centrifuge, Each mL of 1 mol/L sodium hydroxide VS and take out the supernatant liquid. Wash the precipitate ＝108.1 mg of C H O 7 8 with three 10-mL portions of water by centrifuging each Containers and storage Containers—Tight containers. time, combine the supernatant and the washings, and add Storage—Light-resistant. water to make 100 mL. Perform the test with 25 mL this solution as the test solution. Prepare the control solution with 0.42 mL of 0.005 mol/L sulfuric acid VS. To the test Carmellose Calcium solution and the control solution add 1 mL of 3 mol/L hydrochloric acid and 3 mL of barium chloride TS, then add カルメロースカルシウム water to make 50 mL, and mix. Allow to stand for 10 minutes, and compare the turbidity of these solutions: the Change the Description to read: turbidity obtained with the test solution is not more than that obtained with the control solution (not more than Description Carmellose Calcium occurs as a white to 1.0z). yellowish white powder. (4) Heavy metals—Proceed with 1.0 g of Carmellose It is practically insoluble in ethanol (95) and in diethyl Calcium according to Method 2, and perform the test. ether. Prepare the control solution with 2.0 mL of Standard Lead 1760 O‹cial Monographs for Part II Supplement II, JPXIV

Solution (not more than 20 ppm). Content (z) of free acids and bound acetyl group (C2H3O) A ＝ ×0.4304 Change the Residue on ignition to read: W Residue on ignition 10.0 – 20.0z (after drying, 1 g). A: Amount (mL) of 0.1 mol/L sodium hydroxide con- sumed, corrected by the blank determination W: Amount (g) of the test sample, calculated on the Cellulose Acetate Phthalate anhydrous basis Content (z)ofacetylgroup(CH O) 酢酸フタル酸セルロース 2 3 P－0.5182×B ＝ ×100－0.5772×C 100－B Add the following commonly used name: B:Amount(z) of free acids obtained in Purity (2) Free Cellacefate acids C: Content (z) of carboxybenzoyl group Change the origin/limits of the content to read: P: Content (z) of free acids and bound acetyl group

(C2H3O) Cellulose Acetate Phthalate is a reaction product of phthalic anhydride and partially acetylated cellulose. Cellulose Acetate Phthalate, calculated on the Chrysanthemum Flower anhydrous and free acid-free basis, contains not less than 21.5z and not more than 26.0z of acetyl group キクカ (-COCH3: 43.04), and not less than 30.0z and not more than 36.0z of carboxybenzoyl group Change the Extract content to read: (-COC H COOH: 149.12). 6 4 Extract content Dilute ethanol-extract: not less than 30.0z. Change the Description to read: Description Cellulose Acetate Phthalate occurs as a white powder or grain. Add the following: It is freely soluble in acetone, and practically insoluble in water, in methanol and in ethanol (99.5). Cnidium Monnieri Fruit

Change the Identiˆcation to read: Cnidii Monnieris Fructus Identiˆcation Determine the infrared absorption spectrum ジャショウシ of Cellulose Acetate Phthalate as directed in the potassium bromide disk method under the Infrared Spectrophotomet- Cnidium Monnieri Fruit is the fruit of Cnidium ry, and compare the spectrum with the Reference Spectrum monnieri Cusson (Umbelliferae). or spectrum of Cellulose Acetate Phthalate Reference Standard: both spectra exhibit similar intensities of absorp- Description Elliptical cremocarp, often each mericarp tion at the same wave numbers. separated;2–3mminlength,1–2mminwidth;externally light brown to brown, each mericarp usually with ˆve Change the Water to read: winged longitudinal ridges; inner surface of mericarp almost ‰at. Water Not more than 5.0z (1 g, volumetric titration, Odor, characteristic; it gives characteristic aroma, later a direct titration, using a mixture of ethanol (99.5) and slight sensation of numbness on chewing. dichloromethane (3:2) instead of methanol for Karl Fischer Under a microscope, a transverse section reveals one oil method). canal between longitudinal ridges, usually two oil canals in the inner part of mericarp facing to gynophore; longitudinal Change the Assay (2) to read: ridges composed of slightly ligniˆed parenchymatous cells, Assay with vascular bundles in the base; epidermal cells and (2) Acetyl group—Weigh accurately about 0.1 g of parenchymatous cells of longitudinal ridges contain solitary Cellulose Acetate Phthalate, put in a glass-stoppered conical crystals of calcium oxalate; parenchymatous cells of ‰ask, add exactly 25 mL of 0.1 mol/L sodium hydroxide albumen contain oil drops and aleurone grains, and oc- VS, and boil for 30 minutes under a re‰ux condenser. After casionally starch grains. cooling, add 5 drops of phenolphthalein TS, and titrate with Identiˆcation To 1 g of pulverized Cnidium Monnieri 0.1 mol/L hydrochloric acid VS. Perform a blank determi- Fruit add 10 mL of ethyl acetate, shake for 10 minutes, nation. ˆlter, and use the ˆltrate as the sample solution. Separately, Supplement II, JPXIV O‹cial Monographs for Part II 1761 dissolve 1 mg of osthole for thin-layer chromatography in add water to make 20 mL, and use as the control solution. 2 mL of methanol, and use this solution as the standard Put 10 mL each of the test solution and the control solution solution. Perform the test with these solutions as directed in test tubes, add 2 mL of a solution of citric acid (2 in 10) under the Thin-layer Chromatography. Spot 5 mLeachof and 0.1 mL of mercapto acetic acid, and mix. Alkalize with the sample solution and the standard solution on a plate of ammonia solution (28) to litmus paper, add water to make silica gel for thin-layer chromatography, develop the plate 20 mL, and mix. Transfer 10 mL each of these solutions into with a mixture of hexane and ethyl acetate (2:1) to a distance test tubes, allow to stand for 5 minutes, and compare the of about 10 cm, and air-dry the plate. Examine under ultrav- color of these solutions against a white background: the iolet light (main wavelength: 365 nm): one of the spot among color of the test solution is not darker than that of the the several spots from the sample solution has the same color control solution (not more than 10 ppm). tone and the Rf value with the blue-white ‰uorescent spot (2) Oxidizingsubstances—To4.0gofCornStarchadd from the standard solution. 50.0 mL of water, shake for 5 minutes, and centrifuge. To 30.0 mL of the supernatant liquid add 1 mL of acetic acid Loss on drying Not more than 12.0z (6 hours). (100) and 0.5 to 1.0 g of potassium iodide, shake, and allow Total ash Not more than 17.0z. to stand for 25 to 30 minutes at a dark place. Add 1 mL of starch TS, and titrate with 0.002 mol/L sodium thiosulfate Acid-insoluble ash Not more than 6.0z. VS until the color of the solution disappears. Perform a Extract content Not less than 8.0z (dilute ethanol-soluble blank determination and make any necessary correction: the extract). volume of 0.002 mol/L sodium thiosulfate VS consumed is not more than 1.4 mL (not more than 20 ppm, calculated as hydrogen peroxide). Change to read: (3) Sulfur dioxide— (i) Apparatus Use as shown in the following ˆgure. Corn Starch

Amylum Maydis トウモロコシデンプン

Corn Starch consists of starch granules derived from the ripen seeds of Zea mays Linnáe (Gramineae). Description Corn Starch occurs as white to pale yellowish white masses or powder. It is practically insoluble in water and in ethanol (99.5).

Identiˆcation (1) Under a microscope, Corn Starch, preserved in a mixture of water and glycerin (1:1), appears as irregularly polygonal simple grains usually 2 – 23 mmin diameter, or irregularly orbicular or spherical simple grains 25–35mm in diameter; hilum appears as distinct cave or 2 – 5 radial clefts; concentric striation absent; a black cross, its The ˆgures are in mm. intersection point on hilum, is observed when grains are put between two polarizing prisms ˆxed at right angle to each A: Boiling ‰ask (500 mL) other. B: Funnel (100 mL) (2) To 1 g of Corn Starch add 50 mL of water, boil for 1 C: Condenser minute, and allow to cool: a subtle white-turbid, pasty liquid D: Test-tube is formed. E: Tap (3) To 10 mL of the pasty liquid obtained in (2) add 0.04 mL of iodine TS: a orange-red to dark blue-purple (ii) Procedure Introduce 150 mL of water into the color is formed, and the color disappears by heating. boiling ‰ask, close the tap of the funnel, and pass carbon dioxide through the whole system at a rate of 100±5mLper pH Put5.0gofCornStarchinanon-metalvessel,add minute. Pass cooling water through the condenser, and place 25.0 mL of freshly boiled and cooled water, mix gently for 1 10 mL of hydrogen peroxide-sodium hydroxide TS in the minute, and allow to stand for 15 minutes: the pH of the test-tube. After 15 minutes, remove the funnel without solution is between 4.0 and 7.0. interrupting the stream of carbon dioxide, and introduce Purity (1) Iron—To 1.5 g of Corn Starch add 15 mL of 2 through the opening into the ‰ask about 25 g of Corn mol/L hydrochloric acid TS, mix, ˆlter, and use the ˆltrate Starch, accurately weighed, with the aid of 100 mL of water. as the test solution. To 2.0 mL of Standard Iron Solution Apply tap grease to the outside of the connection part of the 1762 O‹cial Monographs for Part II Supplement II, JPXIV funnel, and load the funnel. Close the tap of the funnel, Delete the following Monographs: pour 80 mL of 2 mol/L hydrochloric acid TS into the funnel, open the tap to introduce the hydrochloric acid into Digitalis the ‰ask, and close the tap while several mL of the hydrochloric acid remains, in order to avoid losing sulfur Powdered Digitalis dioxide. Place the ‰ask in a water bath, and heat the mixture for 1 hour. Transfer the contents of the test-tube with the aid of a little water to a wide-necked conical ‰ask. Heat in a waterbathfor15minutes,andcool.Add0.1mLof Add the following: bromophenol blue TS, and titrate with 0.1 mol/L sodium hydroxide VS until the color changes from yellow to violet- Epimedium Herb blue lasting for at least 20 seconds. Perform a blank determi- nation and make any necessary correction. Calculate the Epimedii Herba amount of sulfur dioxide by applying the following formula: インヨウカク it is not more than 50 ppm. V Epimedium Herb is the terrestrial part of Epimedi- Amount (ppm) of sulfur dioxide＝ ×1000×3.203 W um pubescens Maximowicz, Epimedium brevicornum Maximowicz, Epimedium wushanense T. S. Ying, W: Amount (g) of the sample Epimedium sagittatum Maximowicz, Epimedium V: Amount (mL) of 0.1 mol/L sodium hydroxide VS Nakai, Morren consumed koreanum Epimedium grandi‰orum var. thunbergianum Nakai or Epimedium sempervi- Loss on drying Not more than 15.0z (1 g, 1309C, 90 rens Nakai (Berberidaceae). minutes). Description Epimedium Herb is composed of a stem and a Reisdue on ignition Not more than 0.6z (1 g). ternate to triternate compound leaf; lea‰et ovate to broadly ovateorovate-lanceolate,3–20cminlength,2–8cmin Containers and storage Containers—Well-closed contain- width, petiolule 15 – 70 mm in length, apex of lea‰et ers. acuminate, needle hair on margin 0.1 – 0.2 cm in length, base of lea‰et cordate to deeply cordate, lateral lea‰et asym- metry; upper surface green to greenish brown, sometimes Cornus Fruit lustrous, lower surface light green, often pilose, especially サンシュユ on vein densely pilose, papery or coriaceous; petiole and stem cylindrical, light yellowish brown to slightly purplish Change the Identiˆcation to read: and light greenish brown, easily broken. Odor, slight; taste, slightly bitter. Identiˆcation To 1 g of coarse cuttings of Cornus Fruit Under a microscope, a transverse section of the leaf add 10 mL of methanol, shake for 5 minutes, ˆlter, and use reveals 3 – 6 vascular bundles in midvein; mesophyll the ˆltrate as the sample solution. Separately, dissolve 1 mg composed of upper epidermis, single-layered palisade, of loganin for thin-layer chromatography in 2 mL of spongy tissue and lower epidermis; leaf margins orbicular or methanol, and use this solution as the standard solution. oblong, sclerenchymatous; multi-cellular hairs on epidermis; Perform the test with these solutions as directed under the 8 – 20 vascular bundles in petiole and 6 – 15 vascular bundles Thin-layer Chromatography. Spot 10 mLeachofthesample in petiolule. Under a microscope, a transverse section of the solution and the standard solution on a plate of silica gel for stem reveals a single to several-layered hypodermis, cortex thin-layer chromatography. Develop with a mixture of ethyl of 4 – 10 layers of sclerenchymatous cells, vascular bundle acetate, water and formic acid (6:1:1) to a distance of about 13 – 30 in number, oblong to obovate. 10 cm, and air-dry the plate. Spray evenly 4-methoxybenzal- dehyde-sulfuric acid TS on the plate, and heat at 1059Cfor5 Identiˆcation To 2 g of pulverized Epimedium Herb add minutes: one of the spots from the sample solution is the 20 mL of methanol, shake for 15 minutes, ˆlter, and use the same with a red-purple spot from the standard solution in ˆltrate as the sample solution. Separately, dissolve 1 mg of color tone and Rfvalue. icariin for thin-layer chromatography in 1 mL of methanol, and use this solution as the standard solution. Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 mL each of the sample solution and the standard solution on a plate of silica gel with ‰uores- cent indicator for thin-layer chromatography. Develop the plate with a mixture of ethyl acetate, ethanol (99.5) and water (8:2:1) to a distance of about 10 cm, and air-dry the plate. Examine under ultraviolet light (main wavelength: 254 Supplement II, JPXIV O‹cial Monographs for Part II 1763 nm): one spot among the spots from the sample solution and Chromatography according to the following conditions, and a dark purple spot from the standard solution show the same measure the peak areas of geniposide, AT and AS,ofboth color tone and the same Rfvalue. solutions.

Loss on drying Not more than 12.5z (6 hours). A Amount (mg) of geniposide＝W × T ×2 S A Total ash Not more than 8.5z. S W : Amount (mg) of geniposide for component determi- Acid-insoluble ash Not more than 2.0z. S nation Extract content Not less than 17.0z (dilute ethanol-solu- Operating conditions— ble extract). Detector: An ultraviolet absorption photometer (wavelength: 240 nm). Column: A stainless steel column 6 mm in inside diameter Powdered Gardenia Fruit and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 mminparticlediameter). サンシシ末 Column temperature: A constant temperature of about 309C. Change the origin/limits of content to read: Mobile phase: A mixture of water and acetonitrile (22:3). Flow rate: Adjust the ‰ow rate so that the retention time Powdered Gardenia Fruit is the powder of Gardenia Fruit. of geniposide is about 15 minutes. System suitability— It contains not less than 3.0z of geniposide, calcu- System performance: Dissolve 1 mg each of geniposide lated on the basis of dried material. for component determination and caŠeine in methanol to make 15 mL. When the procedure is run with 10 mLofthis Change the Identiˆcation (1) to read: solution under the above operating conditions, caŠeine and Identiˆcation (1) To 1.0 g of Powdered Gardenia Fruit, geniposide are eluted in this order with the resolution previously dried in a desiccator (silica gel) for 24 hours, add between these peaks being not less than 3.5. 100 mL of hot water, warm the mixture between 609Cand System repeatability: When the test is repeated 6 times 709C for 30 minutes with frequent shaking, and ˆlter after with 10 mL of the standard solution under the above operat- cooling. To 1.0 mL of the ˆltrate add water to make 10 mL: ing conditions, the relative standard deviation of the peak the color of the resulting solution is yellow and is not lighter area of geniposide is not more than 1.5z. than that of the following control solution. Control solution: Dissolve 9.8 mg of carbazochrome sodium sulfonate in water to make exactly 10 mL. Pipet 1 Ginger mL of this solution, and add water to make exactly 50 mL. ショウキョウ Add the following next to Identiˆcation: Change the Identiˆcation to read: Loss on drying Not more than 13.0z. Identiˆcation To 2 g of pulverized Ginger add 5 mL of AddthefollowingnexttoTotalash: diethyl ether, shake for 10 minutes, ˆlter, and use the ˆltrate as the sample solution. Separately, dissolve 1 mg of [6]-gin- Component determination Weigh accurately about 0.5 g gerol for thin-layer chromatography in 2 mL of methanol, of Powdered Gardenia Fruit, transfer into a glass-stoppered and use this solution as the standard solution. Perform the centrifuge tube, add 40 mL of diluted methanol (1 in 2), test with these solutions as directed under the Thin-layer shake for 15 minutes, centrifuge, and take the supernatant Chromatography. Spot 10 mL of the sample solution and the liquid. To the residue add 40 mL of diluted methanol (1 in standard solution on a plate of silica gel for thin-layer chro- 2), and perform as the same as above. Combine the extracts matography. Develop the plate with a mixture of ethyl soobtained,andadddilutedmethanol(1in2)tomake acetate and hexane (1:1) to a distance of about 10 cm, and exactly 100 mL. Pipet 5 mL of the solution, add methanol to air-dry the plate. Spray evenly 4-dimethylaminobenzalde- make exactly 20 mL, use this solution as the sample hyde TS on the plate, heat at 1059C for 5 minutes, and allow solution. Separately, weigh accurately about 10 mg of to cool: one of the spots from the sample solution and a geniposide for component determination, previously dried green spot from the standard solution show the same color in a desiccator (in vacuum, phosphorus (V) oxide) for 24 tone and Rfvalue. hours, and dissolve in methanol to make exactly 100 mL. Pipet 5 mL of the solution, add methanol to make exactly 10 mL, and use this solution as the standard solution. Perform the test with exactly 10 mL each of the sample solution and the standard solution as directed under the Liquid 1764 O‹cial Monographs for Part II Supplement II, JPXIV

Speciˆc activity When calculate from the results obtained Powdered Ginger by the Assay and the following test, the speciˆc activity is not less than 3000 chorionic gonadotrophin Units per mg ショウキョウ末 protein. (i) Sample solution—To an exactly amount of Chorionic Change the Identiˆcation to read: Gonadotrophin add water to make a solution so that each mL contains about 500 Units of chorionic gonadotrophin Identiˆcation To 2 g of Powdered Ginger add 5 mL of according to the labeled amount. diethyl ether, shake for 10 minutes, ˆlter, and use the ˆltrate (ii) Standard solution—Weigh accurately about 10 mg as the sample solution. Separately, dissolve 1 mg of [6]-gin- of bovine serum albumin, and dissolve in water to make gerol for thin-layer chromatography in 2 mL of methanol, exactly 20 mL. To a suitable volume of this solution add and use this solution as the standard solution. Perform the water to make four solutions containing exactly 300, 200, test with these solutions as directed under the Thin-layer 100 and 50 mg of the albumin per mL, respectively. Chromatography. Spot 10 mL of the sample solution and the (iii) Procedure—Pipet 0.5 mL each of the sample solu- standard solution on a plate of silica gel for thin-layer chro- tion and the standard solutions, put them in glass test tubes matography. Develop the plate with a mixture of ethyl about 18 mm in inside diameter and about 130 mm in length, acetate and hexane (1:1) to a distance of about 10 cm, and add exactly 5 mL of alkaline copper TS, mix, and allow the air-dry the plate. Spray evenly 4-dimethylaminobenzalde- tubes to stand in a water bath at 309Cfor10minutes.Then hyde TS on the plate, heat at 1059C for 5 minutes, and allow add exactly 0.5 mL of diluted Folin's TS (1 in 2), mix, and to cool: one of the spots from the sample solution and a warm in a water bath at 309C for 20 minutes. Determine the green spot from the standard solution show the same color absorbances of these solutions at 750 nm as directed under tone and Rfvalue. the Ultraviolet-visible Spectrophotometry using a solution obtained in the same manner with 0.5 mL of water as the blank. Chorionic Gonadotrophin Plot the absorbances of the standard solutions on the 胎盤性性腺刺激ホルモン vertical axis and their protein concentrations on the horizon- tal axis to prepare a calibration curve, and determine the Delete the origin/limits of content and add the protein content of the sample solution from its absorbance following: by using this curve. Then calculate the amount of the protein in the sample. Method of preparation Chorionic Gonadotrophin is a dried preparation of gonad-stimulating hormone obtained Change the Assay (i) to read: from the urine of healthy pregnant women after the manufacturing process to remove or inactivate the virus. It Assay (i) Test animals—Select healthy female albino rats contains not less than 2500 chorionic gonadotrophin Units weighing about 45 to 65 g. per mg, and contains not less than 3000 chorionic gonadotrophin Units per mg protein. It contains not less than 80z and not more than 125z of Chorionic Gonadotrophin for the labeled Units of chorionic gonadotrophin. Injection

Change the Description to read: 注射用胎盤性性腺刺激ホルモン Description Chorionic Gonadotrophin occurs as a white to Change the Description to read: light yellow-brown powder. It is freely soluble in water. Description Chorionic Gonadotrophin for Injection occurs as a white to light yellow-brown powder or masses. Delete the Toxicity and Pyrogen, and add the following: Add the following next to Identiˆcation: Bacterial endotoxins Less than 0.03 EU/unit. pH Prepare a solution so that each mL of isotonic sodium chloride solution contains 2 mg of Chorionic Gonadotoro- Abnormal toxicity Dilute Chorionic Gonadotrophin with phin for Injection: the pH of this solution is between 5.0 and isotonic sodium chloride solution so that each mL of the 7.0. solution contains 120 Units, and use this solution as the sample solution. Inject 5.0 mL of the sample solution into Delete the Pyrogaen and add the following: the peritoneal cavity of each of 2 or more of well-nourished, healthy guinea pigs weighing about 350 g, and observe the Bacterial endotoxins Less than 0.03 EU/unit. conditions of the animals for more than 7 days: all the Mass variation When calculate the acceptance value using animals exhibit no abnormalities. the mean of estimated contents of the units tested as M, it Supplement II, JPXIV O‹cial Monographs for Part II 1765 meets the requirements of the Mass Variation Test. solution. Prepare before use. Use within the day of prepara- tion. Foreign insoluble matter Perform the test according to (7) Procedure—Pipet 0.5 mL each of the standard Method 2: it meets the requirements of the Foreign Insoluble solutions and the sample solution in small test tubes, add Matter Test for Injections. 3 mL of the alkaline copper solution to them, and mix. Insoluble particulate matter Perform the test according to Allow them to stand at the room temperature for not less Method 1: it meets the requirements of the Insoluble Par- than 10 minutes, add 0.3 mL of diluted Folin's TS (1 in 2), ticulate Matter Test for Injections. mix immediately, and allow to stand for not less than 30 minutes. Determine the absorbances of these solutions so Sterility Perform the test according to the Membrane obtained at 750 nm as directed under the Ultraviolet-visible ˆltration method: it meets the requirements of the Sterility Spectrophotometry using a solution, prepared in the same Test. manner with 0.5 mL of water, as the blank. Plot the calibra- tion curve from the absorbances obtained with the standard solutions, and determine the amount of protein in the Serum Gonadotrophin sample solution from this curve.

血清性性腺刺激ホルモン Speciˆc activity (unit/mg protein) units per mg, obtained in the Assay ＝ ×100 Change the origin/limits of content to read: amount (z) of protein in the sample Method of preparation Serum Gonadotrophin is a dried preparation of gonad-stimulating hormone, obtained from Change the Toxicity to read: pregnant mares' serum which has adequately inspected Abnormal toxicity Dissolve Serum Gonadotrophin in viruses, and subjected to a suitable process for removal or isotonic sodium chloride solution so that each 5 mL of the inactivation of viruses. It contains not less than 2000 serum solution contains 4000 Units, and use this solution as the gonadotrophin Units per mg. sample solution. Inject 5.0 mL of the sample solution into It contains not less than 80z and not more than 125z of the peritoneal cavity of each of 2 or more of well-nourished, the labeled Units of serum gonadotrophin. healthy guinea pigs weighing about 350 g, and inject 0.5 mL of the sample solution into the peritoneal cavity of each of 2 Change the Description to read: or more of well-nourished, healthy mice aged about 5 weeks. Description Serum Gonadotrophin occurs as a white Observe the conditions of the animals for more than 7 days: powder. all the animals exhibit no abnormalities. It is freely soluble in water. Change the Pyrogen to read: AddthefollowingnexttoPurity: Bacterial endotoxins Less than 0.1 EU/unit. Speciˆc activity When calculated from the results obtained by the Assay and the following test, Serum Gonadotrophin containes not less than 3000 serum gonadotrophin Units per Serum Gonadotrophin for Injection mg of protein. (1) Standard solutions—Dissolve about 3 mg of bovine 注射用血清性性腺刺激ホルモン serum albumin in water to make a solution containing 500 mgofthealbuminineachmL.Tothissolutionadd Change the Description to read: water to make four standard solutions so that each mL Description Serum Gonadotrophin for Injection occurs as contains exactly 200 mg, 150 mg, 100 mgand50mgofthe whitepowderormasses. albumin, respectively. (2) Sample solution—Dissolve about 1 mg of Serum Add the following next to Identiˆcation: Gonadotrophin in water to make a solution containing exactly 180 mgineachmL. pH Dissolve 30 mg of Serum Gonadotrophin for Injection (3) Sodium carbonate solution—Dissolve 2 g of sodium in 20 mL of isotonic sodium chloride solution: the pH of this carbonate (standard reagent) in 0.1 mol/L sodium solution is between 5.0 and 7.0. hydroxide TS to make 100 mL. (4) Sodium tartrate solution—Dissolve about 1 g of Delete the Pyrogen and add the following: sodium tartrate dihydrate in water to make 100 mL. Bacterial endotoxins Less than 0.1 EU/unit. (5) Copper (II) sulfate solution—Dissolve 0.5 g of copper (II) sulfate pentahydrate in the sodium tartrate solution to make 100 mL. (6) Alkaline copper solution—Mix 50 mL of the sodium carbonate solution and 1 mL of the copper (II) sulfate 1766 O‹cial Monographs for Part II Supplement II, JPXIV

Add the following: Add the following: Lindera Root Lonicera Leaf and Stem

Linderae Radix Lonicerae Folium Cum Caulis ウヤク ニンドウ

Lindera Root is the root of Lindera strychnifolia Lonicera Leaf and Stem is the leaves and stems of Fernandez-Villar (Lauraceae). Lonicera japonica Thunberg (Caprifoliaceae). Description Fusiform or rosary-like root, 10 – 15 cm in Description Leaves and opposite leaves on short stem; length, 10 – 25 mm in diameter; externally yellowish brown leaf,ovateandentire,withshortpetiole,3–7cminlength, to brown, with a few scars of rootlets; a transverse section 1–3cm in width; upper surface greenish brown, lower reveals cortex brown, xylem light yellowish brown, concen- surface light grayish green; under a magnifying glass, both tric circles and radially arranged lines brown; dense and hard surfacespubescent.Stem,1–4mmindiameter;externally in texture. grayish yellow-brown to purplish brown, a transverse Odor, camphor-like; taste, bitter. section of stem, round and hollow. Under a microscope, a transverse section of the root with Almost odorless; taste, slightly astringent, followed by a periderm reveals a cork layer several cells thick, partially litter bitterness. consisting of cork stone cells; cortex parenchyma sometimes Under a microscope, a transverse section of leaf reveals contains oil cells and ˆbers; in xylem, vessels-xylem ˆbers the outermost layer of upper and lower surfaces to be com- and rays are arranged alternately; parenchyatous cells of posed of a single-layered epidermis, uni-cellular non-glandu- cortex and xylem contain sandy and columnar crystals of lar hairs and multi-cellular glandular hairs on epidermis; in calcium oxalate, simple starch grains 1 – 15 mmindiameter, midvein, several-layered collenchyma present beneath the and 2- to 4- compound starch grains. epidermis and vascular bundles in the center; in mesophyll, palisade layer adjacent to upper epidermis, spongy tissue ad- Identiˆcation To 3 g of pulverized Lindera Root add jacent to lower epidermis; glandular hairs contain brown 40 mL of hexane, and warm under a re‰ux condenser on a secretion, parenchymatous cells contain aggregate crystals water bath for 30 minutes. After cooling, ˆlter, to the of calcium oxalate, and occasionally starch grains. residue add 10 mL of ammonia TS and 30 mL of a mixture of ethyl acetate and diethyl ether (1:1), shake vigorously for Identiˆcation To 1 g of pulverized Lonicera Leaf and Stem 20 minutes, and centrifuge. Separate the supernatant liquid, add 5 mL of methanol, shake for 5 minutes, centrifuge, and add 10 g of anhydrous sodium sulfate, shake, and ˆlter. use the supernatant liquid as the sample solution. Separate- Evaporate the ˆltrate, dissolve the residue with 0.5 mL of ly, dissolve 1 mg of chlorogenic acid for thin-layer chro- ethanol (99.5), and use this solution as the sample solution. matography in 2 mL of methanol, and use this solution as Perform the test with this solution as directed under the the standard solution (1). Separately, dissolve 1 mg of Thin-layer Chromatography. Spot 20 mLofthesample loganin for thin-layer chromatography in 2 mL of methanol, solution on a plate of silica gel for thin-layer chro- and use this solution as the standard solution (2). Perform matography, develop the plate with a mixture of ethyl the test with these solutions as directed under the Thin-layer acetate, methanol and ammonia water (28) (10:2:1) to a Chromatography. Spot 10 mL each of the sample solution, distance of about 10 cm, and air-dry the plate. Spray evenly the standard solution (1) and the standard solution (2) on a DragendorŠ's TS for spraying on the plate: a yellow-brown plate of silica gel for thin-layer chromatography. Develop spot appears at around Rf0.4. the plate with a mixture of ethyl acetate, water and formic acid (6:1:1) to a distance of about 10 cm, and air-dry the Loss on drying Not more than 14.0z (6 hours). plate. Examine under ultraviolet light (main wavelength: Total ash Not more than 2.5z. 365 nm): one of the spot among the several spots from the sample solution has the same color tone and Rfvaluewith Extract content Not less than 6.0z (dilute ethanol-soluble the blue-white ‰uorescent spot from the standard solution extract). (1). Spray evenly 4-methoxybenzaldehyde-sulfuric acid TS on the plate, and heat at 1059C for 5 minutes: one of the spot among the several spots from the sample solution has thesamecolortoneandRf value with the red-purple spot from the standard solution (2).

Purity Stem—Lonicera Leaf and Stem does not contains the stems larger than 5 mm in diameter.

Loss on drying Not more than 12.0z (6 hours). Supplement II, JPXIV O‹cial Monographs for Part II 1767

Total ash Not more than 9.0z. Add the following: Acid-insoluble ash Not more than 1.0z. Lycium Fruit Extract content Not less than 12.0z (dilute ethanol-solu- ble extract). Lycii Fructus クコシ Add the following: Lycium Fruit is the fruit of Lycium chinense Miller Lycium Bark or Lycium barbarum Linne (Solanaceae). Description Fusiform fruit with acute apex, 6 – 20 mm in Lycii Cortex length, 3 – 8 mm in diameter, pericarp red to dark red, externally roughly wrinkled; under a magnifying glass, a ジコッピ transverse section of fruit reveals two locules containing numerous seeds; seed light brown to light yellowish brown, Lycium Bark is the root bark of Lycium chinense about 2 mm in a diameter, compressed reniform. Miller or Lycium barbarum Linnáe (Solanaceae). Odor, characteristic; taste, sweet, later slightly bitter. Description Tubular to semitubular bark, 1 – 6 mm in Identiˆcation To 1.0 g of powdered Lycium Fruit add 5 thickness; externally light brown to light yellowish brown, mL of ethyl acetate, shake for 15 minutes, ˆlter, and use the periderm peeled easily as scale; internally grayish brown, ˆltrate as the sample solution. Perform the test with this longitudinally striate; brittle in texture; fractured surface, solution as directed under the Thin-layer Chromatography. grayish white, not ˆbrous. Spot 20 mL of the sample solution on a plate of silica gel for Odor, weak and characteristic; taste, slightly sweet at ˆrst. thin-layer chromatography, develop the plate with a mixture Under a microscope, a transverse section reveals periderm of hexane and ethyl acetate (10:1) to a distance of about composed of a cork layer of several layers of thin walled 10 cm, and air-dry the plate: a yellow principal spot appears cork cells; in cortex parenchymatous cells containing sandy at around Rf0.6. crystals of calcium oxalate sparsely distributed, occasionally a few ˆbers observed; parenchymatous cells contain starch Purity Foreign matter—It contains not more than 2.0z of grains, 1 – 10 mm in diameter; stone cells very rare. foreign matter such as peduncle or others.

Identiˆcation To 1.0 g of pulverized Lycium Bark add 10 Total ash Not more than 8.0z. mL of methanol, shake for 15 minutes, ˆlter, and use the Acid-insoluble ash Not more than 1.0z. ˆltrate as the sample solution. Perform the test with this solution as directed under the Thin-layer Chromatography. Extract content Not less than 35.0z (dilute ethanol-solu- Spot 10 mL of the sample solution on a plate of silica gel for ble extract). thin-layer chromatography. Develop the plate with a mix- ture of 1-butanol, water, pyridine and acetic acid (100) (3:1:1:1) to a distance of about 10 cm, and air-dry the plate. Opium Alkaloids Hydrochlorides Spray evenly DragendorŠ's TS for spraying on the plate, heat at 1059C for 3 minutes, then spray evenly sodium nitrite 塩酸アヘンアルカロイド TS: a dark brown principal spot appears at around Rf0.5. Change the origin/limits of content to read: Loss on drying Not more than 11.5z (6 hours).

Total ash Not more than 20.0z. Opium Alkaloids Hydrochlorides consists of the hydrochlorides of some of the main alkaloids obtained Acid-insoluble ash Not more than 3.0 . z from opium. Extract content Not less than 10.0z (dilute ethanol-solu- It contains not less than 47.0z and not more than ble extract). 52.0z of morphine (C17H19NO3: 285.34), and not less than 35.0z and not more than 41.0z of other opium alkaloids.

Change the Description to read: Description Opium Alkaloids Hydrochlorides occur as a white to light brown powder. It is soluble in water, and slightly soluble in ethanol (99.5). It is colored by light. 1768 O‹cial Monographs for Part II Supplement II, JPXIV

Change the Identiˆcation (1) to read: noscapine 3.6 Identiˆcation (1) Dissolve 0.1 g of Opium Alkaloids Operating conditions— Hydrochlorides in 10 mL of diluted ethanol (1 in 2), and use Detector: An ultraviolet absorption photometer this solution as the sample solution. Separately, dissolve (wavelength: 285 nm). 60 mg of Morphine Hydrochloride, 40 mg of Noscapine Column: A stainless steel column 4.6 mm in inside Hydrochloride, 10 mg of Codein Phosphate and 10 mg of diameter and 15 cm in length, packed with octadecyl- Papaverine Hydrochloride in 10 mL each of diluted ethanol silanized silica gel for liquid chromatography (5 mmin (1 in 2), and use these solutions as the standard solutions (1), particle diameter). (2), (3) and (4), respectively. Perform the test with these Column temperature: A constant temperature of about solutions as directed under the Thin-layer Chromatography. 409C. Spot 20 mL each of the sample solution and the standard Mobile phase: Dissolve 1.0 g of sodium lauryl sulfate in solutions on a plate of silica gel with ‰uorescent indicator 500 mL of diluted phosphoric acid (1 in 1000), and adjust for thin-layer chromatography. Develop the plate with a the pH to 3.0 with sodium hydroxide TS. To 240 mL of this mixture of acetone, toluene, ethanol (99.5) and ammonia solution add 70 mL of tetrahydrofuran, and mix. solution (28) (20:20:3:1) to a distance of about 10 cm, and Flow rate: Adjust the ‰ow rate so that the retention time air-dry the plate. Examine under ultraviolet light (main of morphine is about 10 minutes. wavelength: 254 nm): each spot from the sample solution is System suitability— the same in color tone and Rf value with the corresponding System performance: Dissolve 60 mg of Morphine spot from the standard solutions (1), (2), (3) and (4) (mor- Hydrochloride, 10 mg of Codeine Phosphate, 10 mg of phine, noscapine, codeine and papaverine). Papaverine Hydrochloride and 40 mg of Noscapine Hydrochloride in water to make 50 mL. When the procedure Change the Assay to read: isrunwith20mL of this solution under the above operating Assay Weigh accurately about 0.1 g of Opium Alkaloids conditions, morphine, codeine, papaverine and noscapine Hydrochlorides, and dissolve in water to make exactly 50 are eluted in this order with the complete separation between mL, and use this solution as the sample solution. Separately, these peaks and with the resolution between the peaks of weigh accurately about 60 mg of morphine hydrochloride morphine and codeine being not less than 1.5. for assay, dissolve in water to make exactly 50 mL, and use System repeatability: When the test is repeated 6 times this solution as the standard solution. Perform the test with with 20 mL of the standard solution under the above operat- exactly 20 mL each of the sample solution and the standard ing conditions, the relative standard deviation of the peak solution as directed under the Liquid Chromatography ac- area of morphine is not more than 1.0z. cording to the following conditions, and determine the peak areas of morphine, codeine, papaverine, thebaine, narceine and noscapine, AT1, AT2, AT3, AT4, AT5 and AT6, from the Powdered Peony Root sample solution, and the peak area of morphine, AS,from the standard solution. シャクヤク末

Amount (mg) of morphine (C17H19NO3) Change the origin/limits of content to read: A ＝W × T1 ×0.887 S A S Powdered Peony Root is the powder of Peony Root. Amount (mg) of other opium alkaloids It contains not less than 2.0z of paeoni‰orin

＝WS (C23H28O11: 480.46), calculated on the dried basis. A ＋A ×0.29＋A ×0.20＋A ×0.19＋A × T2 T3 T4 T5 T6 AS Change the Assay to read: ×0.887 Assay Weigh accurately about 0.5 g of Powdered Peony

WS: Amount (mg) of morphine hydrochloride for assay, Root, add 50 mL of diluted methanol (1 in 2), heat under a calculated on the anhydrous basis re‰ux condenser on a water bath for 30 minutes, cool, and ˆlter. To the residue add 50 mL of diluted methanol (1 in 2), The relative retention time of codine, papaverine, the- and proceed in the same manner. Combine the ˆltrates, add baine, narceine and noscapine with respect to morphine dilutedmethanol(1in2)tomakeexactly100mL,anduse obtained under the following operating conditions are as this solution as the sample solution. Separately, weigh ac- follows. curately about 10 mg of Paeoni‰orin Reference Standard (separately detarmine the water content), dissolve in diluted Component Relative retention time methanol (1 in 2) to make exactly 100 mL, and use this codeine 1.1 solution as the standard solution. Perform the test with papaverine 1.9 exactly 10 mL each of the sample solution and the standard thebaine 2.5 solution as directed under the Liquid Chromatography narceine 2.8 Supplement II, JPXIV O‹cial Monographs for Part II 1769 according to the following conditions. Determine the peak between two polarizing prisms ˆxed at right angle to each areas, AT and AS, of paeoni‰orin in each solution. other. (2) To 1 g of Potato Starch add 50 mL of water, boil for Amount (mg) of paeoni‰orin (C H O ) 23 28 11 1 minute, and allow to cool: a subtle white-turbid, pasty A ＝W × T liquid is formed. S A S (3) To1mLofthepastyliquidobtainedin(2)add0.05

WS: Amount (mg) of Paeoni‰orin Reference Standard, mL of iodine TS: a orange-red to dark blue-purple color is calculated on the anhydrous basis formed, and the color disappears by heating.

Operating conditions— pH Put5.0gofPotatoStarchinanon-metalvessel,add Detector: An ultraviolet absorption photometer 25.0 mL of freshly boiled and cooled water, mix gently for 1 (wavelength: 232 nm). minute, and allow to stand for 15 minutes: the pH of the Column: A stainless steel column 4.6 mm in inside solution is between 5.0 and 8.0. diameter and 15 cm in length, packed with octadecyl- Purity (1) Iron—To 1.5 g of Potato Starch add 15 mL of silanized silica gel for liquid chromatography (5 mmin 2 mol/L hydrochloric acid TS, mix, ˆlter, and use the ˆltrate particle diameter). as the test solution. To 2.0 mL of Standard Iron Solution Column temperature: A constant temperature of about add water to make 20 mL, and use as the control solution. 209C. Put 10 mL each of the test solution and the control solution Mobile phase: A mixture of water, acetonitrile and in test tubes, add 2 mL of a solution of citric acid (2 in 10) phosphoric acid (850:150:1). and 0.1 mL of mercapto acetic acid, and mix. Alkalize with Flow rate: Adjust the ‰ow rate so that the retention time ammonia solution (28) to litmus paper, add water to make of paeoni‰orin is about 10 minutes. 20 mL, and mix. Transfer 10 mL each of these solutions into System suitability— test tubes, allow to stand for 5 minutes, and compare the System performance: Dissolve 1 mg each of Paeoni‰orin color of these solutions against a white background: the Reference Standard and albi‰orin in diluted methanol (1 in color of the test solution is not darker than that of the 2) to make 10 mL. When the procedure is run with 10 mLof control solution (not more than 10 ppm). this solution under the above operating conditions, albi‰orin (2) Oxidizingsubstances—To4.0gofPotatoStarchadd and paeoni‰orin are eluted in this order with the resolution 50.0 mL of water, shake for 5 minutes, and centrifuge. To between these peaks being not less than 2.5. 30.0 mL of the supernatant liquid add 1 mL of acetic acid System repeatability: When the test is repeated 6 times (100) and 0.5 to 1.0 g of potassium iodide, shake, and allow with the standard solution under the above operating to stand for 25 to 30 minutes at a dark place. Add 1 mL of conditions, the relative standard deviation of the peak area starch TS, and titrate with 0.002 mol/L sodium thiosulfate of paeoni‰orin is not more than 1.5z. VS until the color of the solution disappears. Perform a blank determination and make any necessary correction: the volume of 0.002 mol/L sodium thiosulfate VS consumed is Change to read: not more than 1.4 mL (not more than 20 ppm, calculated as hydrogen peroxide). Potato Starch (3) Sulfur dioxide— (i) Apparatus Use as shown in the following ˆgure. Amylum Solani バレイショデンプン

Potato Starch consists of starch granules derived from the tuber of Solanum tuberosum Linnáe (Solanaceae). Description Potato Starch occurs as a white powder. It is practically insoluble in water and in ethanol (99.5).

Identiˆcation (1) Under a microscope, Potato Starch, preserved in a mixture of water and glycerin (1:1), appears as unevenly ovoid or pyriform simple grains usually 30 – 100 mm, often more than 100 mm in diameter, or spherical simple grains 10 – 35 mm in diameter, rarely 2- to 4-compound grains; ovoid or pyriform simple grains with eccentric hilum, spherical simple grains with non-centric or slightly eccentric hilum; striation distinct in all grains; a black cross, its intersection point on hilum, is observed when grains are put 1770 O‹cial Monographs for Part II Supplement II, JPXIV

minutes).

Residue on ignition Not more than 0.6z (1 g).

Containers and storage Containers—Well-closed contain- ers.

