Food and Drug Administration, HHS Pt. 436
records after the first 2 years of the Subpart F—Chemical Tests for Specific holding period. Antibiotics 436.300 Polarimetric assay of carbenicillin PART 436—TESTS AND METHODS OF indanyl sodium. ASSAY OF ANTIBIOTIC AND AN- 436.301 Thin layer chromatography identity TIBIOTIC-CONTAINING DRUGS test for carbenicillin indanyl. 436.302 Clindamycin vapor phase chroma- tography. Sec. 436.303 Clindamycin content of clindamycin palmitate hydrochloride by vapor phase Subpart A—Definitions; Interpretations; chromatography. Requirements 436.304 Clindamycin phosphate vapor phase chromatography. 436.1 Sterility requirements of items pack- 436.305 Thin layer chromatographic identity aged with sterile antibiotic drugs. test for hetacillin. 436.2 Alternative assay methods. 436.306 Lincomycin gas liquid chroma- tography. Subpart B—Sterility Test Methods 436.307 Spectinomycin vapor phase chroma- tography. 436.20 Sterility test methods and proce- 436.308 Paper chromatography identity test dures. for tetracyclines. 436.309 Anhydrotetracyclines and 4-epian- Subpart C—Biological Test Methods hydrotetracycline. 436.310 Thin layer chromatography identity 436.31 Equipment and diluents for use in bi- test for mitomycin. ological testing. 436.311 Thin layer chromatography identity 436.32 Pyrogen test. test for amoxicillin. 436.35 Depressor substances test. 436.312 Atomic absorption method for deter- mining the zinc content of zinc baci- Subpart D—Microbiological Assay tracin. 436.316 Determination of penicillin G con- Methods tent. 436.100 Laboratory equipment. 436.317 Solubility characteristic test for griseofulvin (ultramicrosize) tablets. 436.101 Solutions. 436.318 Continuous flow thin layer chroma- 436.102 Culture media. tography identity test. 436.103 Test organisms. 436.319 Thin layer chromatography identity 436.104 Penicillin activity. test for bacitracin and bacitracin zinc. 436.105 Microbiological agar diffusion assay. 436.320 Ferric chloride colorimetric assay. 436.106 Microbiological turbidimetric assay. 436.321 Griseofulvin gas liquid chroma- tography. Subpart E—General Chemical Tests for 436.322 High–pressure liquid Antibiotics chromatographic assay for anthracycline antibiotics. 436.200 Loss on drying. 436.323 Continuous flow thin layer chroma- 436.201 Moisture determination. tography identity test for cefamandole 436.202 pH. nafate. 436.203 Crystallinity. 436.324 Polarographic analysis of 436.204 Iodometric assay. cefamandole. 436.205 Hydroxylamine colorimetric assay. 436.325 High pressure liquid chroma- 436.206 Test for metal particles in ophthal- tography assay for vidarabine. mic ointments. 436.326 Thin layer chromatographic identity 436.207 Residue on ignition. test for cefoxitin sodium. 436.327 Thin layer chromatographic identity 436.208 Heavy metals determination. test for cyclacillin. 436.209 Melting range or temperature. 436.328 High pressure liquid 436.210 Specific rotation. chromatographic assay for sulfisoxazole 436.211 Identity tests by infrared acetyl content. spectrophotometry. 436.329 High-pressure liquid 436.212 Disintegration test. chromatographic assay for meclocycline. 436.213 Nonaqueous titrations. 436.330 Thin layer chromatographic identity 436.214 Heat stability. test for bacampicillin. 436.215 Dissolution test. 436.331 High-pressure liquid 436.216 High–performance liquid chromatographic assay for chromatographic assay. dactinomycin. 436.217 Film-coat rupture test. 436.332 High-pressure liquid
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chromatographic assay for moxalactam. free erythromycin content in erythro- 436.333 Thin layer chromatographic identity mycin estolate bulk. test for moxalactam. 436.363 High-performance liquid 436.334 High-pressure liquid chromatographic assay for cefmenoxime. chromatographic assay for piperacillin. 436.364 Atomic absorption test for sodium 436.335 High-pressure liquid carbonate content of cefmenoxime hy- chromatographic assay for chlor- drochloride for injection. amphenicol palmitate. 436.365 Thin layer chromatographic identity 436.336 Thin layer chromatographic identity test for rifampin. test for azlocillin. 436.366 High-performance liquid chroma- 436.337 High-pressure liquid tography assay for determining chromatographic assay for cephradine. chromatographic purity of vancomycin. 436.338 High-pressure liquid 436.367 Thin-layer chromatographic identity chromatographic assay for cefoperazone. test for cephalexin hydrochloride. 436.339 High-pressure liquid 436.368 Thin layer chromatographic identity chromatographic assay for bleomycin test for cefprozil. fractions. 436.369 Thin layer chromatography test for 436.340 High-pressure liquid free N-isobutylpiperidone content in chromatographic assay for tetracycline rifabutin. hydrochloride content and 4– 436.370 Spectrophotometric identity test for epitetracycline hydrochloride content. rifabutin capsules. 436.341 High-pressure liquid chromatographic assay for plicamycin. Subpart G—Chemical Tests for 436.342 High-pressure liquid Nonantibiotic Active Ingredients chromatographic assay for cefazolin. 436.343 High-pressure liquid 436.400 Thin layer chromatographic identity chromatographic assay for cefuroxime. test for iodochlorhydroxyquin. 436.344 Thin layer chromatographic identity test for cefuroxime. Subpart H—Tests for Specific Antibiotic 436.345 High-pressure liquid Dosage Forms chromatographic assay for ceftizoxime. 436.346 High-pressure liquid 436.500 Penicillin in oil and wax. chromatographic assay for cyclosporine. 436.503 Procaine penicillin and buffered 436.347 High-pressure liquid crystalline penicillin for aqueous injec- chromatographic assay for cefoxitin. tion. 436.348 High-pressure liquid 436.504 Penicillin-bacitracin ointment. chromatographic assay for ceforanide. 436.505 Penicillin-streptomycin-bacitracin 436.349 High-pressure liquid ointment; penicillin-dihydrostrepto- chromatographic assay for L-lysine in mycin-bacitracin ointment; penicillin- ceforanide for injection. streptomycin-bacitracin methylene disa- 436.350 High-performance liquid licylate ointment; penicillin-dihydro- chromatographic assay for cefonicid. streptomycin-bacitracin methylene disa- 436.351 High-performance liquid licylate ointment. chromatographic assay for amoxicillin 436.506 Benzathine penicillin G and buffered and clavulanic acid. crystalline penicillin for aqueous injec- 436.352 High-performance liquid tion. chromatographic assay for determining 436.507 Benzathine-procaine-buffered crys- clavam-2-carboxylate content in talline penicillins for aqueous injection. clavulanate potassium. 436.508 Penicillin - bacitracin - neomycin 436.353 High-performance liquid ointment; penicillin-bacitracin-neomycin chromatographic assay for amdinocillin. in oil. 436.354 High-performance liquid 436.509 Procaine penicillin-streptomycin- chromatographic assay for ceftriaxone. polymyxin in oil; procaine penicillin-di- 436.355 High–performance liquid hydrostreptomycin-polymyxin in oil; chromatographic assay for ticarcillin– procaine penicillin-streptomycin-poly- clavulanic acid. myxin ointment; procaine penicillin-di- 436.356 High–performance liquid hydrostreptomycin-polymyxin ointment. chromatographic assay for ceftazidime. 436.510 Penicillin - streptomycin - erythro- 436.357 Atomic absorption test for sodium mycin ointment; penicillin-dihydro- carbonate content. streptomycin - erythromycin ointment. 436.358 High–performance liquid 436.511 Penicillin-streptomycin-bacitracin chromatographic assay for pyridine. methylene disalicylate-neomycin oint- 436.360 Gel permeation chromatographic ment; penicillin-dihydrostreptomycin- assay for high molecular weight polymer. bacitracin methylene disalicylate-neo- 436.361 High–performance liquid mycin ointment. chromatographic assay for aztreonam. 436.512 Procaine penicillin G-novobiocin-ne- 436.362 Thin-layer chromatographic test for omycin-dihydrostreptomycin in oil.
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436.513 Chlortetracycline troches; tetra- graphs of this chapter may be used, cycline hydrochloride troches. provided the results obtained are of 436.514 Chlortetracycline hydrochloride equivalent accuracy. However, only the powder topical; tetracycline hydro- chloride powder topical. results obtained from the official 436.515 Capsules tetracycline and methods designated in the individual oleandomycin phosphate; capsules tetra- monographs are conclusive. cycline and troleandomycin; capsules tetracycline hydrochloride and Subpart B—Sterility Test Methods oleandomycin phosphate; capsules tetra- cycline hydrochloride and troleandomycin. § 436.20 Sterility test methods and pro- 436.516 Tetracycline-neomycin complex cedures. powder topical; tetracycline hydro- (a) Laboratory facilities. The test must chloride-neomycin sulfate powder topi- be performed using aseptic techniques cal. in an area as free from contamination 436.517 Bacitracin-neomycin tablets; zinc bacitracin-neomycin tablets; bacitracin as is possible to achieve. Testing methylene disalicylate-neomycin tab- should not be conducted under direct lets. exposure to ultraviolet light or in 436.542 Acid resistance/dissolution test for areas under aerosol treatment. Envi- enteric-coated erythromycin pellets. ronmental tests to assess the suit- 436.543 Acid resistance test for pellet-filled ability of testing conditions should be doxycycline hyclate capsules. made frequently enough to assure the 436.544 Dissolution test for pellet-filled doxycycline hyclate capsules. validity of test results. 436.545 Acid resistance test for erythro- (b) Equipment and reagents—(1) Bac- mycin particles in tablets. terial membrane filter. The filter has a nominal porosity of 0.45 micron±0.02 AUTHORITY: 21 U.S.C. 357. micron, a diameter of approximately 47 SOURCE: 39 FR 18944, May 30, 1974, unless millimeters, and a flowrate of 55 milli- otherwise noted. liters to 75 milliliters of distilled water passing each square centimeter of fil- Subpart A—Definitions; ter area per minute with a differential Interpretations; Requirements pressure of 70 centimeters of mercury at 25° C. § 436.1 Sterility requirements of items (2) Penicillinase solutions. When the packaged with sterile antibiotic drugs. amount of penicillinase to be used is specified in terms of Levy units, use a (a) Diluents packaged in combination penicillinase solution standardized in with sterile antibiotic drugs. If a sterile terms of Levy units. One Levy unit of antibiotic drug is packaged in com- penicillinase inactivates 59.3 units of bination with an immediate container penicillin G in 1 hour at 25° C. and at a of a diluent, the immediate container pH of 7.0 in a phosphate buffered solu- of diluent shall be sterile when tested tion of a pure alkali salt of penicillin G by the method prescribed in when the substrate is in sufficient con- § 436.20(e)(1). centration to maintain a zero order re- (b) Dispensers packaged in combination action. with sterile antibiotic drugs. If a sterile (c) Culture media. Use ingredients antibiotic drug is packaged in com- that conform to the standards pre- bination with a dispenser, such dis- scribed by the U.S.P. or N.F. In lieu of penser shall be sterile when tested by preparing the media from the individ- the method prescribed in § 436.20(e)(1). ual ingredients, they may be made [39 FR 18944, May 30, 1974, as amended at 41 from dehydrated mixtures which, when FR 46852, Oct. 26, 1976] reconstituted with distilled water, have the same or equivalent composi- § 436.2 Alternative assay methods. tion as such media and have growth- Alternative assay methods (including promoting buffering, and oxygen ten- automated procedures) employing the sion-controlling properties equal to or same basic chemistry or microbiology better than such media. The pH of each as the official methods described in medium should be adjusted with 2N hy- this part and in the individual mono- drochloric acid or sodium hydroxide
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before sterilization, so that after steri- amount of penicillin in the sample or lization and the addition of the penicil- by any other suitable method for linase, if necessary, the pH will fall standardizing the penicillin-inactivat- within the specified range. Dispense ing power of the penicillinase prepara- 90±10 milliliter quantities of the liquid tion. media into individual test tubes (38 (3) Medium C. To each liter of me- millimeters x 200 millimeters). Close dium A add 5.0 milliliters of poly- the tubes with suitable closures, and sorbate 80 before sterilization. To each sterilize in an autoclave at 121° C. for tube of sterilized medium add suffi- 20 minutes. The autoclave temperature cient sterile penicillinase, and proceed should be reached within 10 minutes. as directed for medium B. After sterilization, cool the medium at (4) Medium D. To each liter of me- once to approximately 25° C. and store dium A add 5.0 milliliters of poly- at 20° C. to 30° C. The sterility of each sorbate 80 and sufficient 2N sodium hy- lot of tubes of liquid medium may be droxide so that the pH will be 7.9±0.1 confirmed by incubating an adequate after sterilization. Then add sufficient number of tubes as described in the sterile penicillinase to each tube and test procedures in paragraph (e) of this proceed as directed for medium B. section. (5) Medium E. Use U.S.P. XVIII soy- (1) Medium A. Use U.S.P. fluid bean-casein digest medium. thioglycolate medium I. (6) Medium F. To each liter of me- (2) Medium B. Use U.S.P. fluid dium E add 5.0 milliliters of poly- thioglycolate medium I, with sufficient sorbate 80 before sterilization. To each sterile penicillinase added to inac- tube of sterilized medium add suffi- tivate the penicillin activity in the cient sterile penicillinase to solubilize sample under test. The penicillinase the penicillin in the sample to be test- must be added to individual tubes of ed. sterile medium A, using aseptic tech- (7) Medium G. Prepare as follows: nique. Prior to use, or at the time of the test, a representative number of Peptic digest of animal tissue ...... 6.0 gm. the tubes containing added penicillin- Pancreatic digest of casein ...... 4.0 gm. ase are incubated at 30° –32° C. for 24 Yeast extract ...... 3.0 gm. hours to 48 hours, and are examined for Beef extract ...... 1.5 gm. sterility. If the sample contains peni- Dextrose...... 1.0 gm. Agar...... 15.0 gm. cillin as the only antibiotic, the ability Distilled water, q.s...... 1,000.0 ml. of the penicillinase to inactivate all pH 6.6±0.1 the penicillin in the sample under test is checked as follows: Add to one test Suspend the powder in a liter of dis- tube of medium B the proper amount of tilled water. Allow to stand for 5 min- penicillin from one of the individual utes, then mix thoroughly. Boil for 1 or containers under test. Then add 1.0 2 minutes or until solution is complete. milliliter of a 1:1.000 dilution of an 18– Dispense in suitable flasks and sterilize ° 24 hour culture of Staphylococcus aureus at 121 C. for 15 minutes. Aseptically (American Type Culture Collection pour approximately 25-milliliter quan- 6538–P) 1 in medium A. Typical micro- tities into sterile Petri dish bottoms bial growth must be observable after 24 measuring 20 millimeters x 100 milli- hours incubation at 30°–32° C. If the meters. Cover plates with sterile por- sample contains a mixture of penicillin celain tops, glazed on the outside. plus some other antibiotic or anti- Allow plates to stand at room tempera- bacterial agent the ability of the peni- ture for 48 hours prior to use as a con- cillinase to inactivate all the penicillin trol on the sterility of the plates. in the sample is not tested directly on (8) Medium H. Prepare, sterilize, and the sample under test, but is deter- dispense as described for medium G, ex- mined separately, using an amount of cept as follows: penicillin alone equivalent to the Dextrose ...... 40.0 gm. Peptic digest of animal tissue ...... 10.0 gm. 1 Available from: American Type Culture Agar...... 15.0 gm. Collection, 12301 Parklawn Drive, Rockville, Distilled water q.s...... 1,000.0 ml. MD 20852. pH 5.6±0.1 after sterilization
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(9) Medium I. To each liter of Medium lows: Place 100 milliliters of the iso- A add 1 milliliter of p-tert- propyl myristate sample and 10 milli- octylphenoxy polyethoxyethanol. liters of distilled water into a cen- (10) Medium J. To each liter of Me- trifuge bottle of approximately 250 mil- dium E add 1 milliliter of p-tert- liliters capacity and seal the bottle octylphenoxy polyethoxyethanol. tightly. Place the centrifuge bottle on (11) Medium K. (Rinse medium). Pre- a shaker so that its longest dimension pare as follows: is oriented in the direction of shaker Peptic digest of animal tissue ...... 5.0 gm. movement and shake at 250 cycles per Beef extract ...... 3.0 gm. minute for 1 hour. Centrifuge the bot- p-tert-octylphenoxy polyethoxyethanol tle at 1,800 revolutions per minute for ...... 10.0 gm. 20 minutes. With a suitable vacuum Distilled water, q.s...... 1,000.0 ml. system, remove and discard the upper pH 6.9±0.2 after sterilization layer; then pipet 5 milliliters of the (12) Medium L. To each liter of Me- lower water layer into a beaker and de- dium A add 1 milliliter of p-tert- termine the pH using a standardized pH octylphenoxy polyethoxyethanol and meter. If the water-extract pH is less approximately 10,000 Levy units of pen- than 5.5, pass the isopropyl myristate icillinase. through a glass column packed with (13) Medium M. To each liter of Me- basic aluminum oxide, activity grade dium E add 1 milliliter of p-tert- No. 1. Determine the water-extract pH octylphenoxy polyethoxyethanol and of a portion of the isopropyl myristate approximately 10,000 Levy units of pen- that has been passed through the alu- icillinase. minum oxide column. Sterilize iso- (14) Medium N: propyl myristate by filtration through Pancreatic digest of casein ...... 15.0 gm. a 0.22-micron membrane filter and Peptic digest of soybean meal ...... 5.0 gm. aseptically dispense 100-milliliter por- Sodium chloride...... 5.0 gm. tions into sterile 250-milliliter flasks. Agar...... 15.0 gm. (6) Diluting fluid F. To each liter of Water ...... 1,000.0 ml. diluting fluid A add 20 grams of diso- pH 7.3±0.2 after sterilization dium edetate, and adjust with NaOH so (d) Diluting fluids—(1) Diluting fluid A. that after sterilization the final pH Dissolve 1 gram of U.S.P. peptic digest will be 7.1±0.1. Dispense in flasks and of animal tissue or equivalent in suffi- sterilize as described in paragraph (c) cient distilled water to make 1,000 mil- of this section. liliters. Dispense in flasks and sterilize (7) Diluting fluid G. To each liter of as described in paragraph (c) of this sterile diluting fluid A add 10 grams of section. Final pH=7.1±0.1. sterile L-lysine. (2) Diluting fluid B. To each liter of (8) Diluting fluid H. To each liter of diluting fluid A add 5.0 milliliters of diluting fluid A add 10 grams of sodium polysorbate 80 before sterilization. bicarbonate before sterilization. (3) Diluting fluid C. To each liter of (9) Diluting fluid I. To each liter of di- diluting fluid A add 0.5 gram of sodium luting fluid A add 23.4 grams of sterile thioglycollate, and adjust with NaOH L-arginine base. so that after sterilization the final pH (10) Diluting fluid J. Sterilize 2.0 will be pH 6.6±0.6. Dispense in flasks grams of anhydrous sodium carbonate and sterilize as described in paragraph by dry-heating at 180° C for 2 hours. (c) of this section. Dissolve in 100 milliliters of diluting (4) Diluting fluid D. To each liter of fluid A just prior to use. diluting fluid A add 1 milliliter of p- (e) Conduct of test—(1) Bacterial mem- tert- octylphenoxy polyethoxyethanol. brane filter method—(i) Sample prepara- Dispense in flasks and sterilize as de- tion—(a) Antibiotic drug. From each of scribed in paragraph (c) of this section. 20 immediate containers, aseptically Final pH=7.1±10.1. transfer approximately 300 milligrams (5) Diluting fluid E. Use isopropyl my- of solids if it is not a liquid drug, or 1 ristate that is sterile and that has a milliliter by volume if it is a liquid water-extract pH of 5.5 or greater. De- drug, or the entire contents if the con- termine the water-extract pH of a por- tainer contains less than these tion of the isopropyl myristate as fol- amounts; except that if it is a liquid
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drug containing penicillin in a con- cephalosporin classes of antibiotics, centration greater than 300,000 units add sufficient penicillinase to diluting per milliliter, use the volume that con- fluid A to inactivate the residual anti- tains 300,000 units, into a sterile 500- biotic activity on the membrane after milliliter Erlenmeyer flask containing filtration. By means of a sterile cir- approximately 200 milliliters of dilut- cular blade, paper punch, or any other ing fluid A. (If it is a composite sample suitable sterile device, cut a circular packaged in one immediate container portion (approximately 17.5 millime- in accordance with the requirements of ters in diameter) from the center of the § 431.5(b) of this chapter, transfer the filtering area. Transfer the cut center entire contents, or approximately 6 area to a sterile 38 by 200 millimeter grams, into the Erlenmeyer flask.) (outside dimensions) test tube contain- Stopper the flask and swirl to dissolve ing 90±10 milliliters of sterile medium the drug. As soon as the sample has A. Incubate the tube for 7 days at 30°– completely dissolved, proceed as di- 32° C. Using sterile forceps, transfer the rected in paragraph (e)(1)(ii) of this remaining outer portion of the mem- section. If the pooled portions from 20 brane into a second similar tube con- containers will not dissolve completely taining 90±10 milliliters of medium E. in 200 milliliters of diluting fluid or Incubate the second tube for 7 days at will not filter rapidly, 400 milliliters of 22°–25° C. diluting fluid may be used or two sepa- (2) Direct method. From each of 20 im- rate tests may be performed using a mediate containers, transfer approxi- pool of 10 containers for each test. mately 300 milligrams of solids if it is (b) Diluent packaged in combination not a liquid drug, or 1 milliliter by vol- with a sterile drug. Using the entire ume if it is a liquid drug, or the entire contents from each of 20 immediate contents if it contains less than these containers, proceed as directed in para- amounts, except if it is a liquid drug graph (e)(1)(ii) of this section. containing penicillin in a concentra- (c) Sterile dispensers packaged in tion greater than 300,000 units per mil- combination with a sterile drug. Pre- liliter use that volume that contains pare 20 clean, empty containers of ap- 300,000 units, into individual sterile proximately the same size as those in test tubes (38 millimeters × 200 milli- which the sterile antibiotic drug is meters) containing 90±10 milliliters of packaged. To each container add dilut- medium A. Incubate all tubes at 30° C. ing fluid A in a volume approximately to 32° C. for 7 days. Gently agitate the the same as that of the sterile drug tubes every 1 to 3 days or until com- when it is prepared for dispensing. Cap plete solubilization occurs. At inter- the containers, sterilize by autoclaving vals, examine all tubes for visible at 121° C. for 20 minutes, and then growth. If growth is observed in any allow to cool to room temperature. tube, confirm by microscopic examina- Aseptically open each dispenser pack- tion. From each of the same 20 imme- age and remove each dispenser in turn. diate containers, transfer a second por- Use each aseptically to remove 1 milli- tion (equivalent to that portion ini- liter of the fluid from a separate sterile tially transferred to the tubes contain- container prepared as described above. ing medium A) to individual sterile Aseptically transfer the fluid to a 500- test tubes (38 millimeters × 200 milli- milliliter Erlenmeyer flask containing meters) containing 90±10 milliliters of approximately 200 milliliters of dilut- medium E, except when each container ing fluid A. Stopper the flask and pro- does not have sufficient material to ceed as directed in paragraph (e)(1)(ii) provide for the two similar-size por- of this section. tions, obtain the second portion from (ii) Test procedure. Aseptically filter 20 additional immediate containers. In- the solution through a bacteriological cubate all tubes at 22° C. to 25° C. for 7 membrane filter. All air entering the days. Gently agitate the tubes every 1 filtering system is filtered through air to 3 days or until complete filters capable of removing microorga- solubilization occurs. At intervals, ex- nisms. Filter three 100-milliliter quan- amine all tubes for visible growth. If tities of diluting fluid A through the growth is observed in any tube, con- membrane. For the penicillin and firm by microscopic examination.
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(3) Bacterial membrane filter method for immediate containers. If in the origi- ophthalmic ointments—(i) Ointments that nal test, growth is observed in only one do not contain penicillin. From each of of the two media, test both portions of 10 immediate containers aseptically the cut filter membrane by placing transfer 0.1 gram of the product into a each into a separate tube of the same sterile 250-milliliter flask containing medium. The batch meets the require- 100 milliliters of diluting fluid E which ments if no tube on the second test has previously been heated to a tem- shows growth. If growth is observed in perature of 47° C. Repeat the process, any of the control tubes as well as in using 10 additional containers. Swirl the sample tubes in either the original both of the flasks to dissolve the oint- ment. Immediately aseptically filter or the second test such test is invalid each solution through a separate bac- and must be performed again. In any teriological membrane filter previously event, further tests may be justified if moistened with approximately 0.2 mil- there is sufficient reason to believe liliter of medium K. Filter all air en- that the results obtained in the first tering the system through air filters and second tests may not be valid. In capable of removing microorganisms. such instances, the batch is satisfac- Remove any residual antibiotic from tory if on the final test no tube shows the membranes by rinsing each filter growth. five times with 100 milliliters of me- (2) Direct method. The batch, or the dium K. The membranes should be cov- part of the batch represented by a par- ered with fluid throughout each step of ticular filling operation, meets the re- the filtration procedure until the end quirements of the test if no tube shows of the last filtering step. By means of a growth after incubation. If growth is sterile circular blade, paper punch, or observed in any sample tube, run a sec- other suitable sterile device, cut a cir- ond test in the appropriate medium cular portion (approximately 17.5 milli- using 40 immediate containers. The meters in diameter) from the center of the filtering area of each membrane. batch is satisfactory if, on the second Transfer the center portion of the fil- test, no tube shows growth. If growth is tering area of each filter to a sterile observed in any of the control tubes test tube 38 millimeters × 200 millime- (except inoculated tubes, if the sample ters (outside dimensions) containing 90 is penicillin) as well as in the sample milliliters±10 milliliters of sterile me- tubes in either the original or the sec- dium I. Incubate the tube for 7 days at ond test, such test is invalid and must 30° C. to 32° C. Using sterile forceps be performed again. In any event, fur- transfer the outer portion of each filter ther tests may be justified if there is to a similar test tube containing 90 sufficient reason to believe that the re- milliliters ±10 milliliters of sterile me- sults obtained on the first and second dium J. Incubate this tube for 7 days at tests may not be valid. In such in- 22° C. to 25° C. stances the batch is satisfactory if in (ii) Ointments containing penicillin. the final test no tube shows growth. Proceed as directed in paragraph (e)(3)(i) of this section, except in lieu of [39 FR 18944, May 30, 1974, as amended at 41 sterile medium I use sterile medium L FR 46852, Oct. 26, 1976; 42 FR 27228, May 27, for the center portion of the filtering 1977; 43 FR 43457, Sept. 26, 1978; 49 FR 25846, area of each filter and in lieu of sterile June 25, 1984; 49 FR 40006, Oct. 12, 1984; 50 FR medium J use sterile medium M for the 48397, Nov. 25, 1985; 52 FR 4611, Feb. 13, 1987; remaining outer portion of each filter. 53 FR 13401, Apr. 25, 1988] (f) Evaluation of results—(1) Bacterial membrane-filter method. The batch, or Subpart C—Biological Test the part of the batch represented by a Methods particular filling operation meets the requirements of the test if no sample § 436.31 Equipment and diluents for tube shows growth. If growth is ob- use in biological testing. served in any sample tube, run a sec- (a) Equipment—(1) Temperature-meas- ond test in the appropriate medium, uring devices. Use an accurate clinical except perform it in duplicate, using 40
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thermometer or any other tempera- proximately 50 milliliters of distilled ture-measuring device of equal sen- water. Allow to stand overnight at sitivity that has been tested to deter- room temperature and dilute to 100 mine the time necessary to reach the milliliters with distilled water. Filter maximum reading. through cotton. Store under refrigera- (2) Pyrogen-free glassware. Render all tion. glassware free from pyrogens by heat- (6) Diluent 6 (0.5 percent gum acacia ing at 250° C. for not less than 30 min- in distilled water). 11132 utes or by any other suitable method. (7) Diluent 7 (1.0N hydrochloric acid). (3) Pyrogen-free syringes and needles. (8) Diluent 8 (0.1N hydrochloric acid). Render all syringes and needles free (9) Diluent 9 (0.05N sodium hydrox- from pyrogens by heating at 250° C. for not less than 30 minutes or by any ide). other suitable method. (10) Diluent 10 (1 percent U.S.P. (4) Pyrogen-free sodium chloride. Heat methylcellulose (4,000 centipoises) so- sodium chloride for not less than 2 lution): Dissolve 1 gram of U.S.P. hours at 200° C. methylcellulose (4,000 centipoises) in (5) Pyrogen-free sodium carbonate. 100 milliliters of distilled water. Allow Heat anhydrous sodium carbonate for to stand overnight at room tempera- not less than 4 hours at 170° C. ture or until solution is complete. (b) Diluents. (1) Diluent 1 (pyrogen- Store under refrigeration. free water): Prepare pyrogen-free water (11) Diluent 11 (0.12N sodium hydrox- by collecting freshly distilled water ide). and sterilizing it in an autoclave at (12) Diluent 12 (0.5 percent 121° C. for not less than 20 minutes. methylcellulose (4,000 centipoises) in Pyrogen-free water meets the require- distilled water). Proceed as directed in ments for the absence of pyrogens as paragraph (b)(10) of this section, except described in § 436.32(a)(3) when 10 milli- use 0.5 gram of methylcellulose (4,000 liters per kilogram are administered as centipoises). described in § 436.32(a)(2). In testing (13) Diluent 13 (pyrogen-free sodium water for the absence of pyrogens, the carbonate solution). Dissolve 25.6 aliquot to be tested is made isotonic by grams of anhydrous pyrogen-free so- the addition of pyrogen-free sodium dium carbonate (prepared as described chloride. in paragraph (a)(5) of this section) in (2) Diluent 2 (pyrogen-free saline so- 1,000 milliliters pyrogen-free, distilled lution): Prepare an isotonic solution of water (diluent 1). Pyrogen-free, sodium sodium chloride by dissolving 9.0 grams carbonate solution meets the require- of pyrogen-free sodium chloride (pre- ments for the absence of pyrogens as pared as described in § 436.31(a)(4) ) in described in § 436.32(a)(3) when 1.0 milli- pyrogen-free, distilled water (diluent 1) liter per kilogram is administered as to make 1,000 milliliters. Sterilize in described in § 436.32(a)(2). an autoclave at 121° C. for not less than 20 minutes. Pyrogen-free saline solu- (14) Diluent 14 (0.07M sterile sodium tion meets the requirements for the ab- carbonate solution). Dissolve 7.3 grams sence of pyrogens as described in of sodium carbonate in distilled water § 436.32(a)(3) when 10 milliliters per to make 1,000 milliliters. Sterilize in ° kilogram are administered as described an autoclave at 121 C. for 20 minutes. in § 436.32(a)(2). (15) Diluent 15 (pyrogen-free sodium (3) Diluent 3 (sterile distilled water): carbonate solution): Dissolve 9.9 grams Prepare freshly distilled water. Steri- of anhydrous pyrogen-free sodium car- lize in an autoclave at 121° C. for 20 bonate (prepared as directed in para- minutes. graph (a)(5) of this section) in 1,000 mil- (4) Diluent 4 (sterile saline solution): liliters of pyrogen-free, distilled water Dissolve 9.0 grams of sodium chloride (diluent 1). Pyrogen-free sodium car- in distilled water to make 1,000 milli- bonate solution meets the require- liters. Sterilize in an autoclave at 121° ments for the absence of pyrogens as C. for 20 minutes. described in § 436.32(a)(3) when 1.0 milli- (5) Diluent 5 (10 percent gum acacia): liter per kilogram is administered as Dissolve 10 grams of gum acacia in ap- described in § 436.32(a)(2).
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(16) Diluent 16 (0.13M sterile pyrogen- jection of the material is calculated. If free sodium carbonate solution). Dis- the product is packaged for dispensing solve 14.0 grams of anhydrous pyrogen- and is in a combination package with a free sodium carbonate (prepared as de- container of diluent, dilute the product scribed in paragraph (a)(5) of this sec- as directed in the labeling. Warm the tion) in 1,000 milliliters pyrogen-free, product to be tested to approximately distilled water. Sterilize in an auto- 37° C. Dilute the sample with sterile, clave at 121 °C for 20 minutes. pyrogen-free saline (prepared as de- [39 FR 18944, May 30, 1974, as amended at 40 scribed in § 436.31(b)(2)) to the appro- FR 51625, Nov. 6, 1975; 50 FR 48397, Nov. 25, priate concentration specified in the 1985; 53 FR 13401, Apr. 25, 1988] individual section for each antibiotic to be tested. Inject a test dose of 1 mil- § 436.32 Pyrogen test. liliter of the diluted sample per kilo- (a) Method 1—(1) Test animal. Use gram of rabbit weight into an ear vein healthy, mature rabbits weighing not of each of three rabbits within 30 min- less than 1,800 grams each that have utes subsequent to the control tem- maintained their weight on an anti- perature reading. Record the tempera- biotic-free diet for at least 1 week ture at 1, 2, and 3 hours subsequent to under the environmental conditions the injection. specified in this section. House the ani- (3) Evaluation. If no rabbit shows an mals individually in an area of uniform individual rise in temperature of 0.6° C. temperature (±3° C.) and free from dis- or more above its respective control turbances likely to excite them. Do not temperature, and if the sum of the use animals for pyrogen tests more fre- three temperature rises does not ex- quently than once every 48 hours or ceed 1.4° C., the sample meets the re- prior to 2 weeks following their having quirements for the absence of been given a test sample that was ad- pyrogens. If one or two rabbits show a judged pyrogenic. Before using an ani- temperature rise of 0.6° C. or more, or mal that has not been used for a test if the sum of the temperature rises ex- during the previous 2 weeks, condition ceeds 1.4° C., repeat the test using five it 1 to 3 days prior to pyrogen testing other rabbits. If not more than three of by conducting a sham test as directed the eight rabbits show individual rises in paragraph (a)(2) of this section, in temperature of 0.6° C. or more, and if omitting the injection. the sum of the eight temperature rises (2) Procedure. Using equipment and does not exceed 3.7° C., the sample diluents described in § 436.31, as nec- meets the requirements for the absence essary, perform the test in an area of pyrogens. where the animals are housed or under similar environmental conditions. On (b) Method 2. Proceed as directed in the day of the test: Withhold all food paragraph (a) of this section, except di- from the animals being used until after lute the sample with pyrogen-free completion of the test, except that ac- water (diluent 1). cess to water may be allowed; and de- (c) Method 3. Proceed as directed in termine the ‘‘control temperature’’ of paragraph (a) of this section, except di- each animal by inserting the tempera- lute the sample with pyrogen-free ture-measuring device into the rectum water (diluent 1) and inject a test dose of the test animal to a depth of not less of 2.0 milliliters of the diluted sample than 7.5 centimeters and allowing suffi- per kilogram of rabbit weight. cient time to reach a maximum tem- (d) Method 4. Proceed as directed in perature, as previously determined, be- paragraph (a) of this section, except in- fore taking the reading. In any one test ject a test dose of 0.5 milliliter of the use only those animals whose control diluted sample per kilogram of rabbit temperatures do not deviate by more weight. than 1° C. from each other and do not (e) Method 5. Proceed as directed in use any animal with a temperature ex- paragraph (a) of this section, except di- ceeding 39.8° C. The control tempera- lute the sample with pyrogen-free ture recorded for each rabbit con- water (diluent 1) and inject a test dose stitutes the temperature from which of 0.5 milliliter of the diluted sample any subsequent rise following the in- per kilogram of rabbit weight.