Add the following: Processed Aconite Root

Processi Aconiti Radix ブシ

Processed Aconite Root is the tuberous root of Aconitum carmichaeli Debeaux or Aconitum The ˆgures are in mm. japonicum Thunberg (Ranunculaceae) prepared by the following processes. A: Boiling ‰ask (500 mL) Process 1: Autoclaving. [Processed Aconite Root 1] B: Funnel (100 mL) Process 2: Heating or autoclaving after rinsing in C: Condenser the salt, rock salt or calcium chloride water. [Proc- D: Test-tube essed Aconite Root 2] E: Tap Process 3: Treating with lime after rinsing in the salt water. [Processed Aconite Root 3] (ii) Procedure Introduce 150 mL of water into the There are three kinds of Processed Aconite Root boiling ‰ask, close the tap of the funnel, and pass carbon based on their preparing processes as shown above. dioxide through the whole system at a rate of 100±5mLper Processed Aconite Root 1, Processed Aconite Root minute. Pass cooling water through the condenser, and place 2 and Processed Aconite Root 3 contain the total

10 mL of hydrogen peroxide-sodium hydroxide TS in the alkaloid [as benzoylaconine (C32H45NO10:603.70)]of test-tube. After 15 minutes, remove the funnel without inter- not less than 0.7z and not more than 1.5z, not less rupting the stream of carbon dioxide, and introduce through than 0.1z and not more than 0.6z, and not less than the opening into the ‰ask about 25 g of Potato Starch, 0.5z and not more than 0.9z,calculatedonthedried accurately weighed, with the aid of 100 mL of water. Apply bases, respectively. tapgreasetotheoutsideoftheconnectionpartofthe The label indicates the treating process. funnel, and load the funnel. Close the tap of the funnel, Description pour 80 mL of 2 mol/L hydrochloric acid TS into the Processed Aconite Root 1: Cut pieces irregularly polygonal, funnel, open the tap to introduce the hydrochloric acid into less than 10 mm in diameter; externally dark grayish brown the ‰ask, and close the tap while several mL of the to blackish brown; hard in texture; cut surface ‰at, light hydrochloric acid remains, in order to avoid losing sulfur brown to dark brown, usually horny and lustrous. dioxide. Place the ‰ask in a water bath, and heat the mixture Odor, weak and characteristic. for 1 hour. Transfer the contents of the test-tube with the aid Under a microscope, transverse and longitudinal sections of a little water to a wide-necked conical ‰ask. Heat in a reveal pitted, scaraliform, reticulate and spiral vessels; waterbathfor15minutes,andcool.Add0.1mLof starch grains in parenchymatous cells usually gelatinized but bromophenol blue TS, and titrate with 0.1 mol/L sodium sometimes not gelatinized; starch grains, simple, spherical or hydroxide VS until the color changes from yellow to violet- ellipsoid, 2 – 25 mm in diameter, or 2- to a dozen or so- com- blue lasting for at least 20 seconds. Perform a blank determi- pound, hilum of starch grain distinct. nation and make any necessary correction. Calculate the amount of sulfur dioxide by applying the following formula: Processed Aconite Root 2: Nearly obconical root, 15 – 30 it is not more than 50 ppm. mm in length, 12 – 16 mm in diameter, slices cut longitudi- nallyortransversely,20–60mminlength,15–40mmin V Amount (ppm) of sulfur dioxide＝ ×1000×3.203 width, and 200 – 700 mm in thickness, or cut pieces irregular- W ly polygonal, less than 12 mm in diameter; externally light W: Amount (g) of the sample brown to dark brown or yellowish brown; hard in texture, V: Amount (mL) of 0.1 mol/L sodium hydroxide VS usually without wrinkles; cut surface ‰at, light brown to consumed dark brown or yellowish white to light yellowish brown, usu- ally horny, semi-transparent and lustrous. Loss on drying Not more than 20.0z (1 g, 1309C, 90 Odor, weak and characteristic. Supplement II, JPXIV O‹cial Monographs for Part II 1771

Under a microscope, transverse and longitudinal sections Liquid Chromatography according to the following reveal metaderm, primary cortex, endodermis, secondary conditions, and determine the heights of the peaks corre- cortex, cambium, and xylem; primary cortex contains sponding to aconitine, jesaconitine, hypaconitine and oblong to oblong-square sclerenchymatous cells, 30 – 75 mm mesaconitine, HTA and HSA, HTJ and HSJ, HTH and HSH,and in short axis, 60 – 150 mm in long axis; endodermis single HTM and HSM, respectively, and calculate the amounts of layered, endodermal cells elongated in tangential direction; them by the following formulae: the amounts of aconitine, cambium, star shaped or irregular polygons to orbicular; a jesaconitine, hypaconitine and mesaconitine per g calculated group of vessel in xylem v-shaped; sometimes isolated ring on the dried basis are not more than 60 mg, 60 mg, 280 mgand of cambium appears in secondary cortex or in pith; vessels, 140 mg, respectively, and the total amount of them is not pitted, scaraliform, reticulate and spiral; starch grains in more than 450 mg. parenchymatous cells gelatinized. Amount (mg) of aconitine (C H NO ) Processed Aconite Root 3: Cut pieces irregularly polygonal, 34 47 11 C H less than 5 mm in diameter; externally grayish brown; hard ＝ SA × TA ×10 W H in texture; cut surface ‰at, light grayish brown to grayish SA white, not lustrous. Amount (mg) of jesaconitine (C35H49NO12) Odor, weak and characteristic. C H ＝ SJ × TJ ×10 Under a microscope, transverse and longitudinal sections W HSJ reveal pitted, scaraliform, reticulate and spiral vessels; Amount (mg) of hypaconitine (C H NO ) starch grains, simple, spherical or ellipsoid, 2 – 25 mmin 33 45 10 C H diameter, or 2- to a dozen or so- compound, hilum of starch ＝ SH × TH ×10 W H grain distinct. SH Amount (mg) of mesaconitine (C H NO ) Identiˆcation To 3 g of pulverized Processed Aconite Root 33 45 11 C H add 20 mL of diethyl ether and 2 mL of ammonia TS, shake ＝ SM × TM ×10 W H for 10 minutes, and centrifuge. Evaporate the ether layer to SM dryness under reduced pressure, dissolve the residue in 1 mL CSA: Concentration (mg/mL) of aconitine for purity in the of diethyl ether, and use this solution as the sample solution. aconitum diester alkaloids standard solution for Separately, dissolve 1 mg of benzoylmesaconine hydrochlo- purity ride for thin-layer chromatography in 10 mL of ethanol CSJ: Concentration (mg/mL) of jesaconitine for purity in (99.5), and use this solution as the standard solution. the aconitum diester alkaloids standard solution for Perform the test with these solutions as directed under the purity

Thin-layer Chromatography. Spot 10 mLeachofthesample CSH: Concentration (mg/mL) of hypaconitine for purity in solution and the standard solution on a plate of silica gel for the aconitum diester alkaloids standard solution for thin-layer chromatography, develop the plate with a mixture purity of ethyl acetate, ethanol (99.5) and ammonia water (28) CSM: Concentration (mg/mL) of mesaconitine for purity in (40:3:2) to a distance of about 10 cm, and air-dry the plate. the aconitum diester alkaloids standard solution for Spray evenly DragendorŠ's TS for spraying on the plate, purity air-dry the plate, and spray evenly sodium nitrite TS: one of W: Amount (g) of the sample, calculated on the dried the spot among the several spots from the sample solution basis has the same color tone and Rf value with the yellow-brown Operating conditions— spot from the standard solution. Detector: An ultraviolet absorption photometer Purity Aconitum diester alkaloids (aconitine, jesaconitine, (wavelength: 231 nm for aconitine, hypaconitine and hypaconitine and mesaconitine)—Weigh accurately about mesaconitine; 254 nm for jesaconitine). 0.5 g of pulverized Processed Aconite Root, put in a glass- Column: A stainless steel column 4.6 mm in inside stoppered centrifuge tube, suspend in 3.0 mL of water by diameter and 15 cm in length, packed with octadecyl- shaking, and add 1.0 mL of ammonia TS and 20 mL of silanized silica gel for liquid chromatography (5 mmin diethyl ether. Stopper tightly the tube, shake for 30 minutes, particle diameter). centrifuge, and separate the ether layer. To the residue add Column temperature: A constant temperature of about 1.0 mL of ammonia TS and 20 mL of diethyl ether, and 409C. repeat the above process two times. Combine all extracts, Mobile phase: A mixture of phosphate buŠer solution for evaporate to dryness under reduced pressure at not more aconite root and tetrahydrofuran (183:17). than 409C, and dissolve the residue with exactly 10 mL of a Flow rate: Adjust the ‰ow rate so that the retention time mixture of phosphate buŠer solution for aconite root and of mesaconitine is about 31 minutes. acetonitrile (1:1). Centrifuge this solution, and use the System suitability— supernatant liquid as the sample solution. Perform the test System performance: When the procedure is run with with exactly 20 mL each of the sample solution and the aconi- 20 mL of the aconitum diester alkaloids standard solution tum diester alkaloids standard solution as directed under the for purity under the above operating conditions, using 254 1772 O‹cial Monographs for Part II Supplement II, JPXIV nm, mesaconitine, hypaconitine, aconitine and jesaconitine Root based on their preparing processes as shown are eluted in this order, and each resolution between their above. peaks is not less than 1.5, respectively. Powdered Processed Aconite Root 1 and Powdered System repeatability: To 1 mL of aconitum diester Processed Aconite Root 2 contain the total alkaloid alkaloids standard solution for purity add a mixture of phos- [as benzoylaconine (C32H45NO10: 603.70)] of not less phate buŠer solution for aconite root and acetonitrile (1:1) than 0.4z and not more than 1.2z, and not less than to make 10 mL. When the test is repeated 6 times with 20 mL 0.1z and not more than 0.3z,calculatedonthedried of this solution under the above operating conditions, using bases, respectively. 231 nm, the relative standard deviation of the peak height of The label indicates the treating process. mesaconitine is not more than 1.5z. Description Loss on drying Not more than 15.0z (6 hours). Powdered Processed Aconite Root 1: Powdered Processed Aconite Root 1 occurs as a light grayish brown powder. It Total ash has a characteristic odor. Processed Aconite Root 1: Not more than 4.0z. Under a microscope, Powered Processed Aconite Root 1 Processed Aconite Root 2: Not more than 12.0z. reveals gelatinized starch masses or starch grains and paren- Processed Aconite Root 3: Not more than 19.0z. chymatous cells containing them, fragments of reddish Acid-insoluble ash Not more than 0.9z. brown metaderm, fragments of pitted, scaraliform, reticu- late and spiral vessels; also square to oblong-square scleren- Assay Weigh accurately about 2 g of pulverized Processed chymatous cells, 30 – 150 mm in diameter, 100 – 250 mmin Aconite Root, put in a glass-stoppered centrifuge tube, and length, cell wall of sclerenchymatous cells, 6 – 12 mmin add 1.6 mL of ammonia TS and 20 mL of diethyl ether. thickness; starch grains of ``Processed Aconite Root'', sim- Stopper tightly the tube, shake for 30 minutes, centrifuge, ple, spherical or ellipsoid, 2 – 25 mm in diameter, or 2- to a and separate the ether layer. To the residue add 0.8 mL of dozen or so- compound, hilum of starch grain distinct. ammonia TS and 20 mL of diethyl ether, and proceed as above. Repeat this process more three times. Combine all Powdered Processed Aconite Root 2: Powdered Processed extracts, evaporate to dryness under reduced pressure, Aconite Root 2 occurs as a light yellowish white powder. It dissolve the residue in 5 mL of ethanol (99.5), add 30 mL of has a characteristic odor. freshly boiled and cooled water, and titrate with 0.01 mol/L Under a microscope, Powered Processed Aconite Root 2 hydrochloric acid VS until the color of the solution changes reveals gelatinized starch masses and parenchymatous cells from green to gray-blue through blue-green (indicator: 3 containing them, fragments of reddish brown metaderm, drops of methyl red-methylene blue TS). Perform a blank fragments of pitted, scaraliform, reticulate and spiral determination and make any necessary correction. vessels; also square to oblong-square sclerenchymatous cells, 30–150mm in diameter, 100 – 250 mm in length, cell wall of Each mL of 0.01 mol/L hydrochloric acid VS sclerenchymatous cells, 6 – 12 mm in thickness. ＝6.037 mg of total alkaloid [as benzoylaconine

(C32H45NO10: 603.70)] Identiˆcation To 3 g of Powdered Processed Aconite Root add 2 mL of ammonia TS and 20 mL of diethyl ether, shake for 10 minutes, and centrifuge. Evaporate the ether layer to Add the following: dryness under reduced pressure, dissolve the residue in 1 mL of diethyl ether, and use this solution as the sample solution. Powdered Processed Aconite Root Separately, dissolve 1 mg of benzoylmesaconine hydrochlo- ride for thin-layer chromatography in 10 mL of ethanol Processi Aconiti Radix Pulverata (99.5), and use this solution as the standard solution. Perform the test with these solutions as directed under the ブシ末 Thin-layer Chromatography. Spot 10 mLeachofthesample solution and the standard solution on a plate of silica gel for Powdered Processed Aconite Root is the powder of thin-layer chromatography, develop the plate with a mixture Processed Aconite Root prepared by Process 1 or Process 2, or the powder of the tuberous root of of ethyl acetate, ethanol (99.5) and ammonia water (28) (40:3:2) to a distance of about 10 cm, and air-dry the plate. Aconitum carmichaeli Debeaux or Aconitum japoni- Spray evenly DragendorŠ's TS for spraying on the plate, cum Thunberg (Ranunculaceae) prepared by Process air-dry the plate, and spray evenly sodium nitrite TS: one of 1. the spot among the several spots from the sample solution Occasionally, it contains Corn Starch or Lactose. Process 1: Autoclaving. [Powdered Processed has the same color tone and Rf value with the yellow-brown spot from the standard solution. Aconite Root 1] Process 2: Heating or autoclaving after rinsing in Purity Aconitum diester alkaloids (aconitine, jesaconitine, the salt, rock salt or calcium chloride water. [Pow- hypaconitine and mesaconitine)—Weigh accurately about dered Processed Aconite Root 2] 0.5 g of Powdered Processed Aconite Root, put in a glass- There are two kinds of Powdered Processed Aconite stoppered centrifuge tube, suspend in 3.0 mL of water by Supplement II, JPXIV O‹cial Monographs for Part II 1773 shaking, and add 1.0 mL of ammonia TS and 20 mL of particle diameter). diethyl ether. Stopper tightly the tube, shake for 30 minutes, Column temperature: A constant temperature of about centrifuge, and separate the ether layer. To the residue add 409C. 1.0 mL of ammonia TS and 20 mL of diethyl ether, and Mobile phase: A mixture of phosphate buŠer solution for repeat the above process two times. Combine all extracts, aconite root and tetrahydrofuran (183:17). evaporate to dryness under reduced pressure at not more Flow rate: Adjust the ‰ow rate so that the retention time than 409C, and dissolve the residue with exactly 10 mL of a of mesaconitine is about 31 minutes. mixture of phosphate buŠer solution for aconite root and System suitability— acetonitrile (1:1). Centrifuge this solution, and use the System performance: When the procedure is run with 20 supernatant liquid as the sample solution. Perform the test mL of the aconitum diester alkaloids standard solution for with exactly 20 mL each of the sample solution and the aconi- purity under the above operating conditions, using 254 nm, tum diester alkaloids standard solution as directed under the mesaconitine, hypaconitine, aconitine and jesaconitine are Liquid Chromatography according to the following eluted in this order, and each resolution between their peaks conditions, and determine the heights of the peaks corre- is not less than 1.5, respectively. sponding to aconitine, jesaconitine, hypaconitine and System repeatability: To 1 mL of aconitum diester mesaconitine, HTA and HSA, HTJ and HSJ, HTH and HSH,and alkaloids standard solution for purity add a mixture of

HTM and HSM, respectively, and calculate the amounts of phosphate buŠer solution for aconite root and acetonitrile them by the following formulae: the amounts of aconitine, (1:1) to make 10 mL. When the test is repeated 6 times with jesaconitine, hypaconitine and mesaconitine per g calculated 20 mL of this solution under the above operating conditions, on the dried basis are not more than 55 mg, 40 mg, 55 mgand using 231 nm, the relative standard deviation of the peak 120 mg, respectively, and the total amount of them is not height of mesaconitine is not more than 1.5z. more than 230 mg. Loss on drying Not more than 11.0z (6 hours). Amount (mg) of aconitine (C H NO ) 34 47 11 Total ash C H ＝ SA × TA ×10 Powdered Processed Aconite Root 1: Not more than W H SA 4.0z.

Amount (mg) of jesaconitine (C35H49NO12) Powdered Processed Aconite Root 2: Not more than C H 7.0z. ＝ SJ × TJ ×10 W H SJ Acid-insoluble ash Not more than 0.7z. Amount (mg) of hypaconitine (C H NO ) 33 45 10 Assay Weigh accurately about 2 g of Powdered Processed C H ＝ SH × TH ×10 Aconite Root, put in a glass-stoppered centrifuge tube, and W H SH add 1.6 mL of ammonia TS and 20 mL of diethyl ether.

Amount (mg) of mesaconitine (C33H45NO11) Stopper tightly the tube, shake for 30 minutes, centrifuge, C H and separate the ether layer. To the residue add 0.8 mL of ＝ SM × TM ×10 W HSM ammonia TS and 20 mL of diethyl ether, and proceed as above. Repeat this process more three times. Combine all C : Concentration (mg/mL) of aconitine for purity in the SA extracts, evaporate to dryness under reduced pressure, aconitum diester alkaloids standard solution for dissolve the residue in 5 mL of ethanol (99.5), add 30 mL of purity freshly boiled and cooled water, and titrate with 0.01 mol/L C : Concentration (mg/mL) of jesaconitine for purity in SJ hydrochloric acid VS until the color of the solution changes the aconitum diester alkaloids standard solution for from green to gray-blue through blue-green (indicator: 3 purity drops of methyl red-methylene blue TS). Perform a blank C : Concentration (mg/mL) of hypaconitine for purity in SH determination and make any necessary correction. the aconitum diester alkaloids standard solution for purity Each mL of 0.01 mol/L hydrochloric acid VS

CSM: Concentration (mg/mL) of mesaconitine for purity in ＝6.037 mg of total alkaloid [as benzoylaconine

the aconitum diester alkaloids standard solution for (C32H45NO10: 603.70)] purity W: Amount (g) of the sample, calculated on the dried basis

Operating conditions— Detector: An ultraviolet absorption photometer (wavelength: 231 nm for aconitine, hypaconitine and mesaconitine; 254 nm for jesaconitine). Column: A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecyl- silanized silica gel for liquid chromatography (5 mmin 1774 O‹cial Monographs for Part II Supplement II, JPXIV

Add the following: Total ash Not more than 6.5z. Processed Ginger Acid-insoluble ash Not more than 1.5z. Extract content Not less than 8.0z (dilute ethanol-soluble Zingiberis Processum Rhizoma extract). カンキョウ Rhubarb Processed Ginger is the rhizome of Zingiber o‹cinale Roscoe (Zingiberaceae), after being passed ダイオウ through hot water or being steamed. Change the origin/limits of content to read: Description Irregularly compressed and often branched massive rhizome; branched parts slightly curved ovoid or Rhubarb is usually the rhizome of Rheum palmatum oblong-ovoid,2–4cminlength,and1–2cmindiameter; Linnáe, Rheum tanguticum Maximowicz, Rheum external surface grayish yellow to grayish yellow-brown, o‹cinale Baillon, Rheum coreanum Nakai or their with wrinkles and ring node; fractured surface brown to interspeciˆc hybrids (Polygonaceae). dark brown, transparent and horny; under a magnifying It contains not less than 0.25z of sennosides A glass, a transverse section reveals cortex and stele distinctly (C H O : 862.74), calculated on the basis of dried divided; vascular bundles scattered throughout the surface. 42 38 20 material. Odor, characteristic; taste, extremely pungent. Under a microscope, a transverse section reveals cork layer, cortex and stele in this order from the outside; cortex and stele, divided by a single-layered endodermis, composed Powdered Rhubarb of parenchyma, vascular bundles scattered and surrounded ダイオウ末 by ˆber bundles; oil cells contain yellow oil-like substances, scattered in parenchyma; parenchymatous cells contain Change the origin/limits of content to read: solitary crystals of calcium oxalate, and gelatinized starch.

Identiˆcation To 2 g of pulverized Processed Ginger add 5 Powdered Rhubarb is the powder of Rhubarb. mL of diethyl ether, shake for 10 minutes, ˆlter, and use the It contains not less than 0.25z of sennoside A ˆltrate as the sample solution (1). To the residue add 5 mL of (C42H38O20: 862.74), calculated on the basis of dried methanol, proceed in the same manner as above, and use so materials. obtained solution as the sample solution (2). Separately, dissolve 1 mg of [6]-shogaol for thin-layer chromatography in 2 mL of methanol, and use this solution as the standard Add the following: solution (1). Separately, dissolve 1 mg of Sucrose in 2 mL of methanol, and use this solution as the standard solution (2). Sappan Wood Perform the test with these solutions as directed under the Thin-layer Chromatography. Spot 10 mLeachofthesample Sappan Lignum solution (1) and the standard solution (1) on a plate of silica gel for thin-layer chromatography. Develop the plate with a ソボク mixture of ethyl acetate and hexane (1:1) to a distance of about 10 cm, and air-dry the plate. Spray evenly 4- Sappan Wood is the duramen of Caesalpinia sappan dimethylaminobenzaldehyde TS on the plate, heat at 1059C Linnáe (Leguminosae). for 5 minutes, and allow to cool: one of the spot among the Description Chips, slices or short pieces of wood; yellow- several spots from the sample solution (1) has the same color ish red to grayish yellow-brown, sometimes with light brown tone and Rf value with the green spot from the standard to grayish white splint woods; hard in texture; a transverse solution (1). Spot 10 mL each of the sample solution (2) and section shows a pattern like annual ring. the standard solution (2) on a plate of silica gel for thin-layer Almost odorless; almost tasteless. chromatography, develop the plate with a mixture of 1- Under a microscope, a transverse section reveals ray butanol, water and acetic acid (100) (8:5:3) to a distance of composed of 1 – 2 rows of slender and long cells; the area about 10 cm, and air-dry the plate. Spray evenly 1,3- between rays ˆlled with ˆber cells, and large and oblong naphthalenediol TS on the plate, and heat at 1059Cfor5 vessels scattered there; solitary crystals of calcium oxalate in minutes: one of the spot among the several spots from the parenchymatous cells of the innermost of xylem. sample solution (2) has the same color tone and Rf value with the red-purple spot from the standard solution (2). Identiˆcation To 0.5 g of pulverized Sappan Wood add 10 mL of dilute ethanol, shake, and ˆlter. To 5 mL of the Loss on drying Not more than 15.0z (6 hours). ˆltrate add 2 to 3 drops of sodium hydroxide TS: a dark red Supplement II, JPXIV O‹cial Monographs for Part II 1775 color develops. as the sample solution. Perform the test with this solution as directed under the Thin-layer Chromatography. Spot 5 mL Purity Put a small piece of Sappan Wood in calcium of the sample solution on a plate of silica gel for thin-layer hydroxide TS: no purple-blue color develops. chromatography. Develop the plate with a mixture of ethyl Loss on drying Not more than 11.5z (6 hours). acetate, hexane and acetic acid (100) (70:30:1) to a distance of about 10 cm, and air-dry the plate: a yellow spot appears Total ash Not more than 2.0z. at around Rf0.4. Extract content Not less than 7.0z (dilute ethanol-soluble Loss on drying Not more than 17.0z (6 hours). extract). Total ash Not more than 7.5z. Powdered Scutellaria Root Acid-insoluble ash Not more than 1.0z. Extract content Not less than 9.0z (dilute ethanol-soluble オウゴン末 extract).

Change the origin/limits of content to read: Add the following: Powdered Scutellaria Root is the powder of Scutel- laria Root. Tribulus Fruit It contains not less than 10.0z of baicalin (C H O : 446.36), calculated on the basis of dried 21 18 11 Tribuli Fructus material. シツリシ

Add the following: Tribulus Fruit is the fruit of Tribulus terrestris Linnáe (Zygophyllaceae). Termeric Description Pentagonal star shaped fruit, composed of ˆve mericarps, 7 – 12 mm in diameter, often each mericarp Curcumae Rhizoma separated; externally grayish green to grayish brown; a pair of longer and shorter spines on surface of each mericarp, the ウコン longerspine3–7mminlength,theshorterone2–5mmin length, numerous small processes on midrib; pericarp hard Termeric is the rhizome or being removed the cork in texture, cut surface light yellow; each mericarp contains 1 layer from it of Curcuma longa Linnáe (Zingiberaceae), usually after being passed through hot water. – 3 seeds. Almost odorless; taste, mild at ˆrst, followed by bitter- Description Termeric is a main rhizome or a lateral rhi- ness. zome; main rhizome, nearly ovoid, about 3 cm in diameter, Under a microscope, a transverse section reveals epicarp about 4 cm in length; lateral rhizome, cylindrical, with composed of a single-layered epidermis; mesocarp com- round tips, curved, about 1 cm in diameter, 2 - 6 cm in posed of parenchyma and sclerenchyma layer; endocarp length; both main and lateral rhizomes with cyclic nodes; composed of several-layered ˆber cells; a single-layer of cell rhizome with cork layer, yellowish brown, lustrous; rhizome between mesocarp and endocarp contain solitary crystals of without cork layer, dark yellowish red, with yellowish red calcium oxalate; cotyledons of seed contain oil drops and powdersonsurface;hardintexture,noteasilybroken; aleurone grains, and occasionally starch grains. transversely cut surface yellowish brown to reddish brown, Identiˆcation To 2 g of pulverized Tribulus Fruit add 5 mL lustrous like wax. of methanol, shake for 10 minutes, ˆlter, and use the ˆltrate Odor, characteristic; taste, slightly bitter and stimulant, it as the sample solution. Perform the test with this solution as colors a saliva yellow on chewing. directed under the Thin-layer Chromatography. Spot 10 mL Under a microscope, a transverse section reveals the of the sample solution on a plate of silica gel for thin-layer outermost layer to be composed of a cork layer 4 – 10 cells chromatography, develop the plate with a mixture of ethyl thick; sometimes a cork layer partly remains; cortex and acetate and water (40:1) to a distance of about 10 cm, and stele, divided by a single-layered endodermis, composed of air-dry the plate. Spray evenly dilute sulfuric acid on the parenchyma, vascular bundles scattered; oil cells scattered in plate, heat at 1059C for 5 minutes, and examine under parenchyma; parenchymatous cells contain yellow sub- ultraviolet light (main wavelength: 365 nm): a blue-white stances, sandy and solitary crystals of calcium oxalate, and ‰uorescent spot appears at around Rf0.4. gelatinized starch. Purity (1) Peduncle—Not more than 4.0z. Identiˆcation To 0.5 g of pulverized Termeric add 20 mL (2) Foreign matters—Not more than 1.0z of foreign of methanol, shake for 15 minutes, ˆlter and use the ˆltrate 1776 O‹cial Monographs for Part II Supplement II, JPXIV matters other than peduncle. Operating conditions— Detector: An ultraviolet absorption photometer Loss on drying Not more than 11.0z (6 hours). (wavelength: 245 nm). Total ash Not more than 13.0z. Column: A stainless steel column 4.6 mm in inside diameter and 25 cm in length, packed with octadecyl- Acid-insoluble ash Not more than 1.5z. silanized silica gel for liquid chromatography (5 mmin Extract content Not less than 8.5z (dilute ethanol-soluble particle diameter). extract). Column temperature: A constant temperature of about 409C. Mobile phase: Dissolve 3.85 g of ammonium acetate in Uncaria Thorn 200 mL of water, add 10 mL of acetic acid (100) and water to make 1000 mL, and add 350 mL of acetonitrile. チョウトウコウ Flow rate: Adjust the ‰ow rate so that the retention time of rhynchophylline is about 17 minutes. Change the origin/limits of the content to read: System suitability— System performance: Dissolve 5 mg of rhynchophylline Uncaria Thorn is, usually the prickle, of Uncaria for component determination in 100 mL of a mixture of rhynchophylla Miquel, Uncaria sinensis Haviland or methanol and dilute acetic acid (7:3). To 5 mL of this solu- Uncaria macrophylla Wallich (Rubiaceae). tion add 1 mL of ammonia solution (28), and re‰ux for 10 Uncaria Thorn contains not less than 0.03z of total minutes or warm at about 509C for 2 hours. After cooling, alkaloids (rhynchophylline and hirstine), calculated on to 1 mL of the solution so obtained add a mixture of the dried basis. methanol and dilute acetic acid (7:3) to make 5 mL. When theprocedureisrunwith20mLofthissolutionunderthe Change the Component determination to read: above operating conditions, the peak of isorhynchophylline is appears in addition to the peak of rhynchophylline, and Component determination Weigh accurately about 0.2 g the resolution between these peaks is not less than 1.5. of medium powdered Uncaria Thorn, transfer into a glass- System repeatability: When the test is repeated 6 times stoppered centrifuge tube, add 30 mL of a mixture of with 20 mL of the standard solution (1) under the above methanol and dilute acetic acid (7:3), shake for 30 minutes, operating conditions, the relative standard deviation of the centrifuge, and separate the supernatant liquid. To the peak areas of rhynchophylline is not more than 1.5z. residue add two 10-mL portions of a mixture of methanol and dilute acetic acid (7:3), proceed in the same manner, and combine all of the supernatant liquid. To the combined liquid add a mixture of methanol and dilute acetic acid (7:3) Change to read: to make exactly 50 mL, and use this as the sample solution. Separately, weigh accurately about 5 mg of rhynchophylline Wheat Starch for component determination, previously dried in a desicca- tor (silica gel) for 24 hours, and dissolve in a mixture of Amylum Tritici methanol and dilute acetic acid (7:3) to make exactly 100 コムギデンプン mL. Pipet 1 mL of this solution, add a mixture of methanol and dilute acetic acid (7:3) to make exactly 10 mL, and use Wheat Starch consists of the starch granules this solution as the standard solution (1). Separately, dis- obtained from the seeds of Triticum aestivum Linnáe solve 1 mg of hirsutine in 100 mL of a mixture of methanol (Gramineae). and dilute acetic acid (7:3), and use this solution as the stan- dard solution (2). Perform the test with exactly 20 mLeach Description Wheat Starch occurs as white masses or of the sample solution and the standard solution (1) and (2) powder. as directed under the Liquid Chromatography according to It is practically insoluble in water and in ethanol (99.5). the following conditions, and determine the peak areas, A Ta Identiˆcation (1) Under a microscope, Wheat Starch, and A , of rhynchophylline and hirsutine obtained from the Tb preserved in a mixture of water and glycerin (1:1), appears as sample solution, and the peak area, A , of rhynchophylline S large and small sized simple grains, or quite rarely median from the standard solution (1). sized simple grains; usually, large sized grains usually 10 – 60 Amount (mg) of total alkaloids (rhynchophylline and mm in diameter, from upper view, disc like or quite rarely hirstine) reniform, centric hilum and striation indistinct or hardly

ATa＋1.405ATb 1 distinct, often cleft on marginal portion visible; from lateral ＝WS× × AS 20 view, narrowly ellipsoid or fusiform, hilum recognized as a cleft along with long axis; small sized grains 2 – 10 mmin W : Amount (mg) of rhynchophylline for component S diameter, spherical or polygonal; a black cross, its intersec- determination tion point on hilum, is observed when grains are put between Supplement II, JPXIV O‹cial Monographs for Part II 1777 two polarizing prisms ˆxed at right angle to each other. A: Boiling ‰ask (500 mL) (2) To 1 g of Wheat Starch add 50 mL of water, boil for B: Funnel (100 mL) 1 minute, and allow to cool: a subtle white-turbid, pasty C: Condenser liquid is formed. D: Test-tube (3) To 1 mL of the pasty liquid obtained in (2) add 0.05 E: Tap mL of iodine TS: a dark blue-purple color is formed, and the color disappears by heating. (ii) Procedure Introduce 150 mL of water into the boil- ing ‰ask, close the tap of the funnel, and pass carbon pH Put 5.0 g of Wheat Starch in a non-metal vessel, add dioxide through the whole system at a rate of 100±5mLper 25.0 mL of freshly boiled and cooled water, mix gently for 1 minute. Pass cooling water through the condenser, and place minute, and allow to stand for 15 minutes: the pH of the 10 mL of hydrogen peroxide-sodium hydroxide TS in the solution is between 4.5 and 7.0. test-tube. After 15 minutes, remove the funnel without inter- Purity (1) Iron—To 1.5 g of Wheat Starch add 15 mL of rupting the stream of carbon dioxide, and introduce through 2 mol/L hydrochloric acid TS, mix, ˆlter, and use the ˆltrate the opening into the ‰ask about 25 g of Wheat Starch, as the test solution. To 2.0 mL of Standard Iron Solution accurately weighed, with the aid of 100 mL of water. Apply add water to make 20 mL, and use as the control solution. tap grease to the outside of the connection part of the Put 10 mL each of the test solution and the control solution funnel, and load the funnel. Close the tap of the funnel, intesttubes,add2mLofasolutionofcitricacid(2in10) pour 80 mL of 2 mol/L hydrochloric TS acid into the and 0.1 mL of mercapto acetic acid, and mix. Alkalize with funnel, open the tap to introduce the hydrochloric acid into ammonia solution (28) to litmus paper, add water to make the ‰ask, and close the tap while several mL of the 20 mL, and mix. Transfer 10 mL each of these solutions into hydrochloric acid remains, in order to avoid losing sulfur test tubes, allow to stand for 5 minutes, and compare the dioxide. Place the ‰ask in a water bath, and heat the mixture color of these solutions against a white background: the for 1 hour. Transfer the contents of the test-tube with the aid color of the test solution is not darker than that of the of a little water to a wide-necked conical ‰ask. Heat in a control solution (not more than 10 ppm). water bath for 15 minutes, and cool. Add 0.1 mL of (2) Oxidizingsubstances—To4.0gofWheatStarchadd bromophenol blue TS, and titrate with 0.1 mol/L sodium 50.0 mL of water, shake for 5 minutes, and centrifuge. To hydroxide VS until the color changes from yellow to violet- 30.0 mL of the supernatant liquid add 1 mL of acetic acid blue lasting for at least 20 seconds. Perform a blank determi- (100) and 0.5 to 1.0 g of potassium iodide, shake, and allow nation and make any necessary correction. Calculate the to stand for 25 to 30 minutes at a dark place. Add 1 mL of amount of sulfur dioxide by applying the following formula: starch TS, and titrate with 0.002 mol/L sodium thiosulfate it is not more than 50 ppm. VS until the color of the solution disappears. Perform a V blank determination and make any necessary correction: the Amount (ppm) of sulfur dioxide＝ ×1000×3.203 W volume of 0.002 mol/L sodium thiosulfate VS consumed is not more than 1.4 mL (not more than 20 ppm, calculated as W: Amount (g) of the sample hydrogen peroxide). V: Amount (mL) of 0.1 mol/L sodium hydroxide VS (3) Sulfur dioxide— consumed (i) Apparatus Use as shown in the following ˆgure. Loss on drying Not more than 15.0z (1 g, 1309C, 90 minutes).

Residue on ignition Not more than 0.6z (1 g).

Containers and storage Containers—Well-closed contain- ers.

The ˆgures are in mm. Supplement II, JPXIV Infrared Reference Spectra 1779

Change to read: Part I 1780 Infrared Reference Spectra Supplement II, JPXIV

Add the following: Part I Supplement II, JPXIV Infrared Reference Spectra 1781 1782 Infrared Reference Spectra Supplement II, JPXIV Supplement II, JPXIV Infrared Reference Spectra 1783 1784 Infrared Reference Spectra Supplement II, JPXIV Supplement II, JPXIV Infrared Reference Spectra 1785 1786 Infrared Reference Spectra Supplement II, JPXIV Supplement II, JPXIV Infrared Reference Spectra 1787 1788 Infrared Reference Spectra Supplement II, JPXIV Supplement II, JPXIV Infrared Reference Spectra 1789

Add the following: Part II Supplement II, JPXIV Ultraviolet-visible Reference Spectra 1791

Delete the following Ultraviolet-visible Reference Spectra:

Part I

Furosemide 1 Furosemide 2 1792 Ultraviolet-visible Reference Spectra Supplement II, JPXV

Change to read: Part I Supplement II, JPXV Ultraviolet-visible Reference Spectra 1793

Add the following: Part I 1794 Ultraviolet-visible Reference Spectra Supplement II, JPXV Supplement II, JPXV Ultraviolet-visible Reference Spectra 1795 1796 Ultraviolet-visible Reference Spectra Supplement II, JPXV Supplement II, JPXV Ultraviolet-visible Reference Spectra 1797 1798 Ultraviolet-visible Reference Spectra Supplement II, JPXV Supplement II, JPXV Ultraviolet-visible Reference Spectra 1799 General Information

Change to read: 5. International Harmonization ImplementedintheJapanese Pharmacopoeia Fourteenth Edition

Items for which harmonization has been agreed among the European Pharmacopoeia, the United States Phar­ macopeia and the Japanese Pharmacopoeia are implement­ ed in the Japanese Pharmacopoeia Fourteenth Edition (JP 14). Theyare shown in the tables below. The column headed Harmonized items shows the harmonized items written in the Pharmacopoeial Harmonization Agreement Document, and the column headed JP 14 shows the items as theyappear in JP 14. In the Remarks column, notes on anydiŠerences between JP 14 and the agreement are shown as occasion demands.

Harmonized items JP 14 Remarks

Bacterial Endotoxins Test Bacterial Endotoxins Test Apparatus Apparatus Preparation of Standard Endotoxin Preparation of Standard Endotoxin Stock Solution Stock Solution Preparation of Standard Endotoxin Preparation of Standard Endotoxin Solution Solution Preparation of Sample Solutions Preparation of Sample Solutions Determination of Maximum Valid Di­ Determination of Maximum Valid Di­ lution lution Gel­clot technique Gel­clot techniques (1) Preparatorytesting (1) Preparatorytesting (2) Limit test (2) Limit test (3) Assay (3) Assay Photometric techniques Photometric techniques (1) Turbidimetric technique (1) Turbidimetric technique (2) Chromogenic technique (2) Chromogenic technique (3) Preparatorytesting (3) Preparatorytesting (4) Assay (4) Assay Reagents, Test Solutions Reagents, Test Solutions Amebocyte lysate Lysate reagent Lysate TS Lysate TS Water for bacterial endotoxins test Water for bacterial endotoxins test (BET) (BET)

Note: The method for decision of the limit for bacterial endotoxins was agreed between the three pharmacopoeias, but in the Decision of Limit for Bacterial Endotoxins under the General Information in JP 14, the maximum adult dose is calculated basedonanaveragebodymassofanadultof60kg. 1801 1802 General Information Supplement II, JPXIV

Harmonized items JP 14 Remarks

Residue on IgnitionWSulphated Ash ResidueonIgnitionTest Test (Introduction) (Introduction) Explanation of JP's particular expres­ sions in the introduction: The descrip­ tion, for example, ``not more than 0.10z (1g),'' in a monograph, indi­ cates that the mass of the residue is not more than 1.0 mg per g of the sub­ stance to be tested in which about 1g of the substance is weighed accurately and ignited by the procedure described below, and ``after drying'' indicates that the sample is tested after being dried under the conditions speciˆed in the test for Loss on drying. Procedure Procedure Explanation of JP's particular expres­ sions for taking the sample: When the quantity of the sample to be taken is in­ dicated in a volume, pipet exactly the amount directed in the monograph and transfer to the above crucible. When directed as ``after evaporating,'' heat properly to evaporate the solution.

Harmonized items JP 14 Remarks

Sterility Sterility Test Precautions against microbial contamination Comment 1 Culture media and incubation temperatures Media and rinsing ‰uids Fluid thioglycollate medium Fluid thioglycolate medium Note 1– 5 Soya­bean casein digest medium Soybean­casein digest medium Sterility Sterility of media Growth promotion test of aerobes, anaerobes Growth promotion test and fungi Validation test Validation test Note 6 Membrane ˆltration Membrane ˆltration Direct inoculation Direct inoculation Test for sterility of the product to be examined Test for sterility of the products to be examined Membrane ˆltration Membrane ˆltration Aqueous solutions a) Liquid medicines Note 7 Soluble solids b) Solid medicines Comment 2 Oils and oily solutions c) Oils and oily solutions Ointments and creams d) Ointments and creams Direct inoculation of the culture medium Direct inoculation of the culture medium Oily liquids a) Oily liquids Ointments and creams b) Ointments and creams Supplement II, JPXIV General Information 1803

Catgut and other surgical sutures for veterinary use Comment 3 Observation and interpretation of results Cultivation and observation, Observation and inter­ Note8–9 pretation of results Application of the test to parenteral preparations, Comment 4 ophthalmic and other non­injectable preparations required to comply with the test for sterility Table 2.6.1.­1. Strains of the test micro­organ­ Table 1. Microorganisms for growth promotion isms suitable for use in the Growth Promotion test and the validation test TestandtheValidationTest Table 2.6.1.­2. Minimum quantity to be used for Table 3. Minimum quantity to be used for each each medium medium Table 2.6.1.­3. Minimum number of items to be Table 2. Number of items to be taken from the Note10–11 tested lot

Note: 1) Non­pharmaceutical media: Not to be used. 2) Water content of agar: Not being speciˆed. 3) EŠective period of media: Unnecessary to be validated. 4) EŠective period of media stored in hermetic containers: Usable for maximum one year. 5) Medium for sterility test of the products containing a mercurial preservative: For products that cannot be tested by the membrane­ˆltration method, use ‰uid thioglycolate medium incubated at 20 – 259C instead of soybean­casein digest medium. 6) Periodic growth promotion test for a ready­made powder medium: Not need to test with respect to each batch, provided it is prepared under well­controlled conditions. 7) Amount of the rinsing ‰uid each time in the membrane­ˆltration method: 100 mL per ˆlter. 8) Transferring amount from turbid medium to fresh medium: A suitable amount. 9) Requirements for the retesting in the case when the evidence of microbial growth is found: If the microbial growth is found the product examined does not comply with the test for sterility. However, provided that various factors and/or properties of the contaminant suggest that the test itself was inadequate, the test may be repeated. If no evidence of microbial growth is found in the repeat test the product complies with the Sterility Test. If microbial growth is found in the repeat test the product does not comply with the Sterility Test. 10) The table of 'Number of items to be taken from the lot': Speciˆed as a part of the General Test. 11) Number of large­volume products (more than 100 mL) to be taken from the lot: Maximum 10 containers.

Comment 1: The same statement appears in the introduction. Comment 2: There is a statement in 11. Injections (9) under the General Rules for Preparations that ``Unless otherwise speciˆed, Injections meet the requirements of the Sterility Test. In the case of drugs to be dissolved before use, carry out the test with the solution obtained by dissolving the contents in the attached solvent.'' Comment 3: The items not included in JP. Comment 4: A part of this is directed in the General Test. 1804 General Information Supplement II, JPXIV

Harmonized items JP 14 Remarks

Sodium Chloride Sodium Chloride Deˆnition limits of the content Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Acidity or alkalinity Purity (2) Acidity or alkalinity Bromides Purity (5) Bromides Ferrocyanides Purity (7) Ferrocyanides Iodides Purity (6) Iodides Nitrites not speciˆed Phosphates Purity (4) Phosphates Sulphates Purity (3) Sulfates Aluminium not speciˆed Barium Purity (10) Barium Iron Purity (9) Iron Magnesium and alkaline­earth metals Purity (11) Magnesium and alkaline­ earth materials Potassium not speciˆed Loss on drying Loss on drying Bacterial endotoxins not speciˆed Assay Assay

Harmonized items JP 14 Remarks

Citric Acid Monohydrate Citric Acid Deˆnition limits of the content Identiˆcation Identiˆcation Appearance of solution Purity (1) Clarity and color of solu­ tion Readily carbonisable substances Purity (5) Readily carbonizable sub­ stances Oxalic acid Purity (3) Oxalate Sulphates Purity (2) Sulfates Aluminium not speciˆed Water Water Sulphated ash Residue on ignition Bacterial endotoxins not speciˆed Assay Assay Storage Containers and storage Supplement II, JPXIV General Information 1805

Harmonized items JP 14 Remarks

Citric Acid, Anhydrous Anhydrous Citric Acid Deˆnition limits of the content Identiˆcation Identiˆcation Appearance of solution Purity (1) Clarity and color of solu­ tion Readily carbonisable substances Purity (5) Readily carbonizable sub­ stances Oxalic acid Purity (3) Oxalate Sulphates Purity (2) Sulfates Aluminium not speciˆed Water Water Sulphated ash Residue on ignition Bacterial endotoxins not speciˆed Assay Assay Storage Containers and storage

Harmonized items JP 14 General Infromation Remarks

Carboxymethylcellulose Calcium Carmellose Calcium Deˆnition origin Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Identiˆcation C Identiˆcation (3) Identiˆcation D Identiˆcation (4) Alkalinity Purity (1) Alkali Loss on drying Loss on drying Residue on ignition Residue on ignition Limit of chloride Purity (2) Chloride Limit of sulfate Purity (3) Sulfate 1806 General Information Supplement II, JPXIV

Harmonized items JP 14 General Infromation Remarks

Wheat Starch Wheat Starch Deˆnition origin Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Identiˆcation C Identiˆcation (3) pH pH Iron Purity (1) Iron Total protein not speciˆed Oxidising substances Purity (2) Oxidizing substances Sulphur dioxide Purity (3) Sulfur dioxide Loss on drying Loss on drying Sulphated ash Residue on ignition

Harmonized items JP 14 Remarks

Cellacefate Cellulose Acetate Phthalate Deˆnition limits of the content Identiˆcation Identiˆcation Viscosity Viscosity Limit of free acid Purity (2) Free acids Water Water Residue on ignition Residue on ignition Phthalyl content Assay (1) Carboxybenzoyl Content of acetyl Assay (2) Acetyl group Packaging and storage Containers and storage

Harmonized items JP 14 General Infromation Remarks

Corn Starch Corn Starch Deˆnition origin Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Identiˆcation C Identiˆcation (3) pH pH Loss on drying Loss on drying Residue on ignition Residue on ignition Limit of iron Purity (1) Iron Limit of oxidizing substances Purity (2) Oxidizing substances Sulfur dioxide determination Purity (3) Sulfur dioxide Supplement II, JPXIV General Information 1807

Harmonized items JP 14 General Infromation Remarks

Potato Starch Potato Starch Deˆnition origin Identiˆcation A Identiˆcation (1) Identiˆcation B Identiˆcation (2) Identiˆcation C Identiˆcation (3) pH pH Iron Purity (1) Iron Oxidising substances Purity (2) Oxidizing substances Sulphur dioxide Purity (3) Sulfur dioxide Loss on drying Loss on drying Sulphated ash Residue on ignition

Harmonized items JP 14 Remarks

General Information Sodium Dodecyl Sulphate Poly­ SDS­Polyacrylamide Gel Electrophore­ acrylamide Gel Electrophoresis sis (SDS­PAGE) Characteristics of Polyacrylamide 1. Characteristics of Polyacrylamide Gels Gels Denaturing Polyacrylamide Gel Elec­ 2. Polyacrylamide Gel Electrophore­ trophoresis sis under Denaturing Conditions Reducing conditions 1) Reducing conditions Non­reducing conditions 2) Non­reducing conditions Characteristics of Discontinuous 3. Characteristics of Discontinuous BuŠer System Gel Electrophoresis BuŠer System Gel Electrophoresis Preparing Vertical Discontinuous 4. Preparing Vertical Discontinuous BuŠer SDS Polyacrylamide Gels BuŠer SDS­Polyacrylamide Gels Assembling of the gel moulding cas­ 1) Assembling of the gel moulding sette cassette Preparation of the gel 2) Preparation of the gel Mounting the gel in the electrophore­ 3) Mounting the gel in the electro­ sis apparatus and electrophoretic phoresis apparatus and electro­ separation phoretic separation Detection of Protein in Gels 5. Detection of Proteins in Gels Coomassie staining 1) Coomassie staining Silver staining 2) Silver staining Drying of Stained SDS Poly­ 6. Drying of Stained SDS­ acrylamide Gels Polyacrylamide Gels Molecular­Mass Determination 7. Molecular­Mass Determination Validation of the Test 8. Suitability of the Test Quantiˆcation of Impurities 9. Quantiˆcation of Impurities Reagents, Test Solutions Test Solutions Blocking solution Blocking TS 1808 General Information Supplement II, JPXIV

Coomassie staining solution Coomassie staining TS Destaining solution Destaining TS Developer solution Developer TS Fixing solution Fixing TS Silver nitrate reagent Silver nitrate TS for silver staining Trichloroacetic acid reagent Trichloroacetic acid TS for ˆxing Table 1 | Preparation of resolving Table 1. Preparation of resolving gel gel Table 2 | Preparation of stacking gel Table 2. Preparation of stacking gel

Harmonized items JP 14 General Infromation Remarks

Benzyl Alcohol Benzyl Alcohol Deˆnition limits of the content Identiˆcation Identiˆcation Appearance of solution Purity (1) Clarity and color of solu­ tion Refractive index Refractive index Acidity Purity (2) Acid Benzaldehyde and other related Purity (3) Benzaldehyde and other substances related substances Peroxide value Purity (4) Peroxide value Residue on evaporation Purity (5) Residue on evaporation Assay Assay

Harmonized items JP 14 Remarks

General Information Amino Acid Analysis Amino Acid Analysis Apparatus Apparatus General Precautions General Precautions Reference Standard Material Reference Standard Material Calibration of Instrumentation Calibration of Instrumentation Repeatability Repeatability Sample Preparation Sample Preparation Internal Standards Internal Standards Protein Hydrolysis Protein Hydrolysis Method 1 Method 1 Hydrolysis Solution Hydrolysis Solution Procedure Procedure Method 2 Method 2 Hydrolysis Solution Hydrolysis Solution Vapor Phase Hydrolysis Vapor Phase Hydrolysis Method 3 Method 3 Supplement II, JPXIV General Information 1809

Hydrolysis Solution Hydrolysis Solution Vapor Phase Hydrolysis Vapor Phase Hydrolysis Method 4 Method 4 Oxidation Solution Oxidation Solution Procedure Procedure Method 5 Method 5 Hydrolysis Solution Hydrolysis Solution Liquid Phase Hydrolysis Liquid Phase Hydrolysis Method 6 Method 6 Hydrolysis Solution Hydrolysis Solution Vapor Phase Hydrolysis Vapor Phase Hydrolysis Method 7 Method 7 Reducing Solution Reducing Solution Procedure Procedure Method 8 Method 8 StockSolutions StockSolutions Reducing Solution Reducing Solution Procedure Procedure Method 9 Method 9 StockSolutions StockSolutions Carboxymethylation Solution Carboxymethylation Solution BuŠer Solution BuŠer Solution Procedure Procedure Method 10 Method 10 Reducing Solution Reducing Solution Procedure Procedure Method 11 Method 11 Reducing Solutions Reducing Solutions Procedure Procedure Methodologies of Amino Acid Analysis General Methodologies of Amino Acid Analysis General Principles Principles Method 1­Postcolumn Ninhydrin Detection Method 1­Postcolumn Ninhydrin Detection General Principle General Principle Method 2­Postcolumn OPA Fluorometric Detec­ Method 2­Postcolumn OPA Fluorometric Detec­ tion General Principle tion General Principle Method 3­Precolumn PITC Derivatization Method 3­Precolumn PITC Derivatization General Principle General Principle Method 4­Precolumn AQC Derivatization Gener­ Method 4­Precolumn AQC Derivatization Gener­ al Principle al Principle Method 5­Precolumn OPA Derivatization Gener­ Method 5­Precolumn OPA Derivatization Gener­ al Principle al Principle Method 6­Precolumn DABS­Cl Derivatization Method 6­Precolumn DABS­Cl Derivatization General Principle General Principle Method 7­Precolumn FMOC­Cl Derivatization Method 7­Precolumn FMOC­Cl Derivatization General Principle General Principle 1810 General Information Supplement II, JPXIV

Method 8­Precolumn NBD­F Derivatization Method 8­Precolumn NBD­F Derivatization General Principle General Principle Data Calculation and Analysis Data Calculation and Analysis Calculations Calculations Amino Acid Mole Percent Amino Acid Mole Percent Unknown Protein Samples Unknown Protein Samples Known Protein Samples Known Protein Samples

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General Information Capillary Electrophoresis Capillary Electrophoresis Apparatus Apparatus Capillary Zone Electrophoresis Capillary Zone Electrophoresis Optimisation Optimization Instrumental parameters Instrumental parameters Voltage Voltage Polarity Polarity Temperature Temperature Capillary Capillary Electrolytic solution parameters Electrolytic solution parameters BuŠer type and concentration BuŠer type and concentration BuŠer pH BuŠer pH Organic solvents Organic solvents Additives for chiral separations Additives for chiral separations Capillary Gel Electrophoresis Capillary Gel Electrophoresis Characteristics of Gels Characteristics of Gels Capillary Isoelectric Focusing Capillary Isoelectric Focusing Loading step Loading step loading in one step loading in one step sequential loading sequential loading Focusing step Focusing step Mobilisation step Mobilization step Optimisation Optimization Voltage Voltage Capillary Capillary Solutions Solutions Micellar Electrokinetic Chromatography (MEKC) Micellar Electrokinetic Chromatography (MEKC) Optimisation Optimization Instrumental parameters Instrumental parameters Voltage Voltage Temperature Temperature Capillary Capillary Electrolytic solution parameters Electrolytic solution parameters Supplement II, JPXIV General Information 1811

Surfactant type and concentration Surfactant type and concentration BuŠer pH BuŠer pH Organic solvents Organic solvents Additives for chiral separations Additives for chiral separations Other additives Other additives Quantiˆcation Quantiˆcation Calculations Calculations System Suitability System Suitability Apparent Number of Theoretical Plates Apparent Number of Theoretical Plates Resolution Resolution Symmetry Factor Symmetry Factor Signal­to­noise Ratio Signal­to­noise Ratio

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General Information Total Protein Assay Total Protein Assay Method 1 Method 1 Standard Solution Standard Solution Test Solution Test Solution Procedure Procedure Light­Scattering Light­Scattering Calculations Calculations Method 2 Method 2 Explanatory footnote ``Ex­ Standard Solutions Standard Solutions ample: the Minimum Re­ quirements for Biological Test Solution Test Solution Products and individual Blank Blank monograph of JP'' is added. Reagents and Solutions Reagents and Solutions Copper Sulfate Reagent Copper Sulfate Reagent SDS Solution 5z SDS TS Sodium Hydroxide Solution Sodium Hydroxide Solution Alkaline Copper Reagent Alkaline Copper Reagent Diluted Folin­Ciocalteu's Phenol Ragent Diluted Folin's TS Procedure Procedure Calculations Calculations Interfering Substances Interfering Substances Sodium Deoxycholate Reagent Sodium Deoxycholate Reagent Trichloroacetic Acid Reagent Trichloroacetic Acid Reagent Procedure Procedure Method 3 Method 3 Standard Solutions Standard Solutions Test Solution Test Solution Blank Blank Coomassie Reagent Coomassie Reagent 1812 General Information Supplement II, JPXIV

Procedure Procedure Calculations Calculations Method 4 Method 4 Standard Solutions Standard Solutions Test Solution Test Solution Blank Blank Reagents Reagents and Solutions BCA Reagent BCA Reagent Copper Sulfate Reagent Copper Sulfate Reagent Copper­BCA Reagent Copper­BCA Reagent Procedure Procedure Calculations Calculations Method 5 Method 5 For the preparation of biuret Standard Solutions Standard Solutions reagent, dissolving copper (II) sulfate and trisodium ci­ Test Solution Test Solution trate dihydrate in water, not Blank Blank inhotwater,andwarmingif Biuret Reagent Biuret Reagent necessary. Addition of the handling of Procedure Procedure standard solutions to the Calculations Calculations Procedure. Interfering Substances Interfering Substances Comments Comments Method 6 Method 6 Standard Solutions Standard Solutions Test Solution Test Solution Blank Blank Reagents Reagents and Solutions Borate BuŠer Borate BuŠer Stock OPA Reagent Stock OPA Reagent OPA Reagent OPA Reagent Procedure Procedure Calculations Calculations Method 7 Method 7 Procedure A Procedure A Procedure B Procedure B Calculations Calculations Supplement II, JPXIV General Information 1813

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General Information Isoelectric Focusing Isoelectric Focusing Theoretical Aspects Theoretical Aspects Practical Aspects Practical Aspects Apparatus Apparatus Isoelectric Focusing in Polyacrylamide Isoelectric Focusing in Polyacrylamide Gels: Detailed Procedure Gels: Detailed Procedure Preparation of the Gels Preparation of the Gels 1) 7.5 per cent polyacrylamide gel 7.5z Polyacrylamide gel 2) Preparation of the mould Preparation of the mould Method Method Variations to the Detailed Procedure Variations to the Detailed Procedure (subject to validation) (Subject to Validation) Validation of Iso­electric Focusing Proce­ Validation of Iso­Electric Focusing Pro­ dures cedures Speciˆed Variation to the General Method Speciˆed Variations to the General Method Point to Consider Points to Consider Figure­Mould Figure. Mould Reagents Reagents and Solutions Coomassie staining solution Fixing solution for isoelectric focusing in Fixing solution for isoelectric focusing in and destaining solution are polyacrylamide gel polyacrylamide gel speciˆed. Coomassie staining TS Destaining solution

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General Information Peptide Mapping Peptide Mapping Purpose and Scope Purpose and Scope The Peptide Map The Peptide Map Isolation and Puriˆcation Isolation and Puriˆcation Selective Cleavage of Peptide Bonds Selective Cleavage of Peptide Bonds Pretreatment of Sample Pretreatment of Sample Pretreatment of the Cleavage Agent Pretreatment of the Cleavage Agent Pretreatment of the Protein Pretreatment of the Protein Establishment of Optimal Digestion Conditions Establishment of Optimal Digestion Conditions pH pH Temperature Temperature Time Time Amount of Cleavage Agent Amount of Cleavage Agent Chromatographic Separation Chromatographic Separation Chromatographic Column Chromatographic Column Solvent Solvent 1814 General Information Supplement II, JPXIV

Mobile Phase Mobile Phase Gradient Selection Gradient Selection Isocratic Selection Isocratic Selection Other Parameters Other Parameters Validation Validation Analysis and Identiˆcation of Peptides Analysis and Identiˆcation of Peptides Table 1. Examples of Cleavage Agents. Table 1. Examples of Cleavage Agents Table 2. Techniques Used for the Separation of Table 2. Techniques Used for the Separation of Peptides. Peptides

Add the following: instrumentation be dedicated particularly for amino acid analysis.

20. Amino Acid Analysis General Precautions Background contamination is always a concern for the Amino acid analysis refers to the methodology used to de­ analyst in performing amino acid analysis. High purity termine the amino acid composition or content of proteins, reagents are necessary (e.g., low purity hydrochloric acid peptides, and other pharmaceutical preparations. Proteins can contribute to glycine contamination). Analytical and peptides are macromolecules consisting of covalently reagents are changed routinely every few weeks using only bonded amino acid residues organized as a linear polymer. high­pressure liquid chromatography (HPLC) grade sol­ The sequence of the amino acids in a protein or peptide vents. Potential microbial contamination and foreign determines the properties of the molecule. Proteins are material that might be present in the solvents are reduced by considered large molecules that commonly exist as folded ˆltering solvents before use, keeping solvent reservoirs structures with a speciˆc conformation, while peptides are covered, and not placing amino acid analysis instrumenta­ smaller and may consist of only a few amino acids. Amino tion in direct sunlight. acid analysis can be used to quantify protein and peptides, to Laboratory practices can determine the quality of the determine the identity of proteins or peptides based on their amino acid analysis. Place the instrumentation in a low amino acid composition, to support protein and peptide tra‹c area of the laboratory. Keep the laboratory clean. structure analysis, to evaluate fragmentation strategies for Clean and calibrate pipets according to a maintenance peptide mapping, and to detect atypical amino acids that schedule. Keep pipet tips in a covered box; the analysts may might be present in a protein or peptide. It is necessary to not handle pipet tips with their hands. The analysts may hydrolyze a protein/peptide to its individual amino acid wear powder­free latex or equivalent gloves. Limit the constituents before amino acid analysis. Following pro­ number of times a test sample vial is opened and closed tein/peptide hydrolysis, the amino acid analysis procedure because dust can contribute to elevated levels of glycine, can be the same as that practiced for free amino acids in serine, and alanine. other pharmaceutical preparations. The amino acid con­ A well­maintained instrument is necessary for acceptable stituents of the test sample are typically derivatized for anal­ amino acid analysis results. If the instrument is used on a ysis. routine basis, it is to be checked daily for leaks, detector and lamp stability, and the ability of the column to maintain Apparatus resolution of the individual amino acids. Clean or replace all Methods used for amino acid analysis are usually based on instrument ˆlters and other maintenance items on a routine a chromatographic separation of the amino acids present in schedule. the test sample. Current techniques take advantage of the automated chromatographic instrumentation designed for Reference Standard Material analytical methodologies. An amino acid analysis instru­ Acceptable amino acid standards are commercially ment will typically be a low­pressure or high­pressure liquid available for amino acid analysis and typically consist of an chromatograph capable of generating mobile phase aqueous mixture of amino acids. When determining amino gradients that separate the amino acid analytes on a chro­ acid composition, protein or peptide standards are analyzed matographic column. The instrument must have postcolumn with the test material as a control to demonstrate the integri­ derivatization capability, unless the sample is analyzed using ty of the entire procedure. Highly puriˆed bovine serum precolumn derivatization. The detector is usually an ultrav­ albumin has been used as a protein standard for this iolet­visible or ‰uorescence detector depending on the purpose. derivatization method used. A recording device (e.g., Calibration of Instrumentation integrator) is used for transforming the analog signal from Calibration of amino acid analysis instrumentation the detector and for quantitation. It is preferred that Supplement II, JPXIV General Information 1815 typically involves analyzing the amino acid standard, which variation limits to ensure the best results. Areas to focus on consists of a mixture of amino acids at a number of concen­ to lower the variability of the amino acid analysis include trations, to determine the response factor and range of sample preparation, high background spectral interference analysis for each amino acid. The concentration of each due to quality of reagents and/or laboratory practices, amino acid in the standard is known. In the calibration instrument performance and maintenance, data analysis and procedure, the analyst dilutes the amino acid standard to interpretation, and analyst performance and habits. All several diŠerent analyte levels within the expected linear parameters involved are fully investigated in the scope of the range of the amino acid analysis technique. Then, replicates validation work. at each of the diŠerent analyte levels can be analyzed. Peak Sample Preparation areas obtained for each amino acid are plotted versus the Accurate results from amino acid analysis require puriˆed known concentration for each of the amino acids in the protein and peptide samples. BuŠer components (e.g., salts, standard dilution. These results will allow the analyst to urea, detergents) can interfere with the amino acid analysis determine the range of amino acid concentrations where the and are removed from the sample before analysis. Methods peak area of a given amino acid is an approximately linear that utilize postcolumn derivatization of the amino acids are function of the amino acid concentration. It is important generally not aŠected by buŠer components to the extent that the analyst prepare the samples for amino acid analysis seen with precolumn derivatization methods. It is desirable so that they are within the analytical limits (e.g., linear to limit the number of sample manipulations to reduce working range) of the technique employed in order to obtain potential background contamination, to improve analyte accurate and repeatable results. recovery, and to reduce labor. Common techniques used to Four to six amino acid standard levels are analyzed to remove buŠer components from protein samples include the determine a response factor for each amino acid. The following methods: (1) injecting the protein sample onto a response factor is calculated as the average peak area or reversed­phase HPLC system, eluting the protein with a peak height per nmol of amino acid present in the standard. volatile solvent containing a su‹cient organic component, A calibration ˆle consisting of the response factor for each and drying the sample in a vacuum centrifuge; (2) dialysis amino acid is prepared and used to calculate the concentra­ against a volatile buŠer or water; (3) centrifugal ultraˆltra­ tion of each amino acid present in the test sample. This tion for buŠer replacement with a volatile buŠer or water; calculation involves dividing the peak area corresponding to (4) precipitating the protein from the buŠer using an organic a given amino acid by the response factor for that amino solvent (e.g., acetone); and (5) gel ˆltration. acid to give the nmol of the amino acid. For routine analysis, a single­point calibration may be su‹cient; however, the Internal Standards calibration ˆle is updated frequently and tested by the It is recommended that an internal standard be used to analysis of analytical controls to ensure its integrity. monitor physical and chemical losses and variations during amino acid analysis. An accurately known amount of Repeatability internal standard can be added to a protein solution prior to Consistent high quality amino acid analysis results from hydrolysis. The recovery of the internal standard gives the an analytical laboratory require attention to the repeatability general recovery of the amino acids of the protein solution. of the assay. During analysis of the chromatographic separa­ Free amino acids, however, do not behave in the same way tion of the amino acids or their derivatives, numerous peaks as protein­bound amino acids during hydrolysis because can be observed on the chromatogram that correspond to their rates of release or destruction are variable. Therefore, the amino acids. The large number of peaks makes it neces­ the use of an internal standard to correct for losses during sary to have an amino acid analysis system that can repeat­ hydrolysis may give unreliable results. It will be necessary to edly identify the peaks based on retention time and integrate take this point under consideration when interpreting the the peak areas for quantitation. A typical repeatability results. Internal standards can also be added to the mixture evaluation involves preparing a standard amino acid of amino acids after hydrolysis to correct for diŠerences in solution and analyzing many replicates (i.e., six analyses or sample application and changes in reagent stability and ‰ow more) of the same standard solution. The relative standard rates. Ideally, an internal standard is an unnaturally deviation (RSD) is determined for the retention time and occurring primary amino acid that is commercially available integrated peak area of each amino acid. An evaluation of and inexpensive. It should also be stable during hydrolysis, the repeatability is expanded to include multiple assays its response factor should be linear with concentration, and conducted over several days by diŠerent analysts. Multiple it needs to elute with a unique retention time without assays include the preparation of standard dilutions from overlapping other amino acids. Commonly used amino acid starting materials to determine the variation due to sample standards include norleucine, nitrotyrosine, and a­aminobu­ handling. Often the amino acid composition of a standard tyric acid. protein (e.g., bovine serum albumin) is analyzed as part of the repeatability evaluation. By evaluating the replicate Protein Hydrolysis variation (i.e., RSD), the laboratory can establish analytical Hydrolysis of protein and peptide samples is necessary for limits to ensure that the analyses from the laboratory are amino acid analysis of these molecules. The glassware used under control. It is desirable to establish the lowest practical for hydrolysis must be very clean to avoid erroneous results. 1816 General Information Supplement II, JPXIV

Glove powders and ˆngerprints on hydrolysis tubes may technique will allow the analyst to account for some residue cause contamination. To clean glass hydrolysis tubes, boil destruction. Microwave acid hydrolysis has been used and is tubes for 1 hour in 1 mol/L hydrochloric acid or soaktubes rapid but requires special equipment as well as special in concentrated nitric acid or in a mixture of concentrated precautions. The optimal conditions for microwave hydroly­ hydrochloric acid and concentrated nitric acid (1:1). Clean sis must be investigated for each individual protein/peptide hydrolysis tubes are rinsed with high­purity water followed sample. The microwave hydrolysis technique typically by a rinse with HPLC grade methanol, dried overnight in an requires only a few minutes, but even a deviation of one oven, and stored covered until use. Alternatively, pyrolysis minute may give inadequate results (e.g., incomplete hydrol­ of clean glassware at 5009C for 4 hours may also be used to ysis or destruction of labile amino acids). Complete proteol­ eliminate contamination from hydrolysis tubes. Adequate ysis, using a mixture of proteases, has been used but can be disposable laboratory material can also be used. complicated, requires the proper controls, and is typically Acid hydrolysis is the most common method for hydrolyz­ more applicable to peptides than proteins. ing a protein sample before amino acid analysis. The acid Note: During initial analyses of an unknown protein, hydrolysis technique can contribute to the variation of the experiments with various hydrolysis time and temperature analysis due to complete or partial destruction of several conditions are conducted to determine the optimal condi­ amino acids. Tryptophan is destroyed; serine and threonine tions. are partially destroyed; methionine might undergo oxida­ Method 1 tion; and cysteine is typically recovered as cystine (but Acid hydrolysis using hydrochloric acid containing phenol cystine recovery is usually poor because of partial destruc­ is the most common procedure used for protein/peptide tion or reduction to cysteine). Application of adequate hydrolysis preceding amino acid analysis. The addition of vacuum (Åless than 200 mm of mercury or 26.7 Pa) or phenol to the reaction prevents the halogenation of tyrosine. introduction of an inert gas (argon) in the headspace of the Hydrolysis Solution 6 mol/L hydrochloric acid contain­ reaction vessel can reduce the level of oxidative destruction. ing 0.1z to 1.0z of phenol. In peptide bonds involving isoleucine and valine the amido Procedure— bonds of Ile­Ile, Val­Val, Ile­Val, and Val­Ile are partially Liquid Phase Hydrolysis Place the protein or peptide cleaved; and asparagine and glutamine are deamidated, sample in a hydrolysis tube, and dry. [Note: The sample is d­ resulting in aspartic acid and glutamic acid, respectively. ried so that water in the sample will not dilute the acid used The loss of tryptophan, asparagine, and glutamine during an for the hydrolysis.] Add 200 mLofHydrolysis Solution per acid hydrolysis limits quantitation to 17 amino acids. Some 500 mg of lyophilized protein. Freeze the sample tube in a dry of the hydrolysis techniques described are used to address ice­acetone bath, and ‰ame seal in vacuum. Samples are these concerns. Some of the hydrolysis techniques described typically hydrolyzed at 1109C for 24 hours in vacuum or (i.e., Methods 4­11) may cause modiˆcations to other amino inert atmosphere to prevent oxidation. Longer hydrolysis acids. Therefore, the beneˆts of using a given hydrolysis times (e.g., 48 and 72 hours) are investigated if there is a technique are weighed against the concerns with the tech­ concern that the protein is not completely hydrolyzed. nique and are tested adequately before employing a method Vapor Phase Hydrolysis This is one of the most com­ other than acid hydrolysis. mon acid hydrolysis procedures, and it is preferred for A time­course study (i.e., amino acid analysis at acid microanalysis when only small amounts of the sample are hydrolysis times of 24, 48, and 72 hours) is often employed available. Contamination of the sample from the acid to analyze the starting concentration of amino acids that are reagent is also minimized by using vapor phase hydrolysis. partially destroyed or slow to cleave. By plotting the Place vials containing the dried samples in a vessel that observed concentration of labile amino acids (i.e., serine and contains an appropriate amount of Hydrolysis Solution.The threonine) versus hydrolysis time, the line can be extrapolat­ Hydrolysis Solution does not come in contact with the test ed to the origin to determine the starting concentration of sample. Apply an inert atmosphere or vacuum (Å less than these amino acids. Time­course hydrolysis studies are also 200 mm of mercury or 26.7 Pa) to the headspace of the used with amino acids that are slow to cleave (e.g., isoleu­ vessel, and heat to about 1109C for a 24­hour hydrolysis cine and valine). During the hydrolysis time course, the time. Acid vapor hydrolyzes the dried sample. Any conden­ analyst will observe a plateau in these residues. The level of sation of the acid in the sample vials is minimized. After this plateau is taken as the residue concentration. If the hydrolysis, dry the test sample in vacuum to remove any hydrolysis time is too long, the residue concentration of the residual acid. sample will begin to decrease, indicating destruction by the hydrolysis conditions. Method 2 An acceptable alternative to the time­course study is to Tryptophan oxidation during hydrolysis is decreased by subject an amino acid calibration standard to the same using mercaptoethanesulfonic acid (MESA) as the reducing hydrolysis conditions as the test sample. The amino acid in acid. free form may not completely represent the rate of destruc­ Hydrolysis Solution 2.5 mol/L MESA solution. tion of labile amino acids within a peptide or protein during Vapor Phase Hydrolysis About 1 to 100 mgofthe the hydrolysis. This is especially true for peptide bonds that protein/peptide under test is dried in a hydrolysis tube. The are slow to cleave (e.g., Ile­Val bonds). However, this hydrolysis tube is placed in a larger tube with about 200 mL Supplement II, JPXIV General Information 1817 of the Hydrolysis Solution. The larger tube is sealed in sulfoxide (DMSO). vacuum (about 50 mm of mercury or 6.7 Pa) to vaporize the Hydrolysis Solution 6 mol/L hydrochloric acid contain­ Hydrolysis Solution. The hydrolysis tube is heated to 1709C ing 0.1z to 1.0z of phenol, to which DMSO is added to to 1859C for about 12.5 minutes. After hydrolysis, the obtain a ˆnal concentration of 2z (v/v). hydrolysis tube is dried in vacuum for 15 minutes to remove Vapor Phase Hydrolysis The protein/peptide hydrolysis the residual acid. is conducted at about 1109C for 24 hours. During the hydrolysis, the cysteine­cystine present in the sample is Method 3 converted to cysteic acid by the DMSO present in the Tryptophan oxidation during hydrolysis is prevented by Hydrolysis Solution.Asanapproachtolimitvariabilityand using thioglycolic acid (TGA) as the reducing acid. compensate for partial destruction, it is recommended to Hydrolysis Solution A solution containing 7 mol/L evaluate the cysteic acid recovery from oxidative hydrolyses hydrochloric acid, 10z of tri‰uoroacetic acid, 20z of of standard proteins containing 1 to 8 mol of cysteine. The thioglycolic acid, and 1z of phenol. response factors from protein/peptide hydrolysates are Vapor Phase Hydrolysis About 10 to 50 mgofthe typically about 30z lower than those for nonhydrolyzed protein/peptide under test is dried in a sample tube. The cysteic acid standards. Because histidine, methionine, sample tube is placed in a larger tube with about 200 mLof tyrosine, and tryptophan are also modiˆed, a complete the Hydrolysis Solution. The larger tube is sealed in vacuum compositional analysis is not obtained with this technique. (about 50 mm of mercury or 6.7 Pa) to vaporize the TGA. Thesampletubeisheatedto1669C for about 15 to 30 Method 7 minutes. After hydrolysis, the sample tube is dried in Cysteine­cystine reduction and alkylation is accomplished vacuum for 5 minutes to remove the residual acid. Recovery by a vapor phase pyridylethylation reaction. of tryptophan by this method may be dependent on the Reducing Solution Transfer 83.3 mL of pyridine, 16.7 amount of sample present. mL of 4­vinylpyridine, 16.7 mL of tributylphosphine, and 83.3 mL of water to a suitable container, and mix. Method 4 Procedure Add the protein/peptide (between 1 and 100 Cysteine­cystine and methionine oxidation is performed mg) to a hydrolysis tube, and place in a larger tube. Transfer with performic acid before the protein hydrolysis. the Reducing Solution to the large tube, seal in vacuum Oxidation Solution The performic acid is prepared fresh (about 50 mm of mercury or 6.7 Pa), and incubate at about by mixing formic acid and 30 percent hydrogen peroxide 1009C for 5 minutes. Then remove the inner hydrolysis tube, (9:1), and incubated at room temperature for 1 hour. and dry it in a vacuum desiccator for 15 minutes to remove Procedure The protein/peptide sample is dissolved in residual reagents. The pyridylethylated protein/peptide can 20 mL of formic acid, and heated at 509C for 5 minutes; then then be acid hydrolyzed using previously described 100 mLoftheOxidation Solution is added. The oxidation is procedures. The pyridylethylation reaction is performed allowed to proceed for 10 to 30 minutes. In this reaction, simultaneously with a protein standard sample containing 1 cysteine is converted to cysteic acid and methionine is to 8 mol of cysteine to improve accuracy in the pyridylethyl­ converted to methionine sulfone. The excess reagent is cysteine recovery. Longer incubation times for the removed from the sample in a vacuum centrifuge. This pyridylethylation reaction can cause modiˆcations to the technique may cause modiˆcations to tyrosine residues in the a­amino terminal group and the e­amino group of lysine in presence of halides. The oxidized protein can then be acid the protein. hydrolyzed using Method 1 or Method 2. Method 8 Method 5 Cysteine­cystine reduction and alkylation is accomplished Cysteine­cystine oxidation is accomplished during the by a liquid phase pyridylethylation reaction. liquid phase hydrolysis with sodium azide. Stock Solutions Prepare and ˆlter three solutions: Hydrolysis Solution 6 mol/L hydrochloric acid contain­ 1 mol/L Tris hydrochloride (pH 8.5) containing 4 mmol/L ing 0.2z of phenol, to which is added sodium azide to edetate disodium (Stock Solution A), 8 mol/L guanidine obtain a ˆnal concentration of 0.2z (w/v). The added hydrochloride (Stock Solution B), and 10z of 2­mercap­ phenol prevents halogenation of tyrosine. toethanol in water (Stock Solution C). Liquid Phase Hydrolysis The protein/peptide hydrolysis Reducing Solution Prepare a mixture of Stock Solution is conducted at about 1109C for 24 hours. During the B and Stock Solution A (3:1) to obtain a buŠered solution hydrolysis, the cysteine­cystine present in the sample is of 6 mol/L guanidine hydrochloride in 0.25 mol/L Tris converted to cysteic acid by the sodium azide present in the hydrochloride. Hydrolysis Solution. This technique allows better tyrosine Procedure Dissolve about 10 mg of the test sample in 50 recovery than Method 4, but it is not quantitative for mLoftheReducing Solution, and add about 2.5 mLofStock methionine. Methionine is converted to a mixture of the Solution C. Store under nitrogen or argon for 2 hours at parent methionine and its two oxidative products, methio­ room temperature in the dark. To achieve the pyridylethyla­ nine sulfoxide and methionine sulfone. tion reaction, add about 2 mL of 4­vinylpyridine to the Method 6 protein solution, and incubate for an additional 2 hours at Cysteine­cystine oxidation is accomplished with dimethyl room temperature in the dark. The protein/peptide is 1818 General Information Supplement II, JPXIV desalted by collecting the protein/peptide fraction from a Reducing Solutions Prepare and ˆlter three solutions: a reversed­phase HPLC separation. The collected sample can solution of 10 mmol/L tri‰uoroacetic acid (Solution A), a be dried in a vacuum centrifuge before acid hydrolysis. solution of 5 mol/L guanidine hydrochloride and 10 mmol/L tri‰uoroacetic acid (Solution B), and a freshly Method 9 prepared solution of N,N­dimethylformamide containing Cysteine­cystine reduction and alkylation is accomplished 36 mg of BTI per mL (Solution C). by a liquid phase carboxymethylation reaction. Procedure In a clean hydrolysis tube, transfer about Stock Solutions Prepare as directed for Method 8. 200 mg of the test sample, and add 2 mL of Solution A or Carboxymethylation Solution Prepare a solution con­ Solution B and 2 mL of Solution C. Seal the hydrolysis tube taining 100 mg of iodoacetamide per mL of ethanol (95). in vacuum. Heat the sample at 609C for 4 hours in the dark. BuŠer Solution Use the Reducing Solution, prepared as The sample is then dialyzed with water to remove the excess directed for Method 8. reagents. Extract the dialyzed sample three times with equal Procedure Dissolve the test sample in 50 mLoftheBuŠer volumes of n­butyl acetate, and then lyophilize. The protein Solution, and add about 2.5 mLofStock Solution C.Store can then be acid hydrolyzed using previously described under nitrogen or argon for 2 hours at room temperature in procedures. The a,b­diaminopropionic and a,g­diaminobu­ the dark. Add the Carboxymethylation Solution in a ratio tyric acid residues do not typically resolve from the lysine 1.5 fold per total theoretical content of thiols, and incubate residues upon ion­exchange chromatography based on for an additional 30 minutes at room temperature in the amino acid analysis. Therefore, when using ion­exchange as dark. [Note: If the thiol content of the protein is unknown, the mode of amino acid separation, the asparagine and then add 5 mL of 100 mmol/L iodoacetamide for every 20 glutamine contents are the quantitative diŠerence in the nmol of protein present.] The reaction is stopped by adding aspartic acid and glutamic acid content assayed with excess of 2­mercaptoethanol. The protein/peptide is desalt­ underivatized and BTI­derivatized acid hydrolysis. [Note: ed by collecting the protein/peptide fraction from a rever­ The threonine, methionine, cysteine, tyrosine, and histidine sed­phase HPLC separation. The collected sample can be assayed content can be altered by BTI derivatization; a dried in a vacuum centrifuge before acid hydrolysis. The hydrolysis without BTI will have to be performed if the S­carboxyamidomethyl­cysteine formed will be converted to analyst is interested in the composition of these other amino S­carboxymethylcysteine during acid hydrolysis. acid residues of the protein/peptide.] Method 10 Methodologies of Amino Acid Analysis General Principles Cysteine­cystine is reacted with dithiodiglycolic acid or Many amino acid analysis techniques exist, and the choice dithiodipropionic acid to produce a mixed disulˆde. [Note: of any one technique often depends on the sensitivity The choice of dithiodiglycolic acid or dithiodipropionic acid required from the assay. In general, about one­half of the depends on the required resolution of the amino acid amino acid analysis techniques employed rely on the analysis method.] separation of the free amino acids by ion­exchange Reducing Solution A solution containing 10 mg of chromatography followed by postcolumn derivatization dithiodiglycolic acid (or dithiodipropionic acid) per mL of (e.g., with ninhydrin or o­phthalaldehyde). Postcolumn 0.2 mol/L sodium hydroxide. detection techniques can be used with samples that contain Procedure Transfer about 20 mg of the test sample to a small amounts of buŠer components, such as salts and hydrolysis tube, and add 5 mLoftheReducing Solution. urea, and generally require between 5 and 10 mgofprotein Add 10 mL of isopropyl alcohol, and then remove all of the sample per analysis. The remaining amino acid techniques sample liquid by vacuum centrifugation. The sample is then typically involve precolumn derivatization of the free amino hydrolyzed using Method 1. This method has the advantage acids (e.g., phenyl isothiocyanate; 6­aminoquinolyl­N­ that other amino acid residues are not derivatized by side hydroxysuccinimidyl carbamate or o­phthalaldehyde; reactions, and the sample does not need to be desalted prior (dimethylamino)azobenzenesulfonyl chloride; 9­ to hydrolysis. ‰uorenylmethylchloroformate; and, 7­‰uoro­4­nitrobenzo­ Method 11 2­oxa­1,3­diazole) followed by reversed­phase HPLC. Asparagine and glutamine are converted to aspartic acid Precolumn derivatization techniques are very sensitive and and glutamic acid, respectively, during acid hydrolysis. usually require between 0.5 and 1.0 mg of protein sample per Asparagine and aspartic acid residues are added and analysis but may be in‰uenced by buŠer salts in the samples. represented by Asx, while glutamine and glutamic acid Precolumn derivatization techniques may also result in residues are added and represented by Glx. Proteins/pep­ multiple derivatives of a given amino acid, which compli­ tides can be reacted with bis(1,1­tri‰uoroacetoxy)iodoben­ cates the result interpretation. Postcolumn derivatization zene(BTI)toconverttheasparagineandglutamineresidues techniques are generally in‰uenced less by performance to diaminopropionic acid and diaminobutyric acid residues, variation of the assay than precolumn derivatization tech­ respectively, upon acid hydrolysis. These conversions allow niques. the analyst to determine the asparagine and glutamine The following Methods may be used for quantitative content of a protein/peptide in the presence of aspartic acid amino acid analysis. Instruments and reagents for these and glutamic acid residues. procedures are available commercially. Furthermore, many Supplement II, JPXIV General Information 1819 modiˆcations of these methodologies exist with diŠerent toethanol. The derivatization of primary amino acids are not reagent preparations, reaction procedures, chromatographic noticeably aŠected by the continuous supply of sodium systems, etc. Speciˆc parameters may vary according to the hypochlorite. exact equipment and procedure used. Many laboratories will Separation of the amino acids on an ion­exchange column utilize more than one amino acid analysis technique to is accomplished through a combination of changes in pH exploit the advantages oŠered by each. In each of these and cation strength. After postcolumn derivatization of Methods, the analog signal is visualized by means of a data eluted amino acids with OPA, the reactant passes through acquisition system, and the peak areas are integrated for the ‰uorometric detector. Fluorescence intensity of OPA­ quantiˆcation purposes. derivatized amino acids are monitored with an excitation wavelength of 348 nm and an emission wavelength of Method 1—Postcolumn Ninhydrin Detection General 450 nm. Principle Detection limit is considered to be a few tens of picomole Ion­exchange chromatography with postcolumn nin­ level for most of the amino acid derivatives. Response hydrin detection is one of the most common methods linearity is obtained in the range of a few picomole level to a employed for quantitative amino acid analysis. As a rule, a few tens of nanomole level. To obtain good compositional Li­based cation­exchange system is employed for the data, the starting with greater than 500 ng of sample before analysis of the more complex physiological samples, and the hydrolysis is best suited for the amino acid analysis of faster Na­based cation­exchange system is used for the more protein/peptide. simplistic amino acid mixtures obtained with protein hydrolysates (typically containing 17 amino acid compo­ Method 3—Precolumn PITC Derivatization General nents). Separation of the amino acids on an ion­exchange Principle column is accomplished through a combination of changes Phenylisothiocyanate (PITC) reacts with amino acids to in pH and cation strength. A temperature gradient is often form phenylthiocarbamyl (PTC) derivatives which can be employed to enhance separation. detected with high sensitivity at 245 nm. Therefore, When the amino acid reacts with ninhydrin, the reactant precolumn derivatization of amino acids with PITC has characteristic purple or yellow color. Amino acids, followed by a reversed­phase HPLC separation with UV de­ except imino acid, give a purple color, and show the maxi­ tection is used to analyze the amino acid composition. mum absorption at 570 nm. The imino acids such as proline After the reagent is removed under vacuum, the deriva­ give a yellow color, and show the maximum absorption at tized amino acids can be stored dry and frozen for several 440 nm. The postcolumn reaction between ninhydrin and weeks with no signiˆcant degradation. If the solution for in­ aminoacidelutedfromcolumnismonitoredat440and jection is kept cold, no noticeable loss in chromatographic 570 nm, and the chromatogram obtained is used for the response occurs after three days. determination of amino acid composition. Separation of the PTC­amino acids on a reversed­phase Detection limit is considered to be 10 pmol for most of the HPLC with ODS column is accomplished through a combi­ amino acid derivatives, but 50 pmol for proline. Response nation of changes in concentrations of acetonitrile and linearity is obtained in the range of 20 to 500 pmol with buŠer ionic strength. PTC­amino acids eluted from column correlation coe‹cients exceeding 0.999. To obtain good are monitored at 254 nm. composition data, samples larger than 1 mgbeforehydroly­ Detection limit is considered to be 1 pmol for most of the sis are best suited for this amino acid analysis of pro­ amino acid derivatives. Response linearity is obtained in the tein/peptide. range of 20 to 500 pmol with correlation coe‹cients exceed­ ing 0.999. To obtain good compositional data, samples Method 2—Postcolumn OPA Fluorometric Detection larger than 500 ng of protein/peptide before hydrolysis is General Principle best suited for this amino acid analysis of proteins/peptides. o­Phthalaldehyde (OPA) reacts with primary amines in the presence of thiol compound, to form highly ‰uorescent Method 4—Precolumn AQC Derivatization General isoindole products. This reaction is utilized for the Principle postcolumn derivatization in analysis of amino acids by Precolumn derivatization of amino acids with 6­amino­ ion­exchange chromatography. The rule of the separation is quinolyl­N­hydroxysuccinimidyl carbamate (AQC) followed the same as Method 1. Instruments and reagents for this by reversed­phase HPLC separation with ‰uorometric form of amino acid analysis are available commercially. detection is used. Many modiˆcations of this methodology exist. 6­Aminoquinolyl­N­hydroxysuccinimidyl carbamate Although OPA does not react with secondary amines (AQC) reacts with amino acids to form stable, ‰uorescent (imino acids such as proline) to form ‰uorescent substances, unsymmetric urea derivatives (AQC­amino acids) which are the oxidation with sodium hypochlorite allows secondary readily amenable to analysis by reversed­phase HPLC. amines to react with OPA. The procedure employs a strong­ Therefore, precolumn derivatization of amino acids with ly acidic cation­exchange column for separation of free AQC followed by reversed­phase HPLC separation is used amino acids followed by postcolumn oxidation with sodium to analyze the amino acid composition. hypochlorite and postcolumn derivatization using OPA and Separation of the AQC­amino acids on ODS column is thiol compound such as N­acetyl­L­cysteine and 2­mercap­ accomplished through a combination of changes in concen­ 1820 General Information Supplement II, JPXIV trations of acetonitrile and salt. Selective ‰uorescence detec­ (dimethylamino)azobenzenesulfonyl chloride (DABS­Cl) tion of the derivatives with excitation wavelength at 250 nm followed by reversed­phase HPLC separation with visible and emission wavelength at 395 nm allows for the direct light detection is used. injection of the reaction mixture with no signiˆcant interfer­ (Dimethylamino)azobenzenesulfonyl chloride (DABS­Cl) ence from the only major ‰uorescent reagent by­product, is a chromophoric reagent employed for the labeling of