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(f) Method 6. Proceed as directed in Con- Volume paragraph (a) of this section, except di- Dilu- centra- of test Antibiotic 1 tion of solution lute sample with 0.05N sodium hydrox- ent test so- to be in- ide (diluent 9). lution 2 jected 3 (g) Method 7. Proceed as directed in Tetracycline hydrochloride ..... 4 5.0 .6 paragraph (a) of this section, except di- Tetracycline phosphate 5 ...... 3 5.0 .6 lute sample with sodium carbonate so- Vidarabine monohydrate 5 ...... 4 1.0 1.0 lution (diluent 13). 1 Diluent number as listed in sec. 436.31(b). (h) Method 8. Proceed as directed in 2 Milligrams of activity per milliliter. paragraph (a) of this section, except in- 3 Milliliters per kilogram of body weight. 4 The concentration of the test solution is expressed in units ject a test dose of 2.0 milliliters of the per milliliter in lieu of milligrams of activity per milliliter. diluted sample per kilogram of rabbit 5 To prepare the test solution, proceed as directed in the in- dividual section of the antibiotic drug regulation in this chapter weight. for the antibiotic to be tested. (i) Method 9. Proceed as directed in paragraph (a) of this section, except di- [46 FR 60568, Dec. 11, 1981, as amended at 46 lute sample with pyrogen-free sodium FR 61071, Dec. 15, 1981; 49 FR 5096, Feb. 10, carbonate solution (diluent 15). 1984] (j) Method 10. Proceed as directed in paragraph (a) of this section, except di- Subpart D—Microbiological Assay lute the sample with sodium carbonate Methods solution (diluent 16). [39 FR 18944, May 30, 1974, as amended at 40 § 436.100 Laboratory equipment. FR 51625, Nov. 6, 1975; 45 FR 22921, Apr. 4, Equipment should be selected which 1980; 50 FR 48397, Nov. 25, 1985; 53 FR 13401, Apr. 25, 1988] is adequate for its intended use and should be thoroughly cleansed after § 436.35 Depressor substances test. each use to remove any antibiotic resi- dues. The equipment should be kept Proceed as directed in the USP XX covered when not in use. Clean glass- depressor substances test. Prepare the ware intended for holding and transfer- sample test solution as follows: For ring the test organisms should be steri- each antibiotic listed in the table lized in a hot air oven at 200–220° C. for below, select the appropriate diluent 2 hours. Volumetric flasks, pipettes, or and test dose (concentration and vol- accurately calibrated diluting devices ume). If the product is packaged for should be used when diluting standard dispensing and is in a combination and sample solutions. package with a container of diluent, di- lute the product as directed in the la- (a) Microbiological agar diffusion beling. assay—(1) Cylinders. Use stainless steel cylinders with an outside diameter of 8 Con- Volume millimeters (±0.1 millimeter), an inside Dilu- centra- of test diameter of 6 millimeters (±0.1 millime- Antibiotic 1 tion of solution ent test so- to be in- ter), and a length of 10 millimeters lution 2 jected 3 (±0.1 millimeter). Bleomycin sulfate ...... 4 4 0.5 1.0 (2) Plates. Plastic or glass Petri Capreomycin sulfate ...... 4 3.0 1.0 dishes may be used, having dimensions Chlortetracycline hydro- chloride ...... 3 5.0 .6 of 20 by 100 millimeters. Covers should Clindamycin phosphate ...... 4 5.0 1.0 be of suitable material. Daunorubicin hydrochloride ... 4 1.5 1.0 (b) Microbiological turbidimetric Dihydrostreptomycin sulfate ... 4 3.0 1.0 Doxorubicin hydrochloride ...... 4 1.5 1.0 assay—(1) Tubes. Tubes which give sat- Doxycycline hyclate ...... 4 5.0 1.0 isfactory results and have uniform Lincomycin hydrochloride length and diameter should be used. If monohydrate ...... 4 3.0 1.0 Minocycline hydrochloride ...... 4 5.0 .6 reusable tubes are employed, care must Plicamycin ...... 3 0.050 1.0 be taken to remove not only all anti- Mitomycin ...... 4 0.050 1.0 biotic residues from the previous test Oxytetracycline 5 ...... 3 5.0 .6 Oxytetracycline hydrochloride 4 5.0 .6 but also all traces of cleaning solution. Rolitetracycline ...... 4 5.0 .6 (2) Colorimeter. Use a suitable photo- Rolitetracycline nitrate ...... 4 5.0 .6 electric colorimeter at a wavelength of Sodium colistimethate ...... 4 3.0 1.6 Spectinomycin hydrochloride 4 15.0 1.0 530 millimicrons. Set the instrument at Streptomycin sulfate ...... 4 3.0 1.0 zero absorbance with clear,
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uninoculated broth prepared as de- Monobasic potassium phosphate: 80.0 gm. scribed in the applicable method for Distilled water, q.s: 1,000.0 ml. the antibiotic being assayed. Adjust with 18N phosphoric acid or [39 FR 18944, May 30, 1974, as amended at 41 10N potassium hydroxide to yield a pH FR 34743, Aug. 17, 1976] 5.95 to 6.05 after sterilization. (7)–(9) [Reserved] § 436.101 Solutions. (10) Solution 10 (0.2M potassium phos- (a) Antibiotic assay solutions are phate buffer, pH 10.5). prepared as follows (solution numbers Dibasic potassium phosphate: 35.0 gm. 1, 2, 3, 4, and 6 correspond to those used 10 N potassium hydroxide: 2.0 ml. in ‘‘Assay Methods of Antibiotics,’’ D. Distilled water, q.s: 1,000.0 ml. C. Grove and W. A. Randall, Medical Adjust with 18N phosphoric acid or Encyclopedia, Inc., New York, N.Y. 10N potassium hydroxide to yield a pH (1955), p. 222), which is incorporated by 10.4 to 10.6 after sterilization. reference. Copies are available from (11) Solution 11 (10 percent potassium the Medical Encyclopedia Inc., 30 East phosphate buffer, pH 2.5). 60th St., New York, NY 11220, or avail- able for inspection at the Office of the Monobasic potassium phosphate: 100.0 gm. Federal Register, 800 North Capitol Concentrated hydrochloric acid: 0.2 ml. (ap- Street, NW., suite 700, Washington, DC. proximately). Distilled water, q.s: 1,000.0 ml. (1) Solution 1 (1 percent potassium phosphate buffer, pH 6.0). Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH Dibasic potassium phosphate: 2.0 gm. Monobasic potassium phosphate: 8.0 gm. 2.0 to 2.8 after sterilization. Distilled water, q.s: 1,000.0 ml. (12) Solution 12 (10 percent potassium phosphate buffer, pH 7.0). Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH Monobasic potassium phosphate: 100.0 gm. 5.95 to 6.05 after sterilization. Distilled water, q.s: 1,000.0 ml. (2) Solution 2 (citrate buffer solution pH Adjust with 18N phosphoric acid or 6.3). 10N potassium hydroxide to yield a pH Citric acid: 13.2 gm. 6.95 to 7.05 after sterilization. Sodium hydroxide: 7.06 gm. (13) Solution 13 (0.01N methanolic hy- Sodium citrate: 97.0 gm. drochloric acid). Distilled water, q.s: 1,000.0 ml. 1.0N hydrochloric acid: 10.0 ml. Adjust with 10 percent citric acid so- Methyl alcohol, q.s: 1,000.0 ml. lution or 10N sodium hydroxide to yield (14) Solution 14 (2 percent sodium bicar- pH 6.2 to 6.4 after sterilization. bonate solution). (3) Solution 3 (0.1M potassium phos- phate buffer, pH 8.0). Sodium bicarbonate: 20.0 gm. Distilled water, q.s: 1,000.0 ml. Dibasic potassium phosphate: 16.73 gm. Monobasic potassium phosphate: 0.523 gm. Prepare daily. Distilled water, q.s: 1,000.0 ml. (15) Solution 15 (80 percent isopropyl al- Adjust with 18N phosphoric acid or cohol solution). 10N potassium hydroxide to yield a pH Isopropyl alcohol: 800.0 ml. 7.9 to 8.1 after sterilization. Distilled water, q.s: 1,000.0 ml. (4) ( Solution 4 0.1M potassium phosphate (16) Solution 16 (0.1 M potassium phos- ). buffer, pH 4.5 phate buffer, pH 7.0). Monobasic potassium phosphate: 13.6 gm. Dibasic potassium phosphate: 13.6 gm. Distilled water, q.s: 1,000.0 ml. Monobasic potassium phosphate: 4.0 gm. Adjust with 18N phosphoric acid or Distilled water, q.s.: 1,000.0 ml. 10N potassium hydroxide to yield a pH Adjust with 18 N phosphoric acid or 4.45 to 4.55 after sterilization. 10 N potassium hydroxide to yield a pH (5) [Reserved] 6.8 to 7.2 after sterilization. (6) Solution 6 (10 percent potassium (17) Solution 17 (5 percent methyl alco- phosphate buffer, pH 6.0). hol in 1 percent potassium phosphate Dibasic potassium phosphate: 20.0 gm. buffer, pH 6.0).
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Methyl alcohol: 50.0 ml. Dextrose: 1.0 gm. 1 percent potassium phosphate buffer, pH 6.0, Agar: 15.0 gm. q.s.: 1,000.0 ml. Distilled water, q.s: 1,000.0 ml. pH 6.5 to 6.6 after sterilization. (18) Solution 18 (0.054M sodium phos- phate buffer, pH 6.9). (2) Medium 2. Sodium dihydrogen phosphate Peptone: 6.0 gm. monohydrate: 3.97 gm. Yeast extract: 3.0 gm. Disodium hydrogen phosphate anhydrous: Beef extract: 1.5 gm. 3.55 gm. Agar: 15.0 gm. Distilled water, q.s.: 1,000.0 mL. Distilled water, q.s: 1,000.0 ml. pH 6.5 to 6.6 after sterilization. [39 FR 18944, May 30, 1974, as amended at 40 FR 52004, Nov. 7, 1975; 45 FR 75194, Nov. 14, (3) Medium 3. 1980; 47 FR 9396, Mar. 5, 1982] Peptone: 5.0 gm. § 436.102 Culture media. Yeast extract: 1.5 gm. Beef extract: 1.5 gm. (a) Ingredients. Use ingredients that Sodium chloride: 3.5 gm. conform to the standards, if any, pre- Dextrose: 1.0 gm. scribed by the U.S.P. or N.F. In lieu of Dipotassium phosphate: 3.68 gm. preparing the media from the individ- Potassium dihydrogen phosphate: 1.32 gm. ual ingredients specified, they may be Distilled water, q.s: 1,000.0 ml. made from dehydrated mixtures that, pH 6.95 to 7.05 after sterilization. when reconstituted with distilled (4) Medium 4. water, have the same composition as Peptone: 6.0 gm. such media. Minor modifications of the Yeast extract: 3.0 gm. individual ingredients specified in this Beef extract: 1.5 gm. section are permissible if the resulting Dextrose: 1.0 gm. media possess growth-promoting prop- Agar: 15.0 gm. erties at least equal to the media de- Distilled water, q.s: 1,000.0 ml. scribed. pH 6.5 to 6.6 after sterilization. (b) Description of media. Medium num- (5) Medium 5. Medium 5 is the same as bers 1, 2, 3, 4, 5, 8, 9, 10, 11, and 13 cor- medium 2, except adjust the final pH to respond to those used in ‘‘Assay Meth- 7.8 to 8.0 after sterilization. ods of Antibiotics,’’ D. C. Grove and W. (6)–(7) [Reserved] A. Randall, Medical Encyclopedia, Inc., (8) Medium 8. Medium 8 is the same as New York, N.Y. (1955) p. 220, which is medium 2, except adjust the final pH to incorporated by reference. Copies are 5.8 to 6.0 after sterilization. available from Medical Encyclopedia (9) Medium 9. Inc., 30 East 60th St., New York, NY, or available for inspection at the Office of Pancreatic digest of casein: 17.0 gm. Papaic digest of soybean: 3.0 gm. the Federal Register, 800 North Capitol Sodium chloride: 5.0 gm. Street, NW., suite 700, Washington, DC. Dipotassium phosphate: 2.5 gm. Medium numbers 18 through 21 cor- Dextrose: 2.5 gm. respond to those used in ‘‘Outline of Agar: 20.0 gm. Details for Official Microbiological As- Distilled water, q.s: 1,000.0 ml. says of Antibiotics,’’ A. Kirshbaum and pH 7.2 to 7.3 after sterilization. B. Arret, ‘‘Journal of Pharmaceutical (10) Medium 10. Medium 10 is the same Sciences,’’ vol. 56, No. 4, April 1967, p. as medium 9, except: 512, which is incorporated by reference. Copies are available from the American Agar: 12.0 gm. Polysorbate 80 (add polysorbate 80 after boil- Pharmaceutical Association, 2215 Con- ing the medium to dissolve the agar): 10.0 stitution Ave. NW., Washington, DC ml. 20037, or available for inspection at the pH 7.2 to 7.3 after sterilization. Office of the Federal Register (see ad- dress in this paragraph). (11) Medium 11. Medium 11 is the same (1) Medium 1. as medium 1, except adjust the final pH to 7.8 to 8.0 after sterilization. Peptone: 6.0 gm. (12) [Reserved] Pancreatic digest of casein: 4.0 gm. (13) Medium 13. Yeast extract: 3.0 gm. Beef extract: 1.5 gm. Peptone: 10.0 gm.
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Dextrose: 20.0 gm. Pancreatic digest of casein: 17.0 gm. Distilled water, q.s: 1,000.0 ml. Soybean peptone: 3.0 gm. pH 5.6 to 5.7 after sterilization. Dextrose: 2.5 gm. (14)–(18) [Reserved] Sodium chloride: 5.0 gm. (19) Medium 19. Dipotassium phosphate: 2.5 gm. Distilled water, q.s.: 1,000.0 ml. Peptone: 9.4 gm. pH 7.3 after sterilization. Yeast extract: 4.7 gm. Beef extract: 2.4 gm. (38) Medium 38. Sodium chloride: 10.0 gm. Dextrose: 10.0 gm. Peptone: 15.0 gm. Agar: 23.5 gm. Papaic digest of soybean meal: 5.0 gm. Distilled water, q.s: 1,000.0 ml. Sodium chloride: 4.0 gm. pH 6.0 to 6.2 after sterilization. Sodium sulfite: 0.2 gm. (20)–(31) [Reserved] L-cystine: 0.7 gm. (32) Medium 32. Prepare as medium 1, Dextrose: 5.5 gm. except add 300 milligrams of hydrated Agar: 15.0 gm. Distilled water, q.s.: 1,000.0 ml. manganese sulfate (MnSO4·H2O) to each liter of medium. pH 7.0 after sterilization. (33) Medium 33. Use medium 1, steri- [39 FR 18944, May 30, 1974, as amended at 40 lized and cooled to 50° C. Aseptically FR 52004, Nov. 7, 1975; 42 FR 14092, Mar. 15, add sufficient sterile sodium 1977; 47 FR 9396, Mar. 5, 1982; 47 FR 22514, May novobiocin solution to give a final con- 25, 1982] centration of 10 micrograms of novobiocin activity per milliliter of § 436.103 Test organisms. medium. Sterile sodium novobiocin so- (a) Preparation of test organism suspen- lution is prepared by filtering a solu- sions. For each test organism listed in tion containing 2.5 milligrams of the following table, select the media novobiocin per milliliter of distilled (as listed by medium number in water through a membrane filter of § 436.102(b)), incubation period of the 0.22–micron porosity. Roux bottle, suggested dilution factor, (34) Medium 34. and suggested storage period for the particular test organism and proceed Glycerol: 10.0 gm. Peptone: 10.0 gm. by the appropriate method described in Beef extract: 10.0 gm. paragraph (b) of this section. Test or- Sodium chloride: 3.0 gm. ganism letters A through K, M, and N Distilled water, q.s.: 1,000.0 ml. correspond to those used in ‘‘Outline of pH 7.0 after sterilization. Details for Official Microbiological As- (35) Medium 35. Same as medium 34, says of Antibiotics,’’ A. Kirshbaum and except add 17.0 grams of agar to each B. Arret, ‘‘Journal of Pharmaceutical liter of medium. Sciences,’’ Vol. 56, No. 4, p. 512 (April (36) Medium 36. 1967), which is incorporated by ref- Pancreatic digest of casein ...... 15.0 gm. erence. Copies are available from the Papaic digest of soybean ...... 5.0 gm. American Pharmaceutical Association, Sodium chloride ...... 5.0 gm. 2215 Constitution Ave. NW., Washing- Agar ...... 15.0 gm. Distilled water, q.s ...... 1,000.0 ml. ton, DC 20037, or available for inspec- pH 7.3 after sterilization ...... tion at the Office of the Federal Reg- (37) Medium 37. ister, 800 North Capitol Street, NW., suite 700, Washington, DC.
Medium used for theÐ Suggested Incubation Suggested storage period Test organisms Method period of dilution of suspensions used Slants Roux bot- Roux bot- factor under refrigera- tles tle tion
Test organism AÐStaphylococcus aureus 1 1 1 24 hours 1:20 1 week. (ATCC 6538P) 2. Test organism BÐMicrococcus luteus 1 1 1 24 hours 1:30 2 weeks. (ATCC 7468) 2. Test organism CÐMicrococcus luteus 1 1 1 24 hours 1:40 2 weeks. (ATCC 9341) 2. Test organism DÐStaphyloccocus 1 1 1 24 hours 1 1:14 1 week. epidermidis (ATCC 12228) 2.
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Medium used for theÐ Suggested Incubation Suggested storage period Test organisms Method period of dilution of suspensions used Slants Roux bot- Roux bot- factor under refrigera- tles tle tion
Test organism EÐSaccharomyces 6 19 ...... 1:30 4 weeks. cerevisiae (ATCC 9763) 2. or 7 19 19 48 hours 1:30 4 weeks. Test organism FÐBordetella 1 1 1 24 hours 1:20 2 weeks. bronchiseptica (ATCC 4617) 2. Test organism GÐBacillus cereus var. 3 1 1 1 week ...... 6 months. mycoides (ATCC 11778) 2. Test organism HÐBacillus subtilis (ATCC 1 1 1 24 hours ...... 6 months. 6633) 2. or 2 1 32 5 days ...... 6 months. Test organism IÐKlebsiella pneumoniae 1 1 1 24 hours 1:25 1 week. (ATCC 10031) 2. Test organism JÐEscherichia coli (ATCC 1 1 1 24 hours 1:20 2 weeks. 10536). Test organism KÐStreptococcus faecium 5 ...... 24 hours. (ATCC 10541) 2. Test organism LÐMicrococcus luteus 1 1 1 24 hours 1:35 4 weeks. (ATCC 10240) 2. Test organism OÐStaphylococcus aureus, 1 33 33 24 hours 1:10 4 weeks. resistant to novobiocin (ATCC 12692) 2. Test organism TÐSaccharomyces 7 19 19 48 hours 1:30 4 weeks. cerevisiae (ATCC 2601) 2. Test organism VÐMicrococcusluteus, re- 1 1 1 48 hours 1:35 4 weeks. sistant to dihydrostreptomycin (ATCC 10240A) 2. Test organism WÐPseudomonas 1 1 1 24 hours 1:25 2 weeks. aeruginosa (ATCC 25619) 2. Test organism XÐMycobacterium 8 36 ...... 2 weeks. smegmatis (ATCC 607).. Test organism YÐPseudomonas 9 36 36 24 hours 1:50 1 week. aeruginosa (ATCC 29336) 2. 1 If the antibiotic to be tested is paromomycin, the dilution factor is 1:25. 2 Available from American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD. 20852.
(b) Methods for preparation of test or- mine the amount of suspension to be ganism suspensions—(1) Method 1—(i) added to each 100 milliliters of agar or Preparation of suspension. Maintain or- nutrient broth by the use of test plates ganisms on agar slants containing 10 or test broth. Store the test organism milliliters of the appropriate medium. suspension under refrigeration. Incubate the slants at 32° C.–35° C. for (2) Method 2. Proceed as directed in 24 hours. Using 3 milliliters of sterile paragraph (b)(1) of this section, except U.S.P. saline T.S., wash the growth in lieu of paragraph (b)(1)(ii) thereof, from the agar slant onto a large agar heat-shock and standardize the suspen- surface, such as a Roux bottle, contain- sion as follows: Centrifuge and decant ing 250 milliliters of the appropriate the supernatant liquid. Resuspend the medium. Spread the suspension of or- sediment with 50 to 70 milliliters of ganisms over the entire surface of the sterile U.S.P. saline T.S. and heat the Roux bottle with the aid of sterile suspension for 30 minutes at 70° C. Use glass beads. Incubate the Roux bottle test plates to assure the viability of at 32° C.–35° C. Wash the resulting the spores and to determine the growth from the agar surface with 50 amount of spore suspension to be added milliliters of sterile U.S.P. saline T.S. to each 100 milliliters of agar. Main- (ii) Standardization of suspension. De- tain the spore suspension under refrig- termine the dilution factor that will eration. give a 25–percent light transmission at (3) Method 3. Proceed as directed in a wavelength of 580 millimicrons using paragraph (b)(1) of this section, except a suitable photoelectric colorimeter in lieu of paragraph (b)(1)(ii) thereof, and a 13–millimeter diameter test tube heat-shock and standardize the suspen- as an absorption cell. It may be nec- sion as follows: Heat the suspension for essary to adjust the suspension. Deter-
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30 minutes at 70° C. Wash the spore sus- incubate the slant and Roux bottle at pension three times with 25 to 50 milli- 37° C and wash the resulting growth liters of sterile distilled water. Resus- from the agar surface with 50 milli- pend the organisms in 50 to 70 milli- liters of Medium 37 as described in liters of sterile distilled water and § 436.102(b)(37). heat-shock again for 30 minutes at 70° [39 FR 18944, May 30, 1974, as amended at 40 C. Use test plates to assure the viabil- FR 52004, Nov. 7, 1975; 42 FR 14092, Mar. 15, ity of the spores and to determine the 1977; 42 FR 18058, Apr. 5, 1977; 44 FR 10378, amount of spore suspension to be added Feb. 20, 1979; 47 FR 22514, May 25, 1982; 47 FR to each 100 milliliters of agar. Main- 27552, June 25, 1982] tain the spore suspension under refrig- eration. § 436.104 Penicillin activity. (4) [Reserved] Use penicillin-free equipment and (5) Method 5. Maintain the test orga- glassware. nisms in 100–milliliter quantities of nu- (a) Preparation of inoculated plates. trient broth—Medium 3 as described in Proceed as directed in § 436.105(a), using § 436.102(b)(3). For the test prepare a 10 milliliters of medium 1 for the base fresh subculture by transferring a layer. For the seed layer, use 4 milli- loopful of the stock culture to 100 mil- liters of medium 4, inoculated with the liliters of the same nutrient broth and amount of test organism C which gave incubate for 16 to 18 hours at 37° C. the clearest, sharpest zones of inhibi- Store this broth culture under refrig- tion measuring 17 to 21 millimeters in eration. diameter when standardized as de- (6) Method 6. Maintain the test orga- scribed in § 436.103(b)(1)(ii). Use the nisms on agar slants containing 10 mil- plates the same day they are prepared. liliters of the medium specified in (b) Preparation of working standard paragraph (a) of this section. Incubate stock solutions and standard response the slants at 32° C.–35° C. for 24 hours. lines solutions. Proceed as directed for Inoculate 100 milliliters of nutrient penicillin G in § 436.105(b), except dilute broth—Medium 13 as described in the working standard stock solution to § 436.102(b)(13). Incubate for 16 to 18 a final concentration of 100 units of hours at 37° C. Proceed as directed in penicillin G per milliliter and use the paragraph (b)(1)(ii) of this section. following final concentrations for the (7) Method 7. Proceed as directed in standard response line: 0.005, 0.0125, paragraph (b)(1) of this section, except 0.025, 0.050, 0.100, and 0.200 unit of peni- incubate the slants at 30° C. for 24 cillin G per milliliter. The 0.050 unit of hours and incubate the Roux bottle at penicillin G–per–milliliter solution is 30° C. for 48 hours. the reference concentration of the (8) Method 8. Maintain organisms on assay. agar slants containing 10 milliliters of (c) Sample preparation. Dissolve 1.0 the appropriate medium and transfer gram of the sample in sufficient dis- to a fresh slant about once a week. In- tilled water to make 18 milliliters. Fil- cubate the slants at 37° C for 48 hours. ter if not clear. Transfer 9.0 milliliters Using 3 milliliters of sterile U.S.P. sa- to a separatory funnel, and add 20 mil- line T.S., wash the growth from the liliters of amyl acetate. Add 1 milli- agar slant into a 500–milliliter Erlen- liter of 10 percent potassium phosphate meyer flask containing 100 milliliters buffer, pH 2.5 (solution 11 as described of medium 34, as described in in § 436.101), shake, allow to separate, § 436.102(b) (34), and 50 grams of glass and draw off the aqueous layer into a beads. Agitate the culture by rotation second separatory funnel. Check the pH at a speed of 130 cycles per minute and of the aqueous solution with pH paper, a radius of 3.5 centimeters at 27° C for and readjust with concentrated hydro- 5 days. Determine the amount of sus- chloric acid if the pH is three or above. pension to be added to each 100 milli- Extract again with 20 milliliters or liters of agar by the use of test plates. amyl acetate, discard the aqueous Store the test organism suspension phase, and combine the amyl acetate under refrigeration. extracts. Wash the extracts with 10 (9) Method 9. Proceed as directed in milliliters of 1 percent potassium phos- paragraph (b)(1) of this section, except phate buffer, pH 2.5, and discard the
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buffer wash. Extract the penicillin 45 readings of the 0.050 unit of penicil- from the amyl acetate with a 10–milli- lin G–per–milliliter concentration is liter aliquot of 1 percent potassium 18.5 millimeters and the average of the phosphate buffer, pH 6.0 (solution 1 as 0.050 unit of pencillin G–per milliliter described in § 436.101). This is the assay concentration of this set of three solution. plates is 18.3 millimeters, the correc- (d) Procedure for assay. For the stand- tion is +0.2 millimeters. If the average ard response line, use a total of 15 reading of the 0.025 unit of pencillin G– plates (three plates for each response per–milliliter concentration of these line solution, except the reference con- same three plates is 15.5 millimeters, centration solution, which is included the corrected value is 15.7 millimeters. on each plate). On each set of three Plot these corrected values, including plates, fill three alternate cylinders the average of the 0.050 unit of penicil- with the reference concentration solu- lin G-per-milliliter concentration, on tion and the other three cylinders with semilogarithmic graph paper using the the concentration of the response line pencillin concentration in units per under test. Thus, there will be 45 ref- milliliter on the logarithmic scale and erence concentration zones of inhibi- the diameter of the zone of inhibition tion and nine zones of inhibition for on the arithmetic scale. Draw the line each of the other concentrations of the of best fit through these points. To es- response line. Treat a portion of the timate the sample potency, average the sample solution (2 to 5 milliliters) with zone diameters of the standard and the 0.1 milliliter of penicillinase solution zone diameters of the sample on the and incubate at 37° C. for 1 hour. For three plates used. If the average zone each sample tested, use three plates. diameter of the sample is lower than On each plate fill two cylinders with that of the standard, subtract the dif- the 0.050 unit of penicillin G per milli- ference between them from the ref- liter standard, two cylinders with the erence concentration diameter of the untreated sample, and two cylinders standard response line. From the re- with the penicillinase-treated sample. sponse line, read the concentrations Incubate all plates, including those of corresponding to these corrected values the standard response line, overnight of zone diameters. Multiply the con- ° at 30 C. A zone of inhibition with the centration by the dilution factor to ob- untreated sample and no zone with the tain the units of penicillin G per sam- penicillinase-treated sample are a posi- ple size tested. tive test for penicillin. If a positive test is obtained, measure the diameters [39 FR 18944, May 30, 1974, as amended at 41 of the zones of inhibition using an ap- FR 34743, Apr. 17, 1976] propriate measuring device such as a millimeter rule, calipers, or an optical § 436.105 Microbiological agar diffu- sion assay. projector. (e) Estimation of penicillin G activity. Using the sample solution prepared To prepare the standard response line, as described in the section for the par- average the diameters of the standard ticular antibiotic to be tested, proceed reference concentration and average as described in paragraphs (a), (b), (c), the diameters of the standard response and (d) of this section. line concentration tested for each set (a) Preparation of inoculated plates. of three plates. Average also all 45 di- For each antibiotic listed in the table ameters of the reference concentration. in this paragraph, select the media (as The average of the 45 diameters of the listed by medium number in reference concentration is the correc- § 436.102(b)), the amount of media to be tion point of the response line. Correct used in the base and seed layers, the the average diameter obtained for each test organism (as listed in § 436.103(a)), concentration to the figure it would be and the suggested inoculum and pre- if the average reference concentration pare the inoculated plates as follows: diameter for that set of three plates Prepare the base layer by adding the were the same as the correction point. appropriate amount of melted agar to Thus, if in correcting the 0.025 penicil- each Petri dish (nominal dimensions 20 lin G concentration, the average of the by 100 millimeters). Distribute the agar
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evenly in each dish on a flat, level sur- flask to obtain a homogeneous suspen- face, placing a cover on each plate in sion, and add the appropriate amount turn; if a nonporous cover is used, of the inoculated media to each of the leave it slightly ajar to prevent accu- plates containing the uninoculated mulation of condensed moisture from base agar. Spread evenly over the agar the hot agar base layer. After the agar surface, cover, and allow to harden on hardens, seat the nonporous cover on a flat, level surface. After the agar has each plate. To prepare the seed layer, hardened, place 6 cylinders described in add the suggested inoculum of the test § 436.100(a)(1) on the inoculated agar organism suspension to a sufficient surface so that they are at approxi- amount of agar, which has been melted mately 60° intervals on a 2.8—centi- and cooled to 48° C–50° C. Swirl the meter radius.
Media to be used (as Milliliters of media to Suggested listed by medium be used in the base volume of number in and seed layers standard- § 436.102(b)) ized Incubation Antibiotic Test or- inoculum to Tempera- ganism be added to ture for the Base Seed Base Seed each 100 plates layer layer layer layer milliliters of seed agar
Milliliters Degrees C. Amoxicillin ...... 11 11 21 4 C 0.5 32±35 Amphotericin B ...... None 19 None 8 E 1.0 29±31 Ampicillin ...... 11 11 21 4 C 0.5 32±35 Bacitracin ...... 2 1 21 4 B 0.3 32±35 Bacitracin ...... 2 1 21 4 L 0.3 32±35 Bleomycin ...... 35 35 10 6 X 1.0 32±35 Carbenicillin ...... 9 10 21 4 W 2 0.5 36±37.5 Cefactor ...... 2 1 21 5 A 0.05 36±37.5 Cefadroxil ...... 2 1 21 4 A 0.05 36±37.5 Cefamandole ...... 2 1 21 5 A 0.06 36±37.5 Cefazolin ...... 2 1 21 4 A 0.05 32±35 Cefotaxime ...... 2 1 21 5 A 0.1 36±37.5 Cefoxitin ...... 2 1 21 5 A 0.1 36±37.5 Cephalexin ...... 2 1 21 4 A 0.05 32±35 Cephaloglycin ...... 2 1 21 4 A 0.2 32±35 Cephaloridine ...... 2 1 21 4 A 0.1 32±35 Cephalothin ...... 2 1 21 4 A 0.1 32±35 Cephapirin ...... 2 1 21 4 A 0.08 32±35 Cephradine ...... 2 1 21 4 A 0.05 32±35 Clindamycin ...... 11 11 21 4 C 1.5 36±37.5 Cloxacillin ...... 2 1 21 4 A 0.1 32±35 Colistimethate, sodium ...... 9 10 21 4 F 0.1 36±37.5 Colistin ...... 9 10 21 4 F 0.1 36±37.5 Cyclacillin ...... 11 11 21 4 C 0.5 36±37.5 Dactinomycin ...... 5 5 10 4 H (1) 36±37.5 Dicloxacillin ...... 2 1 21 4 A 0.1 32±35 Dihydrostreptomycin ...... 5 5 21 4 H (1) 36±37.5 Erythromycin ...... 11 11 21 4 C 1.5 32±35 Gentamicin ...... 11 11 21 4 D 0.03 36±37.5 Kanamycin B ...... 5 5 21 4 H (1) 36±37.5 Methicillin ...... 2 1 21 4 A 0.3 32±35 Mitomycin ...... 8 8 10 4 H 0.5 36±37.5 Nafcillin ...... 2 1 21 4 A 0.3 32±35 Natamycin ...... None 19 None 8 E 0.8 29±31 Neomycin ...... 11 11 21 4 A 0.4 32±35 Neomycin ...... 11 11 21 4 D 1.0 36±37.5 Netilmicin ...... 11 11 20 5 D 0.25 36±37.5 Novobiocin ...... 2 1 21 4 D 4.0 34±36 Nystatin ...... None 19 None 8 T 1.0 29±31 Oleandomycin ...... 11 11 21 4 D 1.0 36±37.5 Oxacillin ...... 2 1 21 4 A 0.3 32±35 Paromomycin ...... 11 11 21 4 D 2.0 36±37.5 Penicillin G ...... 2 1 21 4 A 1.0 32±35 Penicillin V ...... 2 1 21 4 A 1.0 32±35 Plicamycin ...... 8 8 10 4 A 0.1 32±35 Polymyxin B ...... 9 10 21 4 F 0.1 36±37.5 Rifampin ...... 2 2 21 4 H 0.1 29±31 Sisomicin ...... 11 11 21 4 D 0.03 36±37.5 Streptomycin ...... 5 5 21 4 H (1) 36±37.5 Ticarcillin ...... 38 38 21 4 Y 1.5 36±37.5
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Media to be used (as Milliliters of media to Suggested listed by medium be used in the base volume of number in and seed layers standard- § 436.102(b)) ized Incubation Antibiotic Test or- inoculum to Tempera- ganism be added to ture for the Base Seed Base Seed each 100 plates layer layer layer layer milliliters of seed agar
Milliliters Degrees C. Vancomycin ...... 8 8 10 4 H (1) 36±37.5 1 Determine the amount of the inoculum by the use of test plates. 2 Use dilution of the suspension that gives 25 percent light transmission in lieu of the stock suspension.