6­aminoquinoline. Excess reagent is rapidly hydrolyzed (t1/2 amino acids. Amino acids labeled with DABS­Cl (DABS­ º15 seconds) to yield 6­aminoquinoline, N­hydroxysuc­ amino acids) are highly stable and show the maximum cinimide and carbon dioxide, and after 1 minute no further absorption at 436 nm. derivatization can take place. DABS­amino acids, all 19 naturally occurring amino acids Peak areas for AQC­amino acids are essentially un­ derivatives, can be separated on an ODS column of a changed for at least 1 week at room temperature, and the reversed­phase HPLC by employing gradient systems con­ derivatives have more than su‹cient stability to allow for sisting of acetonitrile and aqueous buŠer mixture. Separated overnight automated chromatographic analysis. DABS­amino acids eluted from column are detected at Detection limit is considered to be ranging from ca. 40 to 436 nm in the visible region. 320 fmol for each amino acid, except for Cys. Detection This Method can analyze the imino acids such as proline limit for Cys is approximately 800 fmol. Response linearity together with the amino acids at the same degree of sensitivi­ is obtained in the range of 2.5 to 200 mmol/L with correla­ ty, DABS­Cl derivatization method permits the simultane­ tion coe‹cients exceeding 0.999. Good compositional data ous quantiˆcation of tryptophan residues by previous could be obtained from the analysis of derivatized protein hydrolysis of the protein/peptide with sulfonic acids such as hydrolysates containing as little as 30 ng of protein/peptide. mercaptoethanesulfonic acid, p­toluenesulfonic acid or methanesulfonic acid described under Method 2 in ``Protein Method 5—Precolumn OPA Derivatization General Hydrolysis''. The other acid­labile residues, asparagine and Principle glutamine, can also be analysed by previous conversion into Precolumn derivatization of amino acids with o­phthalal­ diaminopropionic acid and diaminobutyric acid, respec­ dehyde (OPA) followed by reversed­phase HPLC separation tively, by treatment of protein/peptide with BTI described with ‰uorometric detection is used. This technique does not under Method 11 in ``Protein Hydrolysis''. detect amino acids that exist as secondary amines (e.g., The non­proteinogenic amino acid, norleucine cannot be proline). used as internal standard in this method, as this compound is o­Phthalaldehyde (OPA) in conjunction with a thiol eluted in a chromatographic region crowded with peaks of reagent reacts with primary amine groups to form highly primary amino acids. Nitrotyrosine can be used as an ‰uorescent isoindole products. 2­Mercaptoethanol or 3­mer­ internal standard, because it is eluted in a clean region. captopropionic acid can be used as the thiol. OPA itself does Detection limit of DABS­amino acid is about 1 pmol. As not ‰uoresce and consequently produces no interfering little as 2 to 5 pmol of an individual DABS­amino acid can peaks. In addition, its solubility and stability in aqueous be quantitatively analysed with reliability, and only 10 to solution, along with the rapid kinetics for the reaction, make 30 ng of the dabsylated protein hydrolysate is required for it amenable to automated derivatization and analysis using each analysis. an autosampler to mix the sample with the reagent. However, lack of reactivity with secondary amino acids has Method 7—Precolumn FMOC­Cl Derivatization General been predominant drawback. This method does not detect Principle amino acids that exist as secondary amines (e.g., proline). Precolumn derivatization of amino acids with 9­ To compensate for this drawback, this technique may be ‰uorenylmethyl chloroformate (FMOC­Cl) followed by combined with another technique described in Method 7 or reversed­phase HPLC separation with ‰uorometric detec­ Method 8. tion is used. Precolumn derivatization of amino acids with OPA is 9­Fluorenylmethyl chloroformate (FMOC­Cl) reacts with followed by a reversed­phase HPLC separation. Because of both primary and secondary amino acids to form highly the instability of the OPA­amino acid derivative, HPLC ‰uorescent products. The reaction of FMOC­Cl with amino separation and analysis are performed immediately follow­ acid proceeds under mild conditions in aqueous solution and ing derivatization. The liquid chromatograph is equipped is completed in 30 seconds. The derivatives are stable, only with a ‰uorometric detector for the detection of derivatized the histidine derivative showing any breakdown. Although amino acids. Fluorescence intensity of OPA­derivatized FMOC­Cl is ‰uorescent itself, the reagent excess and amino acids is monitored with an excitation wavelength of ‰uorescent side­products can be eliminated without loss of 348 nm and an emission wavelength of 450 nm. FMOC­amino acids. Detection limits as low as 50 fmol via ‰uorescence have FMOC­amino acids are separated by a reversed­phase been reported, although the practical limit of analysis HPLC using ODS column. The separation is carried out by remains at 1 pmol. gradient elution varied linearly from a mixture of acetoni­ trile methanol and acetic acid buŠer (10:40:50) to a mixture Method 6—Precolumn DABS­Cl Derivatization General of acetonitrile and acetic acid buŠer (50:50), and 20 amino Principle acid derivatives are separated in 20 minutes. Each derivative Precolumn derivatization of amino acids with Supplement II, JPXIV General Information 1821 eluted from column is monitored by a ‰uorometric detector ed for each Procedure. Calculate the mole percent for each setatanexcitationwavelengthof260nmandanemission amino acid present in the test sample by the formula: wavelength of 313 nm. 100rU/r,

The detection limit is in the low fmol range. A linearity in which rU is the peak response, in nmol, of the amino acid range of 0.1 to 50 mmol/L is obtained for most of the amino under test; and r is the sum of peak responses, in nmol, for acids. all amino acids present in the test sample. Comparison of the mole percent of the amino acids under test to data from Method 8—Precolumn NBD­F Derivatization General known proteins can help establish or corroborate the Principle identity of the sample protein. Precolumn derivatization of amino acids with 7­‰uoro­4­ Unknown Protein Samples This data analysis technique nitrobenzo­2­oxa­1.3­diazole (NBD­F) followed by reversed­ canbeusedtoestimatetheproteinconcentrationofan phase HPLC separation with ‰uorometric detection is used. unknown protein sample using the amino acid analysis data. 7­‰uoro­4­nitrobenzo­2­oxa­1.3­diazole (NBD­F) reacts Calculate the mass, in mg, of each recovered amino acid by with both primary and secondary amino acids to form highly the formula: ‰uorescent products. Amino acids are derivatized with mM /1000, NBD­F by heating to 609C for 5minutes. W in which m is the recovered quantity, in nmol, of the amino NBD­amino acid derivatives are separated on an ODS acid under test; and M is the average molecular weight for column of a reversed­phase HPLC by employing gradient W that amino acid, corrected for the weight of the water elution system consisting of acetonitrile and aqueous buŠer molecule that was eliminated during peptide bond forma­ mixture, and 17 amino acid derivatives are separated in 35 tion. The sum of the masses of the recovered amino acids minutes. e­Aminocaproic acid can be used as an internal will give an estimate of the total mass of the protein analyzed standard, because it is eluted in a clean chromatographic after appropriate correction for partially and completely region. Each derivative eluted from column is monitored by destroyed amino acids. If the molecular weight of the a ‰uorometric detector set at an excitation wavelength of unknown protein is available (i.e., by SDS­PAGE analysis 480 nm and an emission wavelength of 530 nm. or mass spectroscopy), the amino acid composition of the The sensitivity of this method is almost the same as unknown protein can be predicted. Calculate the number of for precolumn OPA derivatization method (Method 5), residues of each amino acid by the formula: excluding proline to which OPA is not reactive, and might m/(1000M/M ), be advantageous for NBD­F against OPA. The detection WT in which m is the recovered quantity, in nmol, of the amino limit for each amino acid is about 10 fmol. Proˆle analysis acid under test; M is the total mass, in mg, of the protein; was achieved for about 1.5 mg of protein hydrolysates in the and M is the molecular weight of the unknown protein. ˆnal precolumn labeling reaction mixture for HPLC. WT Known Protein Samples This data analysis technique Data Calculation and Analysis can be used to investigate the amino acid composition and When determining the amino acid content of a pro­ protein concentration of a protein sample of known molecu­ tein/peptide hydrolysate, it should be noted that the acid lar weight and amino acid composition using the amino acid hydrolysis step destroys tryptophan and cysteine. Serine and analysis data. When the composition of the protein being threonine are partially destroyed by acid hydrolysis, while analyzed is known, one can exploit the fact that some amino isoleucine and valine residues may be only partially cleaved. acids are recovered well, while other amino acid recoveries Methionine can undergo oxidation during acid hydrolysis, may be compromised because of complete or partial destruc­ and some amino acids (e.g., glycine and serine) are common tion (e.g., tryptophan, cysteine, threonine, serine, methio­ contaminants. Application of adequate vacuum (Ånot less nine), incomplete bond cleavage (i.e., for isoleucine and than 200 mm of mercury or 26.7 Pa) or introduction of inert valine) and free amino acid contamination (i.e., by glycine gas (argon) in the headspace of the reaction vessel during and serine). vapor phase hydrolysis can reduce the level of oxidative Because those amino acids that are recovered best destruction. Therefore, the quantitative results obtained for represent the protein, these amino acids are chosen to cysteine, tryptophan, threonine, isoleucine, valine, methio­ quantify the amount of protein. Well­recovered amino acids nine, glycine, and serine from a protein/peptide hydrolysate are, typically, aspartate­asparagine, glutamate­glutamine, may be variable and may warrant further investigation and alanine, leucine, phenylalanine, lysine, and arginine. This consideration. listcanbemodiˆedbasedonexperiencewithone'sown analysis system. Divide the quantity, in nmol, of each of the Calculations well­recovered amino acids by the expected number of Amino Acid Mole Percent This is the number of speciˆc residues for that amino acid to obtain the protein content amino acid residues per 100 residues in a protein. This result based on each well­recovered amino acid. Average the may be useful for evaluating amino acid analysis data when protein content results calculated. The protein content the molecular weight of the protein under investigation is determined for each of the well­recovered amino acids unknown. This information can be used to corroborate the should be evenly distributed about the mean. Discard identity of a protein/peptide and has other applications. protein content values for those amino acids that have an Carefully identify and integrate the peaks obtained as direct­ unacceptable deviation from the mean. Typically »greater 1822 General Information Supplement II, JPXIV than 5z variation from the mean is considered unaccepta­ ez V ble. Recalculate the mean protein content from the remain­ n m E eo eo Ø h »Ø L » ing values to obtain the protein content of the sample. e: dielectric constant of the buŠer, Divide the content of each amino acid by the calculated z: zeta potential of the capillary surface. mean protein content to determine the amino acid composi­ The velocity of the solute (n)isgivenby: tion of the sample by analysis.

Calculate the relative compositional error, in percentage, nnep{neo by the formula: The electrophoretic mobility of the analyte and the 100m/m , S electro­osmotic mobility may act in the same direction or in in which m is the experimentally determined quantity, in opposite directions, depending on the charge of the solute. nmol per amino acid residue, of the amino acid under test; In normal capillary electrophoresis, anions will migrate in and m is the known residue value for that amino acid. The S the opposite direction to the electro­osmotic ‰ow and their average relative compositional error is the average of the velocities will be smaller than the electro­osmotic velocity. absolute values of the relative compositional errors of the Cations will migrate in the same direction as the electro­ individual amino acids, typically excluding tryptophan and osmotic ‰ow and their velocities will be greater than the cysteine from this calculation. The average relative composi­ electro­osmotic velocity. Under conditions in which there is tional error can provide important information on the stabil­ a fast electro­osmotic velocity with respect to the elec­ ity of analysis run over time. The agreement in the amino trophoretic velocity of the solutes, both cations and anions acid composition between the protein sample and the known can be separated in the same run. composition can be used to corroborate the identity and Thetime(t) taken by the solute to migrate the distance (l) purity of the protein in the sample. from the injection end of the capillary to the detection point (capillary eŠective length) is given by the expression:

Add the following: l l~L t  nep{neo (mep{meo)V 21. Capillary Electrophoresis In general, uncoated fused­silica capillaries above pH 3 have negative charge due to ionized silanol groups in the General Principles inner wall. Consequently, the electro­osmotic ‰ow is from Capillary electrophoresis is a physical method of analysis anode to cathode. The electro­osmotic ‰ow must remain based on the migration, inside a capillary, of charged constant from run to run if good reproducibility is to be analytes dissolved in an electrolyte solution, under the obtained in the migration velocity of the solutes. For some in‰uence of a direct­current electric ˆeld. applications, it may be necessary to reduce or suppress the The migration velocity of an analyte under an electric ˆeld electro­osmotic ‰ow by modifying the inner wall of the of intensity E, is determined by the electrophoretic mobility capillary or by changing the concentration, composition of the analyte and the electro­osmotic mobility of the buŠer and/or pH of the buŠer solution. inside the capillary. The electrophoretic mobility of a solute After the introduction of the sample into the capillary, (m ) depends on the characteristics of the solute (electric ep each analyte ion of the sample migrates within the back­ charge, molecular size and shape) and those of the buŠer in ground electrolyte as an independent zone, according to its which the migration takes place (type and ionic strength of electrophoretic mobility. Zone dispersion, that is the spread­ the electrolyte, pH, viscosity and additives). The elec­ ing of each solute band, results from diŠerent phenomena. trophoretic velocity (n ) of a solute, assuming a spherical ep Under ideal conditions the sole contribution to the solute­ shape, is given by the equation: zone broadening is molecular diŠusion of the solute along q V the capillary (longitudinal diŠusion). In this ideal case the n m EØ »Ø » ep ep 6phr L e‹ciency of the zone, expressed as the number of theoretical plates (N), is given by: q:eŠectivechargeofthesolute, h: viscosity of the electrolyte solution, (m {m )~V~L N ep eo r: Stoke's radius of the solute, 2~D~L V: applied voltage, D: molecular diŠusion coe‹cient of the solute in the L: total length of the capillary. buŠer. When an electric ˆeld is applied through the capillary In practice, other phenomena such as heat dissipation, ˆlled with buŠer, a ‰ow of solvent is generated inside the sample adsorption onto the capillary wall, mismatched capillary, called electro­osmotic ‰ow. The velocity of the conductivity between sample and buŠer, length of the injec­ electro­osmotic ‰ow depends on the electro­osmotic mobili­ tion plug, detector cell size and unlevelled buŠer reservoirs ty (m ) which in turn depends on the charge density on the eo can also signiˆcantly contribute to band dispersion. capillary internal wall and the buŠer characteristics. The Separation between two bands (expressed as the resolu­ electro­osmotic velocity (neo) is given by the equation: tion, RS) can be obtained by modifying the electrophoretic Supplement II, JPXIV General Information 1823 mobility of the analytes, the electro­osmotic mobility 1. Capillary Zone Electrophoresis induced in the capillary and by increasing the e‹ciency for Principle thebandofeachanalyte,accordingtotheequation: In capillary zone electrophoresis, analytes are separated in a capillary containing only buŠer without any anticonvective N(m |m ) R  epb epa medium. With this technique, separation takes place because S 4(šm {m ) ep eo the diŠerent components of the sample migrate as discrete

mepa and mepb: electrophoretic mobilities of the two analytes bands with diŠerent velocities. The velocity of each band separated, depends on the electrophoretic mobility of the solute and the

mš ep: mean electrophoretic mobility of the two analytes electro­osmotic ‰ow in the capillary (see General Principles). 1 Coated capillaries can be used to increase the separation mš  (m {mepa). ep 2 epb capacity of those substances adsorbing on fused­silica Apparatus surfaces. An apparatus for capillary electrophoresis is composed Using this mode of capillary electrophoresis, the analysis of: of both small (Mrº2000) and large molecules (2000ºMr —a high­voltage, controllable direct­current power supp­ º100,000) can be accomplished. Due to the high e‹ciency ly, achieved in free solution capillary electrophoresis, separa­ —two buŠer reservoirs, held at the same level, containing tion of molecules having only minute diŠerences in their the prescribed anodic and cathodic solutions, charge­to­mass ratio can be eŠected. This separation mode —two electrode assemblies (the cathode and the anode), also allows the separation of chiral compounds by addition immersed in the buŠer reservoirs and connected to the of chiral selectors to the separation buŠer. power supply, Optimization —a separation capillary (usually made of fused­silica) Optimization of the separation is a complex process where which, when used with some speciˆc types of detectors, several separation parameters can play a major role. The has an optical viewing window aligned with the detec­ main factors to be considered in the development of separa­ tor. The ends of the capillary are placed in the buŠer tions are instrumental and electrolytic solution parameters. reservoirs. The capillary is ˆlled with the solution Instrumental parameters prescribed in the monograph, Voltage: A Joule heating plot is useful in optimizing the —a suitable injection system, applied voltage and column temperature. Separation time is —a detector able to monitor the amount of substances of inversely proportional to applied voltage. However, an interest passing through a segment of the separation increase in the voltage used can cause excessive heat produc­ capillary at a given time. It is usually based on absorp­ tion, giving rise to temperature and, as a result thereof, tion spectrophotometry (UV and visible) or ‰uoromet­ viscosity gradients in the buŠer inside the capillary. This ry, but conductimetric, amperometric or mass spectro­ eŠect causes band broadening and decreases resolution. metric detection can be useful for speciˆc applications. Polarity: Electrode polarity can be normal (anode at the Indirect detection is an alternative method used to de­ inlet and cathode at the outlet) and the electro­osmotic ‰ow tect non­UV­absorbing and non­‰uorescent com­ will move toward the cathode. If the electrode polarity is pounds, reversed, the electro­osmotic ‰ow is away from the outlet —a thermostatic system able to maintain a constant tem­ and only charged analytes with electro­osmotic mobilities perature inside the capillary is recommended to obtain a greater than the electro­osmotic ‰ow will pass to the outlet. good separation reproducibility, Temperature: The main eŠect of temperature is ob­ —a recorder and a suitable integrator or a computer. served on buŠer viscosity and electrical conductivity, and The deˆnition of the injection process and its automation therefore on migration velocity. In some cases, an increase are critical for precise quantitative analysis. Modes of in capillary temperature can cause a conformational change injection include gravity, pressure or vacuum injection and in proteins, modifying their migration time and the e‹ciency electrokinetic injection. The amount of each sample of the separation. component introduced electrokinetically depends on its Capillary: The dimensions of the capillary (length and electrophoretic mobility, leading to possible discrimination internal diameter) contribute to analysis time, e‹ciency of using this injection mode. separations and load capacity. Increasing both eŠective Use the capillary, the buŠer solutions, the preconditioning length and total length can decrease the electric ˆelds method, the sample solution and the migration conditions (working at constant voltage) which increases migration prescribed in the monograph of the considered substance. time. For a given buŠer and electric ˆeld, heat dissipation, The employed electrolytic solution is ˆltered to remove and hence sample band­broadening, depend on the internal particles and degassed to avoid bubble formation that could diameter of the capillary. The latter also aŠects the detection interfere with the detection system or interrupt the electrical limit, depending on the sample volume injected and the contact in the capillary during the separation run. A detection system employed. rigorous rinsing procedure should be developed for each Since the adsorption of the sample components on the analytical method to achieve reproducible migration times capillary wall limits e‹ciency, methods to avoid these of the solutes. interactions should be considered in the development of a 1824 General Information Supplement II, JPXIV separation method. In the speciˆc case of proteins, several 2. Capillary Gel Electrophoresis strategies have been devised to avoid adsorption on the Principle capillary wall. Some of these strategies (use of extreme pH In capillary gel electrophoresis, separation takes place and adsorption of positively charged buŠer additives) only inside a capillary ˆlled with a gel that acts as a molecular require modiˆcation of the buŠer composition to prevent sieve. Molecules with similar charge­to­mass ratios are protein adsorption. In other strategies, the internal wall of separated according to molecular size since smaller molec­ the capillary is coated with a polymer, covalently bonded to ules move more freely through the network of the gel and the silica, that prevents interaction between the proteins and therefore migrate faster than larger molecules. DiŠerent the negatively charged silica surface. For this purpose, biological macromolecules (for example, proteins and DNA ready­to­use capillaries with coatings consisting of neutral­ fragments), which often have similar charge­to­mass ratios, hydrophilic, cationic and anionic polymers are available. can thus be separated according to their molecular mass by Electrolytic solution parameters capillary gel electrophoresis. BuŠer type and concentration: Suitable buŠers for Characteristics of Gels capillary electrophoresis have an appropriate buŠer capacity Two types of gels are used in capillary electrophoresis: in the pH range of choice and low mobility to minimize permanently coated gels and dynamically coated gels. Per­ current generation. manently coated gels, such as cross­linked polyacrylamide, Matching buŠer­ion mobility to solute mobility, whenever are prepared inside the capillary by polymerization of the possible, is important for minimizing band distortion. The monomers. They are usually bonded to the fused­silica wall type of sample solvent used is also important to achieve and cannot be removed without destroying the capillary. If on­column sample focusing, which increases separation the gels are used for protein analysis under reducing condi­ e‹ciency and improves detection. tions, the separation buŠer usually contains sodium dodecyl An increase in buŠer concentration (for a given pH) sulfate and the samples are denatured by heating a mixture decreases electro­osmotic ‰ow and solute velocity. of sodium dodecyl sulfate and 2­mercaptoethanol or BuŠer pH: The pH of the buŠer can aŠect separation by dithiothreitol before injection. When non­reducing condi­ modifying the charge of the analyte or additives, and by tions are used (for example, analysis of an intact antibody), changing the electro­osmotic ‰ow. In protein and peptide 2­mercaptoethanol and dithiothreitol are not used. Separa­ separation, changing the pH of the buŠer from above to tion in cross­linked gels can be optimized by modifying the below the isoelectric point (pI) changes the net charge of the separation buŠer (as indicated in the free solution capillary solute from negative to positive. An increase in the buŠer electrophoresis section) and controlling the gel porosity pH generally increases the electro­osmotic ‰ow. during the gel preparation. For cross­linked polyacrylamide Organic solvents: Organic modiˆers (methanol, acetoni­ gels, the porosity can be modiˆed by changing the concen­ trile, etc.) may be added to the aqueous buŠer to increase the tration of acrylamide and/or the proportion of cross­linker. solubility of the solute or other additives and/or to aŠect the As a rule, a decrease in the porosity of the gel leads to a degree of ionization of the sample components. The decrease in the mobility of the solutes. Due to the rigidity of addition of these organic modiˆers to the buŠer generally these gels, only electrokinetic injection can be used. causes a decrease in the electro­osmotic ‰ow. Dynamically coated gels are hydrophilic polymers, such as Additives for chiral separations: For the separation of linear polyacrylamide, cellulose derivatives, dextran, etc., optical isomers, a chiral selector is added to the separation which can be dissolved in aqueous separation buŠers giving buŠer. The most commonly used chiral selectors are rise to a separation medium that also acts as a molecular cyclodextrins, but crown ethers, polysaccharides and pro­ sieve. These separation media are easier to prepare than teins may also be used. Since chiral recognition is governed cross­linked polymers. They can be prepared in a vial and by the diŠerent interactions between the chiral selector and ˆlled by pressure in a wall­coated capillary (with no electro­ each of the enantiomers, the resolution achieved for the osmotic ‰ow). Replacing the gel before every injection chiral compounds depends largely on the type of chiral generally improves the separation reproducibility. The selector used. In this regard, for the development of a given porosity of the gels can be increased by using polymers of separation it may be useful to test cyclodextrins having a higher molecular mass (at a given polymer concentration) or diŠerent cavity size (a­, b­, or g­cyclodextrin) or modiˆed by decreasing the polymer concentration (for a given poly­ cyclodextrins with neutral (methyl, ethyl, hydroxyalkyl, etc.) mer molecular mass). A reduction in the gel porosity leads to or ionizable (aminomethyl, carboxymethyl, sulfobutyl ether, a decrease in the mobility of the solute for the same buŠer. etc.) groups. When using modiˆed cyclodextrins, batch­to­ Since the dissolution of these polymers in the buŠer gives batch variations in the degree of substitution of the low viscosity solutions, both hydrodynamic and electroki­ cyclodextrins must be taken into account since it will netic injection techniques can be used. in‰uence the selectivity. Other factors controlling the resolu­ 3. Capillary Isoelectric Focusing tion in chiral separations are concentration of chiral selec­ Principle tor, composition and pH of the buŠer and temperature. The In isoelectric focusing, the molecules migrate under the use of organic additives, such as methanol or urea can also in‰uence of the electric ˆeld, so long as they are charged, in modify the resolution achieved. a pH gradient generated by ampholytes having pI values in a Supplement II, JPXIV General Information 1825 wide range (poly­aminocarboxylic acids), dissolved in the ampholyte and the cathode reservoir is ˆlled with a solution separation buŠer. with a pH higher than the pI of the most basic ampholyte. The three basic steps of isoelectric focusing are loading, Phosphoric acid for the anode and sodium hydroxide for the focusing and mobilization. cathode are frequently used. Loading step: Two methods may be employed: Addition of a polymer, such as methylcellulose, in the —loading in one step: the sample is mixed with ampho­ ampholyte solution tends to suppress convective forces (if lytes and introduced into the capillary either by pressure any) and electro­osmotic ‰ow by increasing the viscosity. or vacuum; Commercial ampholytes are available covering many pH —sequential loading: a leading buŠer, then the ampho­ ranges and may be mixed if necessary to obtain an expanded lytes, then the sample mixed with ampholytes, again pH range. Broad pH ranges are used to estimate the isoelec­ ampholytes alone and ˆnally the terminating buŠer are tric point whereas narrower ranges are employed to improve introduced into the capillary. The volume of the sample accuracy. Calibration can be done by correlating migration must be small enough not to modify the pH gradient. time with isoelectric point for a series of protein markers. Focusing step: When the voltage is applied, ampholytes During the focusing step precipitation of proteins at their migrate toward the cathode or the anode, according to their isoelectric point can be prevented, if necessary, using buŠer net charge, thus creating a pH gradient from anode (lower additives such as glycerol, surfactants, urea or zwitterionic pH) to cathode (higher pH). During this step the compo­ buŠers. However, depending on the concentration, urea nents to be separated migrate until they reach a pH corre­ denatures proteins. sponding to their isoelectric point (pI) and the current drops 4. Micellar Electrokinetic Chromatography (MEKC) to very low values. Principle Mobilization step: If mobilization is required for detec­ In micellar electrokinetic chromatography, separation tion, use one of the following methods. Three methods are takes place in an electrolyte solution which contains a sur­ available: factant at a concentration above the critical micellar concen­ —in the ˆrst method, mobilization is accomplished during tration (cmc). The solute molecules are distributed between the focusing step under the eŠect of the electro­osmotic the aqueous buŠer and the pseudo­stationary phase com­ ‰ow; the electro­osmotic ‰ow must be small enough to posed of micelles, according to the partition coe‹cient of allow the focusing of the components; the solute. The technique can therefore be considered as a —in the second method, mobilization is accomplished by hybrid of electrophoresis and chromatography. It is a tech­ applying positive pressure after the focusing step; nique that can be used for the separation of both neutral and —in the third method, mobilization is achieved after the charged solutes, maintaining the e‹ciency, speed and focusing step by adding salts to the cathode reservoir or instrumental suitability of capillary electrophoresis. One of the anode reservoir (depending on the direction chosen the most widely used surfactants in MEKC is the anionic for mobilization) in order to alter the pH in the capilla­ surfactant sodium dodecyl sulfate, although other sur­ ry when the voltage is applied. As the pH is changed, factants, for example cationic surfactants such as the proteins and ampholytes are mobilized in the cetyltrimethylammonium salts, are also used. direction of the reservoir which contains the added salts The separation mechanism is as follows. At neutral and and pass the detector. alkaline pH, a strong electro­osmotic ‰ow is generated and The separation achieved, expressed as DpI, depends on moves the separation buŠer ions in the direction of the the pH gradient (dpH/dx), the number of ampholytes hav­ cathode. If sodium dodecyl sulfate is employed as the sur­ ing diŠerent pI values, the molecular diŠusion coe‹cient factant, the electrophoretic migration of the anionic micelle (D), the intensity of the electric ˆeld (E) and the variation of is in the opposite direction, towards the anode. As a result, the electrophoretic mobility of the analyte with the pH the overall micelle migration velocity is slowed down com­ (|dm/dpH): pared to the bulk ‰ow of the electrolytic solution. In the case D(dpH/dx) of neutral solutes, since the analyte can partition between DPI3 E(|dm/dpH) the micelle and the aqueous buŠer, and has no elec­ trophoretic mobility, the analyte migration velocity will Optimization depend only on the partition coe‹cient between the micelle The main parameters to be considered in the development and the aqueous buŠer. In the electropherogram, the peaks of separations are: corresponding to each uncharged solute are always between Voltage: Capillary isoelectric focusing utilises very high that of the electro­osmotic ‰ow marker and that of the electric ˆelds, 300 V/cm to 1000 V/cm in the focusing step. micelle (the time elapsed between these two peaks is called Capillary: The electro­osmotic ‰ow must be reduced or the separation window). For electrically charged solutes, the suppressed depending on the mobilization strategy (see migration velocity depends on both the partition coe‹cient above). Coated capillaries tend to reduce the electro­osmotic of the solute between the micelle and the aqueous buŠer, and ‰ow. on the electrophoretic mobility of the solute in the absence Solutions: The anode buŠer reservoir is ˆlled with a of micelle. solution with a pH lower than the pI of the most acidic Since the mechanism in MEKC of neutral and weakly 1826 General Information Supplement II, JPXIV ionized solutes is essentially chromatographic, migration of decrease the electric ˆelds (working at constant voltage), thesoluteandresolutioncanberationalizedintermsofthe increase migration time and improve the separation e‹cien­ retention factor of the solute (k?), also referred to as mass cy. The internal diameter controls heat dissipation (for a distribution ratio (Dm),whichistheratioofthenumberof given buŠer and electric ˆeld) and consequently the sample moles of solute in the micelle to those in the mobile phase. band broadening. For a neutral compound, k? is given by: Electrolytic solution parameters Surfactant type and concentration:Thetypeofsur­ t |t V k? R 0 K S factant, in the same way as the stationary phase in chro­ t V t 1| R M matography, aŠects the resolution since it modiˆes separa­ 0Ø t » mc tion selectivity. Also, the log k? of a neutral compound

tR:migration time of the solute, increases linearly with the concentration of surfactant in the

t0:analysis time of an unretained solute (determined by in­ mobile phase. Since resolution in MEKC reaches a maxi­

jecting an electro­osmotic ‰ow marker which does not mum when k? approaches the value of tm/t0, modifying the enter the micelle, for instance methanol), concentration of surfactant in the mobile phase changes the

tmc:micelle migration time (measured by injecting a resolution obtained. micelle marker, such as Sudan III, which migrates BuŠer pH:Although pH does not modify the partition while continuously associated in the micelle), coe‹cient of non­ionized solutes, it can modify the electro­ K:partition coe‹cient of the solute, osmotic ‰ow in uncoated capillaries. A decrease in the buŠer

VS:volume of the micellar phase, pH decreases the electro­osmotic ‰ow and therefore

VM:volume of the mobile phase. increases the resolution of the neutral solutes in MEKC, resulting in a longer analysis time. Likewise, the resolution between two closely­migrating Organic solvents:To improve MEKC separation of solutes (R )isgivenby: S hydrophobic compounds, organic modiˆers (methanol, t propanol, acetonitrile, etc.) can be added to the electrolytic 1|Ø 0 » N a|1 k? tmc solution. The addition of these modiˆers usually decreases R  ~ ~ b ~ S 4 a k? {1 t migration time and the selectivity of the separation. Since b 1{Ø 0 »k? a tmc the addition of organic modiˆers aŠects the critical micellar concentration, a given surfactant concentration can be used N:number of theoretical plates for one of the solutes, only within a certain percentage of organic modiˆer before a:selectivity, the micellization is inhibited or adversely aŠected, resulting k? and k? :retention factors for both solutes, respectively a b in the absence of micelles and, therefore, in the absence of (k? Àk? ). b a partition. The dissociation of micelles in the presence of a

Similar, but not identical, equations give k? and RS values high content of organic solvent does not always mean that for electrically charged solutes. the separation will no longer be possible; in some cases the Optimization hydrophobic interaction between the ionic surfactant The main parameters to be considered in the development monomer and the neutral solutes forms solvophobic of separations by MEKC are instrumental and electrolytic complexes that can be separated electrophoretically. solution parameters. Additives for chiral separations:For the separation of Instrumental parameters enantiomers using MEKC, a chiral selector is included in the Voltage:Separation time is inversely proportional to micellar system, either covalently bound to the surfactant or applied voltage. However, an increase in voltage can cause added to the micellar separation electrolyte. Micelles that excessive heat production that gives rise to temperature have a moiety with chiral discrimination properties include gradients and viscosity gradients of the buŠer in the cross­ salts of N­dodecanoyl­L­amino acids, bile salts, etc. Chiral section of the capillary. This eŠect can be signiˆcant with resolution can also be achieved using chiral discriminators, high conductivity buŠers such as those containing micelles. such as cyclodextrins, added to the electrolytic solutions Poor heat dissipation causes band broadening and decreases which contain micellized achiral surfactants. resolution. Other additives:Several strategies can be carried out to Temperature:Variations in capillary temperature aŠect modify selectivity, by adding chemicals to the buŠer. The the partition coe‹cient of the solute between the buŠer and addition of several types of cyclodextrins to the buŠer can the micelles, the critical micellar concentration and the also be used to reduce the interaction of hydrophobic solutes viscosity of the buŠer. These parameters contribute to the with the micelle, thus increasing the selectivity for this type migration time of the solutes. The use of a good cooling sys­ of compound. tem improves the reproducibility of the migration time for The addition of substances able to modify solute­micelle the solutes. interactions by adsorption on the latter, is used to improve Capillary:As in free solution capillary electrophoresis, the selectivity of the separations in MEKC. These additives the dimensions of the capillary (length and internal di­ may be a second surfactant (ionic or non­ionic) which gives ameter) contribute to analysis time and e‹ciency of separa­ rise to mixed micelles or metallic cations which dissolve in tions. Increasing both eŠective length and total length can the micelle and form co­ordination complexes with the Supplement II, JPXIV General Information 1827 solutes. When appropriate, the resolution may be calculated by measuring the height of the valley (H ) between two partly Quantiˆcation v resolved peaks in a standard preparation and the height of Peak areas must be divided by the corresponding migra­ the smaller peak (H ) and calculating the peak­to­valley tion time to give the corrected area in order to: p ratio: —compensate for the shift in migration time from run to run, thus reducing the variation of the response, H p/n p —compensate for the diŠerent responses of sample Hv constituents with diŠerent migration times. Where an internal standard is used, verify that no peak of Symmetry Factor the substance to be examined is masked by that of the The symmetry factor (AS) of a peak may be calculated internal standard. using the expression: Calculations w From the values obtained, calculate the content of the A  0.05 S 2d component or components being examined. When prescribed, the percentage content of one or more compo­ w0.05: width of the peak at one­twentieth of the peak nents of the sample to be examined is calculated by deter­ height, mining the corrected area(s) of the peak(s) as a percentage of d: distance between the perpendicular dropped from the the total of the corrected areas of all peaks, excluding those peak maximum and the leading edge of the peak at due to solvents or any added reagents (normalization one­twentieth of the peak height. procedure). The use of an automatic integration system Tests for area repeatability (standard deviation of areas or (integrator or data acquisition and processing system) is of the area/migration­time ratio) and for migration time recommended. repeatability (standard deviation of migration time) are System Suitability introduced as suitability parameters. Migration time In order to check the behavior of the capillary electropho­ repeatability provides a test for the suitability of the capilla­ resis system, system suitability parameters are used. The ry washing procedures. An alternative practice to avoid the choice of these parameters depends on the mode of capillary lack of repeatability of the migration time is to use migration electrophoresis used. They are: retention factor (k?)(only time relative to an internal standard. for micellar electrokinetic chromatography), apparent A test for the veriˆcation of the signal­to­noise ratio for a number of theoretical plates (N), symmetry factor (AS)and standard preparation (or the determination of the limit of resolution (RS). In previous sections, the theoretical expres­ quantiˆcation) may also be useful for the determination of sions for N and RS have been described, but more practical related substances. equations that allow these parameters to be calculated from Signal­to­noise Ratio the electropherograms are given below. The detection limit and quantiˆcation limit correspond to Apparent Number of Theoretical Plates signal­to­noise ratios of 3 and 10 respectively. The signal­to­ The apparent number of theoretical plates (N)maybe noise ratio (S/N) is calculated using the expression: calculated using the expression: 2H S/N t 2 h N5.54 R Ø w » h H: height of the peak corresponding to the component

tR: migration time or distance along the baseline from the concerned, in the electropherogram obtained with the point of injection to the perpendicular dropped from prescribed reference solution, measured from the maximum the maximum of the peak corresponding to the compo­ of the peak to the extrapolated baseline of the signal nent, observed over a distance equal to twenty times the width at

wh: width of the peak at half­height. half­height, h: range of the background in an electropherogram ob­ Resolution tained after injection of a blank, observed over a distance The resolution (RS) between peaks of similar height of equal to twenty times the width at the half­height of the peak two components may be calculated using the expression: in the electropherogram obtained with the prescribed refer­ 1.18(t |t ) ence solution and, if possible, situated equally around the R Ø R2 R1 » S wh1{wh2 place where this peak would be found. tR2ÀtR1

tR1 and tR2: migration times or distances along the baseline from the point of injection to the perpendicu­ lars dropped from the maxima of two adjacent peaks,

wh1 and wh2: peak widths at half­height. 1828 General Information Supplement II, JPXIV

Add the following: and/or preparation. Having salt in the sample can be prob­ lematic and it is best to prepare the sample, if possible, in 22. Isoelectric Focusing deionized water or 2 per cent ampholytes, using dialysis or gel ˆltration if necessary. General Principles The time required for completion of focusing in thin­layer Isoelectric focusing (IEF) is a method of electrophoresis polyacrylamide gels is determined by placing a colored that separates proteins according to their isoelectric point. protein (e.g. hemoglobin) at diŠerent positions on the gel Separation is carried out in a slab of polyacrylamide or surface and by applying the electric ˆeld: the steady state is agarose gel that contains a mixture of amphoteric electro­ reached when all applications give an identical band pattern. lytes (ampholytes). When subjected to an electric ˆeld, the In some protocols the completion of the focusing is indicat­ ampholytes migrate in the gel to create a pH gradient. In ed by the time elapsed after the sample application. some cases gels containing an immobilized pH gradient, pre­ The IEF gel can be used as an identity test when the pared by incorporating weak acids and bases to speciˆc migration pattern on the gel is compared to a suitable regions of the gel network during the preparation of the gel, standard preparation and IEF calibration proteins, the IEF are used. When the applied proteins reach the gel fraction gel can be used as a limit test when the density of a band on that has a pH that is the same as their isoelectric point (pI), IEF is compared subjectively with the density of bands their charge is neutralized and migration ceases. Gradients appearing in a standard preparation, or it can be used as a can be made over various ranges of pH, according to the quantitative test when the density is measured using a mixture of ampholytes chosen. densitometer or similar instrumentation to determine the relative concentration of protein in the bands subject to Theoretical Aspects validation. When a protein is at the position of its isoelectric point, it has no net charge and cannot be moved in a gel matrix by the Apparatus electric ˆeld. It may, however, move from that position by An apparatus for IEF consists of: diŠusion. The pH gradient forces a protein to remain in its —a controllable generator for constant potential, current isoelectric point position, thus concentrating it; this concen­ and power. Potentials of 2500 V have been used and are trating eŠect is called ``focusing''. Increasing the applied considered optimal under a given set of operating voltage or reducing the sample load result in improved conditions. Supply of up to 30 Wof constant power is separation of bands. The applied voltage is limited by the recommended, heat generated, which must be dissipated. The use of thin —a rigid plastic IEF chamber that contains a cooled plate, gels and an e‹cient cooling plate controlled by a thermostat­ of suitable material, to support the gel, ic circulator prevents the burning of the gel whilst allowing —a plastic cover with platinum electrodes that are con­ sharp focusing. The separation is estimated by determining nected to the gel by means of paper wicks of suitable the minimum pI diŠerence (DpI), which is necessary to width, length and thickness, impregnated with solutions separate 2 neighboring bands: of anodic and cathodic electrolytes.

D(dpH/dx) Isoelectric Focusing in Polyacrylamide Gels: Detailed DpI3 E(|dm/dpH) Procedure The following method is a detailed description of an IEF D: DiŠusion coe‹cient of the protein procedure in thick polyacrylamide slab gels, which is used dpH/dx:pHgradient unless otherwise stated in the monograph. E: Intensity of the electric ˆeld, in volts per centimeter |dm/dpH: Variation of the solute mobility with the pH in Preparation of the Gels the region close to the pI Mould The mould (see Figure) is composed of a glass plate (A) on which a polyester ˆlm (B) is placed to facilitate Since D and |dm/dpH for a given protein cannot be handling of the gel, one or more spacers (C), a second glass altered, the separation can be improved by using a narrower plate (D) and clamps to hold the structure together. pH range and by increasing the intensity of the electric ˆeld. Resolution between protein bands on an IEF gel prepared with carrier ampholytes can be quite good. Improvements in resolution may be achieved by using immobilized pH gradients where the buŠering species, which are analogous to carrier ampholytes, are copolymerized within the gel matrix. Proteins exhibiting pIs diŠering by as little as 0.02 pH units may be resolved using a gel prepared with carrier ampholytes while immobilized pH gradients can resolve proteins diŠering by approximately 0.001 pH units.

Practical Aspects Special attention must be paid to sample characteristics Figure. Mould Supplement II, JPXIV General Information 1829

7.5z Polyacrylamide gel Dissolve 29.1 g of acrylamide —variations in the sample application procedure, including and 0.9 g of N,N?­methylenebisacrylamide in 100 mL of diŠerent sample volumes or the use of sample application water. To 2.5 volumes of this solution, add the mixture of masks or wicks other than paper, ampholytes speciˆed in the monograph and dilute to 10 —the use of alternate running conditions, including varia­ volumes with water. Mix carefully and degas the solution. tions in the electric ˆeld depending on gel dimensions and Preparation of the mould Place the polyester ˆlm on the equipment, and the use of ˆxed migration times rather lower glass plate, apply the spacer, place the second glass than subjective interpretation of band stability, plateandˆttheclamps.Place7.5z polyacrylamide gel —the inclusion of a pre­focusing step, prepared before use on a magnetic stirrer, and add 0.25 —the use of automated instrumentation, volumes of a solution of ammonium persulfate (1 in 10) and —the use of agarose gels. 0.25 volumes of N,N,N?,N?­tetramethylethylenediamine. Validation of Iso­Electric Focusing Procedures Immediately ˆll the space between the glass plates of the Where alternative methods to the detailed procedure are mould with the solution. employed they must be validated. The following criteria may Method be used to validate the separation: Dismantle the mould and, making use of the polyester —formation of a stable pH gradient of desired characteris­ ˆlm, transfer the gel onto the cooled support, wetted with a tics, assessed for example using colored pH markers of few millilitres of a suitable liquid, taking care to avoid known isoelectric points, forming air bubbles. Prepare the test solutions and reference —comparison with the electropherogram provided with the solutions as speciˆed in the monograph. Place strips of chemical reference substance for the preparation to be paper for sample application, about 10 mm~5 mm in size, examined, on the gel and impregnate each with the prescribed amount —any other validation criteria as prescribed in the mono­ of the test and reference solutions. Also apply the prescribed graph. quantity of a solution of proteins with known isoelectric Speciˆed Variations to the General Method points as pH markers to calibrate the gel. In some protocols Variations to the general method required for the analysis the gel has pre­cast slots where a solution of the sample is of speciˆc substances may be speciˆed in detail in mono­ applied instead of using impregnated paper strips. Cut 2 graphs. These include: strips of paper to the length of the gel and impregnate them —the addition of urea in the gel (3 mol/L concentration is with the electrolyte solutions: acid for the anode and often satisfactory to keep protein in solution but up to 8 alkaline for the cathode. The compositions of the anode and mol/L can be used): some proteins precipitate at their cathode solutions are given in the monograph. Apply these isoelectric point. In this case, urea is included in the gel paper wicks to each side of the gel several millimetres from formulation to keep the protein in solution. If urea is the edge. Fit the cover so that the electrodes are in contact used, only fresh solutions should be used to prevent with the wicks (respecting the anodic and cathodic poles). carbamylation of the protein, Proceed with the isoelectric focusing by applying the electri­ —the use of alternative staining methods, cal parameters described in the monograph. Switch oŠ the —the use of gel additives such as non­ionic detergents (e.g. current when the migration of the mixture of standard octylglucoside) or zwitterionic detergents (e.g., CHAPS proteins has stabilized. Using forceps, remove the sample or CHAPSO), and the addition of ampholyte to the sam­ application strips and the 2 electrode wicks. Immerse the gel ple, to prevent proteins from aggregating or precipitating. in ˆxing solution for isoelectric focusing in polyacrylamide gel. Incubate with gentle shaking at room temperature for 30 Points to Consider minutes. Drain oŠ the solution and add 200 mL of destain­ Samples can be applied to any area on the gel, but to pro­ ing solution. Incubate with shaking for 1 hour. Drain the tect the proteins from extreme pH environments samples gel, add coomassie staining TS. Incubate for 30 minutes. should not be applied close to either electrode. During Destain the gel by passive diŠusion with destaining solution method development the analyst can try applying the protein until the bands are well visualized against a clear back­ in 3 positions on the gel (i.e. middle and both ends); the ground. Locate the position and intensity of the bands in the pattern of a protein applied at opposite ends of the gel may electropherogram as prescribed in the monograph. not be identical. A phenomenon known as cathodic drift, where the pH Variations to the Detailed Procedure (Subject to Validation) gradient decays over time, may occur if a gel is focused too Where reference to the general method on isoelectric long. Although not well understood, electroendoosmosis focusing is made, variations in methodology or procedure and absorption of carbon dioxide may be factors that lead to may be made subject to validation. These include: cathodic drift. Cathodic drift is observed as focused protein —the use of commercially available pre­cast gels and of migrating oŠ the cathode end of the gel. Immobilized pH commercial staining and destaining kits, gradients may be used to address this problem. —the use of immobilized pH gradients, E‹cient cooling (approximately 49C) of the bed that the —the use of rod gels, gel lies on during focusing is important. High ˆeld strengths —the use of gel cassettes of diŠerent dimensions, including used during isoelectric focusing can lead to overheating and ultra­thin (0.2 mm) gels, 1830 General Information Supplement II, JPXIV aŠect the quality of the focused gel. peptides. A test sample is digested and assayed in parallel with a reference standard or a reference material. Complete Reagents and Solutions— cleavage of peptide bonds is more likely to occur when en­ Fixing solution for isoelectric focusing in polyacrylamide zymes such as endoproteases (e.g., trypsin) are used, instead gel Dissolve 35 g of 5­sulfosalicylic acid dihydrate and of chemical cleavage reagents. A map should contain 100 g of trichloroacetic acid in water to make 1000 mL. enough peptides to be meaningful. On the other hand, if Coomassie staining TS Dissolve 125 mg of coomassie there are too many fragments, the map might lose its brilliant blue R­250 in 100 mL of a mixture of water, speciˆcity because many proteins will then have the same methanol and acetic acid (100) (5:4:1), and ˆlter. proˆles. Destaining solution A mixture of water, methanol and acetic acid (100) (5:4:1). Isolation and Puriˆcation Isolation and puriˆcation are necessary for analysis of bulk drugs or dosage forms containing interfering excipients Add the following: and carrier proteins and, when required, will be speciˆed in the monograph. Quantitative recovery of protein from the 23. Peptide Mapping dosage form should be validated. Selective Cleavage of Peptide Bonds Purpose and Scope The selection of the approach used for the cleavage of Peptide mapping is an identity test for proteins, especially peptide bonds will depend on the protein under test. This those obtained by r­DNA technology. It involves the chemi­ selection process involves determination of the type of cal or enzymatic treatment of a protein resulting in the for­ cleavage to be employed —enzymatic or chemical— and the mation of peptide fragments followed by separation and type of cleavage agent within the chosen category. Several identiˆcation of the fragments in a reproducible manner. It cleavage agents and their speciˆcity are shown in Table 1. is a powerful test that is capable of identifying single amino This list is not all­inclusive and will be expanded as other acid changes resulting from events such as errors in the cleavage agents are identiˆed. reading of complementary DNA (cDNA) sequences or point mutations. Peptide mapping is a comparative procedure Table 1. Examples of Cleavage Agents because the information obtained, compared to a reference standard or reference material similarly treated, conˆrms the Type Agent Speciˆcity primary structure of the protein, is capable of detecting Enzymatic Trypsin (EC 3.4.21.4) C­terminal side of whether alterations in structure have occurred, and demon­ Arg and Lys strates process consistency and genetic stability. Each pro­ Chymotrypsin C­terminal side of tein presents unique characteristics which must be well (EC 3.4.21.1) hydrophobic understood so that the scientiˆc and analytical approaches residues (e.g., permit validated development of a peptide map that pro­ Leu, Met, Ala, vides su‹cient speciˆcity. aromatics) This chapter provides detailed assistance in the applica­ Pepsin (EC 3.4.23.1 & 2) Nonspeciˆc digest tion of peptide mapping and its validation to characterize Lysyl endopeptidase C­terminal side of the desired protein product, to evaluate the stability of the (Lys­C Endopeptidase) Lys expression construct of cells used for recombinant DNA (EC 3.4.21.50) products and to evaluate the consistency of the overall proc­ Glutamyl endopeptidase C­terminal side of (from S. aureus strain V8) Glu and Asp ess, to assess product stability as well as to ensure the identi­ (EC 3.4.21.19) ty of the protein product, or to detect the presence of protein Peptidyl­Asp metallo N­terminal side of variant. endopeptidase Asp The Peptide Map (Endoproteinase Asp­N) Peptide mapping is not a general method, but involves (EC 3.24.33) Clostripain (EC 3.4.22.8) C­terminal side of developing speciˆc maps for each unique protein. Although Arg the technology is evolving rapidly, there are certain methods that are generally accepted. Variations of these methods will Chemical Cyanogen bromide C­terminal side of be indicated, when appropriate, in speciˆc monographs. Met A peptide map may be viewed as a ˆngerprint of a protein 2­Nitro­5­thio­cyanobenzoic and is the end product of several chemical processes that acid N­terminal side of provide a comprehensive understanding of the protein being Cys o­Iodosobenzoic acid C­terminal side of analyzed. Four major steps are necessary for the develop­ Trp and Tyr ment of the procedure: isolation and puriˆcation of the Dilute acid Asp and Pro protein, if the protein is part of a formulation; selective BNPS­skatole Trp cleavage of the peptide bonds; chromatographic separation of the peptides; and analysis and identiˆcation of the Supplement II, JPXIV General Information 1831

Pretreatment of Sample Depending on the size or the protein under test will dictate the temperature of the reaction conˆguration of the protein, diŠerent approaches in the milieu, because some proteins are more susceptible to pretreatment of samples can be used. For monoclonal denaturation as the temperature of the reaction increases. antibodies the heavy and light chains will need to be separat­ For example, digestion of recombinant bovine somatropin is ed before mapping. If trypsin is used as a cleavage agent for conducted at 49C, because at higher temperatures it will proteins with a molecular mass greater than 100,000 Da, precipitate during digestion. lysine residues must be protected by citraconylation or Time: If su‹cient sample is available, a time course maleylation; otherwise, many peptides will be generated. study is considered in order to determine the optimum time Pretreatment of the Cleavage Agent Pretreatment of to obtain a reproducible map and avoid incomplete cleavage agents —especially enzymatic agents— might be digestion. Time of digestion varies from 2 to 30 hours. The necessary for puriˆcation purposes to ensure reproducibility reaction is stopped by the addition of an acid which does not of the map. For example, trypsin used as a cleavage agent interfere in the tryptic map or by freezing. will have to be treated with tosyl­L­phenylalanine chlo­ Amount of Cleavage Agent: Although excessive romethyl ketone to inactivate chymotrypsin. Other methods, amounts of cleavage agent are used to accomplish a reasona­ such as puriˆcation of trypsin by HPLC or immobilization bly rapid digestion time (i.e., 6 to 20 hours), the amount of of enzyme on a gel support, have been successfully used cleavage agent is minimized to avoid its contribution to the when only a small amount of protein is available. chromatographic map pattern. A protein to protease ratio Pretreatment of the Protein Under certain conditions, it between 20:1 and 200:1 is generally used. It is recommended might be necessary to concentrate the sample or to separate that the cleavage agent can be added in two or more stages to the protein from added substances and stabilizers used in optimize cleavage. Nonetheless, the ˆnal reaction volume formulation of the product, if these interfere with the remains small enough to facilitate the next step in peptide mapping procedure. Physical procedures used for pretreat­ mapping —the separation step. To sort out digestion ment can include ultraˆltration, column chromatography, artifacts that might be interfering with the subsequent and lyophilization. Other pretreatments, such as the analysis, a blank determination is performed, using a diges­ addition of chaotropic agents (e.g., urea) can be used to tion control with all the reagents, except the test protein. unfold the protein prior to mapping. To allow the enzyme to Chromatographic Separation have full access to cleavage sites and permit some unfolding Many techniques are used to separate peptides for of the protein, it is often necessary to reduce and alkylate the mapping. The selection of a technique depends on the pro­ disulˆde bonds prior to digestion. tein being mapped. Techniques that have been successfully Digestion with trypsin can introduce ambiguities in the used for separation of peptides are shown in Table 2. In this tryptic map due to side reactions occurring during the section, a most widely used reverse­phase High Performance digestion reaction, such as nonspeciˆc cleavage, deamida­ Liquid Chromatographic (RP­HPLC) method is described tion, disulˆde isomerization, oxidation of methionine as one of the procedures of chromatographic separation. residues, or formation of pyroglutamic groups created from the deamidation of glutamine at the N­terminal side of a Table 2. Techniques Used for the Separation of Peptides peptide. Furthermore, peaks may be produced by auto­ hydrolysis of trypsin. Their intensities depend on the ratio of Reverse­Phase High Performance Liquid Chromatography trypsin to protein. To avoid autohydrolysis, solutions of (RP­HPLC) proteases may be prepared at a pH that is not optimal (e.g., Ion­Exchange Chromatography (IEC) at pH 5 for trypsin), which would mean that the enzyme Hydrophobic Interaction Chromatography (HIC) would not become active until diluted with the digest buŠer. Polyacrylamide Gel Electrophoresis (PAGE), nondenaturat­ ing Establishment of Optimal Digestion Conditions Factors Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis that aŠect the completeness and eŠectiveness of digestion of (SDS­PAGE) proteins are those that could aŠect any chemical or enzymat­ Capillary Electrophoresis (CE) ic reactions. Paper Chromatography­High Voltage (PCHV) pH: The pH of the digestion mixture is empirically High­Voltage Paper Electrophoresis (HVPE) determined to ensure the optimization of the performance of the given cleavage agent. For example, when using cyanogen bromide as a cleavage agent, a highly acidic environment The purity of solvents and mobile phases is a critical (e.g., pH 2, formic acid) is necessary; however, when using factor in HPLC separation. HPLC­grade solvents and water trypsin as a cleavage agent, a slightly alkaline environment that are commercially available, are recommended for RP­ (pH 8) is optimal. As a general rule, the pH of the reaction HPLC. Dissolved gases present a problem in gradient milieu should not alter the chemical integrity of the protein systems where the solubility of the gas in a solvent may be during the digestion and should not change during the less in a mixture than in a single solvent. Vacuum degassing course of the fragmentation reaction. and agitation by sonication are often used as useful degass­ Temperature: A temperature between 259Cand379Cis ing procedures. When solid particles in the solvents are adequate for most digestions. The temperature used is drawn into the HPLC system, they can damage the sealing intended to minimize chemical side reactions. The type of of pump valves or clog the top of the chromatographic 1832 General Information Supplement II, JPXIV column. Both pre­ and post­pump ˆltration is also recom­ the development and establishment of system suitability mended. limits. In addition a specimen chromatogram should be Chromatographic Column The selection of a chromato­ included with the Reference Standard or Reference Material graphic column is empirically determined for each protein. for additional comparison purposes. Other indicators may Columns with 100 Å or 300 Å pore size with silica support include visual inspection of protein or peptide solubility, the can give optimal separation. For smaller peptides, octylsi­ absence of intact protein, or measurement of responses of a lane chemically bonded to totally porous silica articles, 3 to digestion­dependent peptide. The critical system suitability 10 mm in diameter (L7) and octadecylsilane chemically parameters for peptide analysis will depend on the particular bonded to porous silica or ceramic micro­particles, 3 to 10 mode of peptide separation and detection and on the data mm in diameter (L1) column packings are more e‹cient than analysis requirements. the butyl silane chemically bonded to totally porous silica When peptide mapping is used as an identiˆcation test, the particles, 5 to 10 mmindiameter(L26)packing. system suitability requirements for the identiˆed peptides Solvent The most commonly used solvent is water with covers selectivity and precision. In this case, as well as when acetonitrile as the organic modiˆer to which less than 0.1z identiˆcation of variant protein is done, the identiˆcation of tri‰uoroacetic acid is added. If necessary, add isopropyl the primary structure of the peptide fragments in the peptide alcohol or n­propyl alcohol to solubilize the digest compo­ map provides both a veriˆcation of the known primary nents, provided that the addition does not unduly increase structure and the identiˆcation of protein variants by com­ the viscosity of the components. parison with the peptide map of the reference stan­ Mobile Phase BuŠered mobile phases containing phos­ dard/reference material for the speciˆed protein. The use of phate are used to provide some ‰exibility in the selection of a digested reference standard or reference material for a pH conditions, since shifts of pH in the 3.0 to 5.0 range given protein in the determination of peptide resolution is enhance the separation of peptides containing acidic residues themethodofchoice.Forananalysisofavariantprotein,a (e.g., glutamic and aspartic acids). Sodium or potassium characterized mixture of a variant and a reference standard phosphates, ammonium acetate, phosphoric acid, and a pH or reference material can be used, especially if the variant between 2 and 7 (or higher for polymer­based supports) have peptide is located in a less­resolved region of the map. The also been used with acetonitrile gradients. Acetonitrile con­ index of pattern consistency can be simply the number of taining tri‰uoroacetic acid is used quite often. major peptides detected. Peptide pattern consistency can be Gradient Selection Gradients can be linear, nonlinear, best deˆned by the resolution of peptide peaks. Chromato­ or include step functions. A shallow gradient is recommend­ graphic parameters —such as peak­to­peak resolution, ed in order to separate complex mixtures. Gradients are maximum peak width, peak area, peak tailing factors, and optimized to provide clear resolution of one or two peaks column e‹ciency— may be used to deˆne peptide resolu­ that will become ``marker'' peaks for the test. tion. Depending on the protein under test and the method of Isocratic Selection Isocratic HPLC systems using a sin­ separation used, single peptide or multiple peptide resolu­ gle mobile phase are used on the basis of their convenience tion requirements may be necessary. of use and improved detector responses. Optimal composi­ The replicate analysis of the digest of the reference tion of a mobile phase to obtain clear resolution of each standard or reference material for the protein under test peak is sometimes di‹cult to establish. Mobile phases for yields measures of precision and quantitative recovery. which slight changes in component ratios or in pH sig­ Recovery of the identiˆed peptides is generally ascertained niˆcantly aŠect retention times of peaks in peptide maps by the use of internal or external peptide standards. The should not be used in isocratic HPLC systems. precision is expressed as the relative standard deviation Other Parameters Temperature control of the column is (RSD). DiŠerences in the recovery and precision of the usually necessary to achieve good reproducibility. The ‰ow identiˆed peptides are expected; therefore, the system rates for the mobile phases range from 0.1 to 2.0 mL per suitability limits will have to be established for both the minute, and the detection of peptides is performed with a recovery and the precision of the identiˆed peptides. These UV detector at 200 to 230 nm. Other methods of detection limits are unique for a given protein and will be speciˆed in have been used (e.g., postcolumn derivatization), but they the individual monograph. are not as robust or versatile as UV detection. Visual comparison of the relative retention times, the peak Validation This section provides an experimental responses (the peak area or the peak height), the number of means for measuring the overall performance of the test peaks, and the overall elution pattern is completed initially. method. The acceptance criteria for system suitability de­ It is then complemented and supported by mathematical pend on the identiˆcation of critical test parameters that analysis of the peak response ratios and by the chromato­ aŠect data interpretation and acceptance. These critical graphic proˆle of a 1:1 (v/v) mixture of sample and parameters are also criteria that monitor peptide digestion reference standard or reference material digest. If all peaks and peptide analysis. An indicator that the desired digestion in the sample digest and in the reference standard or refer­ endpoint was achieved is by the comparison with a ence material digest have the same relative retention times Reference Standard, which is treated exactly as the article and peaks response ratios, then the identity of the sample under test. The use of a reference standard or reference under test is conˆrmed. material in parallel with the protein under test is critical in If peaks that initially eluted with signiˆcantly diŠerent Supplement II, JPXIV General Information 1833 relative retention times are then observed as single peaks in ing and amino acids analysis are used, the analytical separa­ the 1:1 mixture, the initial diŠerence would be an indication tion is scaled up. Since scale­up might aŠect the resolution of of system variability. However, if separate peaks are peptide peaks, it is necessary, using empirical data, to assure observed in the 1:1 mixture, this would be evidence of the that there is no loss of resolution due to scale­up. Eluates nonequivalence of the peptides in each peak. If a peak in the corresponding to speciˆc peptide peaks are collected, 1:1 mixture is signiˆcantly broader than the corresponding vacuum­concentrated, and chromatographed again, if peak in the sample and reference standard or reference necessary. Amino acid analysis of fragments may be limited material digest, it may indicate the presence of diŠerent by the peptide size. If the N­terminus is blocked, it may need peptides. The use of computer­aided pattern recognition to be cleared before sequencing. C­terminal sequencing of software for the analysis of peptide mapping data has been proteins in combination with carboxypeptidase and proposed and applied, but issues related to the validation of MALDITOF­MS can also be used for characterization the computer software preclude its use in a compendial test purposes. in the near future. Other automated approaches have been The use of MS for characterization of peptide fragments is used that employ mathematical formulas, models, and by direct infusion of isolated peptides or by the use of on­ pattern recognition. Such approaches are, for example, the line LC­MS for structure analysis. In general, it includes automated identiˆcation of compounds by IR spectroscopy electrospray and matrix­assisted laser desorption ionization and the application of diode­array UV spectral analysis for coupled to time­of­‰ight analyzer (MALDITOF) as well as identiˆcation of peptides. These methods have limitations fast atom bombardment (FAB). Tandem MS has also been due to inadequate resolutions, co­elution of fragments, or used to sequence a modiˆed protein and to determine the absolute peak response diŠerences between reference type of amino acid modiˆcation that has occurred. The standard or reference material and sample fragments. comparison of mass spectra of the digests before and after The numerical comparison of the retention times and peak reduction provides a method to assign the disulˆde bonds to areas or peak heights can be done for a selected group of the various sulfhydryl­containing peptides. relevant peaks that have been correctly identiˆed in the If regions of the primary structure are not clearly demon­ peptide maps. Peak areas can be calculated using one peak strated by the peptide map, it might be necessary to develop showing relatively small variation as an internal reference, a secondary peptide map. The goal of a validated method of keeping in mind that peak area integration is sensitive to characterization of a protein through peptide mapping is to baseline variation and likely to introduce error in the reconcile and account for at least 95z of the theoretical analysis. Alternatively, the percentage of each peptide peak composition of the protein structure. height relative to the sum of all peak heights can be calculat­ ed for the sample under test. The percentage is then compared to that of the corresponding peak of the reference Add the following: standard/reference material. The possibility of auto­hydrol­ ysis of trypsin is monitored by producing a blank peptide 24. Rapid Identiˆcation of map, that is, the peptide map obtained when a blank solu­ Microorganisms Based on tion is treated with trypsin. The minimum requirement for the qualiˆcation of peptide Molecular Biological Method mapping is an approved test procedure that includes system suitability as a test control. In general, early in the regulato­ This chapter describes the methods for the identiˆcation ry process, qualiˆcation of peptide mapping for a protein is or estimation of microorganisms (bacteria and fungi), found su‹cient. As the regulatory approval process for the protein in in­process control tests or lot release tests of pharmaceuti­ progresses, additional qualiˆcations of the test can include a cal products, at the species or genus level based on their partial validation of the analytical procedure to provide DNA sequence homology. The identiˆcation of isolates assurance that the method will perform as intended in the found in the sterility test or aseptic processing can be helpful development of a peptide map for the speciˆed protein. for investigating the causes of contamination. Furthermore, Analysis and Identiˆcation of Peptides information on microorganisms found in raw materials used This section gives guidance on the use of peptide mapping for pharmaceutical products, processing areas of phar­ during development in support of regulatory applications. maceutical products, and so on is useful in designing The use of a peptide map as a qualitative tool does not measures to control the microbiological quality of drugs. require the complete characterization of the individual For the identiˆcation of microorganisms, phenotypic analy­ peptide peaks. However, validation of peptide mapping in sis is widely used, based on morphological, physiological, support of regulatory applications requires rigorous cha­ and biochemical features and analysis of components. racterization of each of the individual peaks in the peptide Commercial kits based on diŠerences in phenotype patterns map. Methods to characterize peaks range from N­terminal have been used for the identiˆcation of microorganisms, but sequencingofeachpeakfollowedbyaminoacidanalysisto are not always applicable to microorganisms found in raw the use of mass spectroscopy (MS). materials used for pharmaceutical products and in proc­ For characterization purposes, when N­terminal sequenc­ essing areas of pharmaceutical products. In general, the 1834 General Information Supplement II, JPXIV identiˆcation of microorganisms based on phenotypic observe PCR products on a trans­illuminator (312 nm)and analysis needs special knowledge and judgment is often conˆrm the presence of a single band of the targeted size. If subjective. It is considered that the evolutionary history of multiple bands are observed, cut the targeted band out of the microorganisms (bacteria and fungi)is memorized in their gel, and extract DNA by using appropriate commercial ribosomal RNAs (rRNAs), so that systematic classiˆcation DNA extraction kit. and identiˆcation of microorganisms in recent years have 4. Puriˆcation of PCR products been based on the analysis of these sequences. This chapter Remove unincorporated PCR primers and deoxynucleo­ presents a rapid method to identify or estimate microorgan­ side triphosphates (dNTP)from PCR products by using isms based on partial sequences of divergent regions of the appropriate puriˆcation methods. 16S rRNA gene for bacteria and of the internal transcribed 5. Quantiˆcation of puriˆed DNA spacer 1 (ITS1)region located between 18S rRNA and 5.8S When puriˆed DNA is measured by spectrophotometer, rRNA for fungi, followed by comparison of the sequences calculate 1 OD260 nm as 50 mg/mL. with those in the database. Methods described in this chapter 6. Labeling of PCR products with sequencing reagents do not take the place of usual other methods for the identiˆ­ Use an appropriate ‰uorescence­labeled sequencing cation, and can be modiˆed based on the examiner's reagent suitable for the available DNA sequencer or its experience, and on the available equipment or materials. program and label the PCR products according to the Other gene regions besides those mentioned in this chapter instructions provided with the reagent. canbeusedifappropriate. 7. Puriˆcation of sequencing reagent­labeled PCR products Apparatuses Transfer the product in 75 mL of diluted ethanol (7 in 10) (1)DNA sequencer into a 1.5 mL centrifuge tube, keep in an ice bath for 20 min, Various types of sequencers used a gel board or capillary and centrifuge at 15,000 rpm for 20 min. After removal of can be used. supernatant, add 250 mL of diluted ethanol (7 in 10)to the (2)DNA ampliˆer precipitate and centrifuge at 15,000 rpm for 5 min. Remove To amplify target DNA and label ampliˆed (PCR) the supernatant and dry the precipitate. products with sequencing reagents. 8. DNA homology analysis Procedures Place sequencing reagent­labeled PCR products in the The following procedures are described as an example. DNA sequencer and read the nucleotide sequences of the 1. Preparation of template DNA PCR products. Compare the partial nucleotide sequence It is important to use a pure cultivated bacterium or with those in the BLAST database. fungus for identiˆcation. In the case of colony samples, Judgment colonies are picked up with a sterilized toothpick (in the case If sequencing data show over 90z identity with a se­ of fungi, a small fragment of colony sample is picked up), quence in the database, in general, judgment may be made and suspended in 0.3 mL of DNA releasing solution in a 1.5 as follows. mL centrifuge tube. In the case of culture ‰uid, a 0.5 mL 1. In the case of bacteria, compare about 300 nucleotides portion of ‰uid is put in a 1.5 mL centrifuge tube and between positions 50 to 350 in the product obtained with centrifuged at 10,000 rpm for 10 min. After removal of the the 10F primer, with the BLAST database. Higher supernatant, the pellet is suspended in 0.3 mL of DNA ranked species are judged as identiˆed species or closely releasing solution, and then heated at 1009C for 10 min. In related species. general, PCR can be run for bacteria and yeasts heated in 2. In the case of fungi, compare sequencing data for the DNA releasing solution. For fungi, DNA extraction after product obtained with the ITS1F primer, with the treatment with a mixer or ultrasonic generator may be BLAST database. Higher ranked species are judged as necessary before PCR. identiˆed species or closely related species. 2. PCR Add 2 mL of template DNA in PCR reaction solution. Use Reagents, Test Solutions 10F/800R primers for bacteria and ITS1F/ITS1R primers (1) 0.5 mol/L Disodium dihydrogen ethylenediamine for fungi, and then perform 30 ampliˆcation cycles at 949C tetraacetate TS for 30 sec, 559C for 60 sec, and 729C for 60 sec. DNA frag­ Dissolve 18.6 g of disodium dihydrogen ethylenediamine ments are ampliˆed about 800 bp in the case of bacteria and tetraacetate dihydrate in water to make 100 mL. about 150 – 470 bp depending on the strain in the case of (2) 1 mol/L Tris buŠer solution, pH 8.0 fungi. Include a negative control (water instead of the test Dissolve 24.2 g of 2­amino­2­hydroxymethyl­1,3­ solution)in the PCR. propanediol in a suitable amount of water, adjust the pH to 3. Conˆrmation of PCR products 8.0 with 0.2 mol/L hydrochloric acid TS, and add water to Mix 5 mL of PCR product with 1 mL of loading buŠer make 200 mL. solution, place it in a 1.5 w/vz agarose gel well, and carry (3) TE buŠer solution out electrophoresis with TAE buŠer solution (1­fold concen­ Mix 1.0 mL of 1 mol/L tris buŠer solution, pH 8.0 and tration). Carry out the electrophoresis together with 0.2 mL of 0.5 mol/L disodium dihydrogen ethylenediamine appropriate DNA size markers. After the electrophoresis, tetraacetate TS, and add water to make 100 mL. Supplement II, JPXIV General Information 1835

(4) DNA releasing solution Add the following: Divide TE buŠer solution containing Triton X­100 (1 in 100) into small amounts and store frozen until use. 25. Solid and Particle Densities (5) PCR reaction solution 10­fold buŠer solution* 5 mL Density of a solid or a powder as a state of aggregation dNTP mixture** 4 mL has diŠerent deˆnitions depending on the way of including 10 mmol/L Sense primer 1 mL of the interparticulate and intraparticulate voids that exist 10 mmol/L Anti­sense primer 1 mL between the particles or inside the powder.DiŠerent ˆgures Heat­resistant DNA polymerase (1 U/mL) 1 mL are obtained in each case, and there are diŠerent practical Water 36 mL meanings.Generally, there are three levels of deˆnitions of * Being composed of 100 mmol/L 2­amino­2­hydrox­ the solid or powder density. ymethyl­1,3­propanediol hydrochloride, pH 8.4, 500 (1) Crystal density: It is assumed that the system is mmol/L potassium chloride, 20 mmol/L magnesium chlo­ homogeneous with no intraparticulate void.Crystal density ride and 0.1 g/L gelatin. is also called true density. ** A solution containing 2.5 mmol/L each of dGTP (sodi­ (2) Particle density: The sealed pores or the experimen­ um 2?­deoxyguanosine 5?­triphosphate), dATP (sodium 2?­ tally non­accessible open pores is also included as a part of deoxyadenosine 5?­triphosphate), dCTP (sodium 2?­deox­ the volumes of the solid or the powder. ycytidine 5?­triphosphate) and dTTP (sodium 2?­deox­ (3) Bulk density: The interparticulate void formed in the ythymidine 5?­triphosphate).Adequate products containing powder bed is also included as a part of the volumes of the these components as described above may be used. solid or the powder.Bulk density is also called apparent (6) Sequencing reagent density.Generally, the powder densities at loose packing There are many kinds of sequencing methods, such as the and at tapping are deˆned as the bulk density and the tapped dye­primer method for labeling of primer, the dye­termina­ density, respectively. tor method for labeling of dNTP terminator and so on.Use Generally, the densities of liquid or gas are aŠected only an appropriate sequencing reagent kit for the apparatus and by temperature and pressure, but the solid or powder density program to be used. is aŠected by the state of aggregation of the molecules or the (7) 50­Fold concentrated TAEbuŠer solution particles.Therefore, the solid or powder densities naturally Dissolve 242 g of 2­amino­2­hydroxymethyl­1,3­ vary depending on crystal structure or crystallinity of the propanediol in 57.1 mL of acetic acid (100) and 100 mL of substance concerned, and also varies depending on the 0.5 mol/L disodium dihydrogen ethylenediamine tetraa­ method of preparation or handling if the sample is amor­ cetate TS, and add water to make 1000 mL. phous form or partially amorphous.Consequently, even in a (8) 1­Fold concentrated TAEbuŠer solution case that two solids or powders are chemically identical, it Diluted 50­fold concentrated TAE buŠer solution (1 in 50) may be possible that the diŠerent ˆgures of density are prepared before use is referred to as 1­fold concentrated obtained if their crystal structures are diŠerent.As the solid TAE buŠer solution. or powder particle densities are important physical proper­ (9) Agarose gel ties for the powdered pharmaceutical drugs or the powdered Mix 1.5 g of agarose, 2.0 mL of 50­fold concentrated raw materials of drugs, Japanese Pharmacopoeia XIV speci­ TAE buŠer solution, 10 mL of a solution of ethidium ˆes each density determination as ``Powder Particle Density bromide (3,8­diamino­5­ethyl­6­phenylphenanthridinium Determination'' for the particle density and as ``Determina­ bromide) (1 in 100) and 100 mL of water.After dissolving tion of Bulk and Tapped Densities'' for the bulk density. the materials by heating, cool the solution to about 609C, The solid or powder densities are expressed in mass per and prepare gels. unit volume (kg/m3), and generally expressed in g/cm3 (1 (10) Loading buŠer solution (6­fold concentrated) g/cm31000 kg/m3). Dissolve 0.25 g of bromophenol blue, 0.25 g of xylene cyanol FF and 1.63 g of disodium dihydrogen ethylenedia­ Crystal Density mine tetraacetate dihydrate in 50 mL of water, and add The crystal density of a substance is the average mass per 30 mL of glycerol and water to make 100 mL. unit volume, exclusive of all voids that are not a fundamen­ (11) PCR primers tal part of the molecular packing arrangement.It is an intrinsic property concerning the speciˆc crystal structure of For Primer the substance, and is not aŠected by the method of determi­ Bacteria 10F 5?­GTTTGATCCTGGCTCA­3? nation.The crystal density can be determined either by 800R 5?­TACCAGGGTATCTAATCC­3? calculation or by simple measurement. A.The calculated crystal density is obtained using: Fungi ITS1F 5?­GTAACAAGGT(T/C)TCCGT­3? 1) For example, the crystallographic data (volume ITS1R 5?­CGTTCTTCATCGATG­3? and composition of the unit cell) obtained by indexing the perfect crystal X­ray diŠraction data from single crystal or the powder X­ray diŠraction data. 1836 General Information Supplement II, JPXIV

2) Molecular mass of the substance. Japanese Pharmacopoeia XIV speciˆes ``Determination B. The measured crystal density is obtained as the mass to of Bulkand Tapped Densities''. volume ratio after measuring the single crystal mass and A. The bulkdensity is determined by measuring the volume. apparent volume of a known mass of powder sample that has been passed through a screen in a graduated Particle Density cylinder (constant mass method). Separately, Japanese The particle density takes account both the crystal density Pharmacopoeia XIV speciˆes the method of determin­ and the intraparticulate porosity (sealed and/or experimen­ ing bulkdensity by measuring the mass of powder in a tally non­accessible open pores) as a part of the particle vessel having a known volume (constant volume volume. The particle density depends on the value of the method). volume determined, and the volume in turn depends on the B. The tapped density is obtained by mechanically tapping method of measurement. Particle density can be determined a measuring cylinder containing a powder sample. After either by gas displacement pycnometry or mercury porosi­ determining the initial bulkvolume, carry out tapping metry, but Japanese Pharmacopoeia XIV speciˆes the under a ˆxed measurement condition (tapping rate and pycnometry as the ``Powder Particle Density Determina­ drop height), and the measurement is carried out tion''. repeatedly until the bulkvolume variation obtained at A. The pycnometric density is obtained by assuming that consecutive two measurements is within an acceptable the volume of the gas displaced, which is measured with range (constant mass method). Separately, Japanese the gas displacement pycnometer, is equivalent to that Pharmacopoeia XIV speciˆes the method of determin­ of a known mass of the powder. In pycnometric density ing the tapped density by measuring the mass of a ˆxed measurements, any volume with the open pores accessi­ volume of the tapped powder (constant volume ble to the gas is not included as a part of volume of the method). powder, but the sealed pores or pores inaccessible to the gas is included as a part of the volume of the powder. Due to the high diŠusivity of helium which can pene­ Add the following: trate to most open pores, it is recommendable as the measurement gas of particle density. Therefore, the pycnometric particle density of a ˆnely milled powder is 26. Total Protein Assay generally not very diŠerent from the crystal density. Hence, the particle density by this method is the best The following procedures are provided as illustrations of estimate of the true density of an amorphous or partial­ the determination of total protein content in pharmacopoei­ ly crystalline sample, and can be widely used for al preparations. Other techniques, such as HPLC, are also manufacturing control of the processed pharmaceutical acceptable if total protein recovery is demonstrated. Many powder samples. of the total protein assay methods described below can be B. The mercury porosimetric density is also called granular performed successfully using kits from commercial sources. density. This method also includes the sealed pores as a Note: Where water is required, use distilled water. part of the volumes of the solid or the powder, but Method 1 excludes the volume only from the open pores larger Protein in solution absorbs UV light at a wavelength of than some size limit. This pore size limit or minimal 280 nm, due to the presence of aromatic amino acids, mainly access diameter depends on the maximal mercury tyrosine and tryptophan. This property is the basis of this intrusion pressure applied during the measurement and method. Protein determination at 280 nm is mainly a func­ under normal operating pressure, the mercury does not tion of the tyrosine and tryptophan content of the protein. If penetrate the ˆnenest pores accessible to helium. Since the buŠer used to dissolve the protein has a high absorbance this method is capable of measuring the density which relative to that of water, there is an interfering substance in corresponds to the pore size limit at each mercury the buŠer. This interference can be compensated for when intrusion pressure, the various granular densities can be the spectrophotometer is adjusted to zero buŠer absorbance. obtained from one sample. If the interference results in a large absorbance that Bulk Density and Tapped Density challenges the limit of sensitivity of the spectrophotometer, The bulkdensity of a powder includes the contribution of the results may be compromised. Furthermore, at low con­ interparticulate void volume as a part of the volume of the centrations protein can be absorbed onto the cuvette, there­ powder. Therefore, the bulkdensity depends on both the by reducing the content in solution. This can be prevented by powder particle density and the space arrangement of preparing samples at higher concentrations or by using a particles in the power bed. Further, since the slightest nonionic detergent in the preparation. disturbance of the bed may result in variation of the space Note: Keep the Test Solution, the Standard Solution, and arrangement, it is often very di‹cult to determine the bulk the buŠer at the same temperature during testing. density with good reproducibility. Therefore, it is essential Standard Solution Unless otherwise speciˆed in the to specify how the determination was made upon reporting individual monograph, prepare a solution of the reference the bulkdensity. standard or reference material for the protein under test in Supplement II, JPXIV General Information 1837 the same buŠer and at the same concentration as the Test Standard Solutions Unless otherwise speciˆed in the Solution. individual monograph, dissolve the reference standard or Test Solution Dissolve a suitable quantity of the protein reference material for the protein under test in the buŠer under test in the appropriate buŠer to obtain a solution used to prepare the Test Solution. Dilute portions of this having a concentration of 0.2 to 2 mg per mL. solution with the same buŠer to obtain not fewer than ˆve Procedure Concomitantly determine the absorbances of Standard Solutions having concentrations between 5 and theStandardSolutionandtheTestSolutioninquartzcellsat 100 mg of protein per mL, the concentrations being evenly a wavelength of 280 nm, with a suitable spectrophotometer, spaced. using the buŠer as the blank. To obtain accurate results, the Test Solution Dissolve a suitable quantity of the protein response should be linear in the range of protein concentra­ under test in the appropriate buŠer to obtain a solution tions to be assayed. having a concentration within the range of the concentra­ Light­Scattering The accuracy of the UV spectroscopic tions of the Standard Solutions. An appropriate buŠer will determination of protein can be decreased by the scattering produce a pH in the range of 10 to 10.5. of light by the test specimen. If the proteins in solution exist Blank Use the buŠer used for the Test Solution and the as particles comparable in size to the wavelength of the Standard Solutions. measuring light (250 to 300 nm), scattering of the light beam Reagents and Solutions— results in an apparent increase in absorbance of the test Copper Sulfate Reagent Dissolve 100 mg of copper (II) specimen. To calculate the absorbance at 280 nm due to sulfate pentahydrate and 200 mg of sodium tartrate dihy­ light­scattering, determine the absorbances of the Test drate in water, dilute with water to 50 mL, and mix. Dissolve Solution at wavelengths of 320, 325, 330, 335, 340, 345, and 10 g of anhydrous sodium carbonate in water to a ˆnal 350 nm. Using the linear regression method, plot the log of volume of 50 mL, and mix. Slowly pour the sodium car­ the observed absorbance versus the log of the wavelength, bonate solution into the copper sulfate solution with mixing. and determine the standard curve best ˆtting the plotted Prepare this solution fresh daily. points. From the graph so obtained, extrapolate the 5z SDS TS Dissolve 5 g of sodium dodecyl sulfate in absorbance value due to light­scattering at 280 nm. Subtract water, and dilute with water to 100 mL. the absorbance from light­scattering from the total Alkaline Copper Reagent Prepareamixtureof5z SDS absorbance at 280 nm to obtain the absorbance value of the TS, Copper Sulfate Reagent, and Sodium Hydroxide protein in solution. Filtration with a ˆlter having a 0.2­mm Solution (4 in 125) (2:1:1). This reagent may be stored at porosity or clariˆcation by centrifugation may be performed room temperature for up to 2 weeks. to reduce the eŠect of light­scattering, especially if the Diluted Folin's TS Mix 10 mL of Folin's TS with 50 mL solution is noticeably turbid. of water. Store in an amber bottle, at room temperature.

Calculations Calculate the concentration, CU,ofproteinin Procedure To 1 mL of each Standard Solution, the Test the test specimen by the formula: Solution, and the Blank, add 1 mL of Alkaline Copper Reagent, and mix. Allow to stand at room temperature for C C (A /A ), U S U S 10 minutes. Add 0.5 mL of the Diluted Folin's TS to each in which CS is the concentration of the Standard Solution; solution, and mix each tube immediately after the addition, and AU and AS are the corrected absorbances of the Test and allow to stand at room temperature for 30 minutes. Solution and the Standard Solution, respectively. Determine the absorbances of the solutions from the Standard Solutions and the Test Solution at the wavelength Method 2 of maximum absorbance at 750 nm, with a suitable spec­ This method, commonly referred to as the Lowry assay, is trophotometer, using the solution from the Blank to set the based on the reduction by protein of the phosphomolybdic­ instrument to zero. tungstic mixed acid chromogen in the Folin­Ciocalteu's Calculations [Note: The relationship of absorbance to phenol reagent, resulting in an absorbance maximum at protein concentration is nonlinear; however, if the standard 750 nm. The Folin­Ciocalteu's phenol reagent (Folin's TS) curve concentration range is su‹ciently small, it will reacts primarily with tyrosine residues in the protein, which approach linearity.] Using the linear regression method, plot can lead to variation in the response of the assay to diŠerent the absorbances of the solutions from the Standard proteins. Because the method is sensitive to interfering Solutions versus the protein concentrations, and determine substances, a procedure for precipitation of the protein from the standard curve best ˆtting the plotted points. From the the test specimen may be used. Where separation of interfer­ standard curve so obtained and the absorbance of the Test ing substances from the protein in the test specimen is Solution, determine the concentration of protein in the Test necessary, proceed as directed below for Interfering Solution. Substances prior to preparation of the Test Solution. The Interfering Substances In the following procedure, deoxy­ eŠect of interfering substances can be minimized by dilution cholate­trichloroacetic acid is added to a test specimen to provided the concentration of the protein under test remains su‹cient for accurate measurement. Variations of the Lowry test that are indicated in national regulatory docu­ 1) Example: the Minimum Requirements for Biological ments1) can be substituted for the method described below. Products and individual monograph of JP 1838 General Information Supplement II, JPXIV remove interfering substances by precipitation of proteins storage of the reagent. Filter the reagent before use.] before testing. This technique also can be used to concen­ Procedure Add 5 mL of the Coomassie Reagent to 100 mL trate proteins from a dilute solution. of each Standard Solution, the Test Solution, and the Blank, Sodium Deoxycholate Reagent Prepare a solution of and mixby inversion. Avoid foaming, which will lead to sodium deoxycholate in water having a concentration of poor reproducibility. Determine the absorbances of the 150 mg in 100 mL. solutions from the Standard Solutions and the Test Solution Trichloroacetic Acid Reagent Prepare a solution of at 595 nm, with a suitable spectrophotometer, using the trichloroacetic acid in water having a concentration of 72 g Blanktosettheinstrumenttozero. in 100 mL. [Note: Do not use quartz (silica) spectrophotometer cells: Procedure Add 0.1 mL of Sodium Deoxycholate Reagent the dye binds to this material. Because diŠerent protein to 1 mL of a solution of the protein under test. Mixon a species may give diŠerent color response intensities, the vortexmixer,and allow to stand at room temperature for 10 standard protein and test protein should be the same.] There minutes. Add 0.1 mL of Trichloroacetic Acid Reagent, and are relatively few interfering substances, but detergents and mixon a vortexmixer.Centrifuge at 3000 ~gfor30 ampholytes in the test specimen should be avoided. Highly minutes, decant the liquid, and remove any residual liquid alkaline specimens may interfere with the acidic reagent. with a pipet. Redissolve the protein pellet in 1 mL of Calculations [Note: The relationship of absorbance to Alkaline Copper Reagent. Proceed as directed for Test Solu­ protein concentration is nonlinear; however, if the standard tion. [Note: Color development reaches a maximum in 20 to curve concentration range is su‹ciently small, it will 30 minutes during incubation at room temperature, after approach linearity.] Using the linear regression method, plot which there is a gradual loss of color. Most interfering the absorbances of the solutions from the Standard substances cause a lower color yield; however, some deter­ Solutions versus the protein concentrations, and determine gents cause a slight increase in color. A high salt concentra­ the standard curve best ˆtting the plotted points. From the tion may cause a precipitate to form. Because diŠerent standard curve so obtained and the absorbance of the Test protein species may give diŠerent color response intensities, Solution, determine the concentration of protein in the Test the standard protein and test protein should be the same.] Solution.