(b) Preparation of working standard standard stock solution. Store the stock solutions and standard response line working standard stock solution under solutions. For each antibiotic listed in refrigeration and do not use longer the table in this paragraph, select the than the recommended storage time. working standard drying conditions, Further dilute an aliquot of the work- solvent(s), concentrations, and storage ing standard stock solution to the time for the standard solutions and proper concentrations to prepare the proceed as follows: If necessary, dry standard response line solutions. The the working standard as described in reference concentration of the assay is § 436.200; dissolve and dilute an accu- the mid concentration of the response rately weighed portion to the proper line. concentration to prepare the working
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(c) Procedure for assay. For the stand- corrected diameters, including the av- ard response line, use a total of 12 erage of the 36 diameters of the ref- plates—three plates for each response erence concentration on 2-cycle line solution, except the reference con- semilog paper, using the concentration centration solution which is included of the antibotic in micrograms or units on each plate. On each set of three per milliliter as the ordinate (the loga- plates, fill three alternate cylinders rithmic scale), and the diameter of the with the reference concentration solu- zone of inhibition as the abscissa. The tion and the other three cylinders with response line is drawn either through the concentration of the response line these points by inspection or through under test. Thus, there will be 36 ref- points plotted for highest and lowest erence concentration zones of inhibi- zone diameters obtained by means of tion and nine zones of inhibition for the following equation: each of the four other concentrations of the response line. For each sample 3a + 2 b + c− e tested use three plates. Fill three alter- L = nate cylinders on each plate with the 5 standard reference concentration solu- tion and the other three cylinders with the sample reference concentration so- 3e + 2 d + c− a lution. After all the plates have incu- H = bated for 16 to 18 hours at the appro- 5 priate incubation temperature for each where: antibiotic listed in the table in para- L=Calculated zone diameter for the lowest graph (b) of this section, measure the concentration of the standard response diameters of the zones of inhibition line; using an appropriate measuring device H=Calculated zone diameter for the high- such as a millimeter rule, calipers, or est concentration of the standard re- an optical projector. sponse line; (d) Estimation of potency. To prepare c=Average zone diameter of 36 readings of the standard response line, average the the reference point standard solution; a, b, d, e=Corrected average values for the diameters of the standard reference other standard solutions, lowest to concentration and average the diame- highest concentration, respectively. ters of the standard response line con- centration tested for each set of three To estimate the potency of the sample, plates. Average also all 36 diameters of average the zone diameters of the the reference concentration for all four standard and the zone diameters of the sets of plates. The average of the 36 di- sample on the three plates used. If the ameters of the reference concentration average zone diameter of the sample is is the correction point of the response larger than that of the standard, add line. Correct the average diameter ob- the difference between them to the ref- tained for each concentration to the erence concentration diameter of the figure it would be if the average ref- standard response line. If the average erence concentration diameter for that zone diameter of the sample is lower set of three plates were the same as the than that of the standard, subtract the correction point. Thus, if in correcting difference between them from the ref- the highest concentration of the re- erence concentration diameter of the sponse line, the average of the 36 diam- standard response line. From the re- eters of the reference concentration is sponse line, read the concentrations 16.5 millimeters and the average of the corresponding to these corrected values reference concentration of the set of of zone diameters. Multiply the con- three plates (the set containing the centration by the appropriate dilution highest concentration of the response factor to obtain the antibiotic content line) is 16.3 millimeters, the correction of the sample. is +0.2 millimeter. If the average read- [39 FR 18944, May 30, 1974]
ing of the highest concentration of the EDITORIAL NOTE: For FEDERAL REGISTER ci- response line of these same three plates tations affecting § 436.105, see the List of CFR is 16.9 millimeters, the corrected diam- Sections Affected appearing in the Finding eter is then 17.1 millimeters. Plot these Aids section of this volume.
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§ 436.106 Microbiological turbidimetric the working standard as described in assay. § 436.200; dissolve and dilute an accu- Using the sample solution prepared rately weighed portion to the proper as described in the section for the par- concentration for the working standard ticular antibiotic to be tested, proceed stock solution. Store the working as described in paragraphs (a), (b), and standard stock solution under refrig- (c) of this section. eration and do not use longer than the (a) Preparation of working standard recommended storage time. Prepare stock solutions and standard response line the proper concentrations for the solutions. For each antibiotic listed in standard response line solutions by fur- the table in this paragraph, select the ther diluting an aliquot of the working working standard, drying conditions, standard stock solution. The reference solvent(s), concentrations, and storage concentration of the assay is the mid time for the standard solutions and concentration of the standard response proceed as follows: If necessary, dry line.
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§ N ber as listed in ...... N N N N Diluent (solution num- Distilled water Distilled water Distilled water 0.01 Distilled water Distilled water 0.1 Distilled water Distilled water 0.1 Distilled water Distilled water 0.1 Dimethyl sulfoxide 0.1 alcohol U.S.P. XX 13 Distilled water 15 Distilled water Working standard stock solutions ...... Initial solvent g. per ml.) µ Distilled water Ethyl alcohol (10,000 ...... 436.200) § as listed in (method number Drying conditions Not dried 5 ...... Not dried ...... Not dried Not dried Not dried Not dried Not dried Not dried Not dried Not dried Not dried ...... 1 ...... 1 ...... 1 ...... 1 ...... 1 ...... 5 ...... 1 ...... 6 ...... 1 ...... 1 2 ...... Antibiotic Use sterile equipment for all stages of this assay. The gramicidin working standard and the response line concentrations are used for assay of tyrothricin. 1 2 Amikacin Candicidin Capreomycin Chloramphenicol Chlortetracycline Cycloserine Demeclocycline Dihydrostreptomycin Doxycycline Gramicidin Kanamycin Lincomycin Meclocycline Methacycline Oxytetracycline Rolitetracycline Spectinomycin Streptomycin Tetracycline Tobramycin Troleandomycin Tyrothricin
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(b) Procedure for assay. For each anti- hours. The exact length of the incuba- biotic listed in the table in this para- tion period should be determined by ob- graph, select the test organism (as list- servation of growth in the reference ed in § 436.103(a)), nutrient broth (as concentration tube of the standard. Re- listed by medium number in move the tubes from the water bath § 436.102(b)), and suggested inoculum and add 0.5 milliliter of a 12-percent and proceed as follows: Place 1.0 milli- formaldehyde solution to each tube. liter (or 0.1 milliliter in the case of Determine the absorbance value of gramicidin and tyrothricin) of each each tube in a suitable photoelectric concentration of the standard response colorimeter, at a wavelength of 530 line (prepare as described in paragraph millimicrons. Set the instrument at (a) of this section) and of the sample solution in each set of three replicate zero absorbance with an uninoculated tubes (as described in § 436.100(b)(1)). blank composed of the same amounts Fifteen tubes are used for the five- of nutrient broth and formaldehyde point standard response line and three used in the assay. for each sample. To each tube add 9 NOTE: The amount of working standard and milliliters of the inoculated broth and sample solutions may be reduced as long as place immediately in a water bath at all other solutions used are reduced propor- the appropriate temperature for 2 to 4 tionately.
Suggested volume of standard- ized inoculum Test orga- Medium to be Incubation Antibiotic nism (nutrient added to tempera- broth) each 100 ture milliliters of medium (nutrient broth)
Amikacin ...... A 3 0.1 36±37.5 Candicidin 1 ...... E 13 0.2 27±29 Capreomycin ...... I 3 0.05 36±37.5 Chloramphenicol ...... J 3 0.7 36±37.5 Chlortetracycline ...... A 3 0.1 36±37.5 Cycloserine ...... A 3 0.4 36±37.5 Demeclocycline ...... A 3 0.1 36±37.5 Dihydrostreptomycin ...... I 3 0.1 36±37.5 Doxycycline ...... A 3 0.1 36±37.5 Gramicidin ...... K 3 1.0 36±37.5 Kanamycin ...... A 3 0.2 36±37.5 Lincomycin ...... A 3 0.1 36±37.5 Meclocycline ...... A 3 0.2 36±37.5 Methacycline ...... A 3 0.1 36±37.5 Oxytetracycline ...... A 3 0.1 36±37.5 Rolitetracycline ...... A 3 0.1 36±37.5 Spectinomycin ...... J 3 0.1 36±37.5 Streptomycin ...... I 3 0.1 36±37.5 Tetracycline ...... A 3 0.1 36±37.5 Tobramycin ...... A 3 0.15 36±37.5 Troleandomycin ...... I 3 0.1 36±37.5 Tyrothricin ...... K 3 1.0 36±37.5 1 Use sterile equipment for all stages of this assay.
L=Calculated absorbance value for the low- 3a+ 2 b+c− e L = est concentration of the standard re- 5 sponse line. H=Calculated absorbance value for the 3e+ 2 d +c− a H = highest concentration of the standard 5 response line. where:
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a, b, c, d, e=Average absorbance values for Replace the stopper and place the each concentration of the standard re- weighing bottle in a desiccator over a sponse line, lowest to the highest, re- desiccating agent, such as phosphorous spectively. pentoxide or silica gel, allow to cool to (c) Estimation of potency. To prepare room temperature, and reweigh. Cal- the standard response line, plot the av- culate the percent of loss. erage absorbance values for each con- (b) Method 2. Proceed as directed in centration of the standard response paragraph (a) of this section, except line on one-cycle semilogarithmic use a tared weighing bottle or weighing graph paper with the absorbance values tube equipped with a capillary-tube on the arithmetric scale and con- stopper, the capillary having an inside centrations on the logarithmic scale. diameter of 0.20–0.25 millimeter, and The response line is drawn either place it in a vacuum oven without re- through these points by inspection or moving the stopper. through points plotted for highest and (c) Method 3. Proceed as directed in lowest absorbance values obtained by paragraph (a) of this section, except means of the following equations. dry the sample at a temperature of 110° To estimate the potency of the sample, C. and a pressure of 5 millimeters of average the absorbance values for the mercury or less for 3 hours. sample and determine the antibiotic (d) Method 4. Proceed as directed in concentration from the standard re- paragraph (a) of this section, except sponse line. Multiply the concentration dry the sample at a temperature of 40° by the appropriate dilution factor to C. and a pressure of 5 millimeters of obtain the antibiotic content of the mercury or less for 2 hours. sample. (e) Method 5. Proceed as directed in paragraph (a) of this section, except [39 FR 18944, May 30, 1974, as amended at 40 ° FR 57797, Dec. 12, 1975; 41 FR 49483, Nov. 9, dry the sample at a temperature of 100 1976; 44 FR 22057, Apr. 13, 1979; 46 FR 3835, C. and a pressure of 5 millimeters of 3839, Jan. 16, 1981; 46 FR 16682, Mar. 13, 1981; mercury or less for 4 hours. 46 FR 33512, June 30, 1981; 46 FR 61072, Dec. (f) Method 6. Proceed as directed in 15, 1981; 47 FR 22515, May 25, 1982; 47 FR 23708, paragraph (a) of this section, except June 1, 1982; 48 FR 3960, Jan. 28, 1983; 53 FR dry the sample at a temperature of 40° 32607, Aug. 26, 1988] C. and a pressure of 5 millimeters of mercury or less for 3 hours. Subpart E—General Chemical (g) Method 7. Proceed as directed in Tests for Antibiotics paragraph (a) of this section, except dry the sample at a temperature of 25° § 436.200 Loss on drying. C. and a pressure of 5 millimeters of Use the method specified in the indi- mercury or less for 4 hours. vidual section for each antibiotic. (h) Method 8. Proceed as directed in (a) Method 1. In an atmosphere of paragraph (a) of this section, except about 10 percent relative humidity, transfer approximately 300 milligrams grind the sample, if necessary, to ob- of the sample to a tared weighing bot- tain a fine powder. When tablets, tro- tle equipped with a ground-glass stop- ches, or capsules are to be tested, use per; dry the sample at a temperature of four tablets, troches, or capsules in 25 ° C and a pressure of 5 millimeters of preparing the sample. Transfer about mercury or less for 4 hours, and then 100 milligrams of the sample to a tared dry the sample at a temperature of 100 weighing bottle equipped with a °C and a pressure of 5 millimeters of ground-glass stopper. Weigh the bottle mercury or less for 3 additional hours. and place it in a vacuum oven, tilting (i) Method 9. Use a suitable the stopper on its side so that there is thermogravimetric apparatus prepared no closure during the drying period. for vacuum operation. Rapidly and Dry at a temperature of 60° C. and a thoroughly grind a portion of the sam- pressure of 5 millimeters of mercury or ple and promptly transfer 5 to 10 milli- less for 3 hours. At the end of the dry- grams to the sample pan. Place the ing period, fill the vacuum oven with system under vacuum and allow it to air dried by passing it through a drying come to equilibrium before heating. agent such as sulfuric acid or silica gel. Obtain an accurate sample weight and
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continuously record the weight loss as (c) Standardization of reagents—(1) the sample is heated at a rate of 20° per Water equivalence of Karl Fischer rea- minute from room temperature to gent. Standardize the Karl Fischer rea- about 200 ° C. The weight loss plateau, gent no longer than 1 hour before use or inflection, at about 150 ° C is taken by one of the following methods. as the total volatile weight loss. Cal- (i) Accurately weigh 25–35 milligrams culate the percent weight loss on dry- of water into a dry titration vessel and ing. add 20 milliliters of solvent A. Start [39 FR 18944, May 30, 1974, as amended at 50 the stirrer and titrate to the endpoint FR 48397, Nov. 25, 1985; 51 FR 11572, Apr. 4, by adding measured quantities of Karl 1986] Fischer reagent. Calculate the water equivalence of the Karl Fischer reagent § 436.201 Moisture determination. as follows: (a) Equipment—(1) Apparatus. Use a closed system consisting of all glass W e = automatic burettes, platinum elec- − trodes, and a magnetic stirrer con- VVTA nected to a suitable electrometric ap- where: paratus. This apparatus embodies a e=Water equivalence of the Karl Fischer simple electrical circuit which serves reagent in terms of milligrams of water to pass 5 to 10 microamperes of direct per milliliter; current between a pair of platinum W=Milligrams of water; electrodes immersed in the solution to VT=Milliliters of Karl Fischer reagent be titrated. At the endpoint of the ti- used; tration a slight excess of the reagent VA=Milliliters of Karl Fischer reagent increases the flow of current to be- equivalent to the 20 milliliters of solvent A, determined as directed in paragraph tween 50 and 150 microamperes for 30 (c)(3) of this section. seconds or longer, depending upon the solution being titrated. (ii) Accurately weigh about 25–35 mil- (2) Titrating vessel. Use a suitable ligrams of water into a dry titration titrating vessel which has been pre- vessel, add an excess of Karl Fischer re- viously dried at 105° C. and cooled in a agent, start the stirrer, and titrate to desiccator. the endpoint with methanol solution. (b) Reagents—(1) Karl Fischer reagent. Calculate the water equivalence of the Dissolve 125 grams of iodine in 170 mil- Karl Fischer reagent as follows: liliters of pyridine, add 670 milliliters of methanol and cool. To 100 milliliters W e = of pyridine kept in an ice bath, add sul- VV− fur dioxide until the volume reaches TA 200 milliliters. Slowly add this solution where: to the cooled iodine-methanol-pyridine e=Water equivalence of the Karl Fischer mixture and shake well. (A commer- reagent in terms of milligrams of water cially prepared Karl Fischer reagent, per milliliter; pyridine containing or pyridine-free, W=Milligrams of water; may be used.) Preserve the reagent in VT=Milliliters of Karl Fischer reagent glass-stoppered bottles protected from used; V =Milliliters of methanol solution used; light and from moisture in the air. m f=Milliliters of Karl Fischer reagent equiv- (2) Methanol solution. Add sufficient alent to each milliliter of methanol solu- water (usually 2 milligrams per milli- tion determined as directed in paragraph liter) to methanol so that each milli- (c)(2) of this section. liter of the resulting methanol solution is equivalent to about 0.5 milliliter of (2) Karl Fischer reagent equivalence of Karl Fischer reagent. methanol solution. Titrate a known vol- (3) Solvents—(i) Solvent A. Meth- ume of Karl Fischer reagent with anol:chloroform:carbon tetrachloride methanol solution until the endpoint is (1:2:2 by volume). reached. Calculate the milliliters of (ii) Solvent B. Chloroform:carbon tet- Karl Fischer reagent equivalent to rachloride (1:1 by volume). each milliliter of methanol solution as (iii) Solvent C. Anhydrous methanol. follows:
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(ii) Transfer about 1 to 2 grams, accu- W e = rately weighed, into a dry titrating − × (VT V m ) f vessel. Add 10 milliliters of solvent B where: and proceed as directed in paragraph f=Milliliters of Karl Fischer reagent equiv- (e)(2) of this section. alent to each milliliter of methanol solu- (3) Aerosols with propellant. Place the tion; immediate container to be tested in a VT=Milliliters of Karl Fischer reagent suitable freezing unit having a tem- used; perature of not higher than 0° C. for at Vm=Milliliters of methanol solution used. least 2 hours. Remove the container (3) Karl Fischer reagent equivalence of from the freezing unit, puncture it, mix solvents. (i) Solvent A: Use 20 milli- the entire contents by swirling. Pro- liters of solvent A as the sample. Start ceed as directed in paragraph (e)(3) of the stirrer and titrate to the endpoint by adding measured quantities of Karl this section, using an accurately meas- Fischer reagent. ured 10-milliliter aliquot from the con- (ii) Solvent B: Use 10 milliliters of tainer as the sample and allowing the solvent B as the sample. Add an excess solution to warm to at least 10° C. be- of Karl Fischer reagent to the sample fore determining the endpoint. and start the stirrer. Titrate to the (4) Hygroscopic powders. Weigh the endpoint with methanol solution. immediate container. Using a suitable (iii) Solvent C. Use 20 milliliters of dry hypodermic needle and syringe, in- solvent C as the sample. Start the stir- ject 3 milliliters of anhydrous meth- rer and titrate to the endpoint by add- anol into the container and shake to ing measured quantities of Karl Fisch- dissolve the contents. Using the same er reagent. syringe, remove the withdrawable con- (iv) Calculate the Karl Fischer rea- tents and transfer into the titration gent equivalence of the solvents as fol- vessel. Rinse the syringe and needle by lows: drawing in an additional 3 milliliters of VA=VC=VT, anhydrous methanol. Add the rinsings VB=(VT¥Vm) X f to the titration vessel. Titrate the so- where: lution immediately, proceeding as di- VA,VB, and VC=Milliliters of Karl Fischer rected in paragraph (e)(3) of this sec- reagent equivalent to the aliquots used tion. Determine the Karl Fischer of solvents A, B, and C, respectively; equivalent (in milliliters), if any, of VT=Milliliters of Karl Fischer reagent used; the anhydrous methanol by titrating a Vm=Milliliters of methanol solution used; f=Milliliters of Karl Fischer reagent equiv- blank of the same total volume used in alent to each milliliter of methanol solu- preparing the sample and rinsing the tion determined as directed in paragraph syringe and needle. Dry the immediate (c)(2) of this section. container and its closure for three (d) Sample preparation—(1) Powders. In hours at 100° C., cool to room tempera- the case of tablets, grind 4 tablets to a ture in a desiccator, and weigh. Deter- fine powder. In the case of capsules mine the weight of sample tested by containing enteric-coated pellets, grind subtracting the weight obtained from the pellets to a fine power. If the maxi- the original weight of the immediate mum moisture limit is greater than 1 container. percent, accurately weigh about 300 (5) Solutions. Proceed as directed in milligrams of the sample into a dry paragraph (e)(3) of this section, using titrating vessel. If the maximum mois- about 1 to 2 grams of the sample, accu- ture limit is less than 1 percent, accu- rately weighted. rately weigh 1 to 2 grams of the sam- (e) Titration procedures and calcula- ple. Proceed as directed in paragraph tions—(1) Procedure 1. Add 20 milliliters (e)(1) or (2) of this section. of solvent A to the sample. Start the (2) Ointments and oils. (i) Transfer stirrer and titrate to the endpoint by about 1 to 2 grams, accurately weighed, adding measured quantities of Karl into a dry titrating vessel. Proceed as directed in paragraph (e)(1) of this sec- Fischer reagent. Determine the percent tion; or moisture in the sample as follows:
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()− × × V× e ×100 VTA V e 100 Percent moisture in = T Percent moisture = weighed samples Ws Ws where: Percent V× e e=Water equivalence of the Karl Fischer moisture in = T reagent determined as directed in para- × graph (c)(1) of this section; aerosols Milliliters of sample 10 VT=Milliliters of Karl Fischer reagent Percent moisture in hygroscopic powders= used; V =Milliliters of Karl Fischer reagent Percent A ()V− V× e ×100 equivalent to the 20 milliliters of solvent moisture in = T b hygroscopic W A, determined as directed in paragarph powders s (c)(3) of this section; (ii) If titration procedure 2 is used: Ws=Weight of the sample in milligrams. (2) Procedure 2. Add an excess of Karl Percent moisture ()V− V f× e ×100 Fischer reagent to the sample, start in weighed = T m the stirrer, and titrate to the endpoint samples W with methanol solution. Calculate the s percent moisture in the sample as fol- Percent ()V− V f× e lows: moisture in = T m (i) For powders: aerosols Milliliters of sample ×10
()V− V f× e ×100 Percent = T m ()V− V f − V× e ×100 Percent moisture moisture in = T m b Ws hygroscopic powders Ws (ii) For oils and ointments: where: ()V− V f − V× e ×100 VT=Milliliters of Karl Fischer reagent Percent = T m B used; moisture W Vm=Milliliters of methanol solution used; s f=Milliliters of Karl Fischer reagent equiv- where: alent to each milliliter of methanol so- VT=Milliliters of Karl Fischer reagent lution determined as directed in para- used; graph (c)(2) of this section;
Vm=Milliliters of methanol solution used; Vb=Karl Fischer equivalent (in milliliters) f=Milliliters of Karl Fischer reagent equiv- of the methanol used as a sample sol- alent to each milliliter of methanol so- vent; lution determined as directed in para- e=Water equivalence of the Karl Fischer graph (c)(2) of this section. reagent determined as directed in para- VB=Milliliters of Karl Fischer reagent graph (c)(1) of this section. equivalent to the 10 milliliters of sol- vent B determined as directed in para- [39 FR 18944, May 30, 1974, as amended at 48 graph (c)(3) of this section; FR 51292, Nov. 8, 1983; 50 FR 41679, Oct. 15, e=Water equivalence of the Karl Fischer 1985; 51 FR 22071, June 18, 1986; 51 FR 27532, reagent determined as directed in para- Aug. 1, 1986] graph (c)(1) of this section; Ws=Weight of the sample in milligrams. § 436.202 pH. (3) Procedure 3. Add about 20 milli- (a) Apparatus. A suitable potentiom- liters of solvent A to a dry titrating eter fitted with two electrodes, one vessel and proceed as directed in titra- being constructed of glass and sensitive tion procedure 1 or 2. Disregard the to hydrogen ion activity and the other volume of reagents used to determine being a calomel or a silver/silver chlo- the endpoint. Promptly introduce an ride reference electrode. A combination accurately weighed or measured quan- electrode with glass electrode and ref- tity of sample into the titrating vessel erence electrode contained in the same and titrate to the endpoint using either system may be used. titration procedure 1 or 2 without addi- (b) Standardization. Select two stand- tional solvents. Calculate the percent ard buffer solutions such that the ex- moisture in the sample as follows: pected pH value of the sample is within (i) If titration procedure 1 is used: their pH range and is also within 2 pH
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unit of one of the standard buffer solu- larizing microscope. The particles re- tions. Standardize the pH meter with veal the phenomena of birefringence the two buffer solutions. Make any and extinction positions on revolving necessary adjustment of the meter if the microscope stage. the observed pH value of either stand- (b) Method 2. Proceed as directed in ard solution differs by more than 0.05 paragraph (a) of this section, except to pH units of its known value. prepare the sample for examination, (c) Sample preparation. If necessary, mount a few particles in mineral oil, dilute the sample with carbon dioxide- add 1 drop of ethyl alcohol, and allow free distilled water to the concentra- to react for about 30 seconds. tion specified in the individual section for each antibiotic. § 436.204 Iodometric assay. (d) Test procedure. Determine the pH (a) Reagents. (1) 0.01N Sodium °± ° of the sample at 25 2 C. Rinse the thiosulfate (2.482 grams Na2S2O35H2O electrode(s) between determinations and 125 milligrams Na2CO3 per liter). first with distilled water and then with (2) 1.0N Sodium hydroxide. a portion of the next sample to be test- (3) 1.2N Hydrochloric acid. ed. Store electrode(s) with tips im- (4) 0.01N Iodine solution (prepared mersed in water. from 0.1N iodine U.S.P.). [39 FR 18944, May 30, 1974, as amended at 42 (5) Starch iodide paste, T.S. (U.S.P.). FR 29857, June 10, 1977; 42 FR 31449, June 21, (b) Preparation of sample and working 1977] standard solutions—(1) Workingstandard solutions. From the following table, se- § 436.203 Crystallinity. lect the initial solvent, diluent, and Use the method specified in the indi- final concentration as listed for each vidual section for each antibiotic. antibiotic working standard. Dissolve (a) Method 1. To prepare the sample and dilute an accurately weighed por- for examination, mount a few particles tion to the specified final concentra- in mineral oil on a clean glass slide. tion and proceed as directed in para- Examine the sample by means of a po- graphs (c) and (d) of this section.
Final concentration in Diluent (solution num- units or milligrams of Antibiotic Initial solvent ber as listed in activity per milliliter of § 436.101(a)) standard solution
Amoxicillin ...... None ...... Distilled water ...... 1.0 mg. Ampicillin ...... do ...... do ...... 1.25 mg. Cephalexin ...... do ...... do ...... 2 mg. Cephaloridine ...... do ...... do ...... 2 mg. Cephalothin ...... do ...... do ...... 2 mg. Cephapirin ...... do ...... do ...... 2 mg. Cloxacillin ...... do ...... do ...... 1.25 mg. Cyclacillin ...... do ...... do ...... 1.0 mg. Dicloxacillin ...... do ...... 1 ...... 1.25 mg. Methicillin ...... do ...... 1 ...... 1.25 mg. Nafcillin ...... do ...... 1 ...... 1.25 mg. Oxacillin ...... do ...... 1 ...... 1.25 mg. Penicillin G ...... do ...... 1 ...... 2,000 units. Penicillin V potassium ...... do ...... 1 ...... 2,000 units.
(2) Bulk antibiotic solutions. From the following table, select the initial solvent, diluent, and final concentration as listed for each antibiotic. Dissolve an accu- rately weighed aliquot (approximately 30 to 60 milligrams) of the sample, dilute to the appropriate final concentration, and proceed as directed in paragraphs (c) and (d) of this section.
Final concentration in Diluent (solution num- units or milligrams of Antibiotic Initial solvent ber as listed in activity per milliliter of § 436.101(a)) sample
Amoxicillin trihydrate ...... None ...... Distilled water ...... 1.0 mg. Ampicillin ...... do ...... do ...... 1.25 mg. Ampicillin sodium ...... do ...... 1 ...... 1.25 mg. Ampicillin trihydrate ...... do ...... Distilled water ...... 125 mg.
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Final concentration in Diluent (solution num- units or milligrams of Antibiotic Initial solvent ber as listed in activity per milliliter of § 436.101(a)) sample
Bacampicillin hydrochloride ...... None ...... do ...... 1.25 mg.2 Cephalexin ...... do ...... do ...... 2 mg. Cephaloridine ...... do ...... do ...... 2 mg. Cephalothin sodium ...... do ...... do ...... 2 mg. Cephapirin sodium ...... do ...... do ...... 2 mg. Cloxacillin sodium monohydrate ...... do ...... do ...... 1.25 mg. Cloxacillin sodium monohydrate ...... do ...... do ...... 1.25 mg. Dicloxacillin sodium monohydrate ...... do ...... 1 ...... 1.25 mg. Cyclacillin ...... do ...... Distilled water ...... 1.0 mg. Methicillin sodium monohydrate ...... do ...... 1 ...... 1.25 mg. Mezlocillin ...... do ...... Distilled water ...... 2.0 mg. Nafcillin sodium monohydrate ...... do ...... 1 ...... 1.25 mg. Oxacillin sodium monohydrate ...... do ...... 1 ...... 1.25 mg. Penicillin G benzathine blank solution ...... do ...... Distilled water ...... 2,000 units. Penicillin G benzathine inactivated solution ...... do ...... 1N NaOH ...... 2,000 units.1 Penicillin G potassium ...... do ...... 1 ...... 2,000 units. Penicillin G procaine ...... 2 ml methyl alcohol ..... 1 ...... 2,000 units. Penicillin G sodium ...... None ...... 1 ...... 2,000 units. Penicillin V ...... 2 ml methyl alcohol ..... 1 ...... 2,000 units. Penicillin V potassium ...... do ...... 1 ...... 2,000 units. 1 Allow to stand in 1N NaOH for 15 minutes before assaying. 2 The final concentration of bacampicillin hydrochloride is calculated in milligrams of ampicillin activity per milliliter of sample. The ampicillin working standard is used for the assay of bacampicillin hydrochloride.
(3) Finished product solutions. Prepare tration, add 1 drop of the starch iodide the sample for assay as directed in the paste. Continue the titration by the ad- individual section for each antibiotic dition of 0.01- to 0.02-milliliter portions product to be tested by diluting to the of 0.01N sodium thiosulfate, shaking concentration prescribed in the table vigorously after each addition. The in paragraph (b)(2) of this section and endpoint is reached when the blue color proceed as described in paragraphs (c) of the starch-iodine complex is dis- and (d) of this section. charged. Calculate the antibiotic con- (c) Inactivated sample and standard so- tent as described in paragraph (f) of lutions. (1) Transfer 2.0 milliliters each this section. of the sample and the appropriate (f) Calculations—(1) F factor determina- working standard solutions to glass- tion. Using the appropriate working stoppered Erlenmeyer flasks. standard for the particular antibiotic (2) Add 2.0 milliliters of 1N sodium to be tested, assay the standard as di- hydroxide, except if the sample has rected in this section. Calculate the F been diluted in 1N sodium hydroxide, factor (the units of micrograms of ac- and allow to stand at room tempera- tivity equivalent of each milliliter of ture for 15 minutes. 0.01N sodium thiosulfate consumed) by (3) Add 2.0 milliliters of 1.2N hydro- means of the following formula: chloric acid. WP× (4) Add 10.0 milliliters of 0.01N iodine = s solution, stopper, allow to stand at F room temperature for 15 minutes, and Vs proceed as directed in paragraph (e) of where:
this section. Ws=Actual weight in milligrams of stand- (d) Blank determination. Transfer 2.0 ard in the 2.0 milliliters titrated; milliliters each of the sample and the P=Potency of the working standard in appropriate working standard solutions units or micrograms per milligram; to glass-stoppered Erlenmeyer flasks. Vs=Milliliters of sodium thiosulfate used in Add 10.0 milliliters of 0.01N iodine solu- the working standard blank determina- tion and immediately proceed as di- tion minus the milliliters of sodium thiosulfate used in the titration of the rected in paragraph (e) of this section. inactivated working standard solution (e) Titration procedure. Titrate the ex- (the difference is the equivalent of the cess iodine using 0.01N sodium number of milliliters of 0.01N iodine ab- thiosulfate. Toward the end of the ti- sorbed by the inactivated standard).
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(2) Bulk antibiotic. Calculate the po- nents. To 1 volume of this neutralized tency of the sample in units or solution add 8 volumes of distilled micrograms per milligram by means of water and 2 volumes of 95 percent etha- the following formula: nol. This solution should be used for 1 × day only. VFu (4) Ferric ammonium sulfate. Dissolve 272 grams of ferric ammonium sulfate Wu in a mixture of 26 milliliters of con- where: centrated sulfuric acid and sufficient Vu=Milliliters of sodium thiosulfate used distilled water to make 1 liter. This re- in the sample blank determination agent may be used for 1 week when minus the milliliters of sodium thiosulfate used in the titration of the stored in a brown bottle at room tem- inactivated sample solution (the dif- perature. ference is the equivalent of the number (b) Preparation of working standard so- of milliliters of 0.01N iodine absorbed by lutions. From the following table, se- the inactivated sample); lect the diluent and final concentration Wu=Actual weight in miligrams of sample as listed for each antibiotic working in the 2.0 milliliters titrated. standard. Dissolve and dilute an accu- (3) Finished products. Calculate the rately weighed portion to the specified potency of the sample in units or milli- final concentration and proceed as di- grams by means of the appropriate one rected in paragraph (d) of this section. of the following formulas: Final con- V× F × d centration Units of antibiotic = u Diluent (solution in milli- per dose or item Antibiotic number as listed grams 2n per milli- in § 436.101(a)) liter of standard V× F × d solution Milligrams of antibiotic = u per dose or item n× 2 , 000 Amoxicillin ...... Distilled water ... 1.0 where: Ampicillin ...... do ...... 1.25 1 d=Dilution factor for the sample; Cefazolin ...... 1.0 n=Number of doses or items in the sample Cephaloridine ...... Distilled water ... 1.0 Cephalothin ...... do ...... 2.0 assayed. Cephapirin ...... do ...... 1.0 [39 FR 18944, May 30, 1974, as amended at 39 Cloxacillin ...... 1 ...... 1.25 FR 34032, Sept. 23, 1974; 42 FR 59856, Nov. 22, Cyclacillin ...... Distilled water ... 1.25 1977; 44 FR 10378, Feb. 20, 1979; 46 FR 2980, Dicloxacillin ...... 1 ...... 1.25 Jan. 13, 1981; 46 FR 25602, May 8, 1981; 46 FR Methicillin ...... 1 ...... 1.25 46312, Sept. 18, 1981; 46 FR 58298, Dec. 1, 1981; Nafcillin ...... 1 ...... 1.25 46 FR 61072, Dec. 15, 1981; 49 FR 6091, Feb. 17, Oxacillin ...... 1 ...... 1.25 1984] Penicillin G ...... 1 ...... 1.25 Penicillin G procaine ...... 17 ...... 2.0 § 436.205 Hydroxylamine colorimetric Penicillin V Potassium ...... 1 ...... 1.25 assay. 1 To prepare the working standard solution, proceed as di- rected in the individual section of the antibiotic drug regulation (a) Reagents—(1) Hydroxylamine hydro- in this chapter for the antibiotic to be tested. chloride solution. Dissolve 350 grams of hydroxylamine hydrochloride in suffi- (c) Preparation of sample solutions. cient distilled water to make 1 liter. From the following table, select the (2) Buffer. Dissolve 173 grams of so- diluent and final concentration as list- dium hydroxide and 20.6 grams of so- ed for each antibiotic. Dissolve an ac- dium acetate in sufficient distilled curately weighed portion of the sam- water to make 1 liter. ple, dilute to the appropriate final con- (3) Neutral hydroxylamine. Mix 1 vol- centration, and proceed as directed in ume each of hydroxylamine hydro- paragraph (d) of this section; if the chloride solution described in para- product is packaged for dispensing, di- graph (a)(1) of this section and the buff- lute an aliquot of the stock solution er described in paragraph (a)(2) of this (prepared as described in the individual section. Check the pH and if necessary monograph) to the appropriate con- adjust to pH 7.0±0.1 by adding an addi- centration and then proceed as directed tional amount of one of the compo- in paragraph (d) of this section.