Method 3 Method 4 This method, commonly referred to as the Bradford This method, commonly referred to as the bicinchoninic assay, is based on the absorption shift from 470 nm to acid or BCA assay, is based on reduction of the cupric 595 nm observed when the brilliant blue G­250 dye binds to (Cu2{) ion to cuprous (Cu{) ion by protein. The bicincho­ protein. The Coomassie Brilliant Blue G­250 dye binds most ninic acid reagent is used to detect the cuprous ion. The readily to arginyl and lysyl residues in the protein, which can method has few interfering substances. When interfering lead to variation in the response of the assay to diŠerent substances are present, their eŠect may be minimized by proteins. dilution, provided that the concentration of the protein Standard Solutions Unless otherwise speciˆed in the under test remains su‹cient for accurate measurement. individual monograph, dissolve the reference standard or Standard Solutions Unless otherwise speciˆed in the the reference material for the protein under test in the buŠer individual monograph, dissolve the reference standard or used to prepare the Test Solution. Dilute portions of this so­ the reference material for the protein under test in the buŠer lution with the same buŠer to obtain not fewer than ˆve used to prepare the Test Solution. Dilute portions of this so­ Standard Solutions having concentrations between 100 mg lution with the same buŠer to obtain not fewer than ˆve and 1 mg of protein per mL, the concentrations being evenly Standard Solutions having concentrations between 10 and spaced. 1200 mg of protein per mL, the concentrations being evenly Test Solution Dissolve a suitable quantity of the protein spaced. under test in the appropriate buŠer to obtain a solution Test Solution Dissolve a suitable quantity of the protein having a concentration within the range of the concentra­ under test in the appropriate buŠer to obtain a solution tions of the Standard Solutions. having a concentration within the range of the concentra­ Blank Use the buŠer used to prepare the Test Solution and tions of the Standard Solutions. the Standard Solutions. Blank Use the buŠer used to prepare the Test Solution and Coomassie Reagent Dissolve 100 mg of brilliant blue the Standard Solutions. G­2502) in 50 mL of ethanol (95). [Note: Not all dyes have Reagents and Solutions— the same brilliant blue G content, and diŠerent products BCA Reagent Dissolve about 10 g of bicinchoninic acid, may give diŠerent results.] Add 100 mL of phosphoric acid, 20 g of sodium carbonate monohydrate, 1.6 g of sodium dilute with water to 1000 mL, and mix. Filter the solution tartrate dihydrate, 4 g of sodium hydroxide, and 9.5 g of through ˆlter paper (Whatman No.1 or equivalent), and sodium hydrogen carbonate in water. Adjust, if necessary, store the ˆltered reagent in an amber bottle at room temper­ with sodium hydroxide or sodium hydrogen carbonate to a ature. [Note: Slow precipitation of the dye will occur during pH of 11.25. Dilute with water to 1000 mL, and mix. Copper Sulfate Reagent Dissolve about 2 g of copper 2) Dye purity is important in the reagent preparation. (II) sulfate pentahydrate in water to a ˆnal volume of 50 mL. Supplement II, JPXIV General Information 1839

Copper­BCA Reagent Mix 1 mL of Copper Sulfate dilute with water to 200 mL. This Biuret Reagent is stable at Reagent and 50 mL of BCA Reagent. room temperature for 6 months. Do not use the reagent if it Procedure Mix 0.1 mL of each Standard Solution,the Test develops turbidity or contains any precipitate. Solution,andtheBlankwith2mLoftheCopper­BCA Procedure To one volume of the Standard Solutions and a Reagent. Incubate the solutions at 379C for 30 minutes,note solution of the Test Solution add an equal volume of sodium the time,and allow to come to room temperature. Within 60 hydroxide solution (6 in 100),and mix. Immediately add a minutes following the incubation time,determine the absor­ volume of Biuret Reagent equivalent to 0.4 volume of the bances of the solutions from the Standard Solutions and the Test Solution,and mix. Allow to stand at a temperature Test Solution in quartz cells at 562 nm,with a suitable between 159Cand259C for not less than 15 minutes. Within spectrophotometer,using the Blank to set the instrument to 90 minutes after the addition of the Biuret Reagent,deter­ zero. After the solutions are cooled to room temperature, mine the absorbances of the Standard Solutions and the the color intensity continues to increase gradually. If sub­ solution from the Test Solution at the wavelength of maxi­ stances that will cause interference in the test are present, mum absorbance at 545 nm,with a suitable spectrophotom­ proceed as directed for Interfering Substances under Method eter,using the Blank to set the instrument to zero. [Note: 2. Because diŠerent protein species may give diŠerent color Any solution that develops turbidity or a precipitate is not response intensities,the standard protein and test protein acceptable for calculation of protein concentration.] should be the same. Calculations Using the least­squares linear regression Calculations [Note: The relationship of absorbance to pro­ method,plot the absorbances of the Standard Solutions tein concentration is nonlinear; however,if the standard versus the protein concentrations,and determine the stan­ curve concentration range is su‹ciently small,it will dard curve best ˆtting the plotted points,and calculate the approach linearity.] Using the linear regression method,plot correlation coe‹cient for the line. [Note: Within the given the absorbances of the solutions from the Standard range of the standards,the relationship of absorbance to Solutions versus the protein concentrations,and determine protein concentration is approximately linear.] A suitable the standard curve best ˆtting the plotted points. From the system is one that yields a line having a correlation standard curve so obtained and the absorbance of the Test coe‹cient of not less than 0.99. From the standard curve Solution,determine the concentration of protein in the Test and the absorbance of the Test Solution,determine the Solution. concentration of protein in the test specimen,making any necessary correction. Method 5 Interfering Substances To minimize the eŠect of interfer­ This method,commonly referred to as the Biuret assay,is ing substances,the protein can be precipitated from the basedontheinteractionofcupric(Cu2{) ion with protein in initial test specimen as follows. Add 0.1 volume of 50z an alkaline solution and the resultant development of absor­ trichloroacetic acid to 1 volume of a solution of the test bance at 545 nm. specimen,withdraw the supernatant layer,and dissolve the Standard Solutions Unless otherwise speciˆed in the in­ precipitate in a small volume of 0.5 mol/L sodium dividual monograph,prepare a solution of Albumin Human hydroxide TS. Use the solution so obtained to prepare the for which the protein content has been previously deter­ Test Solution. mined by nitrogen analysis (using the nitrogen­to­protein Comments This test shows minimal diŠerence between conversion factor of 6.25) or of the reference standard or equivalent IgG and albumin samples. Addition of the reference material for the protein under test in sodium sodium hydroxide and the Biuret Reagent as a combined chloride solution (9 in 1000). Dilute portions of this solution reagent,insu‹cient mixing after the addition of the sodium with sodium chloride solution (9 in 1000) to obtain not fewer hydroxide,or an extended time between the addition of the than three Standard Solutions having concentrations sodium hydroxide solution and the addition of the Biuret between 0.5 and 10 mg per mL,the concentrations being Reagent will give IgG samples a higher response than evenly spaced. [Note: Low responses may be observed if the albumin samples. The trichloroacetic acid method used to sample under test has signiˆcantly diŠerent level of proline minimize the eŠects of interfering substances also can be than that of Albumin Human. A diŠerent standard protein used to determine the protein content in test specimens at maybeemployedinsuchcases.] concentrations below 500 mgpermL. Test Solution Prepare a solution of the test protein in sodium chloride solution (9 in 1000) having a concentration Method 6 within the range of the concentrations of the Standard This ‰uorometric method is based on the derivatization of Solutions. the protein with o­phthalaldehyde (OPA),which reacts with

Blank Use sodium chloride solution (9 in 1000). the primary amines of the protein (i.e.,NH 2­terminal amino Biuret Reagent Dissolve about 3.46 g of copper (II) sulfate acid and the e­amino group of the lysine residues). The pentahydrate in 10 mL of water,with heating if necessary, sensitivity of the test can be increased by hydrolyzing the and allow to cool (Solution A). Dissolve about 34.6 g of protein before testing. Hydrolysis makes the a­amino group sodium citrate dihydrate and 20.0 g of anhydrous sodium of the constituent amino acids of the protein available for carbonate in 80 mL of water,with heating if necessary,and reaction with the OPA reagent. The method requires very allow to cool (Solution B). Mix Solutions A and B,and small quantities of the protein. 1840 General Information Supplement II, JPXIV

Primary amines, such as tris(hydroxymethyl)amino­ ‰uorescent intensity.] methane and amino acid buŠers, react with OPA and must Calculations The relationship of ‰uorescence to protein be avoided or removed.Ammonia at high concentrations concentration is linear.Using the linear regression method, will react with OPA as well.The ‰uorescence obtained when plot the ‰uorescent intensities of the solutions from the amine reacts with OPA can be unstable.The use of automat­ Standard Solutions versus the protein concentrations, and ed procedures to standardize this procedure may improve determine the standard curve best ˆtting the plotted points. the accuracy and precision of the test. From the standard curve so obtained and the ‰uorescent Standard Solutions Unless otherwise speciˆed in the intensity of the Test Solution, determine the concentration individual monograph, dissolve the reference standard or of protein in the test specimen. the reference material for the protein under test in the buŠer Method 7 used to prepare the Test Solution.Dilute portions of this so­ This method is based on nitrogen analysis as a means of lution with the same buŠer to obtain not fewer than ˆve protein determination.Interference caused by the presence Standard Solutions having concentrations between 10 and of other nitrogen­containing substances in the test protein 200 mg of protein per mL, the concentrations being evenly can aŠect the determination of protein by this method. spaced. Nitrogen analysis techniques destroy the protein under test Test Solution Dissolve a suitable quantity of the protein but are not limited to protein presentation in an aqueous under test in the appropriate buŠer to obtain a solution environment. having a concentration within the range of the concentra­ Procedure A Determine the nitrogen content of the protein tions of the Standard Solutions. under test as directed elsewhere in the Pharmacopoeia. Blank Use the buŠer used to prepare the Test Solution and Commercial instrumentation is available for the Kjeldahl the Standard Solutions. nitrogen assay. Reagents and Solutions— Procedure B Commercial instrumentation is available for Borate BuŠer Dissolve about 61.83 g of boric acid in nitrogen analysis.Most nitrogen analysis instruments use water, and adjust with potassium hydroxide to a pH of 10.4. pyrolysis (i.e., combustion of the sample in oxygen at Dilute with water to 1000 mL, and mix. temperatures approaching 10009C), which produces nitric Stock OPA Reagent Dissolve about 120 mg of o­ oxide (NO) and other oxides of nitrogen (NO ) from the phthalaldehyde in 1.5 mL of methanol, add 100 mL of x nitrogen present in the test protein.Some instruments Borate BuŠer, and mix.Add 0.6mL of polyoxyethylene (23) convert the nitric oxides to nitrogen gas, which is quantiˆed lauryl ether, and mix.This solution is stable at room temper­ with a thermal conductivity detector.Other instruments mix ature for at least 3 weeks. nitric oxide (NO) with ozone (O ) to produce excited nitro­ OPA Reagent To 5 mL of Stock OPA Reagent add 3 gen dioxide (NO .), which emits light when it decays and can 15 mL of 2­mercaptoethanol.Prepare at least 30 minutes 2 be quantiˆed with a chemiluminescence detector.A protein prior to use.This reagent is stable for one day. reference standard or reference material that is relatively Procedure Adjust each of the Standard Solutions and the pure and is similar in composition to the test proteins is used Test Solution to a pH between 8.0 and 10.5. Mix 10 mLof to optimize the injection and pyrolysis parameters and to the Test Solution and each of the Standard Solutions with evaluate consistency in the analysis. 100 mL of OPA Reagent, and allow to stand at room temper­ Calculations The protein concentration is calculated by ature for 15 minutes.Add 3 mL of 0.5mol/L sodium dividing the nitrogen content of the sample by the known hydroxide TS, and mix.Using a suitable ‰uorometer, nitrogen content of the protein.The known nitrogen content determine the ‰uorescent intensities of solutions from the of the protein can be determined from the chemical compo­ Standard Solutions and the Test Solution at an excitation sition of the protein or by comparison with the nitrogen wavelength of 340 nm and an emission wavelength between content of the appropriate reference standard or reference 440 and 455 nm.[Note: The ‰uorescence of an individual material. specimen is read only once because irradiation decreases the INDEX

Page citations refer to the pages of the Supplement I, and to pages of the JP XIV main volume, including those where the text being revised in the Supplement originally appeared. This Supplement II commences with page 1669 and succeeding Supplements will continue to be paged in sequence.

1„1358 Main Volume of JP XIV 1359„1668 Supplement I 1669„1866 Supplement II

Toxoid, 913 Silicate, Synthetic, 235 A Diphtheria Toxoid for Adult Use, Sucrose Sulfate Ester, 766 912 Amantadine Hydrochloride, 235 Absorbent Habu-venom Toxoid, 938 Ambenonium Chloride, 236 Cotton, 851, 1757 Hepatitis B Vaccine, 938 Amidotrizoic Acid, 237 Cotton, Puriˆed, 851, 1757 Puriˆed Pertussis Vaccine, 1005 Amikacin Sulfate, 238 Cotton, Sterile, 852, 1757 Tetanus Toxoid, 1066 Aminoacetic Acid, 931 Cotton, Sterile, Puriˆed, 852, 1757 A‰oqualone, 225 Aminobenzylpenicillin, 246 Gauze, 853, 1757 Agar, 859 Anhydrous, 246 Gauze, Sterile, 854, 1757 Powdered, 859 Sodium, 246 Absorptive Ointment, 855 Ajmaline, 226 Aminophylline, 239 Acacia, 855, 1543 Tablets, 227 Injection, 239 Powdered, 855, 1543 Akebia Stem, 859 Amitriptyline Acebutolol Hydrochloride, 219 Albumin Tannate, 227 Hydrochloride, 240 Aceglutamide Aluminum, 1377 Alcohol, 914 Hydrochloride Tablets, 240 Acetaminophen, 219 Benzyl, 874 Ammonia Water, 241 Acetazolamide, 220 Dehydrated, 914 Amobarbital, 242 Acetic Acid, 856 for Disinfection, 916 Sodium for Injection, 242 Glacial, 856 Isopropyl, 556 Amomum Seed, 863 Acetohexamide, 221, 1687 Stearyl, 1059 Amorphous Insulin Zinc Injection Acetylcholine Chloride for Aldioxa, 228 (Aqueous Suspension), 542 Injection, 222 Alimemazine Tartrate, 229 Amoxapine, 243 Acetylkitasamycin, 223, 1378 Alisma Rhizome, 860 Amoxicillin, 244, 1381 Acetylleucomycin, 223 Powdered, 860 Amphotericin B, 245 Acetylsalicylic Acid, 252 Allopurinol, 229 Ampicillin, 246, 1382 Tablets, 252 Aloe, 860, 1544 Anhydrous, 246, 1384 Acetylspiramycin, 224, 1378 Powdered, 861, 1544, 1757 Ethoxycarbonyloxyethyl Achyranthes Root, 857 Alprazolam, 230 Hydrochloride, 259 Aclarubicin Hydrochloride, 224, 1379 Alpinia O‹cinarum Rhizome, 1544 Sodium, 246, 1385 Acrinol, 224 Alprenolol Hydrochloride, 231 Ampicillinphthalidyl and Zinc Oxide Oil, 857 Alprostadil, 1688 Hydrochloride, 782 and Zinc Oxide Ointment, 858 Alfadex, 231 Amyl Nitrite, 246 Actinomycin D, 225, 1381 Alum, 862 Anemarrhena Rhizome, 864 Adhesive Plaster, 858, 1757 Burnt, 863 Anesthamine, 465 Adrenaline, 447 Solution, 861 Anesthetic Ether, 462 Hydrochloride Injection, 447 Aluminum Angelica Dahurica Root, 864 Hydrochloride Solution, 448 Acetylsalicylate, 253 Anhydrous Adsorbed Aspirin, 253 Aminobenzylpenicillin, 246 Diphtheria-Puriˆed Pertussis- Monostearate, 861 Ampicillin, 246 Tetanus Combined Vaccine, Potassium Sulfate, 862 CaŠeine, 294 1005 Potassium Sulfate, Dried, 863 Citric Acid, 367, 1438, 1698 Diphtheria-Tetanus Combined Silicate, Natural, 233 Dibasic Calcium Phosphate, 879

1841 1842 Index Supplement II, JP XIV

Lactose, 961 Phellodendron, Powdered, 1008 Bromhexine Hydrochloride, 285 Antiformin, Dental, 864 Bearberry Leaf, 870 Bromocriptine Mesilate, 286 Antipyrine, 247 Bear Bile, 869 Bromovalerylurea, 287 Apricot Beclometasone Dipropionate, 264 Bucumolol Hydrochloride, 287 Kernel, 864 Beef Tallow, 870 Bufetolol Hydrochloride, 288 Kernel Water, 865 Beeswax Bufexamac, 289 Arbekacin Sulfate, 247, 1386 White, 871 Cream, 289 Areca, 866, 1545 Yellow, 871 Ointment, 289 Arginine Bekanamycin Sulfate, 265, 1390 Bumetanide, 291 Hydrochloride, 248 Belladonna Bunazosin Hydrochloride, 291 Hydrochloride Injection, 249 Extract, 871 Burdock Fruit, 1546 L-Arginine Root, 872 Bupleurum Root, 876 Hydrochloride, 248 Benidipine Hydrochloride, 1691 Bupranolol Hydrochloride, 292 Hydrochloride Injection, 249 Tablets, 1692 Burnt Alum, 863 Aromatic Castor Oil, 889 Benoxinate Hydrochloride, 661 Busulfan, 293 Arotinolol Hydrochloride, 249 Benserazide Hydrochloride, 265 Butropium Bromide, 294 Arsenical Paste, 866 Bentonite, 873 Butyl Parahydroxybenzoate, 876 Arsenic Trioxide, 250 Benzalkonium Butyrate, Ribo‰avin, 734 Arsenous Acid, 250 Chloride, 266 Artemisia Capillaris Flower, 867 Chloride Solution, 266 C Ascorbic Acid, 250 Chloride Solution 50, Injection, 251 Concentrated, 267 Cacao Butter, 877 Powder, 251 Benzbromarone, 268, 1391 CaŠeine, 294 Asiasarum Root, 867, 1545 Benzethonium and Sodium Benzoate, 296 Asparagus Tuber, 1546 Chloride, 269 Anhydrous, 294 Aspergillus Galactosidase, 921 Chloride Solution, 269 Calciferol, 449 Aspirin, 252 Benzocaine, 465 Calcium Aluminum, 253 Benzoic Acid, 270 Carbonate, Precipitated, 297 Tablets, 252 Benzoin, 873 Carboxymethylcellulose, 885 Aspoxicillin, 254 Benzyl Carmellose, 885 Astragalus Root, 867 Alcohol, 874, 1758 Chloride, 298 Astromicin Sulfate, 255, 1387 Benzoate, 874 Chloride Injection, 298, 1399 Atractylodes Benzylpenicillin CMC, 885 Lancea Rhizome, 869 Benzathine, 1392, 1694 Folinate, 299 Rhizome, 868 Potassium, 270, 1393 Fosfomycin, 494 Atropine Berberine Gluconate, 299 Sulfate, 255 Chloride, 271 Hydroxide, 877 Sulfate Injection, 256, 1688 Tannate, 272 Lactate, 300 Azathioprine, 256 Betahistine Mesilate, 273 Leucovorin, 299 Tablets, 256 Betamethasone, 274 Oxide, 878 Aztreonam, 258 Dipropionate, 275 Pantothenate, 301 Azithromycin Hydrate, 1690 Sodium Phosphate, 276, 1395 Para-aminosalicylate, 301 Valerate, 276, 1395 Para-aminosalicylate Granules, B Bethanechol Chloride, 278 302 Bifonazole, 278 Polystyrene Sulfonate, 303, 1399 Bacampicillin Hydrochloride, 259, Biperiden Hydrochloride, 279 Stearate, 881 1389 Bisacodyl, 280 Calumba, 881 Bacitracin, 1390 Suppositories, 280, 1396 Powdered, 881 Baclofen, 260 Bismuth Camellia Oil, 882 Tablets, 260 Subgallate, 281 Camostat Mesilate, 304 Bamethan Sulfate, 262 Subnitrate, 282 Camphor, Synthetic, 306 Barbital 263 Bitter d-Camphor, 305, 1400 Barium Sulfate, 264 Cardamon, 875 dl-Camphor, 306, 1400 Bark Orange Peel, 875 Capsicum, 882, 1546 Cinnamon, 896 Tincture, 875 and Salicylic Acid Spirit, 884 Cinnamon, Powdered, 897 Bleomycin Powdered, 883, 1546 Magnolia, 970 Hydrochloride, 283, 1396 Tincture, 883 Magnolia, Powdered, 971 Sulfate, 283, 1398 Capsules, 885 Mallotus, 971 Blood, Whole Human, 1080 Cloˆbrate, 373 Moutan, 979 Boric Acid, 284 Flurazepam, 488 Moutan, Powdered, 980 Botulism Antitoxin, Equine, Freeze- Indometacin, 535 Mulberry, 981 dried, 876 Sodium Iodide (123I), 755 Phellodendron, 1007 Bromazepam, 284 Sodium Iodide (131I), 755 Supplement II, JP XIV Index 1843

Sodium Iopodate, 757 Cefozopran Hydrochloride, 337 Oil, 897 Vitamin A, 1075 Cefpiramide Sodium, 338, 1421 Powdered, 1547 Vitamin A Oil, 1074 Cefpirome Sulfate, 338 Cisplatin, 1697 Captopril, 307 Cefpodoxime Proxetil, 1422 Citric Acid, 366, 1437 Carbamazepine, 308 Cefradine, 339 Anhydrous, 367, 1438, 1698 Carbazochrome Sodium Sulfonate, Cefroxadine, 340, 1424 Citrus Unshiu Peel, 898 308, 1400 Cefsulodin Sodium, 340 Clarithromycin, 367, 1699 Carbetapentane Citrate, 674 Ceftazidime, 342 Clemastine Fumarate, 368 Carbetaoebtene Citrete, 674 Cefteram Pivoxil, 344, 1425 Clematis Root, 1547 Carbidopa, 309, 1401 Ceftibuten, 344, 1426 Clindamycin L-Carbocisteine, 310 Ceftizoxime Sodium, 345 Hydrochloride, 1439 Carbolic Acid, 1010 Ceftriaxone Sodium, 346, 1427 Phosphate, 369, 1440 for Disinfection, 1010 Cefuroxime Axetil, 347, 1429, 1696 Clinoˆbrate, 369 Liqueˆed, 1011 Cefuroxime Sodium, 348, 1696 Clocapramine Hydrochloride, 370 Carbon Dioxide, 312 Cellulose Clofedanol Hydrochloride, 371 Carboxymethylcellulose, 885 Acetate Phthalate, 893, 1760 Cloˆbrate, 372 Calcium, 886 Microcrystalline, 889 Capsules, 373 Sodium, 887 Powdered, 892 Clomifene Cardamon, 885 Cetanol, 894 Citrate, 373, 1441 Carmellose, 885 Cetraxate Hydrochloride, 349, 1431 Citrate Tablets, 374 Calcium, 886, 1759 Chloral Hydrate, 350 Clomipramine Hydrochloride, 375 Sodium, 887 Chloramphenicol, 351, 1432 Clonazepam, 375 Carmofur, 313 Palmitate, 1432 Clonidine Hydrochloride, 376 Carnauba Wax, 888 Sodium Succinate, 1434 Cloperastine Hydrochloride, 377 Carteolol Hydrochloride, 313 Chlordiazepoxide, 351 Clotiazepam, 378 Carumonam Sodium, 314, 1401 Powder, 351, 1434 Clotrimazole, 379 Cassia Seed, 888 Tablets, 352, 1435 Clove, 898 Castor Oil, 888 Chlorhexidine Oil, 898 Catalpa Fruit, 889 Gluconate Solution, 353 Powdered, 898 Cefaclor, 314, 1403 Hydrochloride, 354 Cloxacillin Sodium, 380, 1441 Cefadroxil, 314 Chlorinated Lime, 894 Cloxazolam, 380 Cefalexin, 315 Chlormadinone Acetate, 355, 1435 CMC, 885 Cefaloridine, 317, 1405 Chlorobutanol, 894 Calcium, 886 Cefalotin Sodium, 317, 1406 Chlorphenesin Carbamate, 356, 1436 Sodium, 887 Cefamandole Sodium, 317, 1407 Chlorpheniramine Cnidium Cefapirin Sodium, 317 and Calcium Powder, 895 Monnieri Fruit, 1760 Cefatrizine Propylene Glycolate, 318 Maleate, 357 Rhizome, 899 Cefazolin Maleate Injection, 357 Cocaine Hydrochloride, 381 Sodium, 319 Maleate Powder, 358 Coconut Oil, 899 Sodium Hydrate, 321 Maleate Tablets, 359 Codeine Cefbuperazone Sodium, 322, 1409 d-Chlorpheniramine Maleate, 359 Phosphate, 382 Cefcapene Pivoxil Hydrochloride, Chlorpromazine Phosphate Powder, 1, 382, 1442 322 Hydrochloride, 360 Phosphate Powder, 10, 383, 1442 Cefdinir, 324 Hydrochloride Injection, 361 Phosphate Tablets, 384, 1443 Cefditoren Pivoxil, 325 Hydrochloride Tablets, 361 Cod Liver Oil, 900, 1548 Cefepime Dihydrochloride, 326 Chlorpropamide, 362 Coix Seed, 900 for Injection, 1695 Tablets, 362 Colchicine, 384, 1699 Cefetamet Pivoxil Hydrochloride, Cholecalciferol, 363 Colistin 328 Cholera Vaccine, 896 Sodium Methanesulfonate, 385 Ceˆxime, 329 Cholesterol, 896 Sulfate, 1444 Cefmenoxime Hydrochloride, 330, Chorionic Colophonium, 1029 1410 Gonadotrophin, 934, 1764 Compound Cefmetazole Sodium, 321 Gonadotrophin for Injection, 936, Acrinol and Zinc Oxide Oil, 857 Cefminox Sodium, 332 1764 Diastase and Sodium Bicarbonate Cefodizime Sodium, 1412 Chrysanthemum Flower, 1547, 1760 Powder, 907 Cefoperazone Sodium, 332 Ciclacillin, 364, 1437 Hycodenone Injection, 995 Cefoselis Sulfate, 334 Ciclosporin, 364, 1697 Iodine Glycerin, 950 Cefotaxime Sodium, 335, 1413 A, 364 Methyl Salicylate Spirit, 977 Cefotetan, 335, 1415 Cimetidine, 365 Oxycodone and Atropine Cefotiam Cimicifuga Rhizome, 896 Injection, 996 Hexetil Hydrochloride, 335, 1417 Cinchocaine Hydrochloride, 404 Oxycodone Injection, 995 Hydrochloride, 335 Cinnamon Phellodendron Powder for Cefoxitin Sodium, 336, 1419 Bark, 896, 1547 Cataplasm, 1009, 1556 1844 Index Supplement II, JP XIV

Rhubarb and Senna Powder, 1028 Antiformin, 864 Hydrochloric Acid, 515 Salicylic Acid Spirit, 1033 Iodine Glycerin, 951 Iodine Tincture, 950 Scopolia Extract and Diastase Paraformaldehyde Paste, 1001 Diluted Opium Powder, 987 Powder, 1037 Phenol with Camphor, 1011 Dimemorfan Phosphate, 421 Scopolia Extract and Tannic Acid Sodium Hypochlorite Solution, 864 Dimenhydrinate, 422 Ointment, 1039, 1560 Triozinc Paste, 1072 Dimenhydrinate Tablets, 423 Scopolia Extract and Tannic Acid Dermatol, 281 Dimercaprol, 423 Suppositories, 1041, 1560 Deslanoside, 396 Injection, 424 Thianthol and Salicylic Acid Injection, 397 Dimorpholamine, 424, 1705 Solution, 1067 Dexamethasone, 397 Injection, 425, 1705 Vitamin B Powder, 1075 Dextran 906 Dinoprost, 425 Concentrated 40, 398 Dionin, 467 Glycerin, 504 40 Injection, 398 Dioscorea Rhizome, 911 Glycerol, 504 70, 400 Diphenhydramine, 426 Condurango, 900 Sulfate Sodium Sulfur 5, 401 and Bromovalerylurea Powder, Fluidextract, 901 Sulfate Sodium Sulfur 18, 402 911 Coptis Rhizome, 901, 1548 Dextrin, 906 Hydrochloride, 427 Powdered, 1548 Dextromethorphan Hydrobromide, Phenol and Zinc Oxide Liniment, Corn 402 912 Oil, 903 Diagnostic Sodium Citrate Solution, Tannate, 427 Starch, 903, 1761 754 Diphenylhydantoin, 685 Cornus Fruit, 903, 1762 Diastase, 907 Powder, 686 Cortisone and Sodium Bicarbonate Powder, Sodium for Injection, 686 Acetate, 386 907 Tablets, 686 Corydalis Tuber, 904 Diazepam, 403 Diphtheria Cotton, Absorbent, 851 Dibasic Antitoxin, Equine, Freeze-dried, Cream, Bufexamac, 289 Calcium Phosphate, 878 912 Creosote, 904 Sodium Phosphate, 907 Toxoid, 912 Cresol, 905 Dibekacin Sulfate, 404, 1447 Diphtheria-Tetanus Combined Solution, 905 Dibucaine Hydrochloride, 404 Toxoid, 912 Croconazole Hydrochloride, 387 Dichlorphenamide, 406 Dipyridamole, 428, 1451 Crude Glycyrrhiza Extract, 934 Tablets, 407 Disodium Crystalline Diclofenac Sodium, 405 Edetate, 913 Insulin Zinc Injection (Aqueous Diclofenamide, 406, 1448 Ethylenediaminetetraacetate, 913 Suspension), 543 Tablets, 407 Disopyramide, 429 Penicillin G Potassium, 270 Dicloxacillin Sodium, 408 Distigmine Crystal Violet, 617 Diethylcarbamazine Bromide, 429 Cyanamide, 387 Citrate, 408 Bromide Tablets, 430 Cyanocobalamin, 388 Citrate Tablets, 409, 1449 Disulˆram, 430 Injection, 389 Diethylstilbestrol Dobutamine Hydrochloride, 431, Cyclopentolate Hydrochloride, 389 Diphosphate, 493 1452 Cyclophosphamide, 390 Diphosphate Tablets, 494 Dopamine Cycloserine, 390 Difenidol Hydrochloride, 409 Hydrochloride, 432 Cyperus Rhizome, 906 Digenea, 908 Hydrochloride Injection, 433, 1452 Cyproheptadine Hydrochloride, 391 Digitalis, 908, 1762 Dover's Powder, 993 Cytarabine, 392, 1701 Powdered, 910, 1762 Doxapram Hydrochloride, 433 Digitoxin, 410 Doxorubicin Hydrochloride, 434, D Tablets, 411 1453 Digoxin, 414, 1702 Doxycycline Hydrochloride, 434, Dantrolene Sodium, 392 Injection, 415, 1703 1454 Daunorubicin Hydrochloride, 1445 Tablets, 416, 1704 Dried Deferoxamine Mesilate, 393, 1701 Dihydrocodeine Aluminum Hydroxide Gel, 233 Dehydrated Phosphate, 417 Aluminum Hydroxide Gel Fine Alcohol, 915 Phosphate Powder, 1, 417, 1449 Granules, 233 Ethanol, 915 Phosphate Powder, 10, 418, 1450 Aluminum Potassium Sulfate, 863 Dehydrocholate Sodium Injection, Dihydroergotamine Mesilate, 418, Sodium Carbonate, 1055 396 1450 Sodium Sulˆte, 1058 Dehydrocholic Acid, 394 Dihydroergotoxine Mesilate, 412 Thyroid, 1069 Injection, 396 Dihydroxyaluminum Allantoinate, Yeast, 1083 Puriˆed, 394 228 Dromostanolone Demethylchlortetracycline Dilazep Hydrochloride, 419 Propionate, 435 Hydrochloride, 1446 Diltiazem Hydrochloride, 420, 1451 Propionate Injection, 436 Dental Dilute Droperidol, 435, 1456 Supplement II, JP XIV Index 1845

Drostanolone Ethenzamide, 461 Fluorometholone, 484 Propionate, 435, 1456 Ether, 462 Fluorouracil, 485 Propionate Injection, 436, 1456 Anesthetic, 462 Fluoxymesterone, 486 Dydrogesterone, 437 Guaiacol Glyceryl, 505 Fluphenazine Enanthate, 487 Tablets, 438 Polyoxyethylene Lauryl Alcohol, Flurazepam, 487 964 Capsules, 488 E Ethinylestradiol, 463 Hydrochloride, 489 Tablets, 463 Flurbiprofen, 489, 1466 Ecarazine Hydrochloride, 811 Ethionamide, 464, 1707 Foeniculated Ammonia Spirit, 919 Ecothiopate Iodide, 438 Ethosuximide, 465 Folic Acid, 490, 1466 Edrophonium Ethoxybenzamide, 461 Injection, 491 Chloride, 439 Ethyl Tablets, 491 Chloride Injection, 440 Aminobenzoate, 465 Formalin, 919 EDTA Sodium, 913 Cysteine Hydrochloride, 466 Water, 920 Elcatonin, 440 L-Cysteine Hydrochloride, 466 Formoterol Fumarate, 492 En‰urane, 443, 1456 Parahydroxybenzoate, 916 Forsythia Fruit, 920 Enoxacin, 444 Ethylenediamine, 917 Fosfestrol, 493 Enviomycin Sulfate, 1457 Ethylmorphine Hydrochloride, 467 Tablets, 494 Eperisone Hydrochloride, 1706 Etilefrine Fosfomycin Ephedra Herb, 914 Hydrochloride, 468 Calcium, 494, 1713 Ephedrine Hydrochloride Tablets, 468, 1708 Sodium, 496, 1713 Hydrochloride, 444 Etizolam, 1463 Fradiomycin Sulfate, 497, 1467 Hydrochloride Injection, 445 Etoposide, 1709 Freeze-dried Hydrochloride Powder, 10z,446 Eucalyptus Oil, 917 BCG Vaccine (for Percutaneous Hydrochloride Tablets, 446 Eucommia Bark, 1548 Use), 869 Epimedium Herb 1762 Evodia Fruit, 918, 1548 Botulism Antitoxin, Equine, 876 Epinephrine, 447 Exsiccated Gypsum, 938 Diphtheria Antitoxin, Equine, 912 Hydrochloride Injection, 447 Extract Habu Antivenom, Equine, 938 Hydrochloride Solution, 448 Belladonna, 871 Inactivated Tissue Culture Rabies Injection, 447 Crude Glycyrrhiza, 934 Vaccine, 1024 Solution, 448 Glycyrrhiza, 933 Japanese Encephalitis Vaccine, Epirenamine, 447 Nux Vomica, 984 957 Hydrochloride Injection, 447 Live Attenuated Measles Vaccine, Hydrochloride Solution, 448 F 972 Epirizole, 448 Live Attenuated Mumps Vaccine, Epirubicin Hydrochloride, 1458 Famotidine, 469 981 Ergocalciferol, 449, 1460 for Injection, 469, 1464 Live Attenuated Rubella Vaccine, Ergometrine Powder, 469 1029 Maleate, 450 Tablets, 469 Mamushi Antivenom, Equine, 972 Maleate Injection, 451 Faropenem Sodium, 472 Smallpox Vaccine, 1053 Maleate Tablets, 451 Fenbufen, 473 Smallpox Vaccine Prepared in Cell Ergotamine Tartrate, 452 Fennel, 918 Culture, 1053 Erythromycin, 453, 1460 Oil, 919 Tetanus Antitoxin, Equine, 1066 Ethylsuccinate, 453 Powdered, 918 Fritillaria Bulb, 1549 Lactobionate, 1462 Fentanyl Citrate, 473 Fructose, 497 Stearate, 454 Ferrous Sulfate, 474 Injection, 498 Estazolam, 454 Fine Granules, Aluminum Hydroxide Fruit Estradiol Gel, Dried, 233 Catalpa, 889 Benzoate, 455, 1462 Flavin Adenine Dinucleotide Cornus, 903 Benzoate Injection, 456 Sodium, 475, 1710 Evodia, 918 Benzoate Injection (Aqueous Flavoxate Hydrochloride, 476 Forsythia, 920 Suspension), 456 Floctafenine, 477, 1465 Gardenia, 922 Estriol, 457, 1463 Flomoxef Sodium, 478, 1465 Gardenia, Powdered, 923 Injection (Aqueous Suspension), for Injection, 1711 Rose, 1029 458 Flopropione, 478 Rose, Powdered, 1029 Tablets, 458 Capsules, 1712 Schisandra, 1035 Etacrynic Acid, 459 Flower, Artemisia Capillaris, 867 Zanthoxylum, 1083 Tablets, 460 Flucytosine, 478 Zanthoxylum, Powdered, 1084 Ethacridine Lactate, 224 Fludiazepam, 479 Furosemide, 498, 1714 Ethambutol Hydrochloride, 460 Flunitrazepam, 480 Tablets, 1715 Ethanol, 914 Fluocinolone Acetonide, 481 Fursultiamine Hydrochloride, 499 Dehydrated, 915 Fluocinonide, 482 for Disinfection, 916 Fluorescein Sodium, 483 1846 Index Supplement II, JP XIV

G H Hymecromone, 525

Gabexate Mesilate, 500 Habu Antivenom, Equine, Freeze- I b-Galactosidase (Aspergillus), 921 dried, 938 b-Galactosidase (Penicillium), 921 Haloperidol, 507 Ibuprofen, 526 Gallium (67Ga) Citrate Injection, 501 Halothane, 508 Ichthammol, 948 Gambir, 920 Haloxazolam, 509, 1472 Idarubicin Hydrochloride, 527, 1475 Powdered, 920 Hemp Fruit, 1551 Idoxuridine, 527 Gardenia Fruit, 922, 1549 Heparin Ophthalmic Solution, 528, 1475 Powdered 1763 Sodium, 510, 1717 Ifenprodil Tartrate, 529 Gas Gangrene Antitoxin, Equine, 923 Sodium Injection, 511, 1718 Imipenem, 530, 1476 Gastrodia Tuber, 1550 Herb Imipramine Gauze, Absorbent, 853 Ephedra, 914 Hydrochloride, 530 Gel, Aluminum Hydroxide, Dried, Geranium, 926 Hydrochloride Tablets, 531 233 Houttuynia, 939 Immature Orange, 948 Gelatin, 923 Mentha, 974 Immunoglobulin, Human Normal, Puriˆed, 924 Perilla, 1004 949 Gentamicin Sulfate, 502, 1468 Plantago, 1014 Imperata Rhizome, 949, 1551 Gentian, 925 Powdered Geranium, 926 Indenolol Hydrochloride, 532 and Sodium Bicarbonate Powder, Powdered Swertia, 1064 Indigocarmine, 533 926 Swertia, 1063 Injection, 533 Japanese, 957 Homatropine Hydrobromide, 511 Indium (111In) Chloride Injection, 534 Powdered, 925 Homochlorcyclizine Hydrochloride, Indometacin, 534 Powdered, Japanese, 957 512, 1472 Capsules, 535 Geranium Herb, 926 Honey, 938 Suppositories, 536, 1477 Ginger, 927, 1763, 1764 Houttuynia Herb, 939 In‰uenza HA Vaccine, 949 Powdered, 927 Human Normal Immunoglobulin, Injection Ginseng, 927 949 Acetylcholine Chloride for, 222 Powdered, 929 Hycoato Injection, 996 (Aqueous Suspension), Estradiol Glacial Acetic Acid, 856 Hydralazine Benzoate, 456 Glehnia Root, 930 Hydrochloride, 513 (Aqueous Suspension), Estriol, Glibenclamide, 502 Hydrochloride for Injection, 513 458 Glucose, 503 Hydrochloride Powder, 513 (Aqueous Suspension), Insulin Zinc, Injection, 503 Hydrochloride Tablets, 514 542 Glutathione, 1716 Hydrochloric Acid, 514 (Aqueous Suspension), Insulin Zinc, Glycerin, 504, 1469 Dilute, 515 Amorphous, 542 and Potash Solution, 930 Lemonade, 939 (Aqueous Suspension), Insulin Zinc, Concentrated, 504, 1469 Hydrochlorothiazide, 515, 1473 Crystalline, 543 Glycerol, 504 Hydrocortisone, 516 (Aqueous Suspension), Insulin Zinc, Concentrated, 504 Acetate, 517, 1474 Protamine, 544 Glyceryl Monostearate, 931 and Diphenhydramine Ointment, (Aqueous Suspension), Isophane Glycine, 931 939 Insulin, 541 Glycyrrhiza, 932, 1550 Butyrate, 518 Adrenaline Hydrochloride, 447 Extract, 933 Sodium Phosphate, 519, 1474 Aminophylline, 239 Powdered, 933, 1550 Sodium Succinate, 520 Amobarbital Sodium for, 242 Gramicidin, 1470 Succinate, 521 Arginine Hydrochloride, 249 Gonadotrophin Hydrocotarnine Hydrochloride, 522 L-Arginine Hydrochloride, 249 Chorionic, 934 Hydrogenated Oil, 940 Ascorbic Acid, 251 for Injection, Chorionic, 936 Hydrophilic Atropine Sulfate, 256 for Injection, Serum, 937 Ointment, 940 Calcium Chloride, 298 Serum, 936 Petrolatum, 1005 Chlorpheniramine Maleate, 357 Gramicidin 1470 Hydrous Lanolin, 962 Chlorpromazine Hydrochloride, Granules Hydroxocobalamin Acetate, 523 361 Calcium Para-aminosalicylate, 302 Hydroxypropylcellulose, 940 Cyanocobalamin, 389 Pas-calcium, 302 Low Substituted, 942 Dehydrocholate Sodium, 396 Griseofulvin, 505, 1471 Hydroxypropylmethylcellulose Dehydrocholic Acid, 396 Guaiacol Glyceryl Ether, 505 Phthalate, 947 Deslanoside, 397 Guaifenesin, 505 2208, 943 Dextran 40, 399 Guanabenz Acetate, 506 2906, 944 Digoxin, 415 Guanethidine Sulfate, 507 2910, 945 Dimercaprol, 424 Gypsum, 937 Hydroxyzine Dimorpholamine, 425 Exsiccated, 938 Hydrochloride, 524 Diphenylhydantoin Sodium for, Pamoate, 524 686 Supplement II, JP XIV Index 1847

Dromostanolone Propionate, 436 Progesterone, 714 Powdered, 954 Drostanolone Propionate, 436 Protamine Sulfate, 719 Syrup, 955 Edrophonium Chloride, 440 Pyridoxine Hydrochloride, 725 Ipratropium Bromide, 550 Ephedrine Hydrochloride, 445 Reserpine, 730 Iproveratril Hydrochloride, 839 Epinephrine, 447 Ribo‰avin Phosphate, 736 Isepamicin Sulfate, 552 Epinephrine Hydrochloride, 447 Ribo‰avin Sodium Phosphate, L-Isoleucine, 553 Epirenamine Hydrochloride, 447 736 Isoniazid, 553 Ergometrine Maleate, 451 Sodium Bicarbonate, 750 Injection, 554, 1478 Estradiol Benzoate, 456 Sodium Chloride, Isotonic, 752 Tablets, 554, 1478 Folic Acid, 491 Sodium Chloride, 0.9,752 Isophane Insulin Injection (Aqueous Fructose, 498 Sodium Chloride, 10,752 Suspension), 541 Gallium (67Ga) Citrate, 501 Sodium Chromate (51Cr), 752 l-Isoprenaline Hydrochloride, 555 Glucose, 503 Sodium Iodohippurate (131I), 756 Isopropanol, 556 Heparin Sodium, 511 Sodium Pertechnetate (99mTc), 758 Isopropyl Alcohol, 556 Human Serum Albumin, Iodinated Sulfobromophthalein Sodium, 775 Isopropylantipyrine, 556 (131I), 546 Sulpyrine, 777 l-Isoproterenol Hydrochloride, 555 Hycoato, 996 Suxamethonium Chloride, 781 Isosorbide, 557 Hycodenone, Compound, 995 Suxamethonium Chloride for, 781 Dinitrate, 558 Hydralazine Hydrochloride for, Testosterone Enanthate, 787 Dinitrate Tablets, 558 513 Thallium (201Tl) Chloride, 791 Isotonic Indigocarmine, 533 Thiamine Hydrochloride, 794 Salt Solution, 752 Indium (111In) Chloride, 534 Thiamylal Sodium for, 796 Sodium Chloride Injection, 752 Insulin, 539 Thiopental Sodium for, 798 Sodium Chloride Solution, 752 Isoniazid, 554 Tubocurarine Chloride, 831 Levallorphan Tartrate, 570 Tubocurarine Hydrochloride, 831 J Lidocaine, 575 Vasopressin, 837 Lidocaine Hydrochloride, 575 Vinblastine Sulfate for, 841 Japanese

Magnesium Sulfate, 586 Vitamin B12,389 Angelica Root, 956 D-Mannite, 588 Vitamin B1 Hydrochloride, 794 Encephalitis Vaccine, 956 D-Mannitol, 588 Vitamin B2 Phosphate Ester, 736 Encephalitis Vaccine, Freeze-dried, Meglumine Amidotrizoate, 594 Vitamin B6,725 957 Meglumine Iotalamate, 595 Vitamin C, 251 Gentian, 957, Meglumine Sodium Weak Opium Alkaloids and Gentian, Powdered, 1551 Amidotrizote, 596 Scopolamine, 992 Valerian, 957 Meglumine Sodium Iodamide, 597 Xylitol, 845 Josamycin, 559, 1479 Metenolone Enanthate, 608 Insulin, 536 Propionate, 559, 1480 Morphine and Atropine, 978 Insulin Human (Genetical Jujube, 958 Morphine Hydrochloride, 630 Recombination), 537 Seed, 1551 Neostigmine Methylsulfate, 638 Injection, 539 Nicotinic Acid, 645 Zinc Injection (Aqueous K Noradrenaline Hydrochloride, 650 Suspension), 542 Norepinephrine, 650 Zinc Injection (Aqueous Kainic Acid, 560 Norepirenamine Hydrochloride, Suspension), Amorphous, 542 and Santonin Powder, 958 650 Zinc Injection (Aqueous Kallidinogenase, 560, 1718 Operidine, 679 Suspension), Crystalline, 543 Kanamycin Opium Alkaloids and Atropine, Zinc Protamine Injection (Aqueous Monosulfate, 1481 990 Suspension), 544 Sulfate, 563, 1482 Opium Alkaloids and Iodamide, 545 Kaolin, 959 Scopolamine, 991 Iodinated (131I) Human Serum Kernel Opium Alkaloids Hydrochlorides, Albumin Injection, 546 Apricot, 864 989 Iodine, 546 Peach, 1002 Oxycodone and Atropine, Glycerin, Compound, 950 Peach, Powdered, 1002 Compound, 996 Glycerin, Dental, 951 Ketamine Hydrochloride, 563 Oxycodone, Compound, 995 Tincture, 949 Ketoprofen, 564 Oxytocin, 665 Tincture, Dilute, 950 Ketotifen Fumarate, 1483 Paoaverine Hydrochloride, 671 Salicylic Acid and Phenol Spirit, Kitasamycin, 565, 1484 Pethidine Hydrochloride, 679 952 Tartrate, 1485 Phenolsulfonphthalein, 682 Iopamidol, 547 Phenytoin Sodium for, 686 Iopanoic Acid, 548, 1478 L Prednisolone Sodium Succinate for, Tablets, 548, 1478 705 Iotalamic Acid, 549 Lactic Acid, 959 Procainamide Hydrochloride, 709 Iotroxic Acid, 550 Lactose, 960 Procaine Hydrochloride, 710 Ipecac, 953 Anhydrous, 961 1848 Index Supplement II, JP XIV

Lactulose, 566 Lycium Hydrochloride Injection, 602 Lanatoside Bark, 1767 Mequitazine, 602 C, 567 Fruit, 1767 Merbromin, 604 CTablets, 567 Lysine Hydrochloride, 581 Solution, 605 Lanolin Lysozyme Hydrochloride, 1491 Mercaptopurine, 603 Hydrous, 962 L-Lysine Hydrochloride, 581 Mercurochrome, 604 Puriˆed, 963 Solution, 605 Lard, 964 M Meropenem Trihydrate, 605, 1719 Latamoxef Sodium, 569, 1486 Mestranol, 606 Lauromacrogol, 964 Macrogol Metenolone Leaf Ointment, 968 Acetate, 607 Bearberry, 870 400, 965 Enanthate, 607 Senna, Powdered, 1046 1500, 966 Enanthate Injection, 608 Sweet Hydrangea, Powdered, 1063 4000, 967 Methamphetamine Hydrochloride, Senna, 1044 6000, 967 609 Sweet Hydrangea, 1063 20000, 968 L-Methionine, 609 Lenampicillin Hydrochloride, 1488 Magnesium Methotrexate, 610, 1493 L-Leucine, 569 Carbonate, 582 Methoxsalen, 611 Leucomycin, 565 Oxide, 583 Methylbenactyzium Bromide, 611 Levallorphan Silicate, 584 Methylcellulose, 978 Tartrate, 570 Stearate, 969 Methylchlorophenylisoxazolylpenicil- Tartrate Injection, 570 Sulfate, 585 lin Sodium, 380 Levodopa, 571 Sulfate Injection, 586, 1492 Methyldichlorophenylisoxazolylpenicil- Levomepromazine Maleate, 572 Sulfate Mixture, 970 lin Sodium, 408 Levothyroxine Magnolia Bark, 970, 1552 Methyldopa, 612 Sodium, 572 Flower, 1552 Tablets, 613 Sodium Tablets, 573 Powdered, 1552 dl-Methylephedrine Lidocaine, 574 Mallotus Bark, 971 Hydrochloride, 613, 1719 Hydrochloride Injection, 575 Maltose, 586 Hydrochloride Powder, 614 Injection, 575 Mamushi Antivenom, Equine, Freeze- Hydrochloride Powder, 10,614, Light dried, 972 1720 Anhydrous Silicic Acid, 1050 D-Mannite, 587 Methylergometrine Liquid Para‹n, 1001 Injection, 588 Maleate, 615 Lime D-Mannitol, 587, 1718 Maleate Tablets, 615 Chlorinated, 894 Injection, 588, 1492 Methyl Quick, 878 Maprotiline Hydrochloride, 588 Parahydroxybenzoate, 976 Slaked, 877 Meclofenoxate Hydrochloride, 589 Salicylate, 618 Lincomycin Hydrochloride, 576, 1490 Mecobalamin, 590 Methylprednisolone, 616 Lindera Root 1766 Medazepam, 591 Succinate, 1720 Liothyronine Medicinal Methylrosanilinium Chloride, 617 Sodium, 576 Carbon, 972 Methyltestosterone, 618, 1722 Sodium Tablets, 577, 1490 Soap, 973 Tablets, 619, 1722 Liqueˆed Mefenamic Acid, 592 Meticrane, 619, 1493 Carbolic Acid, 1011 Mefruside, 592 Metildigoxin, 620 Phenol, 1011 Tablets, 593 Metoclopramide, 621 Liquid Para‹n, 1000 Meglumine, 974 Tablets, 1723 Lithium Carbonate, 578 Amidotrizoate Injection, 594 Metronidazole, 622 Lithospermum Root, 964 Iotalamate Injection, 595 Metyrapone, 623 Live Sodium Amidotrizoate Injection, Mexiletine Hydrochloride, 623 Attenuated Measles Vaccine, Freeze- 596 Miconazole, 624 dried, 972 Sodium Iodamide Injection, 597 Nitrate, 625 Attenuated Mumps Vaccine, Freeze- Melphalan, 597 Microcrystalline Cellulose, 889 dried, 981 Menatetrenone, 598, 1492 Micronomicin Sulfate, 626, 1494 Attenuated Rubella Vaccine, Freeze- Mentha Herb, 974 Midecamycin, 626 dried, 1029 Oil, 975 Acetate, 627 Oral Poliomyelitis Vaccine, 1015 Water, 975 Migrenin, 627, 1495 Longgu, 965 dl-Menthol, 975 Minocycline Hydrochloride, 628, Lonicera Leaf and Stem 1766 l-Menthol, 976 1495 Loquat Leaf, 1552 Mepenzolate Bromide, 599 Mitomycin C, 629, 1496 Lorazepam, 579 Mepirizole, 448 Mixture, Magnesium Sulfate, 970 Low Substituted Mepitiostane, 600, 1492 Monobasic Calcium Phosphate, 880 Hydroxypropylcellulose, 942 Mepivacaine Monosodium Loxoprofen Sodium, 580 Hydrochloride, 601, 1719 Trichloroethyl Phosphate, 823 Supplement II, JP XIV Index 1849

Trichloroethyl Phosphate Syrup, Nux Injection, 989 824 Vomica, 983 Ipecac Powder, 993 Morphine Vomica Extract, 984 Powdered, 987 and Atropine Injection, 978, 1553 Vomica Extract Powder, 984 Tincture, 988 Hydrochloride, 629, 1497 Vomica Tincture, 985 Orange Hydrochloride Injection, 630, 1497 Nystatin, 656 Immature, 948 Hydrochloride Tablets, 631, 1498 Oil, 994 Moutan Bark, 979 O Peel Syrup, 994 Mulberry Bark, 980, 1554 Peel Tincture, 995 Mupirocin Calcium Hydrate, 631 O‰oxacin, 1500 Orciprenaline Sulfate, 656 Oil Oriental Bezoar, 995 N Acrinol and Zinc Oxide, 857 Oxapium Iodide, 657 Acrinol and Zinc Oxide, Oxaprozin, 658 Nadolol, 633 Compound, 857 Oxazolam, 658 Nalidixic Acid, 634 Aromatic Castor, Oxetacaine, 659 Naloxone Hydrochloride, 634 Camellia, 889 Oxethazaine, 659 Naphazoline Castor, 888 Oxprenolol Hydrochloride, 660 and Chlorpheniramine Solution, Cinnamon, 897 Oxybuprocaine Hydrochloride, 661 981 Clove, 898 Oxycodone Hydrochloride, 662 Hydrochloride, 635 Coconut, 899 Oxydol, 662 Nitrate, 636 Cod Liver, 900 Oxygen, 663 Naproxen, 636 Corn, 903 Oxymetholone, 664 Narcotine, 654 Eucalyptus, 917 Oxytetracycline Hydrochloride, 665, Hydrochloride, 655 Fennel, 919 1501 Natural Aluminum Silicate, 233 Hydrogenated, 940 Oxytocin, 1728 Neomycin Sulfate, 497 Mentha, 975 Injection, 665, 1730 Neostigmine Olive, 986 Oyster Shell, 997 Methylsulfate, 637 Orange, 994 Methylsulfate Injection, 638 Peanut, 1002 P Netilmicin Sulfate, 638 Rape Seed, 1024 Nicardipine Hydrochloride, 639 Sesame, 1048 Panax Rhizome, 998 Hydrochloride Injection, 640 Soybean, 1059 Powdered, 1555 Niceritrol, 641 Turpentine, 1073 Pancreatin, 999 Nicomol, 642 Vitamin A, 1074 Pancuronium Bromide, 667 Tablets, 643 Zinc Oxide, 1085 Panipenem, 667 Nicorandil 1724 Ointment Pantethine, 669 Nicotinamide, 643 Absorptive, 855 Papaverine Nicotinic Acid, 644 Acrinol and Zinc Oxide, 858 Hydrochloride, 670 Injection, 645 Bufexamac, 290 Hydrochloride Injection, 671 Nifedipine, 645 Hydrocortisone and Paracetamol, 219 Nilvadipine, 1725 Diphenhydramine, 939 Para‹n, 999 Tablets, 1726 Hydrophilic, 940 Light Liquid, 1001 Nitrazepam, 646 Macrogol, 968 Liquid, 1000 Nitrogen, 982 Polyethylene Glycol, 968 Paraformaldehyde, 671 Nitroglycerin Tablets, 647 Scopolia Extract and Tannic Acid Paste Nitrous Oxide, 648 Compound, 1039 Arsenical, 866 Noradrenaline, 649 Simple, 1052 Paraformaldehyde, Dental, 1001 Hydrochloride, 650 Sulfur, Salicylic Acid and Triozinc, Dental, 1072 Hydrochloride Injection, Thianthol, 1062 Pas-calcium, 301 Norepinephrine, 649, 1498 White, 1080 Granules, 302 Injection, 650, 1499 Zinc Oxide, 1085 Peach Kernel, 1002 Norepirenamine, 649 Olive Oil, 986 Peanut Oil, 1002 Hydrochloride Injection, 650 Operidine, 678 Peel Norethisterone, 650 Injection, 679 Bitter Orange, 875 Nor‰oxacin, 651 Ophiopogon Tuber, 987 Citrus Unshiu, 898 Norgestrel, 652 Opium Penbutolol Sulfate, 672 and Ethinylestradiol Tablets, 652, Alkaloids and Atropine Injection, Penicillin 1499 990 G Potassium, 270 Nortriptyline Hydrochloride, 654 Alkaloids and Scopolamine Pentazocine, 672 Noscapine, 654 Injection, 991 Pentobarbital Calcium, 673 Hydrochloride, 655 Alkaloids Hydrochlorides, 988, Pentoxyverine Citrate, 674 Notopterygium Rhizome, 1554 1555, 1767 Peony Root, 1003, 1555 Nuphar Rhizome, 983 Alkaloids Hydrochlorides Powdered, 1556, 1768 1850 Index Supplement II, JP XIV

Peplomycin Sulfate, 675, 1503 Piroxicam, 1733 Diastase and Sodium Bicarbonate, Perilla Herb, 1004 Pivmecillinam Hydrochloride, 693, Compound, 907 Perphenazine, 675 1510 Dihydrocodeine Phosphate, 1, Maleate, 677 Plantago 417 Maleate Tablets, 678 Herb, 1014 Dihydrocodeine Phosphate, 10, Tablets, 676 Seed, 1014 418 Pethidine Plaster, Adhesive, 858 Diphenhydramine and Hydrochloride, 678, 1505 Salicylic Acid, Adhesive, 1032 Bromovalerylurea, 911 Hydrochloride Injection, 679 Platycodon Diphenylhydantoin, 686 Petrolatum Fluidextract, 1014 Dover's, 993 White, 1005 Root, 1014 Ephedrine Hydrochloride, 446 Yellow, 1006 Root, Powdered, 1015 Ephedrine Hydrochloride, 10, Petroleum Benzin, 1006 Polyethylene 446 Pharbitis Seed, 1007 Glycol Ointment, 968 for Cataplasm, Phellodendron, Phellodendron Glycol 400, 965 Compound, 1009 Bark, 1007, 1556 Glycol 1500, 966 Gentian and Sodium Bicarbonate, Albumin Tannate and Bismuth Glycol 4000, 967 926 Subnitrate Powder, 1009, 1556 Glycol 6000, 967 Hydralazine Hydrochloride, 513 Powdered, 1556 Glycol 20000, 968 Kainic Acid and Santonin, 958 Powder for Cataplasm, Compound, Polygala dl-Methylephedrine 1556 Root, 1015 Hydrochloride, 614 Phenacetin, 680 Root, Powdered, 1016 dl-Methylephedrine Phenazone, 247 Polygonum Root, 1557 Hydrochloride, 10,614 Phenethicillin Potassium, 1505 Polymixin B Sulfate, 694, 1511 NuxVomicaExtract, 984 Phenobarbital, 681 Polyoxyethylene Lauryl Alcohol Opium Ipecac, 993 Powder, 681 Ether, 964 Opium, Diluted, 987 Powder, 10, 681 Polyoxyl 40 Stearate, 1016 Phellodendron, Albumin Tannate Phenol, 1010 Polyporus Sclerotium, 1016 and Bismuth Subnitrate, 1009 and Zinc Oxide Liniment, 1011 Polysorbate 80, 1017 Phenobarbital, 681 for Disinfection, 1010 Polyvidone, 1020 Phenobarbital, 10,681 Liqueˆed, 1011 Polyvinylpyrrolidone, 1020 Phenovalin and Magnesium Oxide, with Camphor, Dental, 1011 K25, 1020 1012 Phenolated K30, 1020 Phenytoin, 686 Water, 1012 K90, 1020 Reserpine, 731 Water for Disinfection, 1012 Poria Sclerotium, 1017 Reserpine, 0.1, 731 Phenolsulfonphthalein, 682 Potash Soap, 1018 Rhubarb and Senna, Compound, Injection, 682 Potassium Ribo‰avin, 734 Phenovalin and Magnesium Oxide Benzylpenicillin, 270 Salicylated Alum, 1032 Powder, 1012, 1557 Bromide, 694 Scopolia Extract, 1036 L-Phenylalanine, 683 Canrenoate, 694 Scopolia Extract and Carbon, 1037 Phenylbutazone, 684 Carbonate, 1018 Scopolia Extract and Diastase, Phenylephrine Hydrochloride, 684 Chloride, 695 Compound, 1037 Phenytoin, 685 Clavulanate, 696, 1512 Scopolia Extract and Ethyl Powder, 686 Guaiacolsulfonate, 696 Aminobenzoate, 1037 Sodium for Injection, 686 Hydroxide, 1018 Scopolia Extract, Papaverine and Tablets, 686 Iodide, 697 Ethyl Aminobenzoate, 1038 Phytomenadione, 687 Penicillin G, 270 Swertia and Sodium Bicarbonate, Phytonadione, 687, 1507 Penicillin G, Crystalline, 270 1065 Picrasma Wood, 1013 Permanganate, 698 Thiamine Hydrochloride, 794 Pilocarpine Hydrochloride, 687 Sulfate, 1019 Vitamin B, Compound,

Pimaricin, 1507 Potato Starch, 1019, 1769 Vitamin B1 Hydrochloride, 794 Pindolol, 688 Povidone, 1020 Vitamin B2, 734 Pinellia Tuber, 1013 Povidone-Iodine, 698, 1513 Vitamin C, 251 Pipemidic Acid Trihydrate, 689, 1508 Powder Zinc Oxide Starch, 1086 Piperacillin Sodium, 690, 1508 Ascorbic Acid, 251 Powdered Piperazine Chlordiazepoxide, 351 Acacia, 855, 1543 Adipate, 691 Chlorpheniramine and Calcium, Agar, 859 Phosphate, 692 895 Alisma Rhizome, 860 Phosphate Tablets, 692 Chlorpheniramine Maleate, 358 Aloe, 861, 1544, 1757 Pirarubicin, 1509 Codeine Phosphate, 1, 382 Amomum Seed, 863 Pirenoxine, 693, 1731 Codeine Phosphate, 10,383 Atractylodes Lancea Rhizome, 869 Pirenzepine Hydrochloride Hydrate, Diastase and Sodium Bicarbonate, Atractylodes Rhizome, 868 1732 907 Calumba, 881 Supplement II, JP XIV Index 1851

Capsicum, 883, 1546 Hydrochloride, 710 R Cellulose, 892 Hydrochloride Injection, 710, 1514 Cinnamon Bark, 897, 1547 Procarbazine Hydrochloride, 711, Ranitidine Hydrochloride, 1517 Clove, 898 1515 Rape Seed Oil, 1024 Cnidium Rhizome, 899 Procaterol Hydrochloride, 712 Red Ginseng, 1024 Coix Seed, 900 Processed Aconite Root, 1770 Rehmannia Root, 1025 Coptis Rhizome, 902, 1548 Powdered, 1772 Reserpine, 729, 1518 Cyperus Rhizome, 906 Processed Ginger, 1774 Injection, 730 Digitalis, 910, 1762 Prochlorperazine Powder, 731 Dioscorea Rhizome, 911 Maleate, 713 Powder, 0.1,731 Gambir, 920 Maleate Tablets, 713 Tablets, 731 Gardenia Fruit, 923, 1763 Progesterone, 714 Retinol Gentian, 925 Injection, 714 Acetate, 732, 1519 Geranium Herb, 926 Proglumide, 715 Palmitate, 733, 1519 Ginger, 927, 1764 Promethazine Hydrochloride, 716 Rhizome Ginseng, 929 Propantheline Bromide, 716 Alisma, 860 Glycyrrhiza, 933 Propranolol Hydrochloride, 717 Alisma, Powdered, 860 Ipecac, 954 Propylene Anemarrhena, 864 Japanese Angelica Root, 956 Glycol, 1022 Atractylodes, 868 Japanses Gentian, 957, 1551 Propyl Parahydroxybenzoate, 1021 Atractylodes Lancea, 869 Japanese Valerian, 958 Propylthiouracil, 718 Atractylodes Lancea, Powdered, Magnolia Bark, 971, 1552 Tablets, 718 869 Moutan Bark, 980 Propyphenazone, 556 Atractylodes, Powdered, 868 Opium, 987 Prostaglandin Cimicifuga, 896

Oyster Shell, 998 E1,1718 Cnidium, 899 Panax Rhizome, 998, 1555 E1 a-Cyclodextrin Clathrate Cnidium, Powdered, 899 Peach Kernel, 1002 Compound, 231 Coptis, 901

Peony Root, 1004, 1556, 1768 F2a, 425 Coptis, Powdered, 902 Phellodendron Bark, 1008, 1556 Protamine Cyperus, 906 Picrasma Wood, 1013 Sulfate, 719 Cyperus, Powdered, 906 Platycodon Root, 1015 Sulfate Injection, 719 Dioscorea, 911 Polygala Root, 1016 Prothionamide, 720 Dioscorea, Powdered, 911 Polyporus Sclerotium, 1016 Protirelin, 720 Imperate, 949 Poria Sclerotium, 1017 Tartrate, 722 Nuphar, 983 Rhubarb, 1027, 1558, 1774 Prunella Spike, 1023 Panax, 998 Rose Fruit, 1029 Pueraria Root, 1023, 1557 Panax, Powdered, 998 Scutellaria Root, 1043, 1560, 1775 Puriˆed Scopolia, 1040 Senega, 1044 Absorbent Cotton, 851 Rhubarb, 1026, 1558, 1774 Senna Leaf, 1046, 1561 Dehydrocholic Acid, 395 Powdered, 1027, 1558, 1774 Smilax Rhizome, 1053 Gelatin, 924 Ribo‰avin, 733 Sophora Root, 1058 Lanolin, 963 Butyrate, 734 Sweet Hydrangea Leaf, 1063 Shellac, 1049 Phosphate, 735 Swertia Herb, 1064, 1562 Water, 1079 Phosphate Injection, 736 Tragacanth, 1072 Pyrantel Pamoate, 722 Powder, 734 Zanthoxylum Fruit, 1084 Pyrazinamide, 723, 1515, 1734 Sodium Phosphate, 735 Pranoprofen, 699 Pyridostigmine Bromide, 724 Sodium Phosphate Injection, 736 Prazepam, 700 Pyridoxine Ribostamycin Sulfate, 737, 1520 Tablets, 701 Hydrochloride, 724, 1734 Rice Starch, 1028 Precipitated Calcium Carbonate, 297 Hydrochloride Injection, 725, 1735 Rifampicin, 737, 1521 Prednisolone, 701 Pyroxylin, 1023 Ringer's Solution, 1028 Acetate, 703, 1513 Pyrrolnitrin, 1516 Rokitamycin, 737 Sodium Succinate for Injection, Root 705, 1513 Q Achyranthes, 857 Succinate, 704 Angelica Dahurica, 864 Tablets, 702 Quick Lime, 878 Asiasarum, 867 Primidone, 706, 1514 Quinidine Sulfate, 725 Astragalus, 867 Probenecid, 706 Quinine Belladonna, 872 Tablets, 707 Ethyl Carbonate, 726 Bupleurum, 876 Procainamide Hydrochloride, 727 Glehnia, 930 Hydrochloride, 708 Sulfate, 728 Japanese Angelica, 956 Hydrochloride Injection, 709 Japanese Angelica, Powdered, 956 Hydrochloride Tablets, 709 Lithospermum, 964 Procaine Peony, 1003 1852 Index Supplement II, JP XIV

Peony, Powdered, 1004 Coix, 900 Cefoperazone, 332 Platycodon, 1014 Coix, Powdered, 900 Cefotaxime, 335 Polygala, 1015 Pharbitis, 1007 Cefoxitin, 336 Pueraria, 1023 Plantago, 1014 Cefpiramide, 338 Rehmannia, 1025 Senega, 1043 Cefsulodin, 340 Saposhnikovia, 1034 Powdered, 1044 Ceftizoxime, 345 Saussurea, 1034 Syrup, 1044 Cefuroxime, 348 Scutellaria, 1042 Senna Leaf, 1044, 1560 Chloride, 751, 1524 Scutellaria, Powdered, 1043 Powdered, 1046, 1561 Chloride Injection, 0.9, 752 Sophora, 1058 Serrapeptase, 1735 Chloride Injection, 10, 752, 1525 Sophora, Powdered, 1058 Serum Chromate (51Cr) Injection, 752 Trichosanthes, 1072 Gonadotrophin, 936, 1765 Citrate, 753 Rose Fruit, 1029 Gonadotrophin for Injection, 937, Citrate Injection for Transfusion, Rosin, 1029 1765 753 Roxithromycin, 738, 1522 Sesame Oil, 1048 Cloxacillin, 380 Shellac CMC, 887 S Puriˆed, 1049 Cromoglicate, 754 White, 1049 Dantrolene, 392 Saccharated Pepsin, 1030 Shell Diclofenac, 405 Saccharin Sodium, 1030 Oyster, 997 Dicloxacillin, 408 SaŒower, 1031 Oyster, Powdered, 998 EDTA, 913 SaŠron, 1031 Siccaine, 1523 Flavin Adenine Dinucleotide, 475 Salazosulfapyridine, 739 Silicic Acid, Light Anhydrous, 1050 Fluorescein, 483 Salbutamol Sulfate, 740 Silver Fosfomycin, 496 Salicylated Alum Powder, 1032 Nitrate, 746 Fusidate, 1525 Salicylic Acid, 741 Nitrate Ophthalmic Solution, 1041 Heparin, 510 Plaster, Adhesive, 1032 Protein, 1051 Hydrogen Sulˆte, 1054 Spirit, 1033 Protein Solution, 1051 Hydroxide, 1056 Santonin, 742 Simˆbrate, 746, 1524 Iodide, 755 Tablets, 742, 1735 Simple Iodide (123I) Capsules, 755 Saponated Cresol Solution, 905 Ointment, 1052 Iodide (131I) Capsules, 755 Saposhnikovia Root, 1034 Syrup, 1052 Iodide (131I) Solution, 756 Sappan Wood, 1774 Sinomenium Stem, 1052, 1562 Iodohippurate (131I) Injection, 756 Saussurea Root, 1034 Sisomicin Sulfate, 747, 1524 Iopodate, 756, 1526 Schisandra Fruit, 1035 Slaked Lime, 877 Iopodate Capsules, 757, 1526 Schizonepeta Spike, 1035 Smilax Iotalamate Injection, 757 Sclerotium Rhizome, 1053 Latamoxef, 569 Polyporus, Powdered, 1016 Rhizome, Powdered, 1053 Lauryl Sulfate, 1056 Poria, 1017 Soap Levothyroxine, 572 Poria, Powdered, 1017 Medicinal, 973 Liothyronine, 576 Scopolamine Potash, 1018 Loxoprofen, 580 Butylbromide, 743, 1522 Sodium Metabisulˆte, 1057 Hydrobromide, 744 Acetate, 1053 Methylchlorophenylisoxazolyl- Scopolia Aminobenzylpenicillin, 246 penicillin, 380 Extract, 1035 Ampicillin, 246 Methyldichlorophenylisoxazolyl- Extract and Carbon Powder, 1036 Aurothiomalate, 748, 1736 penicillin, 408 Extract and Ethyl Aminobenzoate Benzoate, 749 Pertechnetate (99mTc) Injection, 758 Powder, 1037, 1559 Bicarbonate, 749 Phosphate, Dibasic, 907 Extract and Tannic Acid Bicarbonate and Bitter Tincture Picosulfate, 758 Ointment, Compound, 1560 Mixture, 1054 Piperacillin, 690 Suppositories, 1039 Bicarbonate Injection, 750, 1524 Polystyrene Sulfonate, 759, 1737 Suppositories, Compound, 1560 Bisulˆte, 1054 Prasterone Sulfate, 760 Extract Powder, 1036 Borate, 750 Pyrosulˆte, 1057 Extract, Papaverine and Ethyl Bromide, 750 Saccharin, 1030 Aminobenzoate Powder, 1038 Carbonate, 1055 Salicylate, 761 Rhizome, 1040 Carbonate, Dried, Sulˆte, Dried, 1058 Scutellaria Root, 1042, 1560 Carmellose, 887 Thiosulfate, 762, 1738 Powdered, 1560, 1775 Cefalotin, 317 Thiosulfate Injection, 762 Secretin, 744 Cefamandole, 317 Valproate, 762 Seed Cefapirin, 317 Solution Amomum, 863 Cefazolin, 319 Adrenaline Hydrochloride, 448 Amomum, Powdered, 863 Cefbuperazone, 322 Alum, 861 Cassia, 888 Cefmetazole, 331 Benzalkonium Chloride, 267 Supplement II, JP XIV Index 1853

Benzethonium Chloride, 269 Sulbactam Sodium, 768 Chlorpheniramine Maleate, 359 Chlorhexidine Gluconate, 353 Sulbenicillin Sodium, 769, 1528 Chlorpromazine Hydrochloride, Cresol, 905 Sulfadiazine Silver, 769 361 Cresol, Saponated, 905 Sulfafurazole, 773 Chlorpropamide, 362 Epinephrine, 448 Sulfamethizole, 770 Clomifene Citrate, 374 Epinephrine Hydrochloride, 448 Sulfamethoxazole, 770 Codeine Phosphate, 384 Epirenamine Hydrochloride, 448 Sulfamonomethoxine, 771 Dichlorphenamide, 407 Glycerin and Potash, 930 Sulfasalazine, 739 Diclofenamide, 407 Idoxuridine Ophthalmic, 528 Sulˆnpyrazone, 772 Diethylcarbamazine Citrate, 409 Merbromin, 605 Tablets, 773 Diethylstilbestrol Diphosphate, Mercurochrome, 605 Sulˆsomezole, 770 494 Naphazoline and Sulˆsoxazole, 773 Digitoxin, 411 Chlorpheniramine, 981 Sulfobromophthalein Digoxin, 416 Ringer's, 1028 Sodium, 774 Dimenhydrinate, 423 Salt, Isotonic, 752 Sodium Injection, 775 Diphenylhydantoin, 686 Silver Nitrate Ophthalmic, 1041 Sulfur, 775 Distigmine Bromide, 430 Silver Protein, 1051 and Camphor Lotion, 1062 Dydrogesterone, 438 Sodium Chloride, Isotonic, 752 Salicylic Acid and Thianthol Ephedrine Hydrochloride, 446 Sodium Hypochlorite, Dental, 864 Ointment, 1062 Ergometrine Maleate, 451 Sodium Iodide (131I), 756 Sulpiride, 776 Estriol, 458 D-Sorbitol, 764 Sulpyrine, 776 Etacrynic Acid, 460 Thianthol and Salicylic Acid, Injection, 777 Ethinylestradiol, 463 Compound, 1067 Sultamicillin Tosilate, 778 Etilefrine Hydrochloride, 468 Tolnaftate, 816 Sultiame, 779 Folic Acid, 491 Zinc Sulfate Ophthalmic, 1086 Suppositorise Fosfestrol, 494 50, Benzalkonium Chloride, Bisacodyl, 280 Hydralazine Hydrochloride, 514 Concentrated, 267 Indometacin, 536 Imipramine Hydrochloride, 531 Sophora Root, 1058 Scopolia Extract and Tannic Acid, Iopanoic Acid, 548 Sorbitan Sesquioleate, 1058 1039 Isoniazid, 554 D-Sorbitol, 763 Scopolia Extract and Tannic Acid, Isosorbide Dinitrate, 558 Solution, 764 Compound, 1041 Lanatoside C, 567 Soybean Oil, 1059 Suxamethonium Levothyroxine Sodium, 573 Spectinomycin Hydrochloride, 1526 Chloride, 780 Liothyronine Sodium, 576 Spike Chloride for Injection, 781 Methyldopa, 613 Prunella, 1023 Chloride Injection, 781 Methylergometrine Maleate, 615 Schizonepeta, 1035 Sweet Hydrangea Leaf, 1063 Methyltestosterone, 619 Spirit Swertia Morphine Hydrochloride, 631 Capsicum and Salicylic Acid, 884 and Sodium Bicarbonate Powder, Nitroglycerin, 647 Foeniculated Ammonia, 919 1065, 1563 Norgestrel and Ethinylestradiol, Iodine, Salicylic Acid and Phenol, Herb, 1063, 1562 652 952 Powdered, 1562 Perphenazine, 676 Methyl Salicylate, Compound, 977 Synthetic Perphenazine Maleate, 678 Salicylic Acid, 1033 Aluminum Silicate, 235 Phenytoin, 686 Salicylic Acid, Compound, 1033 Camphor, 306 Piperazine Phosphate, 682 Spironolactone, 765 Syrup Prazepam, 700 Starch Ipecac, 955 Prednisolone, 702 Corn, 903 Orange Peel, 994 Probenecid, 707 Potato, 1019 Senega, 1044 Procainamide Hydrochloride, 709 Rice, 1028 Simple, 1052 Prochlorperazine Maleate, 713 Wheat, 1080 Trichloroethyl Phosphate, Propylthiouracil, 718 Stearic Acid, 1059 Monosodium, 824 Reserpine, 731 Stearyl Alcohol, 1059 Triclofos Sodium, 824 Santonin, 742 Stem Sulˆnpyrazone, 773 Akebia, 859 T Thiamazole, 792 Sinomenium, 1052 Tipepidine Hibenzate, 806 Sterile Tablets Tolbutamide, 814 Absorbent Cotton, 852 Acetylsalicylic Acid, 252 Trihexyphenidyl Hydrochloride, Absorbent Gauze, 853 Ajmaline, 227 825 Puriˆed Absorbent Cotton, 852 Amitriptyline Hydrochloride, 240 Trimethadione, 827 Puriˆed Water, 1079 Aspirin, 252 Warfarin Potassium, 843 Streptomycin Sulfate, 766, 1527 Azathioprine, 258 Talampicillin Hydrochloride, 782, Sucralfate, 766 Baclofen, 260 1529 Sucrose, 1060 Chlordiazepoxide, 352 Talc, 1065 1854 Index Supplement II, JP XIV

Tannalbin, 227 dl-a-Tocopherol, 807 Chloride Injection, 831 Tannic Acid, 782 Acetate, 808 Hydrochloride, 830 Tartaric Acid, 1066 Nicotinate, 810 Hydrochloride Injection, 831 Tegafur, 782, 1530, 1738 Todralazine Hydrochloride, 811 Tulobuterol Hydrochloride, 831 Teicoplanin, 783, 1530 Toˆsopam, 812 Turpentine Oil, 1073 Terbutaline Sulfate, 786 Tolazamide, 813 Termeric 1775 Tolbutamide, 814 U Testosterone Tablets, 814 Enanthate, 787 Tolnaftate, 815 Ubidecarenone, 832 Enanthate Injection, 787 Solution, 816 Ulinastatin, 833 Propionate, 788, 1738 Tolperisone Hydrochloride, 816 Uncaria Thorn, 1563, 1776 Propionate Injection, 788, 1739 Toxoid Urea, 835 Tetanus Antitoxin, Equine, Freeze- Diphtheria, 912 Urokinase, 1073 dried, 1066 Diphtheria-Tetanus, Combined, Ursodesoxycholic Acid, 835 Tetracaine Hydrochloride, 789 913 Uva Ursi Fluidextract, 1074 Tetracycline Hydrochloride, 789 Diphtheria-Tetanus, Combined, Thallium (201Tl) Chloride Injection, Adsorbed, 913 V 791 for Adult Use, Diphtheria, Theophylline, 791, 1531 Adsorbed, 912 Vaccine Thiamazole, 791 Habu-venom, Adsorbed, 938 Cholera, 896 Tablets, 792 Tetanus, Adsorbed, 1066 Diphtheria-Puriˆed Pertussis- Thiamine Tragacanth, 1072 Tetanus Combined, Adsorbed, Hydrochloride, 793, 1532, 1740 Powdered, 1072 1005 Hydrochloride Injection, 794 Tranexamic Acid, 817, 1745 Hepatitis B, Adsorbed, 938 Hydrochloride Powder, 794 Capsules, 1746 In‰uenza HA, 949 Nitrate, 795, 1532 Injection, 1747 Japanese Encephalitis, 956 Thiamylal Tablets, 1747 Japanese Encephalitis, Freeze-dried, Sodium, 796, 1741 Transfusion, Sodium Citrate Injection 957 Sodium for Injection, 796, 1742 for, 753 Live Attenuated Measles, Freeze-d- Thianthol, 1067 Trapidil, 818 ried, 972 Thiopental Trepibutone, 819 Live Attenuated Mumps, Freeze-d- Sodium, 797, 1532 Tretoquinol Hydrochloride, 827 ried, 981 Sodium for Injection, 798 Triamcinolone, 819, 1535 Live Attenuated Rubella, Freeze-d- Thioridazine Hydrochloride, 798 Acetonide, 820 ried, 1029 Thiotepa, 799 Triamterene, 821 Live Oral Poliomyelitis, 1015 L-Threonine, 799 Tribulus Fruit, 1775 Pertussis, Puriˆed, Adsorbed, 1005 Thrombin, 1068 Trichlormethiazide, 822, 1748 Rabies, Inactivated Tissue Culture, Thymol, 1069 Tablets, 1749 Freeze-dried, 1024 Thyroid, Dried, 1069 Trichloroethyl Phosphate, Smallpox, Freeze-dried, 1053 Tiaramide Hydrochloride, 800 Monosodium, 823 Smallpox, Freeze-dried, Prepared in Tablets, 1742 Trichomycin, 823 Cell Culture, 1053 Ticarcillin Sodium, 801 Trichosanthes Root, 1072 Weil's Disease and Akiyami Ticlopidine Hydrochloride, 802 Triclofos Combined, 1080 Timepidium Bromide, 803 Sodium, 823 (for Percutaneous Use), BCG, Tincture Sodium Syrup, 824 Freeze-dried, 869 Bitter, 875 Trichomycin, 1536 L-Valine, 836 Capsicum, 883 Trihexyphenidyl Vancomycin Hydrochloride, 837, Iodine, 949 Hydrochloride, 824 1539 Nux Vomica, 985 Hydrochloride Tablets, 825 Vasopressin Injection, 837, 1751 Opium, 988 Trimebutine Maleate, 1537 Verapamil Hydrochloride, 839 Orange Peel, 995 Trimetazidine Hydrochloride, 826 Vinblastine Tinidazole, 804, 1743 Trimethadione, 827 Sulfate, 840, 1752 Tipepidine Tablets, 827 Sulfate for Injection, 841, 1753 Hibenzate, 804, 1533 Trimetoquinol Hydrochloride, 827, Vincristine Sulfate, 842 Hibenzate Tablets, 806 1538 Vitamin Titanium Oxide, 1070 Tropicamide, 828 A Acetate, 732 Tizanidine Hydrochloride, 1744 L-Tryptophan, 829 A Capsules, 1075 Toad Venom, 1071, 1563 Tuber A Oil, 1074, 1564 Tobramycin, 807, 1533 Corydalis, 904 A Oil Capsules, 1075, 1565 Tocopherol, 807, 1534 Ophiopogon, 987 A Palmitate, 733

Acetate, 808, 1535 Pinellia, 1013 B1 Hydrochloride, 793 Calcium Succinate, 809 Tubocurarine B1 Hydrochloride Injection, 794 Nicotinate, 810 Chloride, 830 B1 Hydrochloride Powder, 794 Supplement II, JP XIV Index 1855

B1 Nitrate, 795 Ammonia, 241 B2,733 Apricot Kernel, 865 X B2 Phosphate Ester, 735 for Disinfection, Phenolated, 1012 B2 Phosphate Ester Injection, 736 for Injection, 1078 Xylitol, 844 B2 Powder, 734 Formalin, 920 Injection, 845, 1540 B6,724 Mentha, 975 B6 Injection, 725 Phenolated, 1012 Y B12,388 Puriˆed, 1079 B12 Injection, 389 Sterile, Puriˆed, 1079 Yeast, Dried, 1083 C, 250 Wax, Carnauba, 888 Yellow CInjection, 251 Weak Opium Alkaloids and Beeswax, 871 CPowder, 251 Scopolamine Injection, 992 Petrolatum, 1006

D2,449 Weil's Disease and Akiyami Combined D3,363 Vaccine, 1080 Z E, 807 Wheat Starch, 1080, 1776 E Acetate, 808 White Zanthoxylum Fruit, 1083 E Calcium Succinate, 809 Beeswax, 871 Zedoary, 1084 E Nicotinate, 810 Ointment, 1080 Zinc

K1,687 Petrolatum, 1005 Chloride, 1084 Shellac, 1049 Oxide, 845 W Soft Sugar, 1061 Oxide Oil, 1085 Whole Human Blood, 1080 Oxide Ointment, 1085 Warfarin Wine, 1081 Oxide Starch Powder, 1086 Potassium, 843 Wood Sulfate, 846 Potassium Tablets, 843 Picrasma, 1013 Sulfate Ophthalmic Solution, 1086 Water, 1076 Picrasma, Powdered, 1013 Zinostatin Stimalamer, 846 INDEX IN JAPANESE

アモキサピン 243 イソプロピルアンチピリン 556 ア アモキシシリン 244, 1381 イドクスウリジン 527 アモバルビタール 242 イドクスウリジン点眼液 528, 1475 亜鉛華デンプン 1086 アラビアゴム 855, 1543 イブプロフェン 526 亜鉛華軟膏 1085 アラビアゴム末 855, 1543 イミペネム 530, 1476 アカメガシワ 971 亜硫酸水素ナトリウム 1054 イレイセン 1547 アクチノマイシン D 225, 1381 アルジオキサ 228 インジゴカルミン 533 アクリノール 224 アルプラゾラム 230 インジゴカルミン注射液 533 アクリノール・亜鉛華軟膏 858 アルプロスタジル 1688 インスリン 536 アクリノール・チンク油 857 アルプロスタジルアルファデクス インスリン亜鉛水性懸濁注射液 542 アザチオプリン 256 231 インスリン注射液 539 アザチオプリン錠 257 アロエ 860, 1544 インチンコウ 867 亜酸化窒素 648 アロエ末 861, 1544, 1757 インドメタシン 534 アジスロマイシン水和物 1690 アロプリノール 228 インドメタシンカプセル 535 アジピン酸ピペラジン 691 アンソッコウ 873 インドメタシン坐剤 536, 1477 アジマリン 226 安息香酸 270 インフルエンザ HA ワクチン 949 アジマリン錠 227 安息香酸エストラジオール 455, インヨウカク 1762 亜硝酸アミル 246 1462 アスコルビン酸 250 安息香酸エストラジオール水性懸濁注 ウ アスコルビン酸散 251 射液 456 アスコルビン酸注射液 251 安息香酸エストラジオール注射液 ウイキョウ 918 アズトレオナム 258 456 ウイキョウ末 918 アスピリン 252 安息香酸ナトリウム 749 ウイキョウ油 918 アスピリンアルミニウム 253 安息香酸ナトリウムカフェイン 296 ウコン 1775 アスピリン錠 252 安息香酸ベンジル 874 ウヤク 1766 アスポキシシリン 254 アンチピリン 247 ウリナスタチン 833 アセグルタミドアルミニウム 1377 アンピシリン 246, 1382 ウルソデオキシコール酸 835 アセタゾラミド 220 アンピシリンナトリウム 246, 1385 ウロキナーゼ 1073 アセチルキタサマイシン 222, 1378 アンモニア・ウイキョウ精 919 ウワウルシ 870 アセチルスピラマイシン 224, 1378 アンモニア水 241 ウワウルシ流エキス 1074 アセトアミノフェン 219 アセトヘキサミド 221, 1687 イ エ アセンヤク 920 アセンヤク末 920 イオウ 775 エイジツ 1029 亜ヒ酸パスタ 866 イオウ・カンフルローション 1062 エイジツ末 1029 アフロクアロン 225 イオウ・サリチル酸・チアントール軟膏 液状フェノール 1011 アヘンアルカロイド・アトロピン注射 1062 エスタゾラム 454 液 990 イオタラム酸 549 エストリオール 457, 1463 アヘンアルカロイド・スコポラミン注 イオタラム酸ナトリウム注射液 757 エストリオール錠 458 射液 991 イオタラム酸メグルミン注射液 595 エストリオール水性懸濁注射液 458 アヘン散 987 イオトロクス酸 550 エタクリン酸 459 アヘンチンキ 988 イオパノ酸 548, 1478 エタクリン酸錠 460 アヘン・トコン散 993 イオパノ酸錠 548, 1478 エタノール 814 アヘン末 987 イオパミドール 547 エチオナミド 464, 1707 アマチャ 1063 イオポダートナトリウム 756, 1526 エチゾラム 1463 アマチャ末 1063 イオポダートナトリウムカプセル エチニルエストラジオール 463 アミドトリゾ酸 237 757, 1526 エチニルエストラジオール錠 463 アミドトリゾ酸ナトリウムメグルミン イクタモール 948 エチルコハク酸エリスロマイシン 注射液 596 イソソルビド 557 453 アミドトリゾ酸メグルミン注射液 イソニアジド 553 エチル炭酸キニーネ 727 594 イソニアジド錠 554, 1478 エチレンジアミン 917 アミノ安息香酸エチル 465 イソニアジド注射液 554, 1478 エデト酸ナトリウム 913 アミノフィリン 239 イソフェンインスリン水性懸濁注射液 エーテル 462 アミノフィリン注射液 239 541 エテンザミド 461 アムホテリシン B245 イソプロパノール 556 エトスクシミド 465