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Final con- (d) Procedure. Using a volume of from centration Diluent (solution in milli- 1 to 2 milliliters of standard or sample Antibiotic number as listed grams solution, add an equal volume of water in § 436.101(a)) per milli- liter of and mix. Add the following reagents in sample the specified volumetric proportions Amoxicillin trihydrate ...... Distilled water ... 1.0 with respect to the sample or standard Ampicillin ...... do ...... 1.25 solutions: Add 1.25 volumes of neutral Ampicillin sodium ...... 1 ...... 1.25 hydroxylamine reagent and allow to Ampicillin trihydrate ...... Distilled water ... 1.25 Bacampicillin hydrochloride ...... do ...... 1 1.2 react for 5 minutes. Add 1.25 volumes of Cefazolin sodium ...... 1 ...... 1.0 ferric ammonium sulfate reagent, mix, Cephaloridine ...... Distilled water ... 1.0 Cephalothin sodium ...... do ...... 2.0 and after 3 minutes determine the ab- Cephapirin sodium ...... do ...... 1.0 sorbance of the resulting solution at Cloxacillin sodium 1 ...... 1.25 the wavelength of 480 millimicrons, monohydrate. Cyclacillin ...... Distilled water ... 1.25 using a suitable spectrophotometer and Dicloxacillin sodium ...... do ...... 1.25 a reagent blank prepared by treating a monohydrate. volume of water in the same manner as Methicillin sodium 1 ...... 1.25 monohydrate. the standard or sample solution. The Nafcillin sodium monohydrate 1 ...... 1.25 time elapsed after the addition of the Oxacillin sodium monohydrate 1 ...... 1.25 ferric ammonium sulfate reagent and Penicillin G potassium ...... 1 ...... 1.25 Penicillin G procaine ...... 17 ...... 2.0 the reading of the absorbance must be Penicillin G sodium ...... 1 ...... 1.25 precisely the same (within 10 seconds) Penicillin V ...... 17 ...... 1.25 Penicillin V potassium ...... 1 ...... 1.25 for each solution. Calculate the po- tency of the sample in units or 1 The final concentration of bacampicillin hydrochloride is calculated in milligrams of ampicillin per milliliter of sample. micrograms per milligram as follows: The ampicillin working standard is used for the assay of bacampicillin hydrochloride.
× A1 Potency (in units or micrograms per Units or micrograms per = milliliter of standard solution) × milligram of sample A2 Milligrams of sample per milliliter of sample solution
A1=Absorbance of sample solution. cool to room temperature without agi- A2=Absorbance of standard solution. tation. Invert each Petri dish on the [39 FR 18944, May 30, 1974, as amended at 39 stage of a suitable microscope adjusted FR 34032, Sept. 23, 1974; 39 FR 44012, Dec. 20, to furnish 30 times magnification and 1974; 42 FR 59856, Nov. 22, 1977; 44 FR 10378, equipped with an eye-piece micrometer Feb. 20, 1979; 45 FR 16474, Mar. 14, 1980; 46 FR disc which has been calibrated at the 2981, Jan. 13, 1981; 46 FR 25602, May 8, 1981; 46 magnification being used. In addition FR 61072, Dec. 15, 1981; 49 FR 34350, Aug. 30, to the usual source of light, direct an 1984] illuminator from above the ointment at a 45° angle. Examine the entire bot- § 436.206 Test for metal particles in tom of the Petri dish for metal par- ophthalmic ointments. ticles. By varying the intensity of the (a) Procedure. Extrude the contents of illuminator from above, such metal each of 10 tubes as completely as prac- particles are recognized by their char- ticable into separate, clear, glass Petri acteristic reflection of light. Count the dishes (60 millimeters in diameter), total number of metal particles exceed- cover the dishes, and heat to 80° C. to ing 50 microns in any single dimension. 85° C. for at least 2 hours or until the (b) Evaluation. The batch is accept- ointment has melted completely and able if (1) a total of not more than 50 evenly in the dishes. A higher tempera- such particles is found in 10 tubes; and ture of 100° C.±2° C. may be used if nec- (2) not more than one tube is found to essary to allow adequate settling of contain more than eight such particles. metal particles. Allow the ointment to If the batch fails the above test, repeat
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the test on 20 additional tubes of oint- milliliters. Prepare and store this solu- ment. The total number of metal par- tion in glass containers free from solu- ticles exceeding 50 microns in any sin- ble lead salts. gle dimension from the 30 tubes tested (5) Standard lead solution. Dilute a 10- shall not exceed 150, with not more milliliter aliquot of the lead nitrate than three tubes containing more than stock solution to 100 milliliters with eight such particles. water. This solution must be freshly [39 FR 18944, May 30, 1974; 40 FR 11869, Mar. prepared each time a heavy metals test 14, 1975] is performed. One milliliter of this standard lead solution represents a § 436.207 Residue on ignition. lead level of 10 parts per million in a Use the method specified in the indi- 1.0-gram sample or 20 parts per million vidual section for each antibiotic. in a 0.5-gram sample. (a) Method 1. Place approximately 1 (b) Preparation of the sample. Use the gram of the sample, accurately sulfated ash obtained as described in weighed, in a tared porcelain crucible § 436.207(a). If the heavy metal limit is and carefully ignite at a low tempera- greater than 30 parts per million, the ture until thoroughly charred. The cru- sulfated ash may be obtained from a cible may be loosely covered with a 0.5-gram sample. Add 2 milliliters of porcelain lid during the charring. Add 2 hydrochloric acid to the sulfated ash milliliters of nitric acid and 5 drops of and slowly evaporate to dryness on a sulfuric acid to the contents of the cru- steam bath. Moisten the residue with 1 cible and cautiously heat until white drop of hydrochloric acid, add 10 milli- fumes are evolved, then ignite, pref- liters of hot water, and digest by heat- erably in a muffle furnace, at 500° C. to ° ing on the steam bath for 2 minutes. 600 C. until the carbon is all burned After cooling to room temperature, add off. Cool the crucible in a desiccator ammonia solution dropwise until a pH and weigh. From the weight of residue of 7.2 is reached, then add 2 milliliters obtained, calculate the sulfated ash of 6 percent acetic acid. Filter the solu- content. tion, if necessary, and wash the cru- (b) Method 2. Proceed as directed in paragraph (a) of this section, except cible and the filter with about 10 milli- use 2 milliliters of sulfuric acid and do liters of water. Combine the washings not use the nitric acid. with the filtrate and dilute to exactly 25 milliliters with water. § 436.208 Heavy metals determination. (c) Procedure. Prepare a series of five (a) Reagents—(1) Ammonia solution. standard lead solutions, in increments Prepare an aqueous solution contain- of 10 parts per million, in which the so- ing not less than 9 grams and not more lution of lowest concentration contains 20 parts of lead per million less than than 10 grams of ammonia (NH3) per 100 milliliters. the maximum limit of heavy metals (2) 6 percent acetic acid. Dilute 60 mil- permitted for the sample. Transfer the liliters of glacial acetic acid with suffi- necessary quantities of standard lead cient water to give a solution of 1,000 solution described in paragraph (a)(5) milliliters. of this section directly into metal-free (3) Hydrogen sulfide solution. Prepare 50-milliliter Nessler tubes of uniform a saturated solution of hydrogen sul- diameter, add 2 milliliters of 6 percent fide by passing hydrogen sulfide into acetic acid to each, and adjust each to cold water for a sufficient time. It is a final volume of 25 milliliters with suitable if it produces an immediate water. Transfer the 25-milliliter solu- copious precipitate when added to an tion of the sample described in para- equal volume of 1N ferric chloride. Pre- graph (b) of this section to another pare a fresh hydrogen sulfide solution Nessler tube. Add 10 milliliters of hy- each time a heavy metals test is to be drogen sulfide solution to each stand- performed. ard and sample solution, mix well, and (4) Lead nitrate stock solution. Dissolve allow to stand for 10 minutes. View 159.8 milligrams of lead nitrate with 100 downward over a white surface; the milliliters of 0.15N nitric acid, and di- color of the solution of the sample lute with water to a volume of 1,000 should be no darker than the standard
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that contains the lead equivalent of § 436.210 Specific rotation. the heavy metals limit of the test. (a) Test procedure. The appropriate solvent, test concentration, and polar- § 436.209 Melting range or tempera- ture. imeter tube length are specified in the section for each antibiotic to be tested. (a) Apparatus. Melting range appara- Accurately weigh the sample to be tus consists of a glass container for a tested in a glass-stoppered volumetric bath of colorless fluid, a suitable stir- flask, dissolve in the appropriate sol- ring device, an accurate thermometer, vent, and dilute to the specified test and a controlled source of heat. Any concentration at 25° C. Maintain the apparatus or method of equal accuracy solution at 25° C. and transfer to the may be used. The accuracy should be appropriate polarimeter tube. Deter- checked periodically by use of melting mine the angular rotation of both sol- point standards, preferably those that vent and sample solution in a suitable melt near the expected melting range polarimeter, using a sodium light of the product to be tested. The bath source or a white light source with a fluid is selected with a view to the tem- 589.3-millimicron filter. The zero cor- perature required, but light paraffin is rection is the average of the blank used generally and certain liquid sili- readings and is subtracted from the av- cones are well adapted to the higher erage observed rotation of the sample temperature ranges. The fluid is deep solution if the two figures are of the enough to permit immersion of the same sign, or is added if they are oppo- thermometer to its specified immer- site in sign, to give the corrected angu- sion depth so that the bulb is still 2 lar rotation of the sample solution. centimeters above the bottom of the The determination must be completed bath. within one-half hour from the time the (b) Sample preparation. If necessary, solution is prepared. reduce the sample to a fine powder and (b) Calculations. Determine the spe- store it in a desiccator over sulfuric cific rotation. [α], by the following for- acid for 24 hours. If a method for loss mula: on drying is included in the section for 100a the antibiotic to be tested, a sample []a t = dried by that method may be used. x lc (c) Test procedure. Use a capillary where: glass tube about 10 centimeters long a=The corrected angular rotation of the and 0.8 to 1.2 millimeters internal di- sample solution in degrees at tempera- ameter with the wall 0.2 to 0.3 millime- ture t using a light source of a wave- ter in thickness. Charge the tube with length of x millimicrons; a sufficient amount of the dry power to l=The length of the polarimeter tube in form a column 2.5 to 3.5 millimeters decimeters; high from the sealed end when packed c=The concentration of the solution ex- pressed as number of grams of sub- down as closely as possible by mod- stance in 100 milliliters of solution. erate tapping on a solid surface. Heat the bath until a temperature 10°±1° C. § 436.211 Identity test by infrared below the expected melting range is spectrophotometry. reached, then introduce the charged (a) Apparatus—(1) Spectrophotometer. °± ° tube, and heat at a rate of rise of 3 0.5 A suitable spectrophotometer capable C. per minute until melting is com- of recording the infrared absorption pleted. The temperature at which the spectrum in the 2 to 15 micron range. column of the sample is observed to (2) Hydraulic press. A 30-ton hydraulic collapse definitely against the side of press with 12-inch square platens. the tube at any point is defined as the (b) Sample preparation methods. Use beginning of melting, and the tempera- the sample preparation method speci- ture at which the sample becomes liq- fied in the individual section for each uid throughout is defined as the end of antibiotic. melting or the melting point. (1) Potassium bromide discs. Quantities [39 FR 18944, May 30, 1974, as amended at 41 of materials specified are for a 13-milli- FR 24883, June 21, 1976] meter die. Appropriate adjustments
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should be made in the quantities of ma- tical manner. To pass the infrared terials when dies of other sizes are identity test, the absorption spectrum used. To prepare a 1.0 percent mixture of the sample should compare quali- weigh approximately 2 milligrams of tatively with that of the authentic the sample and mix thoroughly with sample. 200 milligrams of dried potassium bro- mide (infrared spectrophotometric § 436.212 Disintegration test. quality). For a 0.5 percent potassium (a) Apparatus—(1) Basket-rack assem- bromide mixture, use 1 milligram of bly. The basket-rack assembly consists sample. For a 0.25 percent potassium of 6 open-ended glass tubes, each bromide mixture, use 0.5 milligram of 7.75±0.25 centimeters long and having sample. A mortar and pestle, a ball an inside diameter of approximately mill, or other suitable mixing device 21.5 millimeters and a wall approxi- may be used. Transfer the uniformly mately 2 millimeters thick; the tubes milled mixture to the die, evacuate are held in a vertical position by two gradually while raising the pressure to plastic plates, each about 9 centimeters 3,000 pounds per square inch until evac- in diameter and 6 millimeters in thick- uation is complete, then raise the pres- ness, with six holes, each about 24 mil- sure to 16,000 pounds per square inch, limeters in diameter, equidistant from and hold that pressure for 2 to 3 min- the center of the plate and equally utes. Release the pressure, dismantle spaced from one another. Attached by the die, and recover the potassium bro- screws to the undersurface of the lower mide disc. Mount the disc in a suitable plate is 10-mesh No. 23 (0.025 inch) W. holder and proceed as directed in para- and M. gauge woven stainless steel graph (c) of this section. wire cloth. The glass tubes and the (2) Mineral oil mull. Weigh approxi- mately 20 milligrams of the sample upper plastic plate are secured in posi- into an agate mortar and add 2 drops of tion at the top by means of a stainless mineral oil. Triturate thoroughly with steel plate, about 9 centimeters in di- a pestle until a uniform consistency is ameter and 1 millimeter in thickness, obtained. Use two rock salt plates as having six perforations each about 20 an absorption cell. Place a small drop millimeters in diameter, which coin- of the mull in the center of one of the cide with those of the upper plastic plates. Gently put the other plate on plate and the upper open ends of the the mull and slowly squeeze the plates glass tubes. A central shaft about 8 together to spread the mull uniformly. centimeters in length, the upper end of Clamp the two plates firmly together which terminates in an eye through in a metal holder. Examine the assem- which a string or wire may be inserted, bled cell by holding it up to the light. is attached to the stainless steel plate. It should appear smooth and free of The parts of the apparatus are assem- any air bubbles. Proceed as directed in bled and rigidly held by means of three paragraph (c) of this section. bolts passing through the two plastic (3) 1 percent solution. Prepare a 1 per- plates and the steel plate. The design cent solution of the sample in chloro- of the basket-rack assembly may be form and use 1.0 millimeter matched varied somewhat provided the speci- absorption cells. Proceed as directed in fications for the glass tubes and the paragraph (c) of this section. screen mesh size are maintained. (c) Procedure. Place the sample, pre- (2) Disks. Each tube is provided with pared as directed in paragraph (b) of a slotted and perforated cylindrical this section, in the spectrophotometer. disk 9.5±0.15 millimeters thick and Determine the infrared absorbance 20.7±0.15 millimeters in diameter. The spectrum between the wavelengths of 2 disk is made of a suitable, transparent to 15 microns. To be suitable the spec- plastic material having a specific grav- trum should have a transmittance of ity of between 1.18 and 1.20. Five 2-mil- between 20 and 70 percent at most of limeter holes extend between the ends the wavelengths showing significant of the cylinder, one of the holes being absorption. Compare the spectrum to through the cylinder axis and the oth- that of an authentic sample of the ers parallel with it and equally spaced same antibiotic prepared in an iden- on a 6-millimeter radius from it.
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Equally spaced on the sides of the cyl- ual section for the particular antibiotic inder are four notches that form V- tablet being tested, lift the basket shaped planes perpendicular to the from the fluid and observe the tablets. ends of the cylinder. The dimensions of (2) Plain-coated tablets. Place one tab- each notch are such that the openings let in each of the six tubes of the bas- on the bottom of the cylinder are 1.60 ket, add a disk to each tube, and oper- millimeters square and those on the ate the apparatus, using simulated gas- top are 9.5 millimeters wide and 2.55 tric fluids as the immersion fluid. After millimeters deep. All surfaces of the 30 minutes, lift the basket from the disk are smooth. fluid and observe the tablets. If the (3) Raising and lowering device. Use a tablets have not disintegrated com- device for raising and lowering the bas- pletely, substitute simulated intestinal ket in the immersion fluid at a con- fluid as the immersion fluid and con- stant rate between 28 and 32 cycles per tinue the test for a total period of time minute through a distance of not less (including previous immersion in simu- than 5 centimeters and not more than lated gastric fluid) equal to the time 6 centimeters. limit specified in the individual section (b) Immersion fluids. During the per- for the particular antibiotic tablet formance of the tests all immersion being tested. Lift the basket and ob- fluids are maintained at a temperature serve the tablets. of 37°±2° C. by using a thermostatically (3) Enteric-coated tablets. Place one controlled water bath. tablet in each of the six tubes of the (1) Distilled water. basket and operate the apparatus, (2) Simulated gastric fluid: Dissolve using simulated gastric fluid as the im- 2.0 grams of sodium chloride and 7.0 mersion fluid. One hour later, lift the milliliters of hydrochloric acid in basket from the fluid and observe the about 500 milliliters of water. Dissolve tablets. If the tablets show no distinct 3.2 grams of pepsin in this solution and evidence of dissolution or disintegra- add sufficient water to make 1,000 mil- tion, add a disk to each tube and oper- liliters. This solution has a pH of about ate the apparatus, using simulated in- 1.2. testinal fluid as the immersion fluid, (3) Simulated intestinal fluid: Dis- for a total period of time (including the solve 6.8 grams of monobasic potassium previous immersion in simulated gas- phosphate in 250 milliliters of water, tric fluid) equal to the time limit spec- mix and add 190 milliliters of 0.2N so- ified in the individual section for the dium hydroxide and 400 milliliters of particular antibiotic tablet being test- water. Add 10.0 grams of pancreatin, ed. Lift the basket and observe the tab- mix, and adjust the resulting solution lets. with 0.2N sodium hydroxide to a pH of (4) Pastilles. Place one pastille into 7.5±0.1. Dilute to 1,000 milliliters. each of the six tubes of the basket, add (c) Immersion vessel. Use a suitable a disk to each tube, and operate the ap- vessel, such as a 1-liter beaker. paratus, using distilled water as the (d) Operation. Add enough immersion immersion fluid. At the end of the time fluid to the immersion vessel so that limit specified in the individual section when the basket-rack assembly is for the particular antibiotic pastille placed on the raising and lowering de- being tested, lift the basket from the vice at the highest point of the upward fluid and observe the pastilles. stroke, the wire mesh remains at least (5) Capsules. Place one capsule into 2.5 centimeters below the surface of the each of the six tubes of the basket, add fluid and descends to not less than 2.5 a disk to each tube, and operate the ap- centimeters from the bottom of the im- paratus, using distilled water as the mersion vessel. immersion fluid. At the end of the time (e) Procedure—(1) Uncoated or limit specified in the individual section filmcoated tablets. Place one tablet into for the capsules being tested, lift the each of the six tubes of the basket, add basket from the fluid and observe the a disk to each tube, and operate the ap- capsules. paratus, using simulated gastric fluid (f) Evaluation. Complete disintegra- as the immersion fluid. At the end of tion is defined as the state in which the time limit specified in the individ- any residue of the tablet, pastille, or
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capsule (except fragments of the insol- this section, using the procedure or uble coating) remaining on the screen procedures specified in the individual is a soft mass having no palpably firm section for each antibiotic. core. The tablets, pastilles, or capsules pass the disintegration test if all of the Weight in milli- units tested disintegrate completely Antibiotic grams of Solvent under the conditions and time specified sample in the individual section for the anti- Amoxicillin-acid titration 100 20 milliliters biotic tablet, pastille, or capsule being dimethylsulfoxide and tested. If one or two tablets, pastilles, 30 milliliters methyl or capsules fail to disintegrate com- alcohol.* pletely, repeat the test on 12 additional Amoxicillin-base titra- 100 50 milliliters glacial tion. acetic acid. tablets, pastilles, or capsules. The tab- Ampicillin-acid titration 100 20 milliliters lets, pastilles, or capsules pass the dis- dimethylsulfoxide and integration test if not less than 16 of 30 milliliters methyl the total 18 tested disintegrate com- alcohol.* Ampicillin-base titration 100 50 milliliters glacial pletely. Enteric coated tablets fail the acetic acid. disintegration test if they show any Ampicillin sodium-base 50 Do. distinct evidence of dissolution or dis- titration. integration after 1 hour immersion in Cephaloglycin-base ti- 50 Do. simulated gastric fluid. tration. Cephapirin sodium- 50 50 milliliters glacial [39 FR 18944, May 30, 1974, as amended at 52 base titration. acetic acid. FR 4617, Feb. 13, 1987; 55 FR 19873, May 14, Cyclacillin-acid titration 100 20 milliliters 1990] dimethylsulfoxide and 30 milliliters methyl alcohol.* § 436.213 Nonaqueous titrations. Cyclacillin-base titration 100 50 milliliters glacial (a) Equipment—(1) Apparatus. Use a acetic acid. closed system consisting of a suitable Tobramycin-base titra- 30 50 ml glacial acetic titrimeter equipped with a potentiom- tion. acid. eter, an automatic burette, a chart re- *The methyl alcohol is added after the sample has dis- corder, and a glass calomel combinatin solved in dimethylsulfoxide. electrode (with saturated methanolic (d) Blank determination. Place the potassium chloride as the electrolyte). same volume of solvent used to prepare (2) Titration vessel. Use a 100-milliliter the sample solution into a titration tall form beaker without a spout. vessel and proceed as directed in para- (b) Reagents—(1) Methyl alcohol, rea- graph (e) of this section, using the pro- gent grade, anhydrous. cedure or procedures specified in the (2) Dimethylsulfoxide, A.C.S., rea- individual section for each antibiotic. gent grade. (e) Titration procedures—(1) Acid titra- (3) Glacial acetic acid, A.C.S., rea- tion. Equilibrate the electrode by soak- gent grade. ing it overnight in the solvent used for (4) Lithium methoxide reagent: 0.02N preparing the sample solution. Start lithium methoxide in methyl alcohol, the magnetic stirrer and titrate the standardized against primary grade benzoic acid. sample solution with the lithium meth- (5) Perchloric acid reagent: 0.02N per- oxide reagent. Record the change in po- chloric acid in glacial acetic acid, tential of the solution with the addi- standardized against primary grade po- tion of the titrant. Determine the num- tassium acid phthalate. ber of milliliters of reagent consumed (c) Preparation of sample solutions. Se- at neutralization (the inflection point lect the weight of the sample and the of the titration curve). Calculate the solvent listed for each antibiotic. antibiotic content as directed in the in- Transfer the accurately weighed sam- dividual section. ple to a titration vessel. Add the appro- (2) Base titration. Proceed as directed priate solvent, cover, and stir magneti- in paragraph (e)(1) of this section, ex- cally until the sample is dissolved. cept use the perchloric acid reagent as Proceed as directed in paragraph (e) of the titrant and calculate the antibiotic
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content as directed in the individual § 436.215 Dissolution test. section. (a) Equipment. Use either Apparatus 1 [39 FR 18944, May 30, 1974, as amended at 40 or 2 as described in the United States FR 22251, Apr. 22, 1975; 40 FR 23725, June 2, Pharmacopeia XXI dissolution test. 1975; 40 FR 57797, Dec. 12, 1975; 46 FR 2981, Jan. 13, 1981] (b) Procedure. For each dosage form listed in the table in this paragraph se- § 436.214 Heat stability. lect the appropriate dissolution me- Store an accurately weighed portion dium, rotation rate, sampling time, of the sample of approximately 30 mil- and apparatus, and proceed as set forth ligrams in an unstoppered 50-milliliter in either Apparatus 1 or 2 methodology Erlenmeyer flask for 4 days in an elec- of the United States Pharmacopeia tric oven at 100° C±1° C. At the end of XXI dissolution test. Determine the this period, remove the flask from the amount of drug substance dissolved by oven and allow to cool in a desiccator. performing the assay described in para- Accurately weigh an unheated portion graph (c) of this section. The amount of of the original sample of approxi- dissolution medium removed for sam- mately 30 milligrams. Assay both the pling purposes may be disregarded if heated and unheated samples for po- the amount removed is not more than tency as directed in § 436.204 or § 436.205 15 milliliters. If more than 15 milli- of this chapter. Determine the percent liters is removed, then correct for the loss from the difference in potency be- volume removed. tween the unheated original sample and the heat-treated sample. [42 FR 59856, Nov. 22, 1977]
Appara- Dosage form Dissolution medium Rotation Sampling time(s) rate 1 tus
Amoxicillin trihydrate and clavulanate 900 mL distilled water ...... 75 30 min ...... 2 potassium chewable tablets.. Amoxicillin trihydrate and clavulanate ...... do ...... 75 ...... do ...... 2 potassium tablets. Azithromycin capsules...... 900 mL 0.10 M sodium phosphate 100 45 min ...... 2 buffer, pH 6.0, 0.1 mg/mL trypsin. Bacampicillin hydrochloride tablets...... do ...... 75 ...... do ...... 2 Cefadroxil hemihydrate capsules...... 900 mL distilled water ...... 100 45 min ...... 1 Cefadroxil hemihydrate tablets...... 900 mL distilled water ...... 50 30 min ...... 2 Cefixime tablets ...... 900 mL 0.05 M potassium phos- 100 45 min ...... 1 phate buffer, pH 7.2. Cefpodoxime proxetil tablets ...... 900 mL pH 3.0 glycine buffer ...... 75 30 min ...... 2 Cefprozil tablets...... 900 mL purified water ...... 100 45 min ...... 1 Cefuroxime axetil for oral suspension 900 mL Sorenson's Modified 50 30 min ...... 2 Phosphate Buffer, pH 7.0. Cefuroxime axetil tablets...... 900 mL 0.07N hydrochloric acid ... 55 15 min. and 45 min ...... 2 Cephalexin hydrochloride monohydrate 900 mL distilled water ...... 150 45 min ...... 1 tablets.. Cephradine dihydrate capsules...... 900 mL 0.12N hydrochloric acid ... 75 60 min...... 2 Clarithromycin tablets...... 900 mL 0.10 M sodium acetate 50 30 min...... 2 buffer, pH 5.0. Doxycycline hyclate tablets...... 900 mL distilled water ...... 75 60 min and 90 min ...... 2 Doxycycline monohydrate hydrochloric 900 mL 0.1N hydrochloric acid. .... 75 60 min ...... 2 acid capsules.. Erythromycin particles in tablets ...... 900 mL 0.05M potassium phos- 75 45 min...... 2 phate buffer, pH 6.8. Loracarbef capsules...... 900 mL distilled water...... 50 30 min ...... 2 Oxytetracycline hydrochloride cap- 900 mL distilled water ...... 75 30 min and 60 min ...... 2 sules.. Rifabutin capsules ...... 900 mL 0.01 N hydrochloric acid 100 45 min ...... 1 Tetracycline hydrochloride capsules ...... do ...... 75 ...... do ...... 2 (except 500-mg).. Tetracycline hydrochloride capsules ...... do ...... 75 30 min, 60 min, and 90 min 2 (500-mg).. Tetracycline hydrochloride tablets...... do ...... 75 30 min and 60 min ...... 2 Vancomycin hydrochloride capsules. ... 900 mL distilled water...... 100 45 min ...... 1 1 Rotation rate of basket or paddle stirring element (revolutions per minute).
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(c) Antibiotic drug content—(1) Tetra- (3) Doxycycline hyclate. Proceed as di- cycline hydrochloride—(i) Preparation of rected in paragraph (c)(1) of this sec- working standard solution. Accurately tion, except use the doxycycline work- weigh 20 to 30 milligrams of tetra- ing standard. cycline hydrochloride working stand- (4) Bacampicillin hydrochloride. Use ard into a suitable-sized volumetric the ampicillin working standard as the flask. Dissolve and dilute to volume standard of comparison and assay for with water. Further dilute an accu- ampicillin content by either of the fol- rately measured portion with distilled lowing methods. water to obtain a known concentration (i) Iodometric assay. Proceed as di- of 0.01 to 0.02 milligram of tetracycline rected in § 436.204 of this chapter, ex- hydrochloride per milliliter. cept dilute the working standard to a (ii) Preparation of sample solutions. Di- final concentration of 0.3 milligram of lute an accurately measured portion of ampicillin per milliliter and use the the sample with sufficient distilled sample solution as it is removed from water to obtain a concentration of 0.01 the dissolution vessel without further to 0.02 milligram of tetracycline hydro- dilution. chloride per milliliter (estimated). (ii) Hydroxylamine colorimetric assay. (iii) Procedure. Using a suitable spec- Proceed as directed in § 442.40(b)(1)(ii) trophotometer and water as the blank, of this chapter, except: determine the absorbance of each (a) Buffer. In lieu of the buffer de- standard and sample solution at the scribed in § 442.40(b)(1)(ii)(b)(2) of this absorbance peak at approximately 276 chapter, use the buffer prepared as fol- nanometers. Determine the exact posi- lows: Dissolve 200 grams of primary tion of the absorption peak for the par- standard tris (hydroxymethyl) ticular instrument used. aminomethane in sufficient distilled (iv) Calculation. Determine the total water to make 1 liter. Filter before use. amount of tetracycline hydrochloride (b) Preparation of the working standard dissolved as follows: solution. Dissolve and dilute an accu- rately weighed portion of the ampi- ∗ A ×c × d × 900 cillin working standard with sufficient T = u distilled water to obtain a final con- A centration of 0.3 milligram of ampi- s cillin per milliliter; where: (c) Sample solution. Use the sample so- T=Total milligrams of drug dissolved; lution as it is removed from the dis- Au=Absorbance of sample; solution vessel without further dilu- c=Concentration of standard in milligrams; tion; and d=Dilution factor of sample filtrate; (d) Calculations. Determine the total As=Absorbance of standard. amount of ampicillin dissolved as fol- *If more than 15 mL of dissolution medium lows: is removed, correct for the volume removed. (A )(c)( d )(900*) (2) Oxytetracycline hydrochloride; prep- = u aration of working standard-solution. (i) T Accurately weigh 30 milligrams of oxy- As tetracycline-base working standard where: into a suitable-sized volumetric flask. T=Total milligrams of ampicillin equiva- Add 5 milliliters of 0.1N hydrochloric lent dissolved; acid and swirl the flask to dissolve oxy- Au=Absorbance of sample; tetracycline base. Dilute an accurately c=Concentration of working standard solu- measured portion with distilled water tion in milligrams per milliliter; to obtain a known concentration of 0.01 d=Dilution factor of sample filtrate; to 0.02 milligram of oxytetracycline As=Absorbance of standard. per milliliter. *If more than 15 mL of dissolution medium (ii) Proceed as directed in paragraphs is removed, correct for the volume removed. (c)(1) (ii), (iii), and (iv) of this section (5) Cephradine dihydrate—(i) Prepara- except measure the absorbance at the tion of working standard solution. Accu- absorption peak at approximately 273 rately weigh approximately 40 milli- nanometers. grams of cephradine working standard
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into a suitable-sized volumetric flask. to stand for 5 minutes with intermit- Dissolve and dilute to volume with tent swirling. Add 15.0 milliliters of 0.12N hydrochloric acid. Further dilute 0.25N sodium hydroxide, dilute to vol- with a buffer solution (prepared by dis- ume with sufficient 0.05M potassium solving 27.2 grams of sodium acetate phosphate buffer, pH 6.8, and mix. Heat trihydrate in a mixture of 12 milliliters to 60 °C for 5 minutes and allow to cool. of glacial acetic acid and sufficient dis- Using a suitable spectrophotometer tilled water to make 2 liters) to obtain and a blank (prepared as per the proce- a known concentration of 0.01 to 0.03 dure above except that 2.0 milliliters of milligram of cephradine per milliliter. 0.5N sulfuric acid is substituted for the (ii) Preparation of sample solution. Fil- 2.0 milliliters of water) for each solu- ter the sample and dilute an accurately tion, determine the absorbance of each measured portion of the filtrate with working standard and sample solution sufficient buffer solution, described in at the absorbance peak at approxi- paragraph (c)(5)(i) of this section, to mately 236 nanometers. Determine the obtain a concentration of 0.01 to 0.03 exact position of the absorption peak milligram of cephradine per milliliter for the particular instrument used. (estimated). (iii) Proceed as directed in para- (iv) Calculation. Proceed as directed graphs (c)(1) (iii) and (iv) of this sec- in paragraph (c)(1)(iv) of this section. tion, except measure the absorbance at (9) Cefuroxime axetil tablets and powder the absorption peak at approximately for oral suspension—(i) Preparation of 262 nanometers. working standard solution—(a) (6) Amoxicillin trihydrate. Assay for Cefuroxime axetil tablets. Accurately the amoxicillin content as described in weigh approximately 60 milligrams of § 440.103d of this chapter, except use the cefuroxime axetil working standard sample as it is removed from the dis- into a suitable-sized volumetric flask. solution vessel. Dissolve in 5 milliliters of methanol (7) Vancomycin hydrochloride. Assay and dilute to volume with 0.07N hydro- for the vancomycin content as de- chloric acid to obtain a known con- scribed in § 436.105 of this chapter, ex- centration equivalent to 0.01 to 0.02 cept use the sample as it is removed milligram of cefuroxime activity per from the dissolution test. milliliter. (8) Erythromycin—(i) Preparation of (b) Cefuroxime axetil for oral suspen- working standard solution. Accurately sion. Accurately weigh approximately weigh approximately 140 milligrams of 15 milligrams of cefuroxime axetil erythromycin working standard into a working standard into a 100-milliliter 250-milliliter volumetric flask and dis- volumetric flask. Dissolve in 5 milli- solve in 10 milliliters of methyl alco- liters of methanol and dilute to volume hol. Add water nearly to volume, mix, with Sorenson’s Modified Phosphate and allow the solution to cool. Dilute Buffer, pH 7.0 (4.2 grams of sodium di- to volume with water and mix. On the hydrogen orthophosphate dihydrate day of use, dilute an accurately meas- and 14.3 grams of hydrogen disodium ured aliquot with water to obtain a orthophosphate dodecahydrate per liter known concentration of 0.28 milligram of erythromycin per milliliter (before of water). adjusting for standard potency). (ii) Preparation of sample solution—(a) (ii) Preparation of sample solution. Di- Cefuroxime axetil tablets. Filter through lute an accurately measured portion of a 0.45-micron filter and dilute an accu- the filtered sample with sufficient rately measured portion of the filtrate 0.05M potassium phosphate buffer, pH with sufficient 0.07N hydrochloric acid 6.8, to obtain a concentration of about to obtain a concentration equivalent to 0.28 milligram of erythromycin per 0.01 to 0.02 milligram of cefuroxime ac- milliliter (estimated). tivity per milliliter (estimated). (iii) Procedure. Transfer 5.0-milliliter (b) Cefuroxime axetil for oral suspen- aliquots of the working standard solu- sion. Filter the sample through an 8- tion and sample solution to 25-milli- micron filter. A coarse prefilter may be liter volumetric flasks and treat as fol- used to prevent clogging. Use the fil- lows: Add 2.0 milliliters of water, allow trate solution without further dilution.