1857 1858 Index in Japanese Supplement II, JP XIV

エトポシド 1709 塩酸インデノロール 532 塩酸テトラカイン 789 エナント酸テストステロン 787 塩酸エタンブトール 460 塩酸テトラサイクリン 789 エナント酸テストステロン注射液 塩酸エチルモルヒネ 467 塩酸デメチルクロルテトラサイクリン 787 塩酸エチレフリン 468 1446 エナント酸フルフェナジン 487 塩酸エチレフリン錠 468, 1708 塩酸ドキサプラム 433 エナント酸メテノロン 607 塩酸エピルビシン 1458 塩酸ドキシサイクリン 434, 1454 エナント酸メテノロン注射液 608 塩酸エフェドリン 444 塩酸ドキソルビシン 434, 1453 エノキサシン 444 塩酸エフェドリン散 10 446 塩酸トドララジン 811 エピネフリン 447 塩酸エフェドリン錠 446 塩酸ドパミン 432 エピネフリン液 447 塩酸エフェドリン注射液 445 塩酸ドパミン注射液 433, 1452 エピネフリン注射液 447 塩酸エペリゾン 1706 塩酸ドブタミン 431, 1452 エピリゾール 448 塩酸 L-アルギニン 248 塩酸トリヘキシフェニジル 824 エリスロマイシン 453, 1460 塩酸 L-アルギニン注射液 248 塩酸トリヘキシフェニジル錠 825 L-イソロイシン 553 塩酸 L-エチルシステイン 466 塩酸トリメタジジン 826 エルカトニン 440 塩酸 L-リジン 581 塩酸トリメトキノール 827, 1538 L-カルボシステイン 310 塩酸オキシコドン 662 塩酸トルペリゾン 816 エルゴカルシフェロール 449, 1460 塩酸オキシテトラサイクリン 665, 塩酸ナファゾリン 635 L-トリプトファン 829 1501 塩酸ナロキソン 634 L-トレオニン 799 塩酸オキシブプロカイン 661 塩酸ニカルジピン 639 L-バリン 836 塩酸オクスプレノロール 660 塩酸ニカルジピン注射液 640 L-フェニルアラニン 683 塩酸カルテオロール 313 塩酸ノスカピン 655 L-メチオニン 609 塩酸キニーネ 727 塩酸ノルトリプチリン 654 L-ロイシン 569 塩酸クリンダマイシン 1439 塩酸バカンピシリン 259, 1389 塩化亜鉛 1084 塩酸クロカプラミン 370 塩酸パパベリン 670 塩化アンベノニウム 236 塩酸クロコナゾール 387 塩酸パパベリン注射液 671 塩化インジウム (111In) 注射液 534 塩酸クロニジン 376 塩酸バンコマイシン 837, 1539 塩化エドロホニウム 439 塩酸クロフェダノール 371 塩酸ヒドララジン 513 塩化エドロホニウム注射液 440 塩酸クロペラスチン 377 塩酸ヒドララジン散 513 塩化カリウム 695 塩酸クロミプラミン 375 塩酸ヒドララジン錠 514 塩化カルシウム 298 塩酸クロルプロマジン 360 塩酸ヒドロキシジン 524 塩化カルシウム注射液 298, 1399 塩酸クロルプロマジン錠 361 塩酸ヒドロコタルニン 522 塩化スキサメトニウム 781 塩酸クロルプロマジン注射液 361 塩酸ピブメシリナム 693, 1510 塩化スキサメトニウム注射液 781 塩酸クロルヘキシジン 354 塩酸ビペリデン 279 塩化タリウム (201Tl) 注射液 791 塩酸ケタミン 563 塩酸ピリドキシン 724, 1734 塩化ツボクラリン 830 塩酸コカイン 381 塩酸ピリドキシン注射液 725, 1735 塩化ツボクラリン注射液 831 塩酸シクロペントラート 389 塩酸ピレンゼピン水和物 1732 塩化ナトリウム 751, 1524 塩酸ジフェニドール 409 塩酸ピロカルピン 687 10 塩化ナトリウム注射液 752, 塩酸ジフェンヒドラミン 427 塩酸ベニジピン 1691 1525 塩酸ジブカイン 404 塩酸ベニジピン錠 1692 塩化ベタネコール 278 塩酸シプロヘプタジン 391 塩酸フェニレフリン 684 塩化ベルベリン 271 塩酸ジラゼプ 419 塩酸ブクモロール 287 塩化ベンザルコニウム 266 塩酸ジルチアゼム 420, 1451 塩酸ブナゾシン 291 塩化ベンザルコニウム液 267 塩酸スペクチノマイシン 1526 塩酸ブフェトロール 288 塩化ベンゼトニウム 269 塩酸セトラキサート 349, 1431 塩酸ブプラノロール 292 塩化ベンゼトニウム液 269 塩酸セフェタメト ピボキシル 328 塩酸フラボキサート 476 塩化メチルロザニリン 617 塩酸セフェピム 326 塩酸フルスルチアミン 499 塩化リゾチーム 1491 塩酸セフォゾプラン 337 塩酸フルラゼパム 489 エンゴサク 904 塩酸セフォチアム 335 塩酸ブレオマイシン 283, 1396 塩酸 514 塩酸セフォチアムヘキセチル 335, 塩酸プロカイン 710 塩酸アクラルビシン 224, 1379 1417 塩酸プロカインアミド 708 塩酸アセブトロール 219 塩酸セフカペン ピボキシル 322 塩酸プロカインアミド錠 709 塩酸アヘンアルカロイド 988, 1555, 塩酸セフメノキシム 330, 1410 塩酸プロカインアミド注射液 709 1767 塩酸ダウノルビシン 1445 塩酸プロカイン注射液 710, 1514 塩酸アヘンアルカロイド注射液 989 塩酸タランピシリン 782, 1529 塩酸プロカテロール 712 塩酸アマンタジン 235 塩酸チアミン 793, 1532, 1740 塩酸プロカルバジン 711, 1515 塩酸アミトリプチリン 240 塩酸チアミン散 794 塩酸プロプラノロール 717 塩酸アミトリプチリン錠 240 塩酸チアミン注射液 794 塩酸ブロムヘキシン 285 塩酸アルプレノロール 231 塩酸チアラミド 800 塩酸プロメタジン 716 塩酸アロチノロール 249 塩酸チアラミド錠 1742 塩酸ペチジン 678, 1505 l-塩酸イソプレナリン 555 塩酸チオリダジン 798 塩酸ペチジン注射液 679 塩酸イダルビシン 527, 1475 塩酸チクロピジン 802 塩酸ベラパミル 839 塩酸イミプラミン 530 塩酸チザニジン 1744 塩酸ベンセラジド 265 塩酸イミプラミン錠 531 塩酸ツロブテロール 831 塩酸ホモクロルシクリジン 512, Supplement II, JP XIV Index in Japanese 1859

1472 果糖注射液 498 キ 塩酸マプロチリン 588 カノコソウ 957 塩酸ミノサイクリン 628, 1495 カノコソウ末 958 希塩酸 515 塩酸メキシレチン 623 カフェイン 294 キキョウ 1014 塩酸メクロフェノキサート 589 カプセル 885 キキョウ末 1015 塩酸メタンフェタミン 609 カプトプリル 307 キキョウ流エキス 1014 dl-塩酸メチルエフェドリン 613, 過マンガン酸カリウム 698 キクカ 1547, 1760 1719 b-ガラクトシダーゼ（アスペルギルス） キササゲ 889 dl-塩酸メチルエフェドリン散 10 921 キジツ 948 614, 1720 b-ガラクトシダーゼ（ペニシリウム） キシリトール 844 塩酸メピバカイン 601, 1719 921 キシリトール注射液 845, 1540 塩酸メピバカイン注射液 602 カリジノゲナーゼ 560, 1718 吉草酸ベタメタゾン 277, 1395 塩酸モルヒネ 629, 1497 カリ石ケン 1018 キタサマイシン 565, 1484 塩酸モルヒネ錠 631, 1498 カルナウバロウ 888 牛脂 870 塩酸モルヒネ注射液 630, 1497 カルバゾクロムスルホン酸ナトリウム 吸水軟膏 855 塩酸ラニチジン 1517 308, 1400 キョウカツ 1554 塩酸リモナーデ 939 カルバマゼピン 308 キョウニン 864 塩酸リンコマイシン 576, 1490 カルバミン酸クロルフェネシン 356, キョウニン水 865 塩酸レナンピシリン 1488 1436 希ヨードチンキ 950 エンフルラン 443, 1456 カルビドパ 309, 1401 金チオリンゴ酸ナトリウム 748, カルメロース 885 1736 オ カルメロースカルシウム 886, 1759 カルメロースナトリウム 887 ク オウギ 867 カルモナムナトリウム 314, 1401 オウゴン 1042, 1560 カルモフール 313 グアイフェネシン 505 オウゴン末 1043, 1560, 1775 カロコン 1072 グアヤコールスルホン酸カリウム 黄色ワセリン 1006 カンキョウ 1774 696 オウバク 1007, 1556 カンゾウ 932, 1550 クエン酸 366, 1437 オウバク・タンナルビン・ビスマス散 カンゾウ末 1550 クエン酸ガリウム (67Ga) 注射液 501 1009, 1556 乾燥亜硫酸ナトリウム 1058 クエン酸クロミフェン 373, 1441 オウバク末 1008, 1556 カンゾウエキス 933 クエン酸クロミフェン錠 374 オウレン 901, 1548 乾燥甲状腺 1069 クエン酸ジエチルカルバマジン 408 オウレン末 902, 1548 乾燥酵母 1083 クエン酸ジエチルカルバマジン錠 オキサゾラム 658 乾燥細胞培養痘そうワクチン 1053 409, 1449 オキサプロジン 658 乾燥ジフテリアウマ抗毒素 912 クエン酸ナトリウム 753 オキシトシン 1728 乾燥弱毒生おたふくかぜワクチン クエン酸フェンタニル 473 オキシトシン注射液 665, 1730 981 クエン酸ペントキシベリン 674 オキシドール 662 乾燥弱毒生風しんワクチン 1029 クコシ 1767 オキシメトロン 664 乾燥弱毒生麻しんワクチン 972 クジン 1058 オキセサゼイン 659 乾燥水酸化アルミニウムゲル 233 クジン末 1058 オフロキサシン 1500 乾燥水酸化アルミニウムゲル細粒 苦味重曹水 1054 オリブ油 986 233 苦味チンキ 875 オレンジ油 994 カンゾウ粗エキス 934 クラブラン酸カリウム 696, 1512 オンジ 1015 乾燥組織培養不活化狂犬病ワクチン グラミシジン 1470 オンジ末 1016 1024 クラリスロマイシン 367, 1699 乾燥炭酸ナトリウム 1055 グリシン 931 カ 乾燥痘そうワクチン 1053 グリセオフルビン 505, 1471 乾燥日本脳炎ワクチン 957 グリセリン 504, 1469 カイニン酸 560 乾燥破傷風ウマ抗毒素 1066 グリセリンカリ液 930 カイニン酸・サントニン散 958 乾燥はぶウマ抗毒素 938 クリノフィブラート 369 カオリン 959 乾燥 BCG ワクチン 869 グリベンクラミド 502 カカオ脂 877 乾燥ボツリヌスウマ抗毒素 876 グルコン酸カルシウム 299 加香ヒマシ油 889 カンゾウ末 933 グルコン酸クロルヘキシジン液 353 カゴソウ 1023 乾燥まむしウマ抗毒素 972 グルタチオン 1716 カシュウ 1557 乾燥硫酸アルミニウムカリウム 863 クレオソート 904 ガジュツ 1084 カンテン 859 クレゾール 905 加水ラノリン 962 カンテン末 859 クレゾール水 905 ガスえそウマ抗毒素 923 含糖ペプシン 1030 クレゾール石ケン液 905 ガーゼ 853 d-カンフル 305, 1400 クロキサシリンナトリウム 380, カッコン 1023, 1557 dl-カンフル 306, 1400 1441 過テクネチウム酸ナトリウム (99mTc) 肝油 900, 1548 クロキサゾラム 380 注射液 758 カンレノ酸カリウム 694 クロチアゼパム 378 果糖 497 クロトリマゾール 379 1860 Index in Japanese Supplement II, JP XIV

クロナゼパム 375 ゴマ油 1048 シ クロフィブラート 372 ゴミシ 1035 クロフィブラートカプセル 373 コムギデンプン 1080, 1776 ジアスターゼ 907 クロム酸ナトリウム (51Cr) 注射液 コメデンプン 1028 ジアスターゼ・重曹散 907 752 コリスチンメタンスルホン酸ナトリウ ジアゼパム 403 クロモグリク酸ナトリウム 754 ム 385 シアナミド 387 クロラムフェニコール 351, 1432 コルヒチン 384, 1699 シアノコバラミン 388 クロルジアゼポキシド 351 コレカルシフェロール 363 シアノコバラミン注射液 389 クロルジアゼポキシド散 351, 1434 コレステロール 896 ジオウ 1025 クロルジアゼポキシド錠 352, 1435 コレラワクチン 896 歯科用アンチホルミン 864 クロルフェニラミン・カルシウム散 コロンボ 881 歯科用トリオジンクパスタ 1072 895 コロンボ末 881 歯科用パラホルムパスタ 1001 クロルプロパミド 362 コンズランゴ 900 歯科用フェノール・カンフル 1011 クロルプロパミド錠 362 コンズランゴ流エキス 901 歯科用ヨード・グリセリン 951 クロロブタノール 894 ジギタリス 908 サ ジギタリス末 910 ケ ジギトキシン 410 サイクロセリン 390 ジギトキシン錠 411 ケイガイ 1035 サイコ 876 シクラシリン 364, 1437 経口生ポリオワクチン 1015 サイシン 867, 1545 ジクロキサシリンナトリウム 408 ケイ酸マグネシウム 584 酢酸 856 シクロスポリン 364, 1697 軽質無水ケイ酸 1050 酢酸グアナベンズ 506 ジクロフェナクナトリウム 405 軽質流動パラフィン 1001 酢酸クロルマジノン 355, 1435 ジクロフェナミド 406, 1448 ケイヒ 896, 1547 酢酸コルチゾン 386 ジクロフェナミド錠 407 ケイヒ末 897, 1547 酢酸トコフェロール 808, 1535 シクロホスファミド 390 ケイヒ油 897 酢酸ナトリウム 1053 ジゴキシン 414, 1702 結晶性インスリン亜鉛水性懸濁注射液 酢酸ヒドロキソコバラミン 523 ジゴキシン錠 416, 1704 543 酢酸ヒドロコルチゾン 517, 1474 ジゴキシン注射液 415, 1703 結晶セルロース 889 酢酸フタル酸セルロース 893, 1760 ジコッピ 1767 血清性性腺刺激ホルモン 936, 1765 酢酸プレドニゾロン 703, 1513 シコン 964 ケツメイシ 888 酢酸ミデカマイシン 627 次硝酸ビスマス 282 ケトプロフェン 564 酢酸メテノロン 607 シスプラチン 1697 ケンゴシ 1007 酢酸レチノール 732, 1519 ジスルフィラム 430 ゲンチアナ 925 サッカリンナトリウム 1030 ジソピラミド 429 ゲンチアナ・重曹散 926 サフラン 1031 シタラビン 392, 1701 ゲンチアナ末 925 サラシ粉 894 シッカニン 1523 ゲンノショウコ 926 サラシミツロウ 871 シツリシ 1775 ゲンノショウコ末 926 サラゾスルファピリジン 739 ジドロゲステロン 437 サリチル・ミョウバン散 1032 ジドロゲステロン錠 438 コ サリチル酸 741 ジノスタチン スチマラマー 846 サリチル酸精 1033 ジノプロスト 425 コウカ 1031 サリチル酸ナトリウム 761 ジピリダモール 428, 1451 硬化油 940 サリチル酸絆創膏 1032 ジフェンヒドラミン 426 コウジン 1024 サリチル酸メチル 618 ジフェンヒドラミン・フェノール・亜鉛 合成ケイ酸アルミニウム 235 酸化亜鉛 845 華リニメント 912 コウブシ 906 酸化カルシウム 878 ジフェンヒドラミン・ワレリル尿素散 コウブシ末 906 酸化チタン 1070 911 コウボク 970, 1552 酸化マグネシウム 583 ジフテリアトキソイド 912 コウボク末 970, 1552 サンキライ 1053 ジフテリア破傷風混合トキソイド ゴオウ 995 サンキライ末 1053 913 ゴシツ 857 三酸化ヒ素 250 ジプロピオン酸ベタメタゾン 275 ゴシュユ 918, 1548 サンシシ 922, 1549 シメチジン 365 コハク酸クロラムフェニコールナトリ サンシシ末 923, 1763 ジメルカプロール 423 ウム 1434 サンシュユ 903, 1762 ジメルカプロール注射液 424 コハク酸トコフェロールカルシウム サンショウ 1083 ジメンヒドリナート 422 809 サンショウ末 1084 ジメンヒドリナート錠 423 コハク酸ヒドロコルチゾン 521 酸素 663 次没食子酸ビスマス 281 コハク酸ヒドロコルチゾンナトリウム サントニン 742 ジモルホラミン 424, 1705 520 サントニン錠 742 ジモルホラミン注射液 425, 1705 コハク酸プレドニゾロン 704 サンヤク 911 弱アヘンアルカロイド・スコポラミン コハク酸メチルプレドニゾロン サンヤク末 911 注射液 992 1720 シャクヤク 1003, 1555 ゴボウシ 1546 シャクヤク末 1004, 1556 Supplement II, JP XIV Index in Japanese 1861

ジャショウシ 1760 ステアリン酸ポリオキシル 40 1016 セフジニル 324 シャゼンシ 1014 ステアリン酸マグネシウム 969 セフスロジンナトリウム 340 シャゼンソウ 1014 スピロノラクトン 765 セフタジジム 342 臭化イプラトロピウム 550 スルチアム 779 セフチゾキシムナトリウム 345 臭化カリウム 694 スルバクタムナトリウム 768 セフチブテン 344, 1426 臭化ジスチグミン 429 スルピリド 776 セフテラムピボキシル 344, 1425 臭化ジスチグミン錠 430 スルピリン 776 セフトリアキソンナトリウム 346, 臭化水素酸スコポラミン 744 スルピリン注射液 777 1427 臭化水素酸デキストロメトルファン スルファジアジン銀 769 セフピラミドナトリウム 338, 1421 402 スルファメチゾール 770 セフブペラゾンナトリウム 322, 臭化水素酸ホマトロピン 511 スルファメトキサゾール 770 1409 臭化チメピジウム 803 スルファモノメトキシン 771 セフポドキシムプロキセチル 1422 臭化ナトリウム 751 スルフイソキサゾール 773 セフミノクスナトリウム 332 臭化パンクロニウム 667 スルフィンピラゾン 772 セフメタゾールナトリウム 331 臭化ピリドスチグミン 724 スルフィンピラゾン錠 773 セフラジン 339 臭化ブチルスコポラミン 743, 1522 スルベニシリンナトリウム 769, セフロキサジン 340, 1424 臭化ブトロピウム 294 1528 セフロキシムアキセチル 347, 1429, 臭化プロパンテリン 716 スルホブロモフタレインナトリウム 1696 臭化メチルベナクチジウム 611 774 セフロキシムナトリウム 348, 1696 臭化メペンゾラート 599 スルホブロモフタレインナトリウム注 ゼラチン 923 ジュウヤク 939 射液 775 セラペプターゼ 1735 シュクシャ 863 センキュウ 899 シュクシャ末 863, 1768 セ センキュウ末 899 酒石酸 1066 センコツ 983 酒石酸アリメマジン 229 成人用沈降ジフテリアトキソイド センソ 1071, 1563 酒石酸イフェンプロジル 529 912 センナ 1044, 1560 酒石酸エルゴタミン 452 精製水 1079 センナ末 1046, 1561 酒石酸キタサマイシン 1485 精製ゼラチン 924 センブリ 1063, 1562 酒石酸プロチレリン 721 精製セラック 1049 センブリ・重曹散 1065, 1563 酒石酸レバロルファン 570 精製脱脂綿 851 センブリ末 1064, 1562 酒石酸レバロルファン注射液 570 精製デヒドロコール酸 395 ショウキョウ 927, 1763 精製白糖 1060 ソ ショウキョウ末 927, 1764 精製ラノリン 963 硝酸イソソルビド 558 生理食塩液 752 ソウジュツ 869 硝酸イソソルビド錠 558 石油ベンジン 1006 ソウジュツ末 869 硝酸銀 746 セクレチン 745 ソウハクヒ 981, 1554 硝酸銀点眼液 1041 セスキオレイン酸ソルビタン 1058 ソボク 1774 硝酸チアミン 795, 1532 セタノール 894 ソヨウ 1004 硝酸ナファゾリン 636 セッコウ 937 硝酸ミコナゾール 625 セネガ 1043 タ 常水 1076 セネガシロップ 1044 ショウズク 885 セネガ末 1044 ダイオウ 1026, 1558, 1774 消毒用エタノール 916 セファクロル 314, 1403 ダイオウ末 1026, 1558, 1774 消毒用フェノール 1010 セファゾリンナトリウム 319 ダイズ油 1059 消毒用フェノール水 1012 セファゾリンナトリウム水和物 319 タイソウ 958 ショウマ 896 セファトリジンプロピレングリコール 胎盤性性腺刺激ホルモン 934, 1764 ジョサマイシン 559, 1478 318 タクシャ 860 シンイ 1552 セファドロキシル 314 タクシャ末 860 親水軟膏 940 セファピリンナトリウム 317 脱脂綿 851 親水ワセリン 1005 セファマンドールナトリウム 317, タルク 1065 診断用クエン酸ナトリウム液 754 1407 炭酸カリウム 1018 シンフィブラート 746, 1524 セファレキシン 315 炭酸水素ナトリウム 749 セファロチンナトリウム 317, 1406 炭酸水素ナトリウム注射液 750, ス セファロリジン 317, 1405 1524 セフィキシム 329 炭酸ナトリウム 1055 水酸化カリウム 1018 セフォキシチンナトリウム 336, 炭酸マグネシウム 582 水酸化カルシウム 877 1419 炭酸リチウム 578 水酸化ナトリウム 1056 セフォジジムナトリウム 1412 単シロップ 1052 スクラルファート 766 セフォタキシムナトリウム 335, ダントロレンナトリウム 392 ステアリルアルコール 1059 1413 単軟膏 1052 ステアリン酸 1059 セフォテタン 335, 1415 タンニン酸 782 ステアリン酸エリスロマイシン 454 セフォペラゾンナトリウム 332 タンニン酸アルブミン 227 ステアリン酸カルシウム 881 セフジトレン ピボキシル 325 タンニン酸ジフェンヒドラミン 427 1862 Index in Japanese Supplement II, JP XIV

タンニン酸ベルベリン 272 ツ トラピジル 818 トリアムシノロン 819, 1535 チ ツバキ油 882 トリアムシノロンアセトニド 820 トリアムテレン 821 チアマゾール 791 テ トリクロホスナトリウム 823 チアマゾール錠 792 トリクロホスナトリウムシロップ チアミラールナトリウム 796, 1741 テイコプラニン 783, 1530 824 チアントール 1067 D-ソルビトール 763 トリクロルメチアジド 822, 1748 チオテパ 799 D-ソルビトール液 764 トリクロルメチアジド錠 1749 チオペンタールナトリウム 797, D-マンニトール 587 トリコマイシン 823, 1536 1532 D-マンニトール注射液 588 トリメタジオン 827 チオ硫酸ナトリウム 762, 1738 低置換度ヒドロキシプロピルセルロー トリメタジオン錠 827 チオ硫酸ナトリウム注射液 762 ス 942 トルナフタート 815 チカルシリンナトリウム 801 テオフィリン 791, 1531 トルナフタート液 816 チクセツニンジン 998 テガフール 782, 1530, 1738 トルブタミド 814 チクセツニンジン末 998, 1555 デキサメタゾン 397 トルブタミド錠 814 窒素 982 デキストラン硫酸ナトリウム イオウ トレピブトン 819 チニダゾール 804, 1743 5 401 トロピカミド 828 チモ 864 デキストラン硫酸ナトリウム イオウ ドロペリドール 435, 1456 チモール 1069 18 402 トロンビン 1068 注射用アモバルビタールナトリウム デキストラン 40 398 豚脂 964 242 デキストラン 40 注射液 399 注射用塩化アセチルコリン 222 デキストラン 70 400 ナ 注射用塩化スキサメトニウム 781 デキストリン 906 注射用塩酸セフェピム 1695 デスラノシド 396 ナイスタチン 656 注射用塩酸ヒドララジン 513 デスラノシド注射液 397 ナタネ油 1024 注射用血清性性腺刺激ホルモン 937, デヒドロコール酸 394 ナドロール 633 1765 デヒドロコール酸注射液 396 ナファゾリン・クロルフェニラミン液 注射用コハク酸プレドニゾロンナトリ テレビン油 1073 981 ウム 705, 1513 天然ケイ酸アルミニウム 233 ナプロキセン 636 注射用水 1078 テンマ 1550 ナリジクス酸 634 注射用胎盤性性腺刺激ホルモン 936, テンモンドウ 1546 1764 ニ 注射用チアミラールナトリウム 796, ト 1742 ニガキ 1013 注射用チオペンタールナトリウム トウガラシ 882, 1546 ニガキ末 1013 798 トウガラシ末 883, 1546 ニコチン酸 644 注射用ファモチジン 470, 1464 トウガラシ・サリチル酸精 884 ニコチン酸アミド 643 注射用フェニトインナトリウム 686 トウガラシチンキ 883 ニコチン酸注射液 645 注射用フロモキセフナトリウム トウキ 956 ニコチン酸トコフェロール 810 1711 トウキ末 956 ニコモール 642 注射用硫酸ビンブラスチン 841, トウニン 1002 ニコモール錠 643 1753 トウニン末 1002 ニコランジル 1724 チョウジ 898 トウヒ 875 二酸化炭素 312 チョウジ末 898 トウヒシロップ 994 ニセリトロール 641 チョウジ油 898 トウヒチンキ 995 ニトラゼパム 646 チョウトウコウ 1563, 1776 トウモロコシデンプン 903, 1761 ニトログリセリン錠 647 チョレイ 1061 トウモロコシ油 903 ニフェジピン 645 チョレイ末 1061 トコフェロール 807, 1534 日本脳炎ワクチン 956 チンク油 1085 トコン 953 乳酸 959 沈降ジフテリア破傷風混合トキソイド トコンシロップ 955 乳酸カルシウム 300 913 トコン末 954 乳糖 960 沈降精製百日せきジフテリア破傷風混 トシル酸スルタミシリン 778 尿素 835 合ワクチン 1005 トチュウ 1548 ニルバジピン 1725 沈降精製百日せきワクチン 1005 トフィソパム 812 ニルバジピン錠 1726 沈降炭酸カルシウム 297 トブラマイシン 807, 1533 ニンジン 927 沈降破傷風トキソイド 1066 トラガント 1072 ニンジン末 929 沈降はぶトキソイド 938 トラガント末 1072 ニンドウ 1766 沈降 B 型肝炎ワクチン 938 トラザミド 813 チンピ 898 トラネキサム酸 817, 1745 ノ トラネキサム酸カプセル 1746 トラネキサム酸錠 1747 濃塩化ベンザルコニウム液 50 267 トラネキサム酸注射液 1747 濃グリセリン 504, 1469 Supplement II, JP XIV Index in Japanese 1863

ノスカピン 654 人全血液 1080 複方オキシコドン・アトロピン注射液 ノルエチステロン 650 人免疫グロブリン 949 996 ノルエピネフリン 649, 1498 ヒドロキシプロピルセルロース 940 複方オキシコドン注射液 995 ノルエピネフリン注射液 650, 1499 ヒドロキシプロピルメチルセルロース 複方サリチル酸精 1033 ノルゲストレル 652 フタレート 947 複方サリチル酸メチル精 977 ノルゲストレル・エチニルエストラジ ヒドロキシプロピルメチルセルロース 複方ジアスターゼ・重曹散 907 オール錠 652, 1499 2208 943 複方ダイオウ・センナ散 1028 ノルフロキサシン 651 ヒドロキシプロピルメチルセルロース 複方チアントール・サリチル酸液 2906 944 1067 ハ ヒドロキシプロピルメチルセルロース 複方ビタミン B 散 1075 2910 945 複方ヨード・グリセリン 950 バイモ 1549 ヒドロクロロチアジド 515, 1473 複方ロートエキス・ジアスターゼ散 白色セラック 1049 ヒドロコルチゾン 516 1037 白色軟膏 1080 ヒドロコルチゾン・ジフェンヒドラミ 複方ロートエキス・タンニン坐剤 白色ワセリン 1005 ン軟膏 939 1041, 1560 白糖 1061 ピペミド酸三水和物 689, 1508 複方ロートエキス・タンニン軟膏 バクモンドウ 987 ピペラシリンナトリウム 690, 1508 1039, 1560 バクロフェン 260 ヒベンズ酸チペピジン 804, 1533 ブクリョウ 1017 バクロフェン錠 261 ヒベンズ酸チペピジン錠 806 ブクリョウ末 1017 バシトラシン 1390 ビホナゾール 278 ブシ 1770 バソプレシン注射液 837, 1751 ヒマシ油 888 ブシ末 1772 ハチミツ 938 ピマリシン 1507 フシジン酸ナトリウム 1525 ハッカ 974 ヒメクロモン 525 ブスルファン 293 ハッカ水 975 ビャクシ 864 ブドウ酒 1081 ハッカ油 975 ビャクジュツ 868 ブドウ糖 503 パップ用複方オウバク散 1009, 1556 ビャクジュツ末 868 ブドウ糖注射液 503 パニペネム 667 氷酢酸 856 ブフェキサマク 289 ハマボウフウ 930 ピラジナミド 723, 1515, 1734 ブフェキサマク軟膏 290 パモ酸ヒドロキシジン 524 ピラルビシン 1509 ブフェキサマク乳剤性軟膏 289 パモ酸ピランテル 722 ピレノキシン 693, 1731 フマル酸クレマスチン 368 パラアミノサリチル酸カルシウム ピロ亜硫酸ナトリウム 1057 フマル酸ケトチフェン 1483 301 ピロキシカム 1733 フマル酸ホルモテロール 492 パラアミノサリチル酸カルシウム顆粒 ピロキシリン 1023 ブメタニド 291 302 ピロールニトリン 1516 プラステロン硫酸ナトリウム 760 パラオキシ安息香酸エチル 916 ビワヨウ 1552 プラゼパム 700 パラオキシ安息香酸ブチル 876 ピンドロール 688 プラゼパム錠 701 パラオキシ安息香酸プロピル 1021 ビンロウジ 866, 1545 プラノプロフェン 699 パラオキシ安息香酸メチル 976 フラビンアデニンジヌクレオチドナト パラフィン 999 フ リウム 475, 1710 パラホルムアルデヒド 671 プリミドン 706, 1514 バルビタール 263 ファノバリン・マグネシア散 1557 フルオキシメステロン 486 バルプロ酸ナトリウム 762 ファモチジン 469 フルオシノニド 482 パルミチン酸クロラムフェニコール ファモチジン散 469 フルオシノロンアセトニド 481 1432 ファモチジン錠 471 フルオレセインナトリウム 483 パルミチン酸レチノール 733, 1519 ファロペネムナトリウム 472 フルオロウラシル 485 バレイショデンプン 1019, 1769 フィトナジオン 687, 1507 フルオロメトロン 484 ハロキサゾラム 509, 1472 フェナセチン 680 フルジアゼパム 479 ハロタン 508 フェニトイン 685 フルシトシン 478 ハロペリドール 507 フェニトイン散 686 フルニトラゼパム 480 パンクレアチン 999 フェニトイン錠 686 フルラゼパム 487 ハンゲ 1013 フェニルブタゾン 684 フルラゼパムカプセル 488 絆創膏 858 フェネチシリンカリウム 1505 フルルビプロフェン 489, 1466 パンテチン 669 フェノバリン・マグネシア散 1012 プレドニゾロン 701 パントテン酸カルシウム 301 フェノバルビタール 681 プレドニゾロン錠 702 フェノバルビタール散 10 681 フロクタフェニン 477, 1465 ヒ フェノール 1010 プログルミド 715 フェノール・亜鉛華リニメント 1011 プロゲステロン 714 ピコスルファートナトリウム 758 フェノール水 1012 プロゲステロン注射液 714 ビサコジル 280 フェノールスルホンフタレイン 682 フロセミド 498, 1714 ビサコジル坐剤 280, 1396 フェノールスルホンフタレイン注射液 フロセミド錠 1715 ビタミン A 油 1074, 1564 682 プロタミンインスリン亜鉛水性懸濁注 ビタミン A 油カプセル 1075, 1565 フェンブフェン 473 射液 544 ヒトインスリン（遺伝子組換え） 537 複方アクリノール・チンク油 857 プロチオナミド 720 1864 Index in Japanese Supplement II, JP XIV

プロチレリン 720 ポビドンヨード 699, 1513 ミコナゾール 624 プロテイン銀 1051 ホミカ 983 ミツロウ 871 プロテイン銀液 1051 ホミカエキス 984 ミデカマイシン 626 プロピオン酸ジョサマイシン 559, ホミカエキス散 984 ミョウバン水 861 1480 ホミカチンキ 985 プロピオン酸テストステロン 788, ポリスチレンスルホン酸カルシウム ム 1738 303, 1399 プロピオン酸テストステロン注射液 ポリスチレンスルホン酸ナトリウム 無晶性インスリン亜鉛水性懸濁注射液 788, 1739 759, 1737 542 プロピオン酸ドロスタノロン 435, ポリソルベート 80 1017 無水アンピシリン 246, 1384 1456 ホリナートカルシウム 299 無水エタノール 915 プロピオン酸ドロスタノロン注射液 ホルマリン 919 無水カフェイン 295 436, 1456 ホルマリン水 920 無水クエン酸 367, 1438, 1698 プロピオン酸ベクロメタゾン 264 ボレイ 997 無水乳糖 961 プロピルチオウラシル 718 ボレイ末 998 無水リン酸水素カルシウム 879 プロピルチオウラシル錠 718 ムピロシンカルシウム 水和物 631 プロピレングリコール 1022 マ フロプロピオン 478 メ フロプロピオンカプセル 1712 マイトマイシン C 629, 1496 プロベネシド 706 マオウ 914 メキタジン 602 プロベネシド錠 707 マーキュロクロム 604 メグルミン 974 ブロマゼパム 284 マーキュロクロム液 605 メコバラミン 590 ブロムワレリル尿素 287 マクリ 908 メシル酸ガベキサート 500 フロモキセフナトリウム 478, 1465 マクロゴール軟膏 968 メシル酸カモスタット 304 粉末セルロース 892 マクロゴール 400 965 メシル酸ジヒドロエルゴタミン 418 マクロゴール 1500 966 メシル酸ジヒドロエルゴトキシン ヘ マクロゴール 4000 967 413, 1450 マクロゴール 6000 967 メシル酸デフェロキサミン 393, ベタメタゾン 274 マクロゴール 20000 968 1701 ヘパリンナトリウム 510, 1717 マシニン 1551 メシル酸ブロモクリプチン 286 ヘパリンナトリウム注射液 511, 麻酔用エーテル 462 メシル酸ベタヒスチン 272 1718 マルトース 586 メストラノール 606 ベラドンナエキス 871 マレイン酸エルゴメトリン 450 メダゼパム 591 ベラドンナコン 872 マレイン酸エルゴメトリン錠 451 メチクラン 619, 1493 ペルフェナジン 675 マレイン酸エルゴメトリン注射液 メチラポン 623 ペルフェナジン錠 676 451 メチルジゴキシン 620 ベンジルアルコール 874, 1758 マレイン酸クロルフェニラミン 357 メチルセルロース 978 ベンジルペニシリンカリウム 270, d-マレイン酸クロルフェニラミン メチルテストステロン 618, 1722 1393 359 メチルテストステロン錠 619, 1722 ベンジルペニシリンベンザチン マレイン酸クロルフェニラミン散 メチルドパ 612 1392, 1694 358 メチルドパ錠 613 ベンズブロマロン 268, 1391 マレイン酸クロルフェニラミン錠 メチルプレドニゾロン 616 ペンタゾシン 672 359 メチル硫酸ネオスチグミン 637 ベントナイト 873 マレイン酸クロルフェニラミン注射液 メチル硫酸ネオスチグミン注射液 ペントバルビタールカルシウム 673 357 638 マレイン酸トリメブチン 1537 滅菌ガーゼ 854 ホ マレイン酸プロクロルペラジン 713 滅菌精製水 1079 マレイン酸プロクロルペラジン錠 滅菌精製脱脂綿 852 ボウイ 1052, 1562 713 滅菌脱脂綿 852 ボウコン 949, 1551 マレイン酸ペルフェナジン 677 メトキサレン 611 ホウ砂 750 マレイン酸ペルフェナジン錠 678 メトクロプラミド 621 ホウ酸 284 マレイン酸メチルエルゴメトリン メトクロプラミド錠 1723 抱水クロラール 350 615 メトトレキサート 610, 1493 ボウフウ 1034 マレイン酸メチルエルゴメトリン錠 メトロニダゾール 622 ホスフェストロール 493 615 メナテトレノン 598, 1492 ホスフェストロール錠 494 マレイン酸レボメプロマジン 572 メピチオスタン 600, 1492 ホスホマイシンカルシウム 494, D-マンニトール 587, 1718 メフェナム酸 592 1713 D-マンニトール 587, 1718 メフルシド 592 ホスホマイシンナトリウム 496, D-マンニトール注射液 588, 1492 メフルシド錠 593 1713 メルカプトプリン 603 ボタンピ 979 ミ メルファラン 597 ボタンピ末 980 メロペネム 三水和物 605, 1719 ポビドン 1020 ミグレニン 627, 1495 dl-メントール 975 Supplement II, JP XIV Index in Japanese 1865 l-メントール 976 ラクトビオン酸エリスロマイシン リュウタン 957 1462 リュウタン末 957, 1551 モ ラタモキセフナトリウム 569, 1486 流動パラフィン 1000 ラッカセイ油 1002 リョウキョウ 1544 モクツウ 859 ラナトシド C 567 リンゲル液 1028 モッコウ 1034 ラナトシド C 錠 567 リン酸クリンダマイシン 369, 1440 モノステアリン酸アルミニウム 861 リン酸コデイン 382 モノステアリン酸グリセリン 931 リ リン酸コデイン散 1 382, 1442 モルヒネ・アトロピン注射液 978, リン酸コデイン散 10 383, 1442 1553 リオチロニンナトリウム 576 リン酸コデイン錠 384, 1443 リオチロニンナトリウム錠 577, リン酸ジヒドロコデイン 417 ヤ 1490 リン酸ジヒドロコデイン散 1 417, リドカイン 574 1449 焼セッコウ 938 リドカイン注射液 575 リン酸ジヒドロコデイン散 10 ヤクチ 875 リファンピシン 737, 1521 418, 1450 薬用石ケン 973 リボフラビン 733 リン酸ジメモルファン 421 薬用炭 972 リボフラビン散 734 リン酸水素カルシウム 878 ヤシ油 899 リュウコツ 965 リン酸水素ナトリウム 907 硫酸亜鉛 846 リン酸二水素カルシウム 880 ユ 硫酸亜鉛点眼液 1086 リン酸ヒドロコルチゾンナトリウム 硫酸アストロマイシン 255, 1387 519, 1474 ユウタン 869 硫酸アトロピン 255 リン酸ピペラジン 692 ユーカリ油 917 硫酸アトロピン注射液 256, 1689 リン酸ピペラジン錠 692 輸血用クエン酸ナトリウム注射液 硫酸アミカシン 238 リン酸ベタメタゾンナトリウム 276, 753 硫酸アルベカシン 247, 1386 1395 ユビデカレノン 832 硫酸アルミニウムカリウム 862 リン酸リボフラビンナトリウム 735 硫酸イセパマイシン 552 リン酸リボフラビンナトリウム注射液 ヨ 硫酸エンビオマイシン 1457 736 硫酸オルシプレナリン 656 ヨウ化エコチオパート 438 硫酸カナマイシン 563, 1482 レ ヨウ化オキサピウム 657 一硫酸カナマイシン 1481 ヨウ化カリウム 697 硫酸カリウム 1019 レセルピン 729, 1518 ヨウ化人血清アルブミン (131I) 注射液 硫酸キニジン 725 レセルピン散 0.1 731 546 硫酸キニーネ 728 レセルピン錠 731 ヨウ化ナトリウム 755 硫酸グアネチジン 507 レセルピン注射液 730 ヨウ化ナトリウム (123I) カプセル 硫酸ゲンタマイシン 502, 1468 レボチロキシンナトリウム 572 755 硫酸コリスチン 1444 レボチロキシンナトリウム錠 573 ヨウ化ナトリウム (131I) 液 756 硫酸サルブタモール 740 レボドパ 571 ヨウ化ナトリウム (131I) カプセル 硫酸シソマイシン 747, 1524 レンギョウ 920 755 硫酸ジベカシン 404, 1447 ヨウ化ヒプル酸ナトリウム (131I) 注射 硫酸ストレプトマイシン 766, 1527 ロ 液 756 硫酸セフォセリス 334 葉酸 490, 1466 硫酸セフピロム 338 ロキシスロマイシン 738, 1522 葉酸錠 491 硫酸鉄 474 ロキソプロフェンナトリウム 580 葉酸注射液 491 硫酸テルブタリン 786 ロキタマイシン 737 ヨウ素 546 硫酸ネチルマイシン 630 ロジン 1029 ヨクイニン 900 硫酸バメタン 262 ロートエキス 1035 ヨクイニン末 900 硫酸バリウム 264 ロートエキス・アネスタミン散 1037, ヨーダミド 545 硫酸ビンクリスチン 842 1559 ヨーダミドナトリウムメグルミン注射 硫酸ビンブラスチン 840, 1752 ロートエキス・カーボン散 1037 液 597 硫酸フラジオマイシン 497, 1467 ロートエキス・タンニン坐剤 1039 ヨード・サリチル酸・フェノール精 硫酸ブレオマイシン 283, 1398 ロートエキス・パパベリン・アネスタミ 952 硫酸プロタミン 719 ン散 1038 ヨードチンキ 949 硫酸プロタミン注射液 719 ロートエキス散 1036 ヨードホルム 546 硫酸ベカナマイシン 265, 1390 ロートコン 1040 硫酸ペプロマイシン 675, 1503 ロラゼパム 579 ラ 硫酸ペンブトロール 672 硫酸ポリミキシン B 694, 1511 ワ ラウリル硫酸ナトリウム 1056 硫酸マグネシウム 585 ラウロマクロゴール 964 硫酸マグネシウム水 970 ワイル病秋やみ混合ワクチン 1080 酪酸ヒドロコルチゾン 518 硫酸マグネシウム注射液 586, 1492 ワルファリンカリウム 843 酪酸リボフラビン 734 硫酸ミクロノマイシン 626, 1494 ワルファリンカリウム錠 843 ラクツロース 566 硫酸リボスタマイシン 737, 1520