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(iii) Procedure—(a) Cefuroxime axetil liters of the filtered sample into a 50- tablets. Using a suitable spectro- milliliter volumetric flask. Dilute to photometer and 0.07N hydrochloric acid volume with 0.05 M postassium phos- as the blank, determine the absorbance phate buffer, pH 7.2. of each standard and sample solution (iii) Procedure. Proceed as directed in at the absorbance peak at approxi- paragraphs (c)(1) (iii) and (iv) of this mately 280 nanometers. Determine the section, except measure the absorbance exact position of the absorption peak of the peak at approximately 320 for the particular instrument used. nanometers using 0.05 M potassium (b) Cefuroxime axetil for oral suspen- phosphate buffer, pH 7.2 as the blank. sion. Using a suitable spectrophotom- (11) Cephalexin hydrochloride eter and Sorenson’s Modified Phos- monohydrate. Assay for cephalexin ac- phate Buffer, pH 7.0 (4.2 grams of so- tivity of the cephalexin hydrochloride dium dihydrogen orthophosphate dihy- monohydrate as directed in § 442.28 of drate and 14.3 grams of hydrogen diso- this chapter, and use U.S.P. dissolution dium orthophosphate dodecahydrate apparatus 1 (10 mesh basket). Use the per liter of water) as the blank, deter- sample as it is removed from the dis- mine the absorbance of each standard solution vessel. and sample solution at the absorbance (12) Doxycycline monohydrate. Proceed peak at approximately 280 nanometers. as directed in paragraph (c)(1) of this Determine the exact position of the ab- section, except use the doxycycline sorption peak for the particular instru- standard. ment used. (13) Clarithromycin. Proceed as di- (iv) Calculations. Determine the total rected in § 452.50(b)(1) of this chapter amount of cefuroxime activity dis- except: solved as follows: (i) Dissolution medium. Instead of the A× c × d × 900 mobile phase described in T = u § 452.50(b)(1)(i) of this chapter, use 0.10 M sodium acetate buffer prepared as As follows: Weigh 13.6 grams of sodium ac- where: T = Total milligrams of cefuroxime activity etate trihyrate into a container suffi- dissolved; cient to hold 1 liter of solution. Dis- AU = Absorbance of sample; solve the salt in 750 milliliters of dis- c = Cefuroxime activity of working standard tilled water. Adjust the pH of the solu- solution in milligrams per milliliter; tion to 5.0±0.05 with glacial acetic acid. d = Dilution factor of sample filtrate; and Dilute to 1,000 milliliters with distilled As = Absorbance of standard. water. (10) Cefixime—(i) Preparation of work- (ii) Preparation of the standard and ing standard solution. Accurately weigh sample solutions—(a) Standard solution. approximately 25 milligrams of Dissolve (with shaking or sonication) cefixime working standard into a 500- an accurately weighed portion of the milliliter volumetic flask. Wet the clarithromycin working standard, in powder with 0.5 milliliters of methanol, sufficient methanol to obtain a solu- and dilute to volume with 0.05 M potas- tion having a known concentration of sium phosphate buffer, pH 7.2 (prepared approximately 625 micrograms per mil- by dissolving 6.8 grams of monobasic liliter of clarithromycin. Quan- potassium phosphate in distilled water titatively transfer and dilute an ali- to a volume of one liter. The pH is ad- quot of this solution with mobile phase justed to 7.2 with 1.0N NaOH). Sonicate (described in § 452.50(b)(1)(i) of this to assure dissolution and mix. chapter) and mix to obtain a solution (ii) Preparation of sample solution. of known concentration of approxi- Forty-five minutes after the beginning mately 125 micrograms per milliliter of of the rotation, withdraw and filter a clarithromycin. portion of the solution. For the 400- (b) Sample solution. Use the sample so- milligram tablets, pipet 10.0 milliliters lution as it is removed from the dis- of the filtered sample solution into a solution vessel after diluting and mix- 100-milliliter volumetric flask. For the ing with mobile phase (described in 200-milligram tablets, pipet 10.0 milli- § 452.50(b)(1)(i) of this chapter) 1:2 for
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the 250-milligram tablet and 1:4 for the (b) Sample solution. Filter the sample 500-milligram tablet. solutions through a 0.45-micron filter (c) Calculations. Determine the total before use. Pipet 2.0 milliliters of the amount of clarithromycin activity dis- filtered aliquot into a 25-milliliter vol- solved as follows: umetric flask and dilute to volume with the mobile phase described in × × × § 452.60(b)(1)(i) of this chapter. Pipet 4.0 AU c d 900 T = milliliters of this solution into another As 25-milliliter volumetric flask and bring where: to volume with the mobile phase. The T = Total milligrams of clarithromycin ac- solution is stable at room temperature tivity dissolved; for 24 hours. AU = Area of the clarithromycin peak (at a (c) Calculations. Determine the per- retention time equal to that observed for cent of azithromycin dissolved as fol- the standard) in the chromatogram of lows: the sample; AS = Area of the clarithromycin peak in the APD× × ×100 chromatogram of the clarithromycin Percent azithromycin= U s F standard; dissolved AW× c = Clarithromycin activity in the s u clarithromycin working standard solu- where: tion in milligrams per milliliter; and AU =Area of the azithromycin peak (at a re- d = Dilution factor of sample filtrate. tention time equal to that observed for (14) Azithromycin. Proceed as directed the standard) in the chromatogram of in § 452.60(b)(1) of this chapter, except: the sample; AS =Area of the azithromycin peak in the (i) Dissolution medium. Dissolve 85.2 chromatogram of the azithromycin grams of sodium phosphate dibasic and standard; dilute to volume with ultrapure deion- PS =Azithromycin activity in the ized or high-performance liquid azithromycin working standard solution chromatographic-grade water in a in micrograms per milliliter; stoppered 2-liter graduated cylinder. Dilute this entire solution in an appro- 9001 × 25 × 25 priate, suitably sized container with 4 DF =Dilution factor = liters of ultrapure deionized or high- 2× 4 performance liquid chromatographic- 1 If more than 15 milliliters of dissolution grade water. Adjust the pH to 6.0±0.05 medium are removed, correct for the volume with concentrated hydrochloric acid removed; and (about 40.5 milliliters). Add 600 milli- WU =Theoretical azithromycin content (mg) grams of trypsin and mix well. of capsule. (ii) Preparation of the standard and (15) Cefprozil. Proceed as directed in sample solutions—(a) Standard solution. § 442.80(b)(1) of this chapter except: Accurately weigh approximately 15 (i) Sample solutions. Filter the sample milligrams of the azithromycin work- solutions through a 0.45-micron filter ing standard into a 50-milliliter volu- before use. Use the sample solution as metric flask. Add 25 milliliters of the it is removed from the dissolution ves- dissolution medium and sonicate brief- sel without further dilution for the 250- ly. Dilute to volume with dissolution milligram tablet; prepare the sample medium and mix well. Pipet 2.0 milli- solution for the 500-milligram tablet by liters of this solution into a 25-milli- diluting a 5-milliliter aliquot of the fil- liter volumetric flask and dilute to vol- tered solution to volume in a 10-milli- ume with the mobile phase described in liter volumetric flask with distilled § 452.60(b)(1)(i) of this chapter. Pipet 4.0 water. milliliters of this solution into a 25- (ii) Calculations. Determine the total milliliter volumetric flask and bring to percent of cefprozil dissolved as fol- volume with the mobile phase. lows:
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Total percentage = (mg cefprozil (Z) dissolved + mg cefprozil (E) dissolved) dissolved label claim
c = Concentration of the working standard Milligrams of solution in milligrams per milliliter; and A× c × d × 900 d = Dilution factor of the sample filtrate. cefprozil ( Z ) or = U cefprozil ( E ) dis - A (17) Cefadroxil hemihydrate. Proceed solved s as directed in paragraph (c)(1) of this section, except use the cefadroxil where: working standard and measure the ab- AU = Area of the cefprozil (Z) or cefprozil (E) response in the chromatogram of the sorbance at the absorption peak of ap- sample (at a retention time equal to that proximately 264 nanometers. observed for the standard); (18) Rifabutin—(i) Preparation of the AS = Area of the cefprozil (Z) or cefprozil (E) working standard solution. Accurately response in the chromatogram of the weigh approximately 45 milligrams of cefprozil (Z) or cefprozil (E) standard; c = Concentration of the cefprozil (Z) or the rifabutin working standard into a cefprozil (E) working standard solution suitable-sized volumetric flask. Dis- in milligrams per milliliter; and solve and dilute to volume with 0.01N d = Dilution factor of the sample filtrate. hydrochloric acid (prepared by diluting 5.0 milliliters of hydrochloric acid (37 (16) Loracarbef—(i) Preparation of the percent) to 6 liters with distilled water) working standard solution. Accurately weigh approximately 110 milligrams of to obtain a concentration of approxi- the loracarbef working standard into a mately 13 micrograms rifabutin activ- suitable-sized volumetric flask. Dis- ity per milliliter. solve and dilute to volume with water. (ii) Preparation of sample solutions. Further dilute an accurately measured Forty-five minutes after the beginning portion with distilled water to obtain a of the rotation, withdraw a 10-milli- known concentration of 0.02 milligram liter aliquot from the vessel. Dilute a of loracarbef activity per milliliter. 2-milliliter portion of the sample to 25 (ii) Preparation of sample solutions. Di- milliliters with 0.01N hydrochloric lute an accurately measured portion of acid. the sample with sufficient distilled (iii) Procedure. Using a suitable spec- water to obtain a concentration of 0.02 trophotometer and 0.01N hydrochloric milligram of loracarbef activity per acid as the blank, determine the ab- milliliter (estimated). sorbance of each standard and sample (iii) Procedure. Using a suitable spec- solution at the absorbance maximum trophotometer and water as the blank, at approximately 280 nanometers. De- determine the absorbance of each termine the exact position of the ab- standard and sample solution at the sorbance maximum for the particular absorbance maximum at approxi- instrument used. mately 260 nanometers. Determine the (iv) Calculations. Determine the total exact position of the absorbance maxi- amount of rifabutin dissolved as fol- mum for the particular instrument lows: used. (iv) Calculations. Determine the total A× c × d × 900 T = U amount of loracarbef dissolved as fol- × lows: As 1, 000 × × × where: AU c d 900 T = Total milligrams of rifabutin activity T = dissolved; As AU = Absorbance of sample; where: AS = Absorbance of the standard; T = Total milligrams of loracarbef activity c = Rifabutin activity of the working stand- dissolved; ard solution in micrograms per milli- AU = Absorbance of sample; liter; and AS = Absorbance of the standard; d = Dilution factor of the sample filtrate. 394
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(19) Cefpodoxime proxetil—(i) Dissolu- V = Volume of dissolution fluid used in milli- tion fluid: 0.04 molar glycine buffer, pH liters (900); and 3.0—(A) Stock solution. Dissolve 54.5 F1 = 0.7666 (conversion factor to free acid grams of glycine (aminoacetic acid) equivalents). and 42.6 grams of sodium chloride in (d) Evaluation. Use the dissolution ac- about 500 milliliters of deionized water ceptance table and interpretation in in a 1-liter volumetric flask. Add cau- the United States Pharmacopeia XXI. tiously, and with swirling, 14.2 milli- [44 FR 48188, Aug. 17, 1979] liters of concentrated hydrochloric acid. Cool to room temperature. Dilute EDITORIAL NOTE: For FEDERAL REGISTER ci- to volume with deionized water and tations affecting § 430.215, see the List of CFR Sections Affected appearing in the Finding mix. Check the pH of the solution ob- Aids section of this volume. tained by diluting 50 milliliters of the stock solution to 900 milliliters with § 436.216 High-performance liquid deionized water. The pH should be chromatographic assay. 3.0±0.1. If necessary, adjust the pH of (a) Equipment. A suitable high-per- the stock solution with 50 percent so- formance liquid chromatograph dium hydroxide or concentrated hydro- equipped with: chloric acid. Recheck that the pH of (1) A suitable detection system speci- the working solution is 3.0±0.1. fied in the monograph for the drug (B) Working solution. Dilute 50 milli- being tested; liters of stock solution to 900 milli- (2) A suitable recording device of at liters with deionized water. least 25-centimeter deflection; (ii) Preparation of the working stand- (3) A suitable chromatographic data ard solutions. Accurately weigh ap- managing system; and proximately 28 milligrams for the 100- (4) An analytical column, 3 to 30 cen- milligram tablets and 56 milligrams for timeters long, packed with a material the 200-milligram tablets of the as defined in the monograph for the cefpodoxime proxetil working standard drug being tested; and if specified in and dissolve in 10 milliliters of meth- that monograph, the inlet of this col- anol. Dilute to 200 milliliters with dis- umn may be connected to a guard col- solution fluid. Prepare fresh daily. umn, 3 to 5 centimeters in length, (iii) Sample solutions. Filter the sam- packed with the same material of 40 to ple solutions through a 0.45-micron fil- 60 micrometers particle size. ter before use. Use the sample solution (b) Procedure. Perform the assay and as it is removed from the dissolution calculate the drug content using the vessel without further dilution. temperature, instrumental conditions, (iv) Procedure. Using a suitable spec- flow rate, and calculations specified in trophotometer and water as the blank, the monograph for the drug being test- determine the absorbance of each ed. Use a detector sensitivity setting standard and sample solution at the that gives a peak height for the work- absorbance peak at approximately 259 ing standard solution that is at least 50 nanometers. Determine the exact posi- percent of scale with typical chart tion of the absorption peak for the par- speed of not less than 2.5 millimeters ticular instrument used. per minute. Use the equipment de- (v) Calculations. Determine the per- scribed in paragraph (a) of this section. cent of label dissolved as follows: Use the reagents, working standard so- Percent dissolved = (Asam/Astd) X (Cs/L) lution, and sample solution described X V X P X F1 in the monograph for the drug being where: tested. Equilibrate and condition the Asam = Absorbance of the sample at 259 column by passage of 10 to 15 void vol- nanometers; umes of mobile phase followed by five Astd = Absorbance of the working standard so- replicate injections of the same volume lution at 259 nanometers; of the working standard solution. Cs = Concentration of the working standard Allow an operating time sufficiently preparation in milligrams per milliliter; L = Tablet strength, in milligrams per tab- long to obtain satisfactory separation let; and elution of the expected components P = Purity of the reference standard in per- after each injection. Record the peak cent; responses and calculate the prescribed
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system suitability requirements de- (3) Resolution. Calculate the resolu- scribed for the system suitability test tion (R) as follows: in paragraph (c) of this section. (c) System suitability test. Select the − 2()tRj t Ri system suitability requirements speci- R = fied in the monograph for the drug + being tested. Then, using the equip- wi w j ment and procedure described in this where: section, test the chromatographic sys- tRj=Retention time of a solute eluting after i tem for assay as follows: (tRj is larger than tRi´) (1) Trailing factor or asymmetry factor. tRi=Retention time for any solute; Calculate either the trailing factor (T), wi=Width of peak at baseline for any solute; from distances measured along the hor- and izontal line at 5 percent of the peak wj=Width of peak at baseline for any solute eluting after i. height above the baseline or the asym- metry factor (As) measured at a point (4) Coefficient of variation (relative 10 percent of the peak height from the standard deviation). Calculate the coef- baseline; whichever is required in the ficient of variation (SR in percent) as appropriate monograph, as follows: follows:
W n 1 2 T = 0. 05 − 2 ∑(XXi ) 2 f 100 S = i=1 where: R X N −1 W0.05=Width of peak at 5 percent height; and f=Horizontal distance from point of ascent to a point coincident with maximum peak where: height. X¯ is the mean of N of individual measure- ments of Xi. = a+b As If the complete operating system 2a meets the system suitability require- where: ments of the monograph for the drug a=Horizontal distance from point of ascent being tested, proceed as described in to point of maximum peak height; and paragraph (b) of this section, except al- b=Horizontal distance from the point of max- ternate injections of the working imum peak height to point of descent. standard solution with injections of (2) Efficiency of the column. Calculate the sample solution. the number of theoretical plates (n) of (5) Capacity factor. Calculate the ca- the column as follows: pacity factor (k), if required in the monograph as follows: t t− t n = 5.545 R k = r m Wh tm where: where: n=Efficiency, as number of theoretical plates tr=Retention time of solute; and for column; tm=Retention time of solvent or unretained tR=Retention time of solute; and substance, calculated as follows: Wh=Peak width at half-height. Calculate the absolute efficiency of the col- (31416 . )(DL2 )( )(0.75) umn, (reduced plate height) (hr t = m 4F = (L )(10 , 000 ) where: hr D=Column diameter in centimeters; ()n)( d p L=Column length in centimeters; 0.75=Average total column porosity; and where: F=Flow rate in milliliters per minute. L=Length of column in centimeters; n=Number of theoretical plates; and [51 FR 11572, Apr. 4, 1986, as amended at 54 dp FR 47351, Nov. 14, 1989] 396
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§ 436.217 Film-coat rupture test. (c) Preparation of carbenicillin indanyl sodium sample and working standard so- (a) Immersion fluid. Dilute 6.0 milli- lutions. Accurately weigh approxi- liters of hydrochloric acid to 1,000 mil- mately 125 milligrams of the liliters with water. During the per- carbenicillin indanyl sodium sample or formance of the test maintain the im- working standard into a 25-milliliter mersion fluid at a temperature of 37±0.5 volumetric flask. Dissolve and dilute °C by using a thermostatically con- to volume with distilled water. Trans- trolled water bath. fer a 5-milliliter aliquot to a 50-milli- (b) Immersion vessel. Use a suitable liter glass-stoppered centrifuge tube. vessel, such as a 1-liter beaker. Add 15 milliliters of the phosphate-cit- (c) Operation. Add 750 milliliters of rate buffer and 20 milliliters of 4-meth- immersion fluid to the immersion ves- yl-2-pentanone; stopper and shake the sel. tube for 10 seconds. Centrifuge at 2,000 (d) Procedure. Drop a tablet into the revolutions per minute for 10 minutes immersion fluid and record the time to separate the phases. Remove about for the tablet coat to rupture. Repeat 15 milliliters of the upper (4-methyl-2- the test with a further 19 tablets, test- pentanone solvent) phase and proceed ing not more than 10 tablets with a as directed in paragraph (e) of this sec- given volume of immersion fluid. tion. (e) Evaluation. The tablets pass the (d) Preparation of the blank. Place a 5- film-coat rupture test if the mean coat milliliter aliquot of distilled water into rupture time does not exceed 20 sec- a 50-milliliter glass-stoppered cen- onds and not more than 2 tablets have trifuge tube, add 15 milliliters of phos- a coat rupture time exceeding 40 sec- phate-citrate buffer and 20 milliliters onds. of 4-methyl-2-pentanone; stopper and [52 FR 42432, Nov. 5, 1987] shake the tube for 10 seconds. Cen- trifuge at 2,000 revolutions per minute for 10 minutes to separate the phases. Subpart F—Chemical Tests for Remove about 15 milliliters of the Specific Antibiotics upper phase and proceed as directed in paragraph (e) of this section. § 436.300 Polarimetric assay of (e) Procedure. Fill the polarimeter carbenicillin indanyl sodium. tube with the blank solution prepared (a) Equipment. Polarimeter capable of as described in paragraph (d) of this measuring optical rotatory activity at section. Place the tube in the polarim- 365 nanometers: Perkin-Elmer Model eter. Adjust the polarimeter to zero ro- 141 or equivalent, with a suitable 1-dec- tation using a light source with a imeter polarimeter tube. wavelength of 365 nanometers. Use the (b) Reagents—(1) 4-methyl-2-pentanone. same procedure to determine the opti- Meets ACS specifications. cal rotation of both the sample solu- (2) Phosphate-citrate buffer. Dissolve tion and the working standard solution 61.0 grams of anhydrous disodium phos- prepared as directed in paragraph (c) of phate and 11.0 grams of citric acid in this section. 950 milliliters of distilled water. Adjust (f) Calculations. Calculate the the pH to 6.0 with 6N hydrochloric acid. carbenicillin content (potency) of the Dilute to 1,000 milliliters with distilled sample on an anhydrous basis as fol- water. lows:
Degrees of rotation of sample solution× weight of working standard×100 × micrograms of carbenicillin in each Micrograms of milligram of the working standard carbenicillin per = Degrees of rotation of working standard solution × milligram of sample weight of sample×(100 − m )
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where: m=moisture content of the sample. chromatography tank. Allow the sol- vent front to travel about 15 centi- § 436.301 Thin layer chromatography meters from the starting line and then identity test for carbenicillin remove the plate from the tank. Heat indanyl. the plate for 30 minutes at 80° C. in a Using the sample solution prepared circulating air oven and then allow the as described in the section for the anti- plate to cool to room temperature. biotic drug to be tested, proceed as de- Place the plate in the iodine vapor scribed in paragraphs (a), (b), (c), and chamber for about 30 seconds, remove (d) of this section. the plate and spray it with the ferric (a) Equipment—(1) Chromatography chloride-potassium ferricyanide rea- tank. A rectangular tank, approxi- gent. Carbenicillin indanyl appears as a × × mately 9 9 3.5 inches lined with blue spot on a yellow-green background Whatman’s 3MM chromatographic at an Rf of about 0.5. The test is satis- paper (0.3 millimeters) or equivalent. factory if the sample compares quali- (2) Iodine vapor chamber. A rectangu- tatively with the standard. lar tank approximately 9 × 9 × 3.5 inches, with a suitable cover, contain- [39 FR 18944, May 30, 1974, as amended at 41 ing iodine crystals. FR 18509, May 5, 1976] (3) Plates. Use 20 × 20 centimeters thin layer chromatography plates coated § 436.302 Clindamycin vapor phase with silica gel G or equivalent to a chromatography. thickness of 250 microns. (a) Equipment. Gas chromatograph (b) Reagents—(1) Extraction solvent. equipped with a flame ionization detec- Mix ethyl acetate, acetone, pyridine, tor: Barber-Colman 5,000 or equivalent. water, and acetic acid in volumetric (b) Reagents. (1) Pyridine, reagent proportions of 100:200:25:75:1.5 respec- grade, dried over sodium sulfate. tively. (2) Chloroform, reagent grade. (2) Developing solvent. Mix ethyl ace- (3) Acetic anhydride, reagent grade, tate, acetone, pyridine, water, and ace- used as acteylating agent. tic acid in volumetric proportions of (4) Internal standard: Prepare a solu- 300:400:25:75:2 respectively. tion containing 3 milligrams of choles- (3) Ferric chloride-potassium ferricya- tane per milliliter in pyridine. nide reagent. Immediately before use, (c) Typical conditions. (1) Column: 4 mix 100 milliliters of a 1 percent ferric feet × 4 millimeters ID, glass, with 1 chloride solution in 1 percent hydro- percent SE–30 on Diatoport S (60/80 chloric acid with 100 milliliters of a 1 mesh), or equivalent. percent potassium ferricyanide solu- (2) Temperatures: Column 200° C.; de- tion and 75 milliliters of methanol. tector 215° C.; injection port, ambient (c) Preparation of working standard so- temperature. lution. Weigh an amount of the carbenicillin indanyl working standard (3) Carrier gas: Helium approxi- equivalent to approximately 10 milli- mately 120 milliliters per minute. grams of carbenicillin into a 50-milli- (4) Detector: Hydrogen flame—hydro- liter Erlenmeyer flask. Dissolve the gen at 120 pounds per square inch, air material in sufficient extraction sol- at 40 pounds per square inch. vent to make a solution containing 1 (5) Sensitivity: 1,000; attenuation, 2 milligram carbenicillin per milliliter. for clindamycin, 1 for internal stand- (d) Procedure. Pour developing sol- ard: 2×10¥8 amperes. vent into the bottom of the chroma- (d) Preparation of clindamycin sample tography tank. Cover and seal the and working standard solutions. Accu- tank. Allow it to equilibrate for 1 hour. rately weigh approximately 15 milli- Prepare a plate as follows: On a line 2 grams of sample or working standard centimeters from the base of the silica into a glass-stoppered conical 15-milli- gel plate, and at intervals of 2 centi- liter centrifuge tube. Add 1.0 milliliter meters, spot 10 microliters of the of chloroform, 1.0 milliliter of internal standard solution and the sample solu- standard solution, and 0.6 milliliter of tion. The plate should be air dried for acetic anhydride. Agitate the tubes to 30 minutes. Place the plate into the insure dissolution of the sample and
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complete mixing of the liquids. Pro- (2) Pyridine, reagent grade. ceed as directed in paragraph (e) of this (3) Chloroform, reagent grade. section. (4) Internal standard: Prepare a solu- (e) Procedure. Cover the top of each tion containing 5 milligrams of centrifuge tube with a plastic cap. cholesteryl benzoate per milliliter in Punch a small hole in the top of each chloroform. cap to allow vapor to escape. Place the (c) Typical conditions. (1) Column: 6 ° tubes in a 100 C. drying oven for 2.5 feet × 2 millimeters ID, glass, with 1 hours. Remove the tubes from the oven percent UC–W98 on Chromosorb WHP and allow to cool. Take the plastic cap (80/100 mesh) or equivalent. from each tube and replace with the (2) Temperatures: Column 275° C.; de- glass stopper. Centrifuge 10–15 minutes ° ° at 2,000–2,500 r.p.m. to separate the tector 290 C.; injection port 280 C. white solid from the liquid in the tube. (3) Carrier gas: Helium approxi- Inject 0.5 microliter of the clear liquid mately 60 milliliters per minute. into the gas chromatograph. Use the (4) Detector: Hydrogen flame ioniza- conditions and materials listed in para- tion—hydrogen at 12 pounds per square graphs (a), (b), and (c) of this section. inch, air at 32 pounds per square inch. The conditions should be adequate to (5) Sensitivity: 1,000; attenuation, 16; maintain a stable baseline and provide 1 × 10¥ 9 amperes. at least 60 percent deflection of the re- (d) Preparation of clindamycin corder scale by the clindamycin peak. palmitate hydrochloride sample and work- The resolution of the peaks should be ing standard solutions. Accurately weigh complete. The elution order is: Internal approximately 15 milligrams of both standard, clindamycin, and the sample and the working standard epiclindamycin (if present). Calculate into separate glass-stoppered, conical the clindamycin content as directed in 15-milliliter centrifuge tubes. Add 1.0 paragraph (f) of this section. milliliter of internal standard solution, (f) Calculations. Calculate the 1.0 milliliter of pyridine, and 0.5 milli- clindamycin content of the sample as liter of acetic anhydride to each tube. follows: Agitate the tubes to insure dissolution and complete mixing of the liquids. R× W × f Micrograms of clindamycin= u s Proceed as directed in paragraph (e) of per milligram R ×W this section. s u (e) Procedure. Cover the top of each where: centrifuge tube with a plastic cap. Ru=Area of the clindamycin sample peak Punch a small hole in the top of each (at a retention time equal to that ob- served for the clindamycin standard)/ cap to allow vapor to escape. Place the Area of internal standard peak; tubes in a 100° C. drying oven for 2.5 Rs=Area of the clindamycin standard peak/ hours. Remove the tubes from the oven Area of internal standard peak; and allow to cool. Take the plastic cap Ws=Weight of the clindamycin working from each tube and replace with the standard in milligrams; glass stopper. Centrifuge 10–15 minutes Wu=Weight of the sample in milligrams; at 2,000–2,500 r.p.m. to separate the f=Potency of the clindamycin working standard in micrograms per milligram. white solid from the liquid in the tube. Inject 1 microliter of the clear liquid § 436.303 Clindamycin content of into the gas chromatograph. Use the clindamycin palmitate hydro- conditions and materials listed in para- chloride by vapor phase chroma- graphs (a), (b), and (c) of this section. tography. The conditions should be adequate to (a) Equipment. Gas chromatograph maintain a stable baseline and provide equipped with a flame ionization detec- at least 40 percent deflection of the re- tor: Hewlett-Packard 7606 4 or equiva- corder scale by the clindamycin lent. palmitate peak. The resolution of the (b) Reagents. (1) Acetic anhydride, re- peaks should be complete. The internal agent grade. standard will be eluted before the clindamycin palmitate. Calculate the 4 Available from: Hewlett Packard Co., P.O. clindamycin content as directed in Box 301, Loveland, CO 80537. paragraph (f) of this section.
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(f) Calculations. Calculate the (4) Detector: Hydrogen flame—hydro- clindamycin content of the sample as gen flow at 40 milliliters per minute. follows: Air flow at 400 milliliters per minute. (5) Sensitivity: 1 × 10¥ 9 amperes. R× W × f Micrograms of clindamycin= u s (d) Preparation of clindamycin phos- per milligram × phate sample solution. Accurately weigh RsW u approximately 12 milligrams of the where: clindamycin phosphate sample into a
Ru=Area of the sample peak (at a retention 50-milliliter glass-stoppered centrifuge time equal to that observed for the tube. Pipet 25 milliliters of the pH 9.0 clindamycin palmitate hydrochloride borate buffer into the centrifuge tube. standard)/Area of internal standard Add 10 milliliters chloroform and peak; shake vigorously for 15 minutes. Cen- Rs=Area of the clindamycin palmitate hy- trifuge the resulting mixture and pipet drochloride standard peak/Area of in- a 20-milliliter aliquot of the aqueous ternal standard peak; phase into a 35-milliliter centrifuge Ws=Weight of the clindamycin palmitate hydrochloride working standard in mil- tube. Add a weighed amount of intes- ligrams; tinal alkaline phosphatase equivalent 5 Wu=Weight of the sample in milligrams; to 50 units of activity and allow the f=Micrograms of clindamycin activity per solution to stand until the enzyme has milligram of clindamycin palmitate completely dissolved. Place the tube hydrochloride working standard. into a water bath at 37° C.±2° C. for 2.5 hours. After the 2.5-hour hydrolysis, § 436.304 Clindamycin phosphate allow the solution to cool and proceed vapor phase chromatography. as directed in paragraph (f) of this sec- (a) Equipment. Gas chromatograph tion. equipped with an electronic integrator (e) Preparation of the clindamycin hy- and with a flame ionization detector drochloride standard solution. Accu- that has a sensitivity of at least 1 × rately weigh approximately 9 milli- 10¥ 1 0 amperes: Hewlett-Packard 7600 4 grams of the clindamycin hydro- or equivalent. chloride working standard into a 35- (b) Reagents. (1) Trifluoroacetic anhy- milliliter glass-stoppered centrifuge dride. tube and dissolve in 20 milliliters of pH (2) Intestinal alkaline phosphatase. 9.0 borate buffer. Proceed as directed in (3) pH 9.0 borate buffer: Transfer 3.1 paragraph (f) of this section. grams of boric acid into a 1-liter volu- (f) Procedure. Add 10 milliliters of the metric flask containing 500 milliliters internal standard solution to each sam- of water, mix, and add 21 milliliters of ple and standard solution. Shake the 1.0N sodium hydroxide and 10 milli- centrifuge tubes vigorously for 30 min- liters of 0.1M magnesium chloride. Di- utes and centrifuge. Remove the aque- lute to volume with water and mix ous layer and discard. Shake the tubes well. again; mix in an ultrasonic mixer for 2 (4) Internal standard: Prepare a chlo- minutes, then centrifuge. No emulsion roform solution containing approxi- should be present at this stage. Re- mately 0.45 milligram hexacosane per move the remaining aqueous layer by milliliter. suction and transfer a 3-milliliter ali- quot of the chloroform layer to a 1- (5) Anhydrous sodium carbonate. dram tablet vial containing approxi- (c) Typical conditions. (1) Column: 2 mately 1 gram of anhydrous sodium feet × 3 millimeters ID, glass, with 1 sulfate. Swirl the vial to dry the chlo- percent SE–30 on Diatoport S (80/100 roform and transfer a 1-milliliter ali- mesh), or equivalent. quot to another 1-dram tablet vial. ° (2) Temperatures: Column, 180 C., de- Using a 0.25-milliliter pipet, add 0.25 ° tector, 215 C., injection port, ambient milliliter of trifluoracetic anhydride to temperature. (3) Carrier gas: Helium approxi- mately 60 milliliters per minute. 5 Defined such that 50 units hydrolyzes at least 20 micromoles of a clindamycin phos- phate authentic sample under the assay con- 4 See footnote 4 to § 436.303(a). ditions described in this section.
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each of the vials and place into a water (d) Preparation of spotting solutions— bath at 45° C.±2° C. for 30 minutes. Re- (1) Sample solution. Use the sample solu- move the vials from the bath, add tion prepared as described in the sec- about 10 granules of anhydrous sodium tion for the particular product to be carbonate to each vial, and allow to tested. stand for approximately 30 minutes. (2) Reference solutions. Prepare a solu- Centrifuge the vials for approximately tion containing 10 milligrams of an au- 10 minutes at 5,000 r.p.m. Inject 2 thentic hetacillin sample per milliliter microliters of each of the resulting so- in a 4:1 solution of acetone and 0.1N hy- lutions into the gas chromatograph. drochloric acid, and a solution of ampi- Use the conditions and materials listed cillin standard at 1 mg/ml in the same in paragraphs (a), (b), and (c) of this solvent. section. The elution order is: (e) Procedure. Spot a plate as follows: Epiclindamycin (if present), Apply approximately 10 microliters of clindamycin B (if present), the sample solution, 1 µ l, of the ref- clindamycin, and internal standard. erence hetacillin solution, and 1 µ l, of Calculate the clindamycin content as the ampicillin reference solution on a directed in paragraph (g) of this sec- line 1.5 centimeters from the base of tion. the silica gel plate and at intervals of (g) Calculations. Calculate the not less than 2.0 centimeters. Pour de- clindamycin content of the sample as veloping solvent into the glass trough follows: in the bottom of the chromatography tank. After all spots are thoroughly Micrograms of clindamycin per milligram= dry, place the silica gel plate directly × × into the glass trough of the chroma- Ru W s f × tography tank. Cover and seal the RWs u tank. Allow the solvent front to travel where: about 11.5 centimeters from the bottom Ru=Area of the clindamycin sample peak of the plate, remove the plate from the (at a retention time equal to that ob- tank, and allow to air dry. Apply the served for the clindamycin standard)/ spray solution (do not saturate) and Area of internal standard peak; place immediately into an oven main- Ru=Area of the clindamycin standard peak/ tained at 90° C. Heat 15 minutes. Area of internal standard peak; (f) Evaluation. Measure the distance Ws=Weight of the clindamycin working standard in milligrams; the solvent front traveled from the starting line and the distance the spots Wu=Weight of the sample in milligrams; f=Potency of the clindamycin working are from the starting line. Calculate standard in micrograms per milligram. the Rf value by dividing the latter by the former. The sample and standard [39 FR 18944, May 30, 1974, as amended at 41 should have spots of corresponding R FR 24704, June 18, 1976] f values. § 436.305 Thin layer chromatographic [39 FR 18944, May 30, 1974, as amended at 45 identity test for hetacillin. FR 16472, Mar. 14, 1980] (a) Equipment—(1) Chromatography tank. A rectangular tank, approxi- § 436.306 Lincomycin gas liquid chro- mately 9 × 9 × 3.5 inches with a glass matography. solvent trough on the bottom. (a) Equipment. Gas chromatograph (2) Plates. Use 20 × 20 centimeter thin equipped with a flame ionization detec- layer chromatography plates coated tor; Barber-Colman 5000 or equivalent. with Silica Gel G or equivalent to a (b) Reagents. (1) Pyridine, reagent thickness of 250 microns. grade, kept over potassium hydroxide. (b) Developing solvent. Mix 650 milli- (2) Methanol, reagent grade, anhy- liters acetone with 100 milliliters dis- drous. tilled water, 100 milliliters benzene, (3) Ethanol, absolute, reagent grade. and 25 milliliters acetic acid. (4) Internal standard: Prepare a solu- (c) Spray solution. Dissolve 300 milli- tion containing 2 milligrams of grams of ninhydrin in 100 milliliters of tetraphenylcyclopentadienone per mil- ethanol. liliter in pyridine.
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(5) Silylating reagent: Mix (9+1) of Rs=Area of the lincomycin standard peak/ hexamethyldisilazane and trimethyl Area of internal standard peak; chlorosilane. Ws=Weight of the lincomycin working (c) Typical conditions. (1) Column: 4 standard in milligrams; × Wu=Weight of the sample in milligrams; feet 3 millimeters ID, glass, with 3 f=Potency of lincomycin working standard percent SE–30 on Gas-Chrom Q (100/120 in micrograms per milligram. mesh), or equivalent. ° (2) Temperatures: Column 225 C.; de- Percent lincomycin B tector 280° C., injector 270° C. = ×100 (3) Carrier gas: Helium at 15 pounds B content A+ B per square inch. where: (4) Detector: Hydrogen flame ioniza- A=Area of lincomycin peak of the sample; tion—hydrogen at 20 pounds per square B=Area of lincomycin B peak of the sample inch, air at 40 pounds per square inch. corrected for the attenuation adjust- (5) Sensitivity: 100; attenuation 2; ment. current 2 × 10¥ 8 amperes. [39 FR 18944, May 30, 1974, as amended at 46 (d) Preparation of lincomycin sample FR 3839, Jan. 16, 1981] and working standard solutions. Prepare the sample and working standard as § 436.307 Spectinomycin vapor phase follows: Weigh accurately an aliquot of chromatography. about 40 milligrams into a 10-milliliter (a) Equipment. Gas chromatograph volumetric flask, add sufficient pyri- equipped with a flame ionization detec- dine to dissolve, and make to mark. tor; Barber-Colman 5,000 or equivalent. Transfer a 1-milliliter aliquot to a (b) Reagents. (1) Dimethylformamide, glass-stoppered conical centrifuge tube reagent grade, kept dry over anhydrous and proceed as directed in paragraph sodium sulfate. (e) of this section. (2) Internal standard: Prepare a solu- (e) Procedure. Add 0.2 milliliter of the tion containing 2 milligrams of silylating reagent to each centrifuge triphenylantimony per milliliter in dry tube and allow to stand at least 30 min- dimethylformamide. utes. Then add exactly 1 milliliter of (3) Silylating reagent: Hexamethyl- the internal standard, shake well, and disilazane. centrifuge. Inject 5 microliters of the (c) Typical conditions. (1) Column: 4 supernatant into the gas chro- feet by 4 millimeters ID, glass, with 5 matograph. Use the typical conditions percent SE–52 on Diatoport S (80/100 and materials listed in paragraphs (a), mesh), or equivalent. (b), and (c) of this section. The condi- (2) Temperatures: Column 215° C.; de- tions should be adequate to provide at tector 270° C.; injection port 265° C. least 60 percent scale deflection with (3) Carrier gas: Helium 93 milliliters the lincomycin peak and to maintain a per minute at 15 pounds per square stable base line. The resolution of the inch. peaks should be complete. The elution (4) Detector: Hydrogen flame—hydro- order is lincomycin B, lincomycin, and gen at 20 pounds per square inch, air at the internal standard. If necessary, ad- 40 pounds per square inch. just the current setting for the linco- (5) Sensitivity: 1,000; attenuation, 10 mycin B peak to give a satisfactory re- for both spectinomycin and internal sponse relative to that of the linco- standard; 2 × 10¥ 6 amperes. mycin peak. Calculate the lincomycin (d) Preparation of spectinomycin sample content and lincomycin B content as and working standard—(1) Working directed in paragraph (f) of this sec- standard and bulk antibiotic solutions. (i) tion. Accurately weigh approximately 30 (f) Calculations. milligrams of sample or working stand- ard into separate glass-stoppered 25- Lincomycin content R× W × f milliliter Erlenmeyer flasks. of sample in micrograms = u s (ii) Add 10 milliliters of the internal × per milligram RWs u standard solution and 1.0 milliliter of where: hexamethyldisilazane to each flask. Ru=Area of the lincomycin sample peak/ Agitate the flasks to insure dissolution Area of internal standard peak; of the sample and working standard
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and complete mixing of the liquids. (b) Preparation of spotting solutions. Shake the flasks intermittently for 1 Prepare solutions of the working hour. Proceed as directed in paragraph standard and sample as follows: Accu- (e) of this section. rately weigh a portion of the working (2) Finished product solutions. Prepare standard and sample and dilute with the sample for assay as directed in the methanol to obtain a concentration of individual section for each antibiotic 1 milligram per milliliter of antibiotic product to be tested. to be tested. (e) Procedure. Inject 2.5 microliters of (c) Procedure. Fill the chamber to a each solution into the gas chro- depth of 0.6 centimeter with freshly matograph. Use the conditions and ma- prepared solvent. Draw a starting line terials listed in paragraphs (a), (b), and about 2.5 centimeters from and parallel (c) of this section. The conditions to the bottom of the sheet. Wet the should be adequate to maintain a sta- sheet thoroughly with the pH 3.5 buffer ble base line and provide at least 60 and blot it firmly between sheets of ab- percent deflection of the recorder scale sorbent paper. Starting about 5 centi- by the spectinomycin peak. The resolu- meters from the edge of the sheet and tion of the peaks should be complete. at 1.5-centimeter intervals, apply to The internal standard will be eluted be- the starting line 2 microliters each of fore spectinomycin. Calculate the standard solution, sample solution, and spectinomycin content as directed in a 1:1 mixture of the standard and sam- paragraph (f) of this section. ple solutions. Allow a few minutes for (f) Calculations. Calculate the the sheet to dry partially, and while spectinomycin content of the sample as still damp place it in the chamber with follows: the bottom edge touching the solvent. Micrograms When the solvent front has risen about R× W × f 10 centimeters, remove the sheet from of spectinomycin = u s × the chamber. Expose the paper to am- per milligram RWs u monia vapor. Examine the dried sheet where: under a strong source of ultraviolet Ru=Area of spectinomycin sample peak (at light and record the position of any flu- a retention time equal to that observed orescent spots. Measure the distance for the spectinomycin standard)/Area of internal standard peak; the solvent front traveled from the starting line and the distance that the Rs=Area of the spectinomycin standard peak/Area of internal standard peak; fluorescent spots are from the starting Ws=Weight of the spectinomycin working line. Calculate the Rf value by dividing standard in milligrams; the latter by the former. Wu=Weight of the sample in milligrams; f=Potency of the spectinomycin working [39 FR 18944, May 20, 1974, as amended at 44 standard in micrograms per milligram. FR 30333, May 5, 1979; 45 FR 16472, 16474, Mar. 14, 1980] § 436.308 Paper chromatography iden- tity test for tetracyclines. § 436.309 Anhydrotetracyclines and 4- epianhydrotetracycline. (a) Equipment—(1) Sheet (chromatographic). Whatman No. 1 filter Determination of 4- paper for chromatography, 20 × 20 cen- epianhydrotetracycline and timeters. anhydrotetracyclines in tetracycline, (2) Chamber (chromatographic). Cylin- tetracycline hydrochloride, tetra- drical glass chromatographic jar, 25 cycline phosphate, and in dosage forms centimeters high by 12 centimeters in thereof is as follows: diameter, with a ground-glass lid. (a) Screening procedure for total (3) Preparation of solutions—(i) pH3.5 anhydrotetracyclines content—(1) Sample buffer. Mix 13.93 volumes of 0.1M citric solution preparation—(i) Bulk packaged acid with 6.07 volumes of 0.2M of diso- for repacking or for use in the manufac- dium phosphate. ture of another drug. Accurately weigh (ii) Solvent (organic phase). Mix chlo- approximately 50 milligrams of the roform, nitromethane, and pyridine in sample into a 50-milliliter volumetric volumetric proportions of 10:20:3, re- flask and add 10 milliliters of 0.1N hy- spectively. drochloric acid. Shake until sample is
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completely dissolved, and then dilute liters of 0.1N hydrochloric acid and to volume with water. shake on a mechanical shaker for 5 (ii) Sterile dispensing containers. Pro- minutes. Dilute to volume with water ceed as directed in paragraph (a)(1)(i) and filter through a fluted filter paper. of this section. Discard the first 20 milliliters of fil- (iii) Capsules. Transfer a representa- trate and collect the next 20 milli- tive quantity of capsule contents liters. equivalent to 250 milligrams of tetra- (v) Oral powders and suspensions. Pro- cycline hydrochloride to a 250-milli- ceed as described in paragraph (b) of liter volumetric flask. Add 50 milli- this section. liters of 0.1N hydrochloric acid and (2) Test procedure. Using a suitable shake on a mechanical shaker for 5 spectrophotometer, determine the ab- minutes. Dilute to volume with water sorbance of the sample solution pre- and filter through a fluted filter paper. pared as directed in paragraph (a)(1) of Discard the first 20 milliliters of fil- this section at 430 millimicrons using trate and collect the next 20 milli- 0.02N hydrochloric acid as a blank. liters. Then accurately dilute 1.0 milliliter of (iv) Tablets. Grind a representative the sample solution to 100 milliliters number of tablets to a fine powder. with 0.02N hydrochloric acid and deter- Transfer an amount of the powder mine the absorbance of this solution at equivalent to 250 milligrams of tetra- 356 millimicrons, using 0.02N hydro- cycline hydrochloride to a 250-milli- chloric acid as a blank. liter volumetric flask. Add 50 milli- (3) Calculations.
[]a430−(.) a 356 × 0 0019× 100 Percent anhydrotetracyclines = 195
where: anhydrotetracyclines and 4- a430=Absorptivity (1%, 1 cm.) of sample at epianhydrotetracycline described in 430 millimicrons; paragraph (b) of this section. If the re- sults of the test described in paragraph For bulk, Absorbance ×50 × 10 = (a) of this section for total absorptivity anhydrotetracyclines content are with- Milligrams of sample in the required limits in the case of For sterile dispensing containers, capsules, bulks, injectables, tablets, and cap- and tablets; absorptivity=Absorb- sules, these results may be submitted × ance 10; a356=Absorptivity (1%, 1 cm.) in lieu of the results of the test for 4- of sample at 356 millimicrons; epianhydrotetracycline and that test Absorbance ×50 × 1000 as described in paragraph (b) of this For bulk, section need not be performed. absorptivity = Milligrams of sample (b) Determination of For sterile dispensing containers, capsules anhydrotetracyclines content and 4- and tablets; absorptivity=Absorb- epianhydrotetra-cycline content—(1) Ap- ance×1,000; 0.0019×Absorbance ratio paratus and reagents—(i) (A430/A356) observed with tetracycline; Chromatographic tubes (15 millimeters 195=Absorptivity (1%, 1 cm.) of ID × 170 millimeters long having an anhydrotetracycline hydrochloride at outlet tube 4 millimeters ID × 50 milli- 430 millimicrons. meters long). (4) Evaluation. If the total (ii) pH meter standardized at pH 7.0 anhydrotetracyclines content deter- and at pH 10.0. mined by the screening procedure de- (iii) Diatomaceous earth, acid- scribed in paragraph (a) of this section washed (Celite 545 or equivalent). exceeds 2 percent for bulks and 3 per- (iv) EDTA buffer. Dissolve 0.1 mole cent for injectables, tablets, and cap- ethylenediaminetetraacetic acid diso- sules, perform the determination for dium salt in 800 milliliters of water.
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Adjust to pH 7.8 with ammonium hy- chloride into a 50-milliliter beaker and droxide, reagent grade, and dilute to 1 add sufficient 0.1N hydrochloric acid to liter with water. make 10 milliliters. Quickly adjust the (v) Chloroform, spectrophotometric pH to 7.8 with the diluted ammonium grade. hydroxide, and if necessary, with 1N (vi) Diluted ammonium hydroxide: hydrochloric acid and 0.1N hydro- Mix 1 volume of ammonium hydroxide, chloric acid. Quantitatively transfer reagent grade, with 9 volumes of dis- this solution to a 25-milliliter flask by tilled water. rinsing the beaker with EDTA buffer, (vii) 0.1N hydrochloric acid. fill to volume with EDTA buffer, and (viii) 1.0N hydrochloric acid. shake well. Use this solution without (2) Preparation of support phase. Add 5 delay to prepare a column as directed milliliters of EDTA buffer to 10 grams in paragraph (b)(4) of this section. of diatomaceous earth and mix until (v) Oral powders. Reconstitute as di- the diatomaceous earth is uniformly rected in the labeling and proceed as moistened. It will no longer be free- directed in paragraph (b)(3)(iv) of this flowing. section. (3) Preparation of sample solutions. (vi) Sterile dispensing containers. Pro- Prepare the sample solutions as fol- ceed as directed in paragraph (b)(3)(i) lows: of this section. (i) Tetracycline, tetracycline phosphate (4) Column preparation. Pack support complex, and tetracycline hydrochloride phase into the chromatographic tube bulk packaged for repacking or for use in by increments and firmly tamp down the manufacture of another drug. Place each increment. Do not use any glass an amount of sample equivalent to 250 wool in the column outlet. Add enough milligrams of tetracycline hydro- support phase to the column to reach a chloride into a 50-milliliter beaker and height of 9 to 11 centimeters; then add dissolve in 10 milliliters of 0.1N hydro- 1 milliliter of sample solution to 1 chloric acid. Immediately adjust the gram of diatomaceous earth in a small pH to 7.8 with the diluted ammonium beaker, and mix thoroughly. Pack the hydroxide, and if necessary, with 1.0N sample: diatomaceous earth mixture on hydrochloric acid and 0.1N hydro- top of the column. Dry wash the beaker chloric acid. Quantitatively transfer with support phase and pack an addi- this solution to a 50-milliliter volu- tional 1-centimeter layer of support metric flask by rinsing the beaker with phase on top of the sample layer. EDTA buffer, fill to volume with EDTA (5) Column elution and fraction collec- buffer and shake well. Use this solution tion. Within 30 minutes after preparing without delay to prepare a column as the column, elute with chloroform. directed in paragraph (b)(4) of this sec- Collect 5 successive fractions of 5 milli- tion. liters, 5 milliliters, 10 milliliters, 10 (ii) Capsules. Proceed as directed in milliliters, and 5 milliliters. During paragraph (b)(3)(i) of this section, ex- elution, two clear separate yellow cept pool the contents of a representa- bands will appear on the column. The tive number of capsules and use an first band is anhydrotetracyclines and amount of the pooled capsule contents will almost always elute in the first 5- equivalent to 250 milligrams of tetra- milliliter fraction, but occasionally in cycline hydrochloride. the first and second 5-milliliter frac- (iii) Tablets. Proceed as directed in tions. The second band is 4- paragraph (b)(3)(i) of this section, ex- epianhydrotetracycline and will elute cept grind tablets to a powder in a in the remaining fractions. Label the small mortar and use an amount of fraction or fractions containing the powder equivalent to 250 milligrams of first yellow band anhydrotetracyclines. tetracycline hydrochloride. Label the fractions after the first yel- (iv) Oral suspension and pediatric low band 4-epianhydrotetracycline. De- drops. Place 5 milliliters of oral suspen- termine the absorbance of each frac- sion equivalent to 125 milligrams of tion at a wavelength of 438 nanometers tetracycline hydrochloride or 2 milli- using a suitable spectrophotometer liters of pediatric drops equivalent to equipped with a 1.0-centimeter cell and 200 milligrams of tetracycline hydro- chloroform as the blank. If necessary,
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make appropriate dilutions with (6) Calculations—(i) Percent choloroform to obtain a readable value. anhydrotetracyclines. Calculate the per- cent anhydrotetracyclines as follows:
× × Number of milligrams of anhydrotetracyclines in each = A b c fraction containing anhydrotetracyclines 20.28
where: tetracycline phosphate complex bulk A=Absorbance of the sample solution at 438 packaged for repacking or for use in nanometers; the manufacture of another drug = b=Volume of fraction in milliliters; c=Dilution factor of the fraction (for exam- Total weight of anhydrotetracyclines ple, if 2 milliliters of the fraction are in the sample diluted to 10 milliliters for reading, c 100 × will be 5). Weight of the sample 20.28=Absorptivity (1 milligram per milli- Percent anhydrotetracyclines in dosage liter, 1 centimeter) of anhydrotetra- forms= cyclines in chloroform at 438 nanometers. Total weight of anhydrotetracyclines Total weight of anhydrotetracyclines in in the sample the sample=Sum of weights of 100 × anhydrotetracyclines in the fractions Tetracycline content of the sample labeled anhydrotetracyclines × Number of milliliters in the sample solution (ii) Percent 4-epianhydrotetracycline. Percent anhydrotetracyclines in tetra- Calculate the percent 4- cycline, tetracycline hydrochloride, epianhydrotetracycline as follows:
× × Number of milligrams of 4-epianhydrotetracycline = A b c in each fraction labeled 4-epianhydrotetracycline 20.08
where: Total weight of 4-epianhydrotetracycline A=Absorbance of the sample solution at 438 in the sample nanometers; 100 × b=Volume of the fraction in milliliters; Weight of the sample c=Dilution factor of the fraction (for exam- ple, if 2 milliliters of the fraction are Percent 4-epianhydrotetracycline in dos- diluted to 10 milliliters for reading, c age forms= will be 5); Total weight of 4-epianhydrotetracycline 20.08=Absorptivity (1 milligram per milli- in the sample liter, 1 centimeter) of 4- 100 × epianhydrotetracycline in chloroform Tetracycline content of the sample at 438 nanometers. Total weight of 4-epianhydrotetracycline [39 FR 18944, May 30, 1974, as amended at 40 in the sample=Sum of weights of 4- FR 22251, May 22, 1975; 43 FR 11153, Mar. 17, epianhydrotetracycline in the fractions 1978] labeled 4-epianhydrotetracycline × Number of milliliters in the sample so- § 436.310 Thin layer chromatography lution identity test for mitomycin. Percent 4-epianhydrotetracycline in tetra- (a) Equipment—(1) Chromatography cycline, tetracycline hydrochloride, tank. A rectangular tank, approxi- tetracycline phosphate complex bulk mately 9 × 9 × 3.5 inches, lined with fil- packaged for repacking or for use in the manufacture of another drug = ter paper and with a solvent trough on the bottom.
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(2) Plates. Use 20 by 20 centimeter mately 23 centimeters long, 23 centi- thin layer chromatography plates coat- meters high, and 9 centimeters wide, ed with silica gel G or equivalent, to a equipped with a glass solvent trough in thickness of 250 microns. the bottom and a tight-fitting cover for (b) Reagents—(1) Developing solvent. the top. Line the inside walls of the Mix n-butanol, glacial acetic acid, and tank with Whatman’s 3MM water in volumetric proportions of chromatographic paper (0.33 millime- 4:2:1, respectively. ters) or equivalent. (2) Spray solution. Prepare a one-per- (2) Plates. Use 20- by 20-centimeter cent solution of ninhydrin in ethanol. thin layer chromatography plates coat- (c) Preparation of spotting solutions. ed with Silica Gel G or equivalent to a Prepare solutions of the sample and thickness of 250 microns. working standard, each containing 1 (b) Reagents—(1) Developing solvent. milligram of mitomycin per milliliter, Mix methyl alcohol, chloroform, pyri- in water. dine, and distilled water in volumetric (d) Procedure. Pour the developing proportions of 90:80:10:30, respectively. solvent into the solvent trough on the bottom of the tank and onto the paper (2) Spray solution. Dissolve 300 milli- lining the walls of the tank. Cover and grams of ninhydrin in 100 milliliters of seal the tank. Allow it to equilibrate ethyl alcohol. for 30 minutes. Prepare a plate as fol- (c) Preparation of working standard. lows: Apply spotting solutions on a line Weigh an amount of the amoxicillin 2.5 centimeters from the base of the working standard equivalent to 200 silica gel plate and at points 2.0 centi- milligrams of amoxicillin into a 50-mil- meters apart. Apply approximately 2 liliter volumetric flask and bring to microliters of the working standard so- volume with 0.1N hydrochloric acid. lution to points 1 and 3. When these (d) Procedure. Pour the developing spots are dry, apply approximately 2 solvent into the glass trough on the microliters of sample solution to bottom of the tank and onto the paper points 2 and 3. After all spots are thor- lining the walls of the tank. Cover and oughly dry, place the silica gel plate seal the tank. Allow it to equilibrate into the trough in the chromatography for at least 2 hours. Spot duplicate tank. Cover and seal the tank tightly. plates by applying approximately 5 Allow the solvent front to travel about microliters each of standard and sam- 10 centimeters from the starting line. ple solutions on a line 1.5 centimeters Remove the plate and allow it to air from the base of the plate and at inter- dry. After the plate is dry, spray light- vals of not less than 2.0 centimeters. ly with the spray solution. Heat the All solutions must be spotted within 10 plate in an oven at 110° C. for 10–15 minutes of preparation. Place spotted minutes. Mitomycin appears as a pink plate in a desiccator until solvent has spot. evaporated from spots. Place the plate (e) Evaluation. The sample and stand- into the glass trough at the bottom of ard should have spots of corresponding the chromatography tank. Cover the Rf value (approximately 0.51), and tank. Allow the solvent to reach the 15- standard and sample combined should centimeter scored mark, remove the appear as a single spot of correspond- plate from the tank and dry with a cur- ing Rf value. rent of warm air until there is no de- [39 FR 18944, May 30, 1974, as amended at 49 tectable solvent odor. Apply the ninhy- FR 2242, Jan. 19, 1984] drin spray solution to the plate—do not saturate—and place immediately into § 436.311 Thin layer chromatography an oven maintained at 110° C for 15 identity test for amoxicillin. minutes. Using the sample solution prepared (e) Evaluation. Measure the distance as described in the section for the anti- the solvent front traveled from the biotic drug to be tested, proceed as de- starting line and the distance the spots scribed in paragraphs (a) through (e) of are from the starting line. Calculate this section. the Rf value by dividing the latter by (a) Equipment—(1) Chromatography the former. Amoxicillin has an Rf value tank. A rectangular tank, approxi- of about 0.53. The sample and standard
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should have spots of corresponding Rf the percent zinc in the sample as fol- values. lows: [39 FR 34032, Sept. 23, 1974; 48 FR 11427, Mar. C ×100, 000 18, 1983, as amended at 49 FR 2242, Jan. 19, Percent zinc = 1984] Milligrams of sample ×(100 − m) § 436.312 Atomic absorption method for determining the zinc content of where: zinc bacitracin. C=Concentration of zinc in the sample so- lution in micrograms per milliliter; (a) Equipment. An atomic absorbance m=Percent moisture in the sample. spectrophotometer equipped with a zinc hollow-cathode discharge lamp, an [40 FR 15088, Apr. 4, 1975] air-acetylene flame, a nebulizer-burner system for introducing the sample so- § 436.316 Determination of penicillin G content. lution into the flame, an optical dis- persing device (such as a (a) Reagents. The reagents are freshly monochromator) for isolating a reso- prepared every three days and are of nance line of zinc from others produced such quality that when used in this by the emission source, and a suitable procedure with an authentic sample of radiation detector and recorder. penicillin G, not less than 97 percent of (b) Preparation of working standard penicillin G is recovered. and sample solutions—(1) (1) Amyl acetate (iso-amyl acetate) solu- Workingstandard solutions. Prepare a tion. Saturate the amyl acetate (boil- standard stock solution containing 10 ing range 138.5° C—141.5° C) with the N- milligrams of zinc per milliliter as fol- ethylpiperidine salt of penicillin G by lows: Weigh 3.11 grams of zinc oxide adding 2 milligrams of the salt for each into a 250-milliliter volumetric flask, 1.0 milliliter of the solvent. Cool this add 80 milliliters of 1N HCl, warm to solution to 0° C—8° C and filter it dissolve, cool to room temperature, through a sintered-glass filter imme- and dilute to volume with water. Di- diately before use. lute aliquots of this standard stock so- (2) Acetone solution. Saturate reagent lution with 0.001N HCl to obtain three grade acetone with the N- working standard solutions containing ethylpiperidine salt of penicillin G respectively 0.5, 1.5, and 2.5 micrograms using 3 milligrams of salt for each 1 of zinc per milliliter. milliliter of acetone. Cool this solution (2) Sample solution. Accurately weigh to 0° C—8° C and filter it through a sin- approximately 200 milligrams of the tered-glass filter immediately before sample into a 100-milliliter volumetric use. flask. Dissolve and dilute to volume (3) N-ethylpiperidine solution. N- with 0.01N HCl. Transfer a 2.0-milliliter ethylpiperidine (boiling range 129.5° C— aliquot of this solution to a 200-milli- ° liter volumetric flask and dilute to vol- 131.0 C) should be stored in brown bot- ume with 0.001N HCl. tles in a refrigerator. Dilute 1.0 milli- (c) Procedure. Using 0.001N HCl as the liter of this reagent with 4.0 milliliters blank, adjust the absorbance of the in- of amyl acetate. Saturate this solution strument to zero at a detection wave- with the N-ethylpiperidine salt of peni- length of 213.8 nanometers. Determine cillin G, using about 3 milligrams of the absorbance of each standard solu- the salt for each 1.0 milliliter of solu- tion and the sample solution at 213.8 tion. Cool this solution to 0° C—8° C nanometers. and filter it through a sintered-glass (d) Calculations. Plot the absorbance filter immediately before use. versus the concentration of each of the (4) Phosphoric acid solution. Prepare working standard solutions. Draw a by dissolving 1.0 milliliter of reagent straight response line of best fit grade phosphoric acid (85 percent) in 4.0 through these points. Read the con- milliliters of water. Cool to 0° C—8° C centration of zinc in micrograms per and shake before using. milliliter corresponding to the absorb- (5) Silica gel. Use dry silica gel (mesh ance of the sample solution. Calculate size 6–16, Tyler standard). Place about
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0.5 gram of the silica gel in a micro fil- this section. Allow the amyl acetate to ter funnel (approximately 10-millime- remain in contact with the silica gel ter diameter) having a fritted-glass for exactly 20 seconds, then apply suc- disc of medium porosity. tion and collect the filtrate in a small (b) Procedure. Accurately weigh from test tube placed in a suction flash sur- 60 to 70 milligrams of the sample to be rounded by cracked ice. Pipet a 1.0-mil- tested, except if penicillin G procaine liliter aliquot of the amyl acetate fil- is to be tested weigh 90 to 100 milli- trate into a tared flat-bottom glass grams of sample, into a glass test tube tube (approximately 15 x 50 millime- or glass vial of approximately 10–milli- ters) containing 1.0 milliliter of ace- liter capacity. Add 2.0 milliliters of tone solution and 0.5 milliliter of N- water to dissolve or suspend (procaine) ethylpiperidine solution. The time ° ° the penicillin and cool to 0 C—5 C. elapsing between acidification and the Add 2.0 milliliters of amyl acetate so- addition of the filtrate to the above re- lution and 0.5 milliliter of phosphoric agents should not be more than 3 min- acid solution, stopper and shake the utes. Place the glass tube containing container vigorously for approximately 15 seconds. For penicillin G procaine, the mixture into a large weighing bot- add a second 0.5-milliliter portion of tle, stopper the bottle and allow to phosphoric acid solution and shake vig- stand for not less than 2 hours in a re- ° ° orously. Centrifuge to obtain a clear frigerator at 0 C—8 C. Remove the liq- separation of the two layers (approxi- uid from the precipitate by means of a mately 20 seconds). If any penicillin tared micro filter stick and wash with procaine remains undissolved, add a a total of 1.0 milliliter of acetone solu- third 0.5-milliliter portion of phos- tion adding the latter by means of a phoric acid solution, shake the con- hypodermic syringe equipped with a tainer vigorously, and centrifuge. After fine needle. Place the filter stick inside centrifuging, remove as much of the the glass tube, dry under vacuum at amyl acetate layer as possible, usually room temperature for not less than 1 about 1.7 milliliters to 1.8 milliliters, hour, and weigh. (The N- with a suitable hypodermic needle and ethylpiperidine penicillin G residues syringe and place the portion removed can be saved for saturating reagents). into the filter funnel containing silica (c) Calculations. Calculate the percent gel, described in paragraph (a)(5) of penicillin G content as follows:
Percent penicillin Milligrams N- ethylpiperidine penicillin pre cipitate ×149. 4 = G content Weight of sample in milligrams
[42 FR 59857, Nov. 22, 1977] (i) Circulating pump of a centrifugal, immersion type. Tubing approximately § 436.317 Solubility characteristic test 1 centimeter outside diameter and 46 for griseofulvin (ultramicrosize) centimeters in length is attached to tablets. the pump outlet producing a flow rate (a) Apparatus—(1) Vessel. A cylin- of approximately 1,600 milliliters per drical glass tank. The approximate di- minute when operated as described. mensions are 40 centimeters in diame- (ii) A ‘‘4-element stirrer’’ consisting ter and at least 23 centimeters in of a motor and a shaft approximately height. 45 centimeters long and 8 millimeters (2) Heating system. A 1,500-watt im- in diameter. The motor rotates the ver- mersion heating element connected to tical shaft in a clockwise direction at a partial immersion, contact thermom- approximately 180 revolutions per eter and an appropriate control relay. minute. There are 4 elements or sets of (3) Circulating system components. The stirring blades on the shaft. One set, circulating system consists of three located at the bottom of the shaft, is a different circulating devices: 3-bladed element of 2.5 centimeters
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overall radius with circular blades, 1.8 proximately 3 centimeters from the top centimeters in diameter and 1 to 2 mil- of the fluid in the tank and 5 to 6 centi- limeters in thickness, pitched at an meters from the wall of the tank. The angle of approximately 45 degrees from pump’s outlet hose is held by a clamp the horizontal plane, so that fluid is so that hose makes a clockwise arc propelled downward when the shaft is around the inside wall of the tank, de- rotated in a clockwise direction. The scending to a point near the bottom of three remaining sets of stirring blades the tank and 5 to 6 centimeters from have 4 blades each, symmetrically posi- the wall, which is 180 degrees from the tioned about the shaft. Each set of pump inlet. blades is 3.2 centimeters in overall ra- (b) Dissolution medium. Distilled dius. Each blade is rectangular in water. shape, 2.4 centimeters in length, 1.2 (c) Procedure. Place 24 liters of dis- centimeters in height, and 1 to 2 milli- solution medium into the vessel and meters in thickness. The four sets of maintain the temperature at 37±0.5° C blades are located at 5 centimeter in- by means of the heater, circulating tervals on the shaft, the top three pump, and the 4-element stirrer. With- being fixed in a staggered configura- draw a 25-milliliter portion of the dis- tion. solution medium as a sample-blank so- (iii) A rotating basket device consist- lution. Place one tablet into the bas- ing of a motor capable of constant ket, and lower it into its proper posi- speed of 100±5 revolutions per minute in tion in the tank. Rotate the basket at a clockwise direction, a shaft, and a cy- 100±5 revolutions per minute in a clock- lindrical basket. The shaft and the bas- wise direction. After 60 minutes, with- ket are fabricated from Type 316 stain- draw a second 25-milliliter portion as less steel. The shaft is 6 millimeters in the sample solution. Filter the sample- diameter and approximately 30 centi- blank solution and the sample solution meters in length. It must run true on through water-washed glass wool, or an the motor axis so that the basket ro- equivalent filter, discarding the first 10 tates smoothly and without perceptible to 15 milliliters of each filtrate. Deter- wobble. The basket consists of two mine the amount of griseofulvin dis- parts, one of which, the top, is at- tached to the shaft. It is of solid metal solved as directed in paragraph (d)(2) of except for a 2-millimeter round vent, this section. and is fitted with three spring clips (d) Griseofulvin assay—(1) Preparation that allow the removal of the lower of standard solution and standard-blank part, or the basket proper, to admit the solution. Accurately weigh approxi- test sample. The detachable part of the mately 50 milligrams of griseofulvin basket is fabricated of welded seam working standard and place into a 100- stainless steel, 40 mesh woven wire milliliter volumetric flask. Dissolve cloth, formed into a cylinder 3.66 centi- and dilute to volume with methyl alco- meters high and 2.5 centimeters in di- hol. Transfer 2.0 milliliters of this solu- ameter, with a narrow rim of sheet tion to a 200-milliliter volumetric flask metal around the top. and dilute to volume with distilled (4) Circulating system configuration. water. This is the standard solution. All three circulating devices are lo- Transfer a 2.0-milliliter portion of cated in one half of the tank. In clock- methyl alcohol to a 200-milliliter volu- wise order they are the circulating metric flask and dilute to volume with pump, the rotating basket, and the 4- distilled water. This is the standard- element stirrer. There is a distance of blank solution. Filter the standard- 12 to 13 centimeters between each of blank solution and the standard solu- the three devices. The rotating basket tion through water-washed glass wool, shaft and the stirring shaft are located or an equivalent filter, discarding the 9 to 10 centimeters from the tank wall. first 10 to 15 milliliters of each filtrate. The 4-element stirrer is positioned 1 to (2) Procedure. Using a suitable spec- 1.5 centimeters from the bottom of the trophotometer and distilled water as tank. The rotating basket is fixed at 7 the blank, determine the absorbance of to 8 centimeters from the bottom. The the four filtered solutions at the ab- circulating pump intake is located ap- sorbance peak at approximately 295
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nanometers, using suitable spectro- and concentrated ammonium hydrox- photometer cells with a 1-centimeter ide in volumetric proportions of light path. Determine the exact posi- 25:60:30, respectively. tion of the absorbance peak for the par- (2) Spray solution. Dissolve 1 gram of ticular instrument used. ninhydrin in 100 milliliters of n- buta- (3) Calculation. Determine the per- nol and add 1 milliliter of pyridine. centage of griseofulvin dissolved as fol- (c) Preparation of spotting solutions. lows: Prepare solutions of the sample and Percent griseofulvin AWV× × ×10 working standard, each containing 6 = u s milligrams of antibiotic to be tested dissolved × per milliliter in distilled water. APs where: (d) Procedure. Prepare a plate as fol- Au=Absorbance of the sample solution lows: On a line 2 centimeters from the minus the absorbance of the sample- base of the silica gel plate, and at in- blank solution; tervals of 1 centimeter, spot 3 micro- Ws=Weight of the working standard in mil- liters each of the standard solution and ligrams; V=Volume of the dissolution medium in li- the sample solution. In addition, pre- ters; pare one spot composed of 3 microliters As=Absorbance of the standard solution of the sample solution and 3 micro- minus the absorbance of the standard- liters of the standard solution. Place blank solution; the supporting platform in the bottom P=Labeled potency of the sample in milli- of the tank and place the solvent grams of griseofulvin per tablet. trough on it, near the front of the (e) Evaluation. The tablet passes the tank. Place a piece of Whatman #3 MM solubility characteristic test if it dis- filter paper or equivalent, measuring solves to the extent of not less than 50 20x3 centimeters and folded in half, percent at 60 minutes. If the tablet lengthwise, over the front edge of the fails to meet this requirement, repeat tank to form a cushion and drying the test on five additional tablets. The wick for the plate. Place the plate in batch passes the solubility characteris- tic test if not less than 5 of 6 tablets the solvent trough with the coated side meet the requirement. toward the front of the tank and lean- ing against the filter paper at the top. [40 FR 41522, Sept. 8, 1975; 40 FR 45426, Oct. 2, Pour the developing solvent into the 1975] trough and bottom of the tank. Cover § 436.318 Continuous flow thin layer the tank. The plate should extend ap- chromatography identity test. proximately 1 centimeter beyond the (a) Equipment—(1) Chromatography top of the tank and through the slot in tank. A rectangular tank, approxi- the cover. Seal all the openings in the mately 23 centimeters long, 23 centi- tank with masking tape, except where meters high, and 9 centimeters wide the plate leans against the filter paper. equipped with a glass solvent trough in Remove the plate from the tank after the bottom. 5.5 hours. Allow the plate to air dry (2) Plates. Use a 20 × 20 centimeter and then heat it for 15 minutes at 110° thin-layer chromatography plate coat- C in an oven. Remove the plate from ed with Silica Gel G or equivalent to a the oven and immediately spray it with thickness of 250 micrometers. the spray solution. The compound ap- (3) Cover. A stainless steel cover with pears as a pink spot. a slot, measuring 21 × 0.6 centimeters, (e) Evaluation. The sample and stand- cut in the front edge. ard should have traveled the same dis- (4) Supporting platform. A platform tance from the origin, and the standard that can be placed in the bottom of the and sample combined should appear as chromatography tank so that the sol- a single spot that has traveled the vent trough is elevated about 3.75 cen- same distance as the sample and stand- timeters. ard individually. (b) Reagents—(1) Developing solvent. Mix chloroform, redistilled methanol [40 FR 57797, Dec. 12, 1975]
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§ 436.319 Thin layer chromatography perature. The development may be identity test for bacitracin and bac- speeded up by warming the plate in a itracin zinc. 110° C oven. (a) Equipment—(1) Chromatography (e) Evaluation. The sample and stand- tank. A rectangular tank approxi- ard should have spots of corresponding mately 23 centimeters long, 23 centi- Rf value (approximately 0.26) and meters high, and 9 centimeters wide, standard and sample combined should equipped with a glass solvent trough in appear as a single spot of correspond- the bottom and a tight-fitting cover for ing Rf value. the top. Line the inside walls of the [42 FR 27228, May 27, 1977] tank with Whatman 3MM chromatographic paper or equivalent. § 436.320 Ferric chloride colorimetric (2) Plates. Use a 20- by 20-centimeter assay. thin layer chromatography plate coat- ed with silica gel G or equivalent to a (a) Reagents. (1) 1N hydrochloric acid. thickness of 250 micrometers. Activate (2) 0.01N hydrochloric acid. the plate by heating for 20 minutes at (3) Ferric chloride stock solution. 110° C. Allow to cool to room tempera- Quickly weigh (very hygroscopic) 5.0 ture and use immediately. grams of FeCl3·6H2O into a 100-milli- (b) Reagents—(1) Developing solvent. liter beaker. Add approximately 10 mil- Mix n- butanol, water, pyridine, glacial liliters of 1N hydrochloric acid and stir acetic acid, and ethyl alcohol in volu- to dissolve. Quantitatively transfer to metric proportions of 60:10:6:15:5, re- a 50-milliliter glass-stoppered amber spectively. volumetric flask and make up to vol- (2) Spray solution. Dissolve 1 gram of ume with water. ninhydrin in a mixture of 1 milliliter of (4) Ferric chloride working reagent. pyridine and sufficient n- butanol to Pipette 10.0 milliliters of ferric chlo- make 100 milliliters. ride stock solution into a 2-liter volu- (c) Preparation of spotting solutions. metric flask, add 20 milliliters 1N hy- Prepare solutions of the sample and drochloric acid, and bring to volume working standard, each containing 6.0 with water. Check the pH; it should be milligrams of bacitracin per milliliter between 2.0 and 2.1. in 1 percent disodium ethylenediamine (b) Standard solution. Accurately tetraacetic acid in water. weigh approximately 50 milligrams of (d) Procedure. Pour the developing the working standard of the antibiotic solvent into the glass trough on the to be tested and dissolve with 25 milli- bottom of the tank and onto the paper liters of 0.1N hydrochloric acid. Quan- lining the walls of the tank. Cover and titatively transfer to a 250-milliliter seal the tank. Allow it to equilibrate volumetric flask and dilute to volume for at least 30 minutes. Prepare a plate with distilled water. Keep in a glass- as follows: On a line 2.0 centimeters stoppered flask and store under refrig- from the base of the silica gel plate, eration. Discard solution after 7 days. and at intervals of 2.0 centimeters, spot (c) Sample solution. Accurately weigh approximately 1.0 microliter of the approximately 50 milligrams of the standard solution to points 1 and 3. sample and dissolve with 25 milliliters When these spots are dry, apply ap- of 0.1N hydrochloric acid. Quan- proximately 1.0 microliter of sample titatively transfer to a 250-milliliter solution to points 2 and 3. After all volumetric flask and dilute to volume spots are thoroughly dry, place the with distilled water. base of the silica gel plate directly into (d) Procedure. Pipette exactly 10.0 the glass trough in the chroma- milliliters of the standard solution and tography tank. Cover and seal the of the sample solution into separate tank. Allow the solvent front to travel test tubes. To each tube add exactly 10 approximately 13 centimeters from the milliliters of ferric chloride working starting line. Remove the plate from reagent, mix, and allow to stand 15 the tank, and allow it to air dry. After minutes. Determine the absorbance of the plate is dry, spray lightly with the each solution at 490 nanometers in a spray solution. The plate may take 1 suitable spectrophotometer against a hour or more to develop at room tem- blank prepared from 10.0 milliliters of
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0.01N hydrochloric acid and 10.0 milli- (e) Estimation of potency. Calculate liters of ferric chloride working rea- the potency as follows: gent.
Absorbance Milligrams Micrograms of antibiotic =of sample ×of standard × Potency of standards in per milligram Absorbance Milligrams micrograms per milligram of standard of sample
[43 FR 11154, Mar. 17, 1978; 43 FR 34456, Aug. matograph. Use the typical conditions 4, 1978] and materials listed in paragraphs (a), (b), and (c) of this section. The resolu- § 436.321 Griseofulvin gas liquid chro- tion of the peaks should be complete. matography. The griseofulvin peak will elute before (a) Equipment. Gas chromatograph the internal standard peak. Calculate equipped with an electronic integrator the griseofulvin content as directed in and with a flame ionization detector: paragraph (f) of this section. Hewlett Packard 7600 or equivalent. (f) Calculations. Calculate the griseo- (b) Reagents. (1) Chloroform, reagent fulvin content of the sample as follows: grade. (2) Internal standard solution: Pre- Micrograms of R× W × f pare a solution containing 1.0 milli- griseofulvin = u s gram of tetraphenylcyclopentadienone RW× per milliliter in chloroform. per milligram s u (c) Typical conditions—(1) Column. 1.2 where: meters by 4 millimeters ID, glass, Ru=Area of the griseofulvin sample peak packed with 1 percent OV–17 on Gas (at a retention time equal to that ob- Chrom Q (100/120 mesh), or equivalent. served for the griseofulvin standard)/ (2) Temperatures. Column 245° C; de- Area of the internal standard peak; tector 260° C; injection port 260° C. Rs=Area of the griseofulvin working stand- (3) Carrier gas. Helium approximately ard peak/Area of the internal standard peak; 60 millimeters per minute and 40 Ws=Weight of the griseofulvin working pounds per square inch (1.7 kilograms standard in milligrams; per square centimeter). Wu=Weight of the sample in milligrams; (4) Detector. Hydrogen flame ioniza- f=Potency of the griseofulvin working tion-hydrogen at 12 pounds per square standard in micrograms per milligram. inch (0.5 kilogram per square centi- [44 FR 20660, Apr. 6, 1979] meter), air at 34 pounds per square inch (1.43 kilograms per square centimeter). § 436.322 High-pressure liquid (5) Sensitivity. Adjusted to obtain chromatographic assay for peak heights greater than 50 percent anthracycline antibiotics. full scale deflection. (d) Preparation of griseofulvin sample (a) Equipment. A suitable high-pres- and working standard solutions. Accu- sure liquid chromatograph, such as a 1 rately weigh approximately 40 milli- Waters Associates Model 244 or equiv- grams of both the sample and the alent equipped with: working standard into separate 25-mil- (1) A low dead volume cell 8 to 20 liliter volumetric flasks. Add sufficient microliters; internal standard solution to dissolve (2) A light path length of 1 centi- the contents of each flask with vigor- meter; ous mixing and then dilute to volume (3) A suitable ultraviolet detection with internal standard solution and system operating at a wavelength of mix. Proceed as directed in paragraph 254 nanometers; (e) of this section. (e) Procedure. Inject 1.0 microliter of 1 Available from Waters Associates, Inc., this solution into the gas chro- Maple St., Milford, Mass. 10757.
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(4) A suitable recorder of at least 25.4 the individual monograph for the drug centimeter deflection; being tested. (5) A suitable integrator; [43 FR 44836, Dec. 29, 1978] (6) A 30-centimeter column having an inside diameter of 4.6 millimeters and § 436.323 Continuous flow thin layer packed with a suitable reverse phase chromatography identity test for packing such as: Waters Associates, cefamandole nafate. Micro-Bondapak C18.1 (a) Equipment—(1) Chromatography (b) Reagents. (1) Solvent mixture: tank. Use a rectangular tank approxi- Water: acetonitrile (69:31). mately 23 centimeters long, 23 centi- (2) Mobile phase: Water: acetonitrile meters high, and 9 centimeters wide (69:31) adjusted to pH 2 with phosphoric equipped with a glass solvent trough in acid. Filter the mobile phase through a the bottom. suitable glass fiber filter or equivalent (2) Plates. Use a 20 x 20 centimeter that is capable of removing particulate thin-layer chromatography plate coat- contamination to 1 micron in diameter. ed with silica gel G or equivalent to a Degas the mobile phase just prior to its thickness of 250 micrometers. introduction into the chromatograph (3) Cover. A stainless steel cover with pumping system. a slot measuring 21 x 0.6 centimeters, cut in the front edge. (3) Internal standard solution: Pre- (4) Supporting platform. A platform pare a 2.0-milligram-per-milliliter solu- that can be placed in the bottom of the tion of 2-naphthalenesulfonic acid in chromatography tank so that the sol- the solvent mixture. vent trough is elevated about 3.75 cen- (c) Operating conditions. Perform the timeters. assay at ambient temperature with a (b) Reagents—(1) Developing solvent. typical flow rate of 1.5 milliliters per Mix n- butanol, glacial acetic acid, and minute. Use a detector sensitivity set- water in volumetric proportions of ting that gives a peak height for the 4:1:1, respectively. reference standard that is at least 50 (2) Spray solution. Mix starch iodide percent of scale. The minimum be- solution, glacial acetic acid, and 0.1 N tween peaks must be no more than 2 iodine test solution, U.S.P. in volu- millimeters above the initial baseline. metric proportions of 50:3:1. Prepare (d) Procedure. Use the standard and the starch iodide solution by mixing sample solutions prepared as directed starch iodide paste test solution, in the individual monographs for the U.S.P. and water in volumetric propor- drug being tested. Use the equipment, tions of 1:1. reagents, and operating conditions list- (c) Preparation of spotting solutions. ed in paragraphs (a), (b), and (c) of this Prepare solutions of the sample and section. Inject 5 microliters of the working standard, each containing 1 standard solution into the chro- milligram of cefamandole nafate per matograph. Allow an elution time suf- milliliter in distilled water. ficient to obtain satisfactory separa- (d) Procedure. Prepare a plate as fol- tion of expected components (ordi- lows: On a line 2 centimeters from the narily this time is 20 minutes). After base of the silica gel plate, and at in- separation of the standard solution has tervals of 1 centimeter, spot 5 micro- been completed, inject 5 microliters of liters each of the standard solution and the sample solution into the chro- the sample solution. In addition, pre- matograph and repeat the procedure pare one spot composed of 5 microliters described for the standard solution. of the sample solution and 5 micro- The elution order is: Void volume, in- liters of the standard solution. Place ternal standard, doxorubicin, the supporting platform in the bottom dihydrodaunomycin, daunomycin, of the tank and place the solvent adriamycinone, trough on it, near the front of the dihydrodaunomycinone, tank. Place a piece of Whatman #3 MM bromodaunomycin, daunomycinone, filter paper or equivalent, measuring 20 and bis-anhydrodaunomycinone. x 3 centimeters and folded in half, (e) Calculations. Calculate the lengthwise, over the front edge of the anthracycline content as directed in tank to form a cushion and drying
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wick for the plate. Place the plate in (b) Reagent. pH 2.3 Buffer: Dissolve 3.6 the solvent trough with the coated side grams of dibasic sodium phosphate, 39.4 toward the front of the tank and lean- grams of citric acid, and 70.8 grams of ing against the filter paper at the top. potassium chloride in sufficient dis- Pour the developing solvent into the tilled water to make 1 liter. trough and bottom of the tank. Cover (c) Operating conditions—(1) Operating the tank. The plate should extend ap- mode: Differential pulse. proximately 1 centimeter beyond the (2) Scan range: ¥0.3 volt to ¥1.05 top of the tank and through the slot in volts. the cover. Seal all the openings in the (3) Scan rate: ¥2 millivolts per sec- tank with masking tape, except where ond. the plate leans against the filter paper. (4) Sensitivity: 10 to 20 microamperes Remove the plate from the tank after 4 or equivalent to keep peak on scale. hours. Allow the plate to air dry and (5) Mercury drop time: 1 second per then heat it in an oven for 15 minutes drop. at 110° C. Remove the plate from the (6) Modulation amplitude: 25 oven and immediately spray it with the millivolts. spray solution. The compound appears (7) Display direction: + as a white spot on a purple back- (8) Damping: None. ground. (d) Preparation of sample and working (e) Evaluation. The sample and stand- standard solutions. Use the cefamandole ard should have traveled the same dis- lithium working standard. Accurately tance from the origin, and the com- weigh approximately 12 milligrams of bined standard and sample should ap- sample or working standard into a 50- pear as a single spot that has traveled milliliter volumetric flask. Dissolve the same distance as the sample and the sample or working standard in 4 standard individually. milliliters of distilled water. Imme- [44 FR 20664, Apr. 6, 1979] diately prior to polarography, add 30 milliliters of pH 2.3 buffer, dilute to § 436.324 Polarographic analysis of volume with distilled water, and mix. cefamandole. (e) Procedure. Transfer a portion of (a) Equipment—(1) Polarograph. Use a the sample or working standard solu- polarograph equipped with a dropping tion to the polarographic cell. Pass a mercury indicating electrode, a plati- stream of nitrogen through the solu- num auxilliary electrode, and a satu- tion for 5 minutes to remove the dis- rated calomel reference electrode, such solved oxygen. After 5 minutes, dis- as Princeton Applied Research Model perse the nitrogen above the sample. 174 1 or equivalent. Start the mercury dropping from the (2) X–Y plotter. Use a suitable X–Y mercury dropping electrode, and, using plotter, such as Houston Omnigraphic the operating conditions described in Model 2200–3–3 2 or equivalent. paragraph (c) of this section, record the (3) Nitrogen. Use a nitrogen tank polarogram. Compare the polarogram equipped with a pressure-reducing reg- of the sample to that of the working ulator and a filter to remove traces of standard. oxygen, such as an oxisorb filter 1 or (f) Calculations. Calculate the potency equivalent. of cefamandole as follows:
A ×Milligrams of working standard × Potency of Micrograms of cefamandole = working standard in micrograms per milligram per milligram B × Milligrams of sample
where: A=The peak height of the sample;
1 Available from Princeton Applied Re- 2 Available from Houston Instrument, 8500 search Corporation, P.O. Box 2565, Princeton, Cameron Road, Austin, TX 78753. NJ 08540. 415
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B=The peak height of the working stand- milliliters of distilled water and heat ard. on a steam bath for 10 minutes. Shake The peak height is obtained from the until all the powder is dissolved. Cool polarogram by measuring the vertical to room temperature and dilute to vol- distance from the peak to the baseline ume with distilled water. of the sample or working standard. (e) Procedure. Using the equipment, mobile phase, and operating conditions [44 FR 20664, Apr. 6, 1979, as amended at 47 listed in paragraphs (a), (b), and (c) of FR 20756, May 14, 1982] this section, inject 10 microliters of the § 436.325 High pressure liquid chroma- sample or working standard solution tography assay for vidarabine. prepared as directed in paragraph (d) of (a) Equipment. A suitable high pres- this section into the chromatograph. sure liquid chromatograph, such as a Allow an elution time sufficient to ob- Waters Associates Model 244 1 or equiv- tain satisfactory separation of ex- alent, equipped with: pected components. The elution order (1) A low dead volume cell 8 to 20 is void volume, 9-β-D- microliters; arabinofuranosylhypoxanthine (if (2) A light path length of 1 centi- present), vidarabine, and adenine (if meter; present). (3) A suitable ultraviolet detection (f) Calculations. Calculate the system operating at a wavelength of vidarabine content as follows: 254 nanometers; A× W × f (4) A suitable recorder of at least 25.4 Micrograms of vidarabine = s centimeter deflection; per milligram × (5) A 30-centimeter column having an BWu inside diameter of 4 millimeters and where: packed with a suitable octadecyl bond- A=Area of the vidarabine sample peak (at a ed silica phase packing such as Waters retention time equal to that observed Associates, Micro-Bondapak C18.1 for the standard); (b) Mobile phase. (1) Transfer 2.2 B= Area of the standard peak; grams of sodium dioctyl sulfosuccinate Ws=Weight of standard in milligrams; and 10 milliliters of glacial acetic acid Wu=Weight of sample in milligrams; and to a 1-liter volumetric flask. Dissolve f=Potency of standard in micrograms per milligram. with 500 milliliters of methanol, dilute to volume with distilled water, and [44 FR 30334, May 25, 1979, as amended at 47 mix. Filter the mobile phase through a FR 23708, June 1, 1982] suitable glass fiber filter or equivalent that is capable of removing particulate § 436.326 Thin layer chromatographic identity test for cefoxitin sodium. contamination to 1 micron in diameter. (2) De-gas the mobile phase just be- Using the sample solution prepared fore its introduction into the chro- as described in the section for the anti- matograph pumping system. biotic drug to be tested, proceed as de- (c) Operating conditions. Perform the scribed in paragraphs (a), (b), (c), (d), assay at ambient temperature with a and (e) of this section. typical flow rate of 1.5 milliliters per (a) Equipment—(1) Chromatography minute. Use a detector sensitivity set- tank. A rectangular tank, approxi- ting that gives a peak height for the mately 23 centimeters long, 23 centi- reference standard that is at least 50 meters high, and 9 centimeters wide, percent of scale. The minimum be- equipped with a glass solvent trough in tween peaks must be no more than 2 the bottom and a tight-fitting cover for millimeters above the initial baseline. the top. Line the inside walls of the (d) Preparation of sample and working tank with Whatman #3 MM, standard solutions. Accurately weigh chromatographic paper or equivalent. approximately 24 milligrams of sample (2) Plates. Use a 20×20 centimeter thin or working standard into a 200-milli- layer chromatography plate coated liter volumetric flask. Add about 150 with silica gel G or equivalent to a thickness of 250 micrometers. 1 Available from Waters Associates, Inc., (b) Developing solvent. Mix ethyl ace- Maple St., Milford, MA 10757. tate, pyridine, n-butanol, acetic acid,
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and water in volumetric proportions of with Silica Gel G or equivalent to a 42:21:21:6:10, respectively. thickness of 250 microns. (c) Spray solution. Immediately before (b) Reagents—(1) Developing solvent. use, mix 100 milliliters of a 1-percent One percent ammonium formate aque- ferric chloride solution in 1 percent hy- ous solution. drochloric acid with 100 milliliters of a (2) Spray solution. Dilute starch io- 1-percent potassium ferricyanide solu- dide paste TS (U.S.P. XIX) with an tion and 75 milliliters of methanol. equal volume of water. Mix diluted (d) Preparation of working standard so- starch iodide paste, glacial acetic acid, lution. Prepare a solution containing and 0.1N iodine in volumetric propor- approximately 2.5 milligrams per milli- tions of 50:3:1, respectively. liter of cefoxitin working standard in (c) Assay solutions—(1) Preparation of distilled water. working standard solution. Accurately (e) Procedure. Pour the developing weigh an amount of cyclacillin work- solvent into the glass trough on the ing standard and dissolve the material bottom of the tank and onto the paper with sufficient 0.1N sodium hydroxide lining the walls of the tank. Cover and to obtain a solution containing 1 milli- seal the tank. Allow it to equilibrate gram per milliliter. Allow the solution for 1 hour. Prepare a plate as follows: to stand for 15 minutes before using. On a line 2 centimeters from the base (2) Preparation of sample solution. of the silica gel plate, and at intervals Using the sample solution prepared as of 2 centimeters, spot 10 microliters described in the section for the anti- each of the standard solution and the biotic to be tested, proceed as de- sample solution. After all spots are scribed in paragraphs (d) and (e) of this thoroughly dry, place the silica gel section. plate directly into the glass trough. (d) Procedure. Pour the developing Cover and seal the tank. Allow the sol- solvent into the glass trough on the vent front to travel about 15 centi- bottom of the tank. Cover and seal the meters from the starting line. Remove tank. Allow it to equilibrate. Prepare a the plate from the tank and heat it for plate as follows: On a line 2 centi- 1 hour at 60° C in a circulating air meters from the base of the thin layer oven. Remove the plate from the oven chromatography plate and at intervals and allow it to cool at room tempera- of 2 centimeters, spot 5 microliters ture. Apply the spray solution and each of the working standard solution allow it to air dry. After approximately and sample solution. Dry the spots 15 minutes, the compound appears as a thoroughly with a stream of dry air. blue spot on a yellow-green back- Place the plate in the trough in the ground. chromatography tank. Cover and seal (f) Evaluation. Measure the distance the tank. Allow the solvent front to the solvent front traveled from the travel about 15 centimeters from the starting line and the distance the spots starting line and then remove the plate are from the starting line. Calculate from the tank. Dry the plate by heat- the Rf value by dividing the latter by ing for 30 minutes at 80° C in a circulat- the former. The sample and standard ing air oven. Visualize the spots by ap- should have spots of corresponding Rf plying the spray solution. values. (e) Evaluation. Measure the distance [44 FR 10373, Feb. 20, 1979, as amended at 49 the solvent front traveled from the FR 2242, Jan. 19, 1984] starting line, and the distance the spots are from the starting line. Divide § 436.327 Thin layer chromatographic the latter by the former to calculate identity test for cyclacillin. the Rf value. The sample and standard (a) Equipment—(1) Chromatography should appear as white spots against a tank. Use a rectangular tank approxi- blue background at an Rf of approxi- mately 23 x 23 x 9 centimeters, with a mately 0.6. The test is satisfactory if glass solvent trough on the bottom and the Rf value of the sample compares a tight-fitting cover. with that of the working standard. (2) Plates. Use 20 x 20 centimeter thin [46 FR 2981, Jan. 13, 1981, as amended at 49 layer chromatography plates coated FR 2242, Jan. 19, 1984]
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§ 436.328 High pressure liquid erence standard that is at least 50 per- chromatographic assay for cent of scale. The minimum between sulfisoxazole acetyl content. peaks must be no more than 2 millime- (a) Equipment. A suitable high pres- ters above the baseline. sure liquid chromatograph, such as a (d) Preparation of the working standard Waters Associates Model 244 1 or equiv- and sample solutions—(1) Working stand- alent equipped with: ard solution. Prepare a solution con- (1) A low dead volume cell 8 to 20 taining 1.0 milligram per milliliter of microliters; sulfisoxazole acetyl in the internal (2) A light path length of 1 centi- standard solution. meter; (2) Sample solution. Reconstitute the (3) A suitable ultraviolet detection sample as directed in the labeling. system operating at a wavelength of Allow to stand for 1 hour. Shake gently 254 nanometers; and transfer 5.0 milliliters of the sam- (4) A suitable recorder of at least 25.4 ple to a separatory funnel. Extract the centimeter deflection; suspension three times with 75-milli- (5) A 30-centimeter column having an liter portions of chloroform. Collect inside diameter of 4.0 millimeters and the chloroform layers in a 250-milli- packed with a suitable reverse phase liter volumetric flask. Dilute the flask packing such as: Waters Associates, to volume with chloroform and mix. Micro-Bondapak C18; 1 and Filter a portion of the solution through (6) A suitable integrator. a suitable glass fiber filter or equiva- (b) Reagents—(1) Mobile phase. Mix lent which is capable of removing par- acetonitrile (high pressure liquid chro- ticulate contamination to 1 micron in matography grade): water (40:60). Filter diameter. Transfer a 4.0-milliliter ali- the mobile phase through a suitable quot of the filtrate into a 25-milliliter glass fiber filter or equivalent which is glass-stoppered flask and evaporate to capable of removing particulate con- dryness under a stream of dry air. Dis- tamination to 1 micron in diameter. solve the residue in 10.0 milliliters of De-gas the mobile phase just prior to the internal standard solution, stopper, its introduction into the chro- and mix. matograph pumping system. (e) Procedure. Using the equipment, (2) Internal standard solution. Dissolve reagents, and operating conditions list- 0.33 milligram of benzanilide per milli- ed in paragraphs (a), (b), and (c) of this liter in acetonitrile (high pressure liq- section, inject 5 microliters of sample uid chromatography grade). Filter the or working standard solution prepared solution through a suitable glass fiber as described in paragraph (d) of this filter or equivalent which is capable of section, into the chromatograph. Allow removing particulate contamination to an elution time sufficient to obtain 1 micron in diameter. satisfactory separation of expected (c) Operating conditions. Perform the components. The elution order is void assay at ambient temperature with a volume, sulfisoxazole acetyl and typical flow rate of 1.2 milliliters per benzanilide. minute. Use a detector sensitivity set- (f) Calculations. Calculate the ting that gives a peak height for ref- sulfisoxazole content as follows:
A × Concentration of the standard solution in × × Milligrams of sulfisoxazole per = milligrams per milliliter 125 0. 864 milliliter of sample B
where: vided by the area of the internal stand- A=Area of sample peak (at a retention ard peak; time equal to that of the standard) di- B=Area of the standard peak divided by the area of the internal standard peak;
1 Available from: Waters Associates, Inc., Maple Street, Milford, MA 10757. 418
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0.864=The molecular weight of sulfisoxazole the mobile phase through a suitable divided by the molecular weight of glass fiber filter or equivalent that is sulfisoxazole acetyl. capable of removing particulate con- [46 FR 2990, Jan. 13, 1981] tamination to 1 micron in diameter. Degas the mobile phase just prior to its § 436.329 High-pressure liquid introduction into the chromatograph chromatographic assay for pumping system. meclocycline. (c) Operating conditions. Perform the (a) Equipment. A suitable high-pres- assay at ambient temperature with a sure liquid chromatograph, such as a typical flow rate of 0.8 milliliter per 1 Waters Associates Model 244 or equiv- minute. Use a detector sensitivity set- alent equipped with: ting that gives a peak height for the (1) A low dead volume cell 8 to 20 reference standard that is at least 50 microliters; percent of scale. The minimum be- (2) A light path of 1 centimeter; tween peaks must be no more than 2 (3) A suitable ultraviolet detection millimeters above the initial baseline. system operating at a wavelength of 340 nanometers; (d) Preparation of sample and working (4) A suitable recorder of at least 25.4 standard solutions. Accurately weigh an centimeter deflection; amount of sample or working standard (5) A suitable integrator; equivalent to approximately 25 milli- (6) A column approximately 25 centi- grams of meclocycline into a 50-milli- meters in length having an inside di- liter volumetric flask. Dissolve and di- ameter of approximately 4 millimeters lute to volume with methanol and mix. and packed with a suitable reverse- Transfer exactly 3.0 milliliters of this phase packing such as: 10 micrometer solution to a 25-milliliter volumetric silica gel particles bonded to octadecyl flask, dilute to volume with mobile silane, Vydac 201 TP Reverse Phase 2 or phase, and mix. equivalent. (e) Procedure. Using the equipment, (b) Reagents—(1) 0.001M Ammonium reagents, and operating conditions list- (ethylenedinitrilo) tetraacetate. Moisten ed in paragraphs (a), (b), and (c) of this 293 milligrams of (ethylenedinitrilo) section, inject 10 microliters of the tetraacetic acid with 1 milliliter of sample or working standard solution methanol and dissolve in 7 milliliters prepared as described in paragraph (d) of concentrated ammonium hydroxide. of this section into the chromatograph. Dilute to 900 milliliters with distilled Allow an elution time sufficient to ob- water, adjust the pH to 6.6 with glacial tain satisfactory separation of ex- acetic acid, and dilute to 1,000 milli- pected components. The elution order liters with distilled water. is void volume, oxytetracycline (if (2) Mobile phase. Mix 150 milliliters of present), demeclocycline (if present), tetrahydrofuran (high-pressure liquid methacycline (if present), and chromatography grade) with 850 milli- meclocycline. liters of 0.001M ammonium (f) Calculations. Calculate the (ethylenedinitrilo) tetraacetate. Filter meclocycline content as follows:
A ×Milligrams of working standard × Potency of the Micrograms of meclocycline = working standard in micrograms per milligram per milligram B × Milligrams of sample
where: B= Area or peak height of the standard peak. A= Area or peak height of the sample peak [46 FR 3836, Jan. 16, 1981] (at a retention time equal to that ob- served for the standard);
1 Available from: Waters Associates, Inc., 2 Available from: The Separations Group, Maple St., Milford, MA 10757. 16640 Spruce St., Hesperia, CA 92345.
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§ 436.330 Thin layer chromatographic (e) Evaluation. Measure the distance identity test for bacampicillin. the solvent front traveled from the (a) Equipment—(1) Chromatography starting line, and the distance the tank. Use a rectangular tank approxi- spots are from the starting line. Divide mately 23 × 23 × 9 centimeters, with a the latter by the former to calculate glass solvent trough on the bottom and the Rf value. Bacampicillin appears as a tight-fitting cover, lined with a purple spot at an Rf value of approxi- Whatman’s 3MM chromatographic mately 0.52. The test is satisfactory if paper (0.3 millimeter) or equivalent. the Rf value of the sample compares (2) Plates. Use 20 × 20 centimeter thin with that of the working standard. The layer chromatography plates coated combined spot should appear as a sin- with Silica Gel 60F 254 or equivalent to gle spot of corresponding Rf value. a thickness of 250 microns. [46 FR 25602, May 8, 1981, as amended at 49 (b) Reagents—(1) Developing solvent. FR 2242, Jan. 19, 1984] Mix methylene chloride, chloroform, and 95 percent ethyl alcohol in volu- § 436.331 High-pressure liquid metric proportions of 100:10:10, respec- chromatographic assay for tively. dactinomycin. (2) Spray solution. Dissolve 1 gram of (a) Equipment. A suitable high-pres- ninhydrin in 100 milliliters of n-buta- sure liquid chromatograph equipped nol and add 1 milliliter of pyridine. with: (c) —(1) Spotting solutions Preparation (1) A low dead volume cell 8 to 20 Dissolve of working standard solution. microliters; and dilute a weighed amount of the (2) A light path length of 1 centi- bacampicillin hydrochloride working meter; standard with sufficient 95 percent ethyl alcohol to obtain a solution con- (3) A suitable ultraviolet detection taining 2 milligrams per milliliter. system operating at a wavelength of (2) Preparation of sample solution. Dis- 254 nanometers; solve and dilute a weighed amount of (4) A suitable recorder of at least the sample with sufficient 95 percent 25.4-centimeter deflection; ethyl alcohol to obtain a solution con- (5) A suitable integrator; and taining 2 milligrams per milliliter. (6) A 30-centimeter column having an Proceed as described in paragraphs (d) inside diameter of 4.0 millimeters and and (e) of this section. packed with octadecyl silane chemi- (d) Procedure. Pour the developing cally bonded to porous silica or ce- solvent into the glass trough on the ramic microparticles, 5 micrometers to bottom of the tank and onto the paper 10 micrometers in diameter, U.S.P. XX. lining the walls of the tank. Cover and (b) Mobile phase. Mix acetonitrile seal the tank. Allow it to equilibrate (high-pressure liquid chromatography for one hour. Prepare a plate as fol- grade): water (60:40). Filter the mobile lows: On a line 2.5 centimeters from the phase through a suitable glass fiber fil- base of the thin layer chromatography ter or equivalent that is capable of re- plate and at intervals of 2.0 centi- moving particulate contamination to 1 meters, spot 5 microliters of the work- micron in diameter. Degas the mobile ing standard solution to positions 1 and phase just prior to its introduction 3. When these spots are dry, apply 5 into the chromatograph pumping sys- microliters of the sample solution to tem. points 2 and 3. After all the spots are (c) Operating conditions. Perform the thoroughly dry, place the plate into assay at ambient temperature with a the trough in the bottom of the tank. typical flow rate of 2.5 milliliters per Cover and tightly seal the tank, allow minute. Use a detector sensitivity set- the solvent front to travel about 15 ting that gives a peak height for the centimeters from the starting line working standard that is at least 50 (about 30 minutes) and then remove the percent of scale. The minimum be- plate from the tank. Air dry the plate. tween peaks must be no more than 2 Visualize the spots by spraying with millimeters above the initial baseline. spray solution and heating in an oven (d) Preparation of working standard at 100° C for approximately 10 minutes. and sample solutions—(1) Preparation of
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working standard solution. Prepare a so- § 436.332 High-pressure liquid lution containing 0.25 milligram per chromatographic assay for milliliter of dactinomycin in mobile moxalactam. phase. (a) Equipment. A suitable high-pres- (2) Preparation of sample solution. Pre- sure liquid chromatograph equipped pare the sample solution as described with: in the individual monograph for the (1) A low dead volume cell 8 to 20 drug being tested. microliters; (e) Procedure. Use the equipment, mo- (2) A light path length of 1 centi- bile phase, operating conditions, and meter; working standard and sample solutions (3) A suitable ultraviolet detection described in paragraphs (a), (b), (c), and system operating at a wavelength of (d) of this section, and proceed as di- 254 nanometers; rected in paragraph (e)(1) of this sec- (4) A 30-centimeter column having an tion. inside diameter of 4.0 millimeters and (1) System suitability test. Equilibrate packed with octadecyl silane chemi- and condition the column by passage of cally bonded to porous silica or ce- about 10 to 15 void volumes of mobile ramic microparticles, 5 to 10 microm- phase followed by two or more rep- eters in diameter, U.S.P. XX; licate injections of 10 microliters each (5) A suitable recorder of at least 25.4 of the working standard solution. centimeter deflection; Allow an elution time sufficient to ob- (6) A suitable integrator. tain satisfactory separation of ex- (b) Mobile phase. Mix 0.01M ammo- pected components after each injec- nium acetate:methanol (19:1). Filter tion. Record the peak responses and, the mobile phase through a suitable calculate the relative standard devi- glass fiber filter or equivalent that is ation as described for system suit- capable of removing particulate con- ability tests in the U.S.P. XX General tamination to 1 micron in diameter. Chapter 621 chromatography. Proceed Degas the mobile phase just prior to its as directed in paragraph (e)(2) of this introduction into the chromatograph section if the minimum performance pumping system. requirement for the relative standard (c) Operating conditions. Perform the deviation is not more than 1.0 percent. assay at ambient temperature with a If the minimum performance require- typical flow rate of 0.5 milliliter per ment is not met, adjustment must be minute. Use a detector sensitivity set- made to the system to obtain satisfac- ting that gives a peak height for the tory operation before proceeding as de- working standard that is at least 50 scribed in paragraph (e)(2) of this sec- percent of scale. tion. (d) Preparation of working standard so- (2) Determination of the chromatogram. lution. Transfer the contents of an am- Inject 10 microliters of the working poule of working standard to a tared standard solution into the chro- weighing bottle. Place the unstoppered matograph. Allow an elution time suf- weighing bottle in a desiccator con- ficient to obtain satisfactory separa- taining a saturated aqueous solution of tion of the expected components. After potassium carbonate to provide an at- separation of the working standard so- mosphere of 42 percent relative humid- lution has been completed, inject 10 ity. Allow the moisture content of the microliters of the sample solution into working standard to equilibrate for 16 the chromatograph and repeat the pro- hours. Determine the moisture content cedure described for the working stand- as described in § 436.201 of this chapter. ard solution. Equilibrated standard material must be kept in a closed weighing bottle and (f) Calculations. Calculate the used within 36 hours of equilibration. dactinomycin content as described in Dissolve approximately 50 milligrams the individual monograph for the drug of the working standard, accurately being tested. weighed and corrected for moisture, [49 FR 24017, June 11, 1984, as amended at 50 with sufficient distilled water to obtain FR 5749, Feb. 12, 1985] a solution containing 0.5 milligram of
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moxalactam per milliliter. Use the pre- (g) Calculations. (1) Calculate the pared solution immediately. moxalactam content in micrograms (e) Preparation of sample solution. Mix per milligram as follows: contents of vial thoroughly. Dissolve RWP× × an accurately weighed portion of ap- Microgramsof moxalactam = u s proximately 50 milligrams of sample per milligram of sample × with distilled water to obtain a con- RWs u centration of 0.5 milligram per milli- where: liter (estimated); also, reconstitute the Ru=Sum of the areas of the moxalactam sample as directed in the labeling. sample R-isomer and the S-isomer peaks; Then using a suitable hypodermic nee- Rs=Sum of the areas of the moxalactam dle and syringe, remove all of the working standard R-isomer and the S- isomer peaks; withdrawable contents if it is rep- Wu=Weight of the sample in milligrams; resented as a single dose container; or, Ws=Weight of the moxalactam working if the labeling specifies the amount of standard in milligrams; potency in a given volume of the re- P=Potency of the moxalactam working sultant preparation, remove an accu- standard in micrograms per milligram, rately measured representative portion corrected for moisture. from each container. Further dilute an (2) Calculate the moxalactam con- aliquot of this solution with distilled tent of the vial as follows: water to obtain a concentration of 0.5 milligram per milliliter (estimated). Milligrams of RWP× × × d = u s Use the prepared solution immediately. moxalactam per vial × (f) Procedure. Using the equipment, Rs 100, 000 reagents, and operating conditions as where: listed in paragraphs (a), (b), and (c) of Ru=Sum of the areas of the moxalactam R- this section, inject 5 microliters of the isomer and the S-isomer peaks; working standard solution into the Rs=Sum of the areas of the moxalactam working standard R-isomer and the S- chromatograph. Allow an elution time isomer peaks; sufficient to obtain satisfactory sepa- Ws=Weight of the moxalactam working ration of the expected components. standard in milligrams; After separation of the working stand- P=Potency of the moxalactam working ard solution has been completed, inject standard in micrograms per milligram, 5 microliters of the sample solution corrected for moisture; into the chromatograph and repeat the d=Dilution factor. procedure described for the working (3) Calculate the ratio of R-isomer to standard solution. S-isomer as follows:
Area of the R-isomer peak Ratio of R-isomer to S-isomer = Area of the S-isomer peak
[46 FR 61069, Dec. 15, 1981] (2) Plates. Use a 20x20 centimeter thin layer chromotagraphy plate coat- § 436.333 Thin layer chromatographic ed with silca gel G or equivalent to a identity test for moxalactam. thickness of 250 micrometers. (a) Equipment—(1) Chromatography (b) Developing solvent. Mix ethyl ace- tank. A rectangular tank, approxi- tate, glacial acetic acid, acetonitrile, mately 23 centimeters long, 23 centi- and water in volumetric proportions of meters high, and 9 centimeters wide, 42:14:14:18, respectively. equipped with a glass solvent trough in (c) Preparation of spotting solutions. the bottom and a tight-fitting cover for Prepare solutions of the sample and the top. Line the inside walls of the working standard, each containing 10 tank with Whatman #3MM milligrams per milliliter of chromatographic paper or equivalent. moxalactam in distilled water.
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(d) Procedure. Pour the developing (2) 10 percent tetrabutylammonium solvent into the glass trough on the hydroxide in water. bottom of the tank and onto the paper (3) Ampicillin-piperacillin solution: lining the walls of the tank. Cover and Dissolve and dilute 25 milligrams of seal the tank. Allow it to equilibrate ampicillin and 5 milligrams of for 1 hour. Prepare a plate as follows: piperacillin monohydrate with suffi- On a line 2 centimeters from the base cient mobile phase to obtain 100 milli- of the silica gel plate, and at intervals liters, and mix. of 2 centimeters, spot 10 microliters (c) Mobile phase. Methanol:water:0.2M each of the standard solution and the monobasic sodium phosphate:10 per- sample solution. After all spots are cent tetrabutylammonium hydroxide thoroughly dry, place the silica gel (450 : 447 : 100:3) adjusted to pH 5.5 ±0.02 plate directly into the glass trough. with phosphoric acid. The concentra- Cover and seal the tank. Allow the sol- tion of reagents may be varied to ob- vent front to travel about 15 centi- tain acceptable operation of the sys- meters from the starting line. Remove tem. De-gas the mobile phase just prior the plate from the tank and air dry. to its introduction into the chro- Expose the plate to iodine vapors for 40 matograph pumping system. minutes. Immediately circumscribe all (d) Preparation of working standard spots using a suitable marker. and sample solutions—(1) Working stand- (e) Evaluation. Measure the distance ard solution. Place approximately 20 the solvent front traveled from the milligrams of the working standard, starting line and the distance the spots accurately weighed, into a 50-milliliter are from the starting line. Calculate volumetric flask. Add 25 to 30 milli- the Rf value by dividing the latter by liters of mobile phase. Shake until dis- the former. The sample and standard solved. Dilute to volume with mobile should have spots of corresponding Rf phase. values and intensity. (2) Sample solution—(i) Micrograms per milligram. Place approximately 20 milli- [46 FR 61070, Dec. 15, 1981, as amended at 49 grams of the sample, accurately FR 2242, Jan. 19, 1984] weighed, into a 50-milliliter volumetric § 436.334 High-pressure liquid flask. Add 25 to 30 milliliters of mobile chromatographic assay for phase. Shake until dissolved. Dilute to piperacillin. volume with mobile phase. (ii) Milligrams per vial. Reconstitute (a) Equipment. A high-pressure liquid as directed in the labeling. Withdraw chromatograph equipped with: the total contents and dilute with mo- (1) A low dead volume cell 8 to 20 bile phase to a concentration of 0.4 mil- microliters; ligram of piperacillin per milliliter. (2) A light path length of 1 centi- (e) Procedure. Use the equipment, re- meter; agents, mobile phase, and working (3) A suitable ultraviolet detection standard and sample solutions de- system operating at a wavelength of scribed in paragraphs (a), (b), (c), and 254 nanometers; (d) of this section and proceed as di- (4) A suitable recorder of at least rected in paragraph (e) of this section. 25.4-centimeter deflection; (1) Systems suitability test. Chro- (5) A suitable integrator; matograph three replicate samples of (6) A 25-centimeter column having an ampicillin-piperacillin solution as di- inside diameter of 4.6 millimeters and rected in paragraph (e)(2) of this sec- packed with octadecyl silane chemi- tion. Allow an elution time sufficient cally bonded to porous silica or ce- to obtain satisfactory separation of ex- ramic microparticles, 5 to 10 microm- pected components after each injec- eters in diameter (United States Phar- tion. Record the peak responses and macopeia XX). calculate the resolution factor as de- (b) Reagents. (1) 0.2M monobasic so- scribed for system suitability tests in dium phosphate: Dissolve 27.60 grams the United States Pharmacopeia XX of monobasic sodium phosphate with General Chapter 621 for gas chroma- sufficient water to make 1,000 milli- tography. The resolution factor be- liters. tween ampicillin and piperacillin is not
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less than 15. If the resolution factor lished. Inject 10 microliters of the does not meet this limit, adjustments working standard solution into the must be made to the system to obtain chromatograph. Allow an elution time satisfactory operation before proceed- sufficient to obtain separation of the ing as described in paragraph (e)(2) of expected components. After separation this section. of the working standard solution has (2) Determination of the chromatogram. been completed, inject 10 microliters of Operate the high-pressure liquid chro- the sample solution into the chro- matograph at ambient temperature at matograph and repeat the procedure a flow rate of one milliliter per minute. described for the working standard so- Use a detector sensitivity setting that lution. gives a peak height for the reference standard that is at least 50 percent of (f) Calculations—(1) Calculate the scale. Purge the column with mobile piperacillin content in micrograms per phase until a steady baseline is estab- milligram as follows:
A ×Weight of standard in milligrams × Potency of Micrograms of piperacillin = working standard in micrograms per milligram per milligram of sample B × Weight of sample in milligrams
where: B=Area of the standard peak. A=Area of the sample peak (at a retention time equal to that observed for the (2) Calculate the piperacillin content standard); in grams per vial as follows:
A × Milligrams of standard in milligrams per milliliter × × Gramsof piperacillin = Potencyof workingstandard in microgramsper milligram d per vial B ×1,000 ×1, 000
where: (6) A 30-centimeter column having an A=Area of the sample peak (at a retention inside diameter of 4.0 millimeters and time equal to that observed for the packed with octadecyl silane chemi- standard); cally bonded to porous silica or ce- B=Area of the standard peak; d=Dilution factor. ramic microparticles, 5 to 10 microm- eters in diameter, U.S.P. XX. [47 FR 15768, Apr. 13, 1982; 47 FR 33493, Aug. (b) Mobile phase. Mix meth- 3, 1982] anol:water:glacial acetic acid (170:30:1). § 436.335 High-pressure liquid Degas the mobile phase just prior to its chromatographic assay for chlor- introduction into the chromatograph amphenicol palmitate. pumping system. (a) Equipment. A suitable high-pres- (c) Operating conditions. Perform the sure liquid chromatograph equipped assay at ambient temperature with a with: typical flow rate of 2.0 milliliters per (1) A low dead volume cell 8 to 20 minute. Use a detector sensitivity set- microliters; ting that gives a peak height for the (2) A light path of 1 centimeter; reference standard that is at least 50 (3) A suitable ultraviolet detection percent of scale. The minimum be- system operating at a wavelength of tween peaks must be no more than 2 280 nanometers; millimeters above the initial baseline. (4) A suitable recorder of at least (d) Preparation of sample and working 25.4-centimeter deflection; standard solutions. Accurately weigh (5) A suitable integrator; and approximately 65 milligrams of sample
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or chloramphenicol palmitate working tilled water to make 1,000 milliliters standard each into a 50-milliliter volu- (solution A). Dissolve 17.88 grams of so- metric flask. Add approximately 35 dium phosphate, dibasic, heptahydrate milliliters of methanol and 1 milliliter (Na2HPO4.7H2 O) in sufficient distilled of glacial acetic acid. Place in an ultra- water to make 1,000 milliliters (solu- sonic bath for 10 minutes and dilute to tion B). Place 12.1 milliliters of solu- volume with methanol. tion B into a 100-milliliter volumetric (e) Procedure. Using the equipment, flask and dilute to volume with solu- mobile phase, and operating conditions tion A. listed in paragraphs (a), (b), and (c) of (2) Developing solvent. Place 50 milli- this section, inject 10 microliters of the liters of n-butyl acetate, 9 milliliters of working standard solution into the n-butanol, 25 milliliters of glacial ace- chromatograph. Allow an elution time tic acid, and 15 milliliters of buffer into sufficient to obtain satisfactory sepa- a separatory funnel. Shake well and ration of expected components. After allow the layers to separate. Discard separation of the working standard so- the lower phase and use the upper lution has been completed, inject 10 phase as the developing solvent. microliters of the sample solution into (c) Preparation of spotting solutions. the chromatograph and repeat the pro- Prepare solutions of the sample and cedure described for the working stand- working standard, each containing 20 ard solution. milligrams of azlocillin per milliliter (f) Calculations. Calculate the chlor- in distilled water. amphenicol content as follows: (d) Procedure. Pour developing sol- vent into the glass trough on the bot- Micrograms of (A)( W )( f ) tom of the chromatography tank to a = s chloramphenicol depth of about 1 centimeter. Use the per milligram (BW )( u ) chamber immediately. Prepare plate as where: follows: Apply spotting solutions on a A=Area of chloramphenicol palmitate sam- line 2.5 centimeters from the base of ple peak (at a retention time equal to that the silica gel plate and at points 2.0 observed for the standard); centimeters apart. Apply approxi- B=Area of the working standard peak; mately 10 microliters of the working Ws=Weight of standard in milligrams; Wu=Weight of sample in milligrams; and standard solution to points 1 and 3. f=Micrograms of chloramphenicol activity When these spots are dry, apply ap- per milligram of chloramphenicol proximately 10 microliters of sample palmitate working standard. solution to points 2 and 3. Place spot- [49 FR 6091, Feb. 17, 1984] ted plate in a desiccator until solvent has evaporated from spots. Place the § 436.336 Thin layer chromatographic plate into the glass trough at the bot- identity test for azlocillin. tom of the chromatography tank. (a) Equipment—(1) Chromatography Cover the tank. Allow the solvent to tank. A rectangular tank, approxi- travel about 15 centimeters from the mately 23 centimeters long, 23 centi- starting line. Remove the plate from meters high, and 9 centimeters wide, the tank and allow to air dry. Warm equipped with a glass solvent trough in the iodine vapor chamber to vaporize the bottom and a tight-fitting cover for the iodine crystals and place the dry the top. plate in the iodine vapor chamber until (2) Iodine vapor chamber. A rectangu- the spots are visible, usually about 10 lar tank approximately 23 centimeters minutes. long, 23 centimeters high, and 9 centi- (e) Evaluation. Measure the distance meters wide, with a suitable cover, the solvent front traveled from the containing iodine crystals. starting line and the distance the spots (3) Plates. Use 20 x 20 centimeter thin are from the starting line. Calculate layer chromatography plates coated the Rf value by dividing the latter by with Silica Gel G or equivalent to a the former. The azlocillin sample and thickness of 250 microns. the standard should have spots of cor- (b) Reagents—(1) Buffer. Dissolve 9.078 responding Rf values (approximately grams of potassium phosphate, 0.4), and standard and sample combined monobasic (KH2PO4) in sufficient dis- should appear as a single spot for 425
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azlocillin. The penicilloate and suitably sized container. Add 5.0 milli- penilloate of azlocillin as well as ampi- liters of distilled water and place in an cillin appear as additional spots with ultrasonic bath to facilitate dissolu- Rf values of approximately 0.15, 0.3, and tion. Dilute with a sufficient amount of 0.25, reectively. mobile phase to obtain a solution con- [47 FR 53348, Nov. 26, 1982] taining 0.8 milligram of cephradine ac- tivity per milliliter. § 436.337 High-pressure liquid (2) Preparation of cephalexin working chromatographic assay for standard solution. Dissolve an accu- cephradine. rately weighed portion of the (a) Equipment. A suitable high-pres- cephalexin working standard with mo- sure liquid chromatograph equipped bile phase to obtain a solution contain- with: ing 0.02 milligram of cephalexin activ- (1) A low dead volume cell 8 to 20 ity per milliliter. Place in an microliters; untrasonic bath to facilitate dissolu- (2) A light path length of 8 millime- tion. ters; (3) Preparation of sample solutions—(i) (3) A suitable ultraviolet detection Product not packaged for dispensing system operating at a wavelength of (micrograms of cephradine per milligram). 254 nanometers; Dissolve an accurately weighed portion (4) A suitable recorder that is com- of the sample with mobile phase to ob- patible with the detector output; tain a solution containing 0.8 milli- (5) A suitable integrator (optional); gram per milliliter. Place in an ultra- and sonic bath to facilitate dissolution. (6) A 25-centimeter column having an Using this sample solution, proceed as inside diameter of 4.6 millimeters and directed in paragraph (f)(1) of this sec- packed with octadecyl silane chemi- tion. cally bonded to porous silica or ce- (ii) Product packaged for dispensing. ramic microparticles, 10 micrometers Determine both micrograms of in diameter, U.S.P. XX. cephradine per milligram of the sample (b) Reagents. (1) 4 percent glacial ace- and milligrams of cephradine per con- tic acid. tainer. Use separate containers for (2) 3.86 percent sodium acetate. preparation of each sample solution as (c) Mobile phase. 4 percent glacial described in paragraphs (e)(3)(ii) (a) acetic acid:3.86 percent sodium ace- and (b) of this section. tate:methanol:distilled water (a) Micrograms of cephradine per milli- (3:15:200:782). Filter the mobile phase gram. Dissolve an accurately weighed through a suitable glass fiber filter or portion of the sample with mobile equivalent that is capable of removing phase to obtain a solution containing particulate contamination to 1 micron 0.8 milligram per milliliter. Place in an in diameter. Degas the mobile phase ultrasonic bath to facilitate dissolu- prior to its introduction into the chro- tion. Using this sample solution, pro- matograph pumping system. The dis- ceed as directed in paragraph (f)(1) of tilled water:methanol ratio may be this section. varied to obtain acceptable operation (b) Milligrams of cephradine per con- of the system. tainer. Reconstitute the sample as di- (d) Operating conditions. Perform the rected in the labeling. Then, using a assay at ambient temperature with a suitable hypodermic needle and sy- typical flow rate of 1.2 milliliters per ringe, remove all of the withdrawable minute. Use a detector sensitivity set- contents if it is represented as a single- ting that gives a peak height for the dose container; or, if the labeling speci- cephradine in the cephradine working fies the amount of potency in a given standard that is about 75 percent of full volume of the resultant preparation, scale. remove an accurately measured rep- (e) Preparation of working standard resentative portion from each con- and sample solutions—(1) Preparation of tainer. Dilute the solution thus ob- cephradine working standard solution. tained with mobile phase to obtain a Place an accurately weighed portion of solution containing 0.8 milligram per the cephradine working standard into a milliliter. Using this sample solution,
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proceed as directed in paragraph (f)(1) × × of this section. Milligrams of = Au P s d cefoperazone per vial × (f) Procedure—(1) Cephradine content. As 1, 000 Using the equipment, reagents, mobile where: phase, and operating conditions as list- Au=Area of the cephradine peak in the chro- ed in paragraphs (a), (b), (c), and (d) of matogram of the sample (at a retention this section, inject 10 microliters of the time equal to that observed for the cephradine working standard solution standard);
into the chromatograph. Allow an As=Area of the chephradine peak in the Chro- elution time sufficient to obtain satis- matogram of the cephradine working factory separation of the expected standard; components. After separation of the Ps=Cephradine activity in the cephradine working standard solution has been working standard solution in completed, inject 10 microliters of the micrograms per milliliter; sample solution prepared as described Cs=Milligrams of the standard per milliliter; in paragraph (e)(3)(i) of this section and into the chromatograph and repeat the d=Dilution factor of the sample. procedure described for the working (3) Calculate the percent cephalexin standard solution. The elution order is content of the sample as follows: void volume, cephalexin, and cephradine. If the sample is packaged × × × Percent AWPa b b 10 for dispensing, repeat the procedure for = cephalexin × × − each sample solution prepared as de- Ab W u (100 m ) scribed in paragraphs (e)(3)(ii) (a) and where: (b) of this section. Aa=Area of the cephalexin peak in the chro- (2) Cephalexin content. Proceed as di- matogram of the sample (at a retention rected in paragraph (f)(1) of this sec- time equal to that observed for the tion, except: standard);
(i) Use a detector sensitivity setting Ab=Area of the cephalexin peak in the chro- that gives a peak height for the matogram of the cephalexin working cephalexin in the cephalexin working standard; standard that is about 75 percent of full Wb=Milligrams of cephalexin per milliliter of scale; and cephalexin working standard solution; (ii) Use the cephalexin working Wu=Milligrams of cephradine per milliliter of standard in lieu of the cephradine sample solution; working standard. Pb=Micrograms of cephalexin per milligram of cephalexin working standard; and (g) Calculations. (1) Calculate the m=Percent moisture content of the sample. micrograms of cephradine per milli- gram of sample as follows: [49 FR 47483, Dec. 5, 1984] § 436.338 High-pressure liquid Micrograms of AP× ×100 cefoperazone per milligram = u s chromatographic assay for × × − cefoperazone. milligram As C u ()100 m where: (a) Equipment. A suitable high-pres- sure liquid chromatograph equipped Au=Area of the cephradine peak in the chro- matogram of the sample (at a retention with: time equal to that observed for the (1) A low dead volume cell 8 to 20 standard); microliters; As=Area of the cephradine peak in the chro- (2) A light path length of 1 centi- matogram of the cephradine working meter; standard; (3) A suitable ultraviolet detection Ps=Cephradine activity in the cephradine working standard solution in system operating at a wavelength of micrograms per milliliter; 254 nanometers; Cu=Milligrams of sample per milliliter of (4) A suitable recorder of at least 25.4 sample solution; and centimeter deflection; m= Percent moisture content of the sample. (5) A suitable integrator; (2) Calculate the cephradine content (6) A 30-centimeter column having an of the vial as follows: inside diameter of 4.0 millimeters and
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packed with octadecyl silane chemi- suitable hypodermic needle and sy- cally bonded to porous silica or ce- ringe, remove all of the withdrawable ramic microparticles, 5 to 10 microm- contents if it is represented as a single- eters in diameter, United States Phar- dose container; or, if the labeling speci- macopeia XX. fies the amount of potency in a given (b) Mobile phase. Mix 1.2 milliliters volume of the resultant preparation, 1M triethylammonium acetate, 2.8 mil- remove an accurately measured rep- liliters 1M acetic acid, and 120 milli- resentative portion from each con- liters acetonitrile in a one liter flask tainer. Further dilute and aliquot of and dilute to volume with distilled this solution with mobile phase to a water. Filter the mobile phase through concentration of 0.16 milligram of a suitable glass fiber filter or equiva- cefoperazone activity per milliliter. lent that is capable of removing partic- Using this sample solution, proceed as ulate contamination to 1 micron in di- directed in paragraph (f) of this sec- ameter. Degas the mobile phase just tion. prior to its introduction into the (f) Procedure. Using the equipment, chromatographic pumping system. reagents, and operating conditions as (c) Operating conditions. Perform the listed in paragraphs (a), (b), and (c) of assay at ambient temperature with a this section, inject 10 microliters of the typical flow rate of 2.0 milliliters per working standard solution into the minute. Use a detector sensitivity set- chromatograph. Allow an elution time ting that gives a peak height for the sufficient to obtain satisfactory sepa- working standard that is at least 50 ration of the expected components. percent of scale. After separation of the working stand- (d) Preparation of working standard so- ard solution has been completed, inject lution. Dissolve approximately 40 milli- 10 microliters of the sample solution grams of working standard, accurately prepared as described in paragraph weighed, with mobile phase to obtain a (e)(1) of this section into the chro- solution containing 0.16 milligram of matograph and repeat the procedure cefoperazone activity per milliliter. described for the working standard so- (e) Preparation of sample solutions—(1) lution. If the sample is packaged for Product not packaged for dispensing dispensing, repeat the procedure for (micrograms of cefoperazone per milli- each sample solution prepared as de- gram). Dissolve an accurately weighed scribed in paragraphs (e)(2)(i) and (ii) of portion of the sample with sufficient this section. mobile phase to obtain a solution con- (g) Calculations—(1) Calculate the taining 0.16 milligram of cefoperazone micrograms of cefoperazone per milli- activity per milliliter. Using this sam- gram of sample as follows: ple solution, proceed as directed in paragraph (f) of this section. Micrograms of × × APu s 100 (2) Product packaged for dispensing. cefoperazone per milligram = × × − Determine both micrograms of milligram As C u ()100 m cefoperazone per milligram of the sam- where: ple and milligrams of cefoperazone per Au=Area of the cefoperazone sample peak (at container. Use separate containers for a retention time equal to that observed preparation of each sample solution as for the standard); described in paragraph (e)(2)(i) and (ii) As=Area of the cefoperazone working stand- of this section. ard peak; Ps=Cefoperazone activity in the cefoperazone (i) Micrograms of cefoperazone per mil- working standard solution in ligram. Dissolve and accurately weighed micrograms per milliliter; portion of the sample with sufficient Cu=Milligrams of sample per milliliter of mobile phase to obtain a solution con- sample solution; and taining 0.16 milligram of cefoperazone m= Percent moisture content of the sample. activity per milliliter. Using this sam- (2) Calculate the cefoperazone con- ple solution, proceed as directed in tent of the vial as follows: paragraph (f) of this section. (ii) Milligrams of cefoperazone per con- × × Milligrams of = Au P s d tainer. Reconstitute the sample as di- cefoperazone per vial × rected in the labeling. Then using a As 1, 000
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where: (c) Preparation of sample solution. Re- Au=Area of the cefoperazone sample peak (at constitute the vial with 6 milliliters of a retention time equal to that observed deaerated water. for the standard); (d) Procedure. Using the equipment As=Area of the cefoperazone working stand- ard peak; and reagents listed in paragraphs (a) and (b) of this section, start pumping Ps=Cefoperazone activity in the cefoperazone working standard solution in the mobile solvent at the initial condi- micrograms per milliliter; and tions. Inject 10 microliters of the sam- d=Dilution factor of the sample. ple solution into the chromatograph [48 FR 789, Jan. 7, 1983; 48 FR 7439, Feb. 22, and begin the linear gradient pumping 1983; 48 FR 28250, June 21, 1983] program. After the final mobile phase conditions are reached (1 hour) con- § 436.339 High-pressure liquid tinue to pump the solvent mixture for chromatographic assay for an additional 20 minutes or until the bleomycin fractions. demethylbleomycin A2 is eluted. The (a) Equipment. A high-pressure liquid elution order is void volume, chromatograph equipped with: bleomycinic acid, bleomycin A2, (1) Two solvent pumps; bleomycin A5, bleomycin B2, bleomycin (2) A solvent programmer; B4, and demethylbleomycin A2. (3) A low dead volume cell 8 to 20 (e) Calculations. Calculate the per- microliters; centage of each bleomycin by compar- (4) A light path length of 1 centi- ing its peak area contribution to that meter; of the total response of all the (5) A suitable ultraviolet detection bleomycins. system operating at a wavelength of [48 FR 51912, Nov. 15, 1983] 254 nanometers; (6) A suitable recorder; § 436.340 High-pressure liquid (7) A suitable integrator; and chromatographic assay for tetra- (8) A suitable-sized column approxi- cycline hydrochloride content and mately 25 centimeters in length having 4-epitetracycline hydrochloride an inside diameter of 4.6 millimeters content. and packed with octadecyl silane (a) Equipment. A suitable high-pres- chemically bonded to porous silica or sure liquid chromatograph equipped ceramic microparticles, 5 to 10 mi- with: crometers in diameter, USP XX. (1) A low dead volume cell 8 to 20 (b) Reagents—(1) 0.005M 1- microliters; pentanesulfonic acid in 0.5 percent acetic (2) A light path length of 1 centi- acid adjusted to pH 4.3 with concentrated meter; ammonium hydroxide. Filter and degas (3) A suitable ultraviolet detection before using. system operating at a wavelength of (2) Methanol, spectrophotometric grade. 254 nanometers; Filter and degas before using. (4) A suitable recorder of at least (3) Mobile phase. Adjust the solvent 25.4-centimeter deflection; programmer for linear gradient devel- (5) A suitable integrator; and opment starting with a mixture of 0.005M 1-pentanesulfonic acid:methanol (6) A 30-centimeter column having an (9:1) and ending with a mixture of inside diameter of 4.0 millimeters and 0.005M 1-pentanesulfonic acid:methanol packed with octadecyl silane chemi- (6:4) in 1 hour at a flow rate of 1.2 milli- cally bonded to porous silica or ce- liters per minute. Minor flow rate and ramic microparticles. gradient changes can be made as nec- (b) Mobile phase. Dissolve 0.55 gram of essary depending on column and in- monobasic ammonium phosphate in 900 strument conditions. Disodium milliliters of water. Adjust the pH to ethylenediaminetetraacetic acid USP 1.8 with concentrated phosphoric acid at a concentration of 0.005M may be and dilute to 1 liter with water. Mix 800 added to the mobile phase if necessary milliliters of this solution with 200 mil- for satisfactory performance. liliters of methanol. Filter the mobile
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phase through a suitable glass fiber fil- Transfer 10.0 milliliters of the reconsti- ter that is capable of removing particu- tuted sample into a 50-milliliter volu- late contamination to 1 micron in di- metric flask and dilute to volume with ameter. Degas the mobile phase just a methanol:water mixture (7:18). prior to its introduction into the chro- (e) Procedure. Using the equipment, matography pumping system. reagents, and operating conditions as (c) Operating conditions. Perform the listed in paragraphs (a), (b), and (c) of assay at ambient temperature with a this section, inject 10 microliters of the typical flow rate of 1.0 milliliter per working standard solution into the minute. Use a detector sensitivity set- ting that gives a peak height for the 4- chromatograph. Allow an elution time epitetracycline peak that is at least 50 sufficient to obtain separation of the percent of scale. expected components. After separation (d) Preparation of working standard of the working standard solution has and sample solutions—(1) Working stand- been completed, inject 10 microliters of ard solution. Accurately weigh approxi- the sample solution into the chro- mately 18 milligrams of the tetra- matograph and repeat the procedure cycline hydrochloride working stand- described for the working standard so- ard into a 50-milliliter volumetric lution. The elution order is 4- flask. Into the same flask, accurately epitetracycline followed by tetra- weigh approximately 38 milligrams of cycline. the 4-epitetracycline working standard. (f) Calculations. Calculate the tetra- Dissolve and dilute to volume with a cycline hydrochloride and 4- methanol:water mixture (7:18). epitetracycline hydrochloride content (2) Sample solution. Reconstitute the as follows: sample as directed in the labeling.
Milligrams of tetracycline hydrochloride AWBWC[(× ) + ( × )] = 1 1 e per milliliter of sample × A2 1, 000
AWDWE[(× ) + ( × )] Milligrams of 4-epitetracycline hydrochloride = 3 e t per milliliter of sample × A4 1, 000
where: in the 4-epitetracycline working stand- A1=Area of the tetracycline sample peak (at ard; and a retention time equal to that observed E=Percent 4-epitetracycline hydrochloride in for tetracycline in the tetracycline work- the tetracycline working standard. ing standard); [48 FR 51290, Nov. 8, 1983] A2=Area of the tetracycline peak in the tet- racycline working standard; A3=Area of the 4-epitetracycline sample peak § 436.341 High-pressure liquid (at a retention time equal to that ob- chromatographic assay for served for the 4-epitetracycline peak in plicamycin. the 4-epitetracycline working standard); (a) Equipment. A suitable high-pres- A4=Area of the 4-epitetracycline peak in the 4-epitetracycline working standard; sure liquid chromatograph equipped Wt=Milligrams of the tetracycline working with: standard; (1) A low dead volume cell 8 to 20 We=Milligrams of the 4-epitetracycline work- microliters; ing standard; (2) A light path length of 1 centi- B=Percent tetracycline hydrochloride in the meter; tetracycline working standard; C=Percent tetracycline hydrochloride in the (3) A suitable ultraviolet detection 4-epitetracycline working standard; system operating at a wavelength of D=Percent 4-epitetracycline hydrochloride 280 nanometers;
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(4) A suitable recorder of at least Chapter 621 chromatography. Proceed 25.4-centimeter deflection; as directed in paragraph (e)(2) of this (5) A suitable integrator; and section if the minimum performance (6) A 25-centimeter column having an requirement for the relative standard inside diameter of 4.6 millimeters and deviation is not more than 2.0 percent. packed with octadecyl silane chemi- If the minimum performance require- cally bonded to porous silica or ce- ment is not met, adjustment must be ramic microparticles, 5 micrometers to made to the system to obtain satisfac- 10 micrometers in diameter, U.S.P. XX. tory operation before proceeding as de- (b) Reagents—(1) 0.01M phosphoric scribed in paragraph (e)(2) of this sec- acid. tion. (2) Mobile phase. Mix acetonitrile (2) Determination of the chromatogram. (high-pressure liquid chromatography Inject 10 microliters of the working grade):0.01M phosphoric acid (350:650). standard solution into the chro- Filter the mobile phase through a suit- matograph. Allow an elution time suf- able glass fiber filter or equivalent ficient to obtain satisfactory separa- that is capable of removing particulate tion of expected components. After sep- contamination to 1 micron in diameter. aration of the working standard has Degas the mobile phase just prior to its been completed, inject 10 microliters of introduction into the chromatograph the sample solution into the chro- pumping system. matograph and repeat the procedure (c) Operating conditions. Perform the described for the working standard so- assay at ambient temperature with a lution. typical flow rate of 1.0 milliliter per (f) Calculations. Calculate the minute. Use a detector sensitivity set- plicamycin content as described in the ting that gives a peak height for the individual monograph for the drug working standard that is at least 50 being tested. percent of scale. [49 FR 24017, June 11, 1984, as amended at 50 (d) Preparation of working standard FR 5749, Feb. 12, 1985] and sample solutions—(1) Preparation of working standard solution. Place ap- § 436.342 High-pressure liquid proximately 5 milligrams of the chromatographic assay for plicamycin working standard, accu- cefazolin. rately weighed, into a 50-milliliter, (a) Equipment. A suitable high-pres- amber volumetric flask and dilute to sure liquid chromatograph equipped volume with mobile phase and mix. with: (2) Preparation of sample solution. Pre- (1) A low dead volume cell 8 to 20 pare the sample solution as described microliters; in the individual monograph for the (2) A light path length of 1 centi- drug being tested. meter; (e) Procedure. Use the equipment, re- (3) A suitable ultraviolet detection agents, operating conditions, and system operating at a wavelength of working standard and sample solutions 254 nanometers; described in paragraphs (a), (b), (c), and (4) A suitable recorder of at least 25.4 (d) of this section, and proceed as di- centimeter deflection; and rected in paragraph (e)(1) of this sec- (5) A 30-centimeter column having an tion. inside diameter of 4.0 millimeters and (1) System suitability test. Equilibrate packed with octadecyl silane chemi- and condition the column by passage of cally bonded to porous silica or ce- about 10 to 15 void volumes of mobile ramic microparticles, 5 to 10 microm- phase followed by two or more rep- eters in diameter, USP XX. licate injections of the working stand- (b) Reagents—(1) Buffer solution, pH ard solution. Allow an elution time suf- 3.6. Transfer 0.9 gram of sodium phos- ficient to obtain satisfactory separa- phate, dibasic USP and 1.298 grams of tion of expected components after each citric acid USP to a 1-liter volumetric injection. Record the peak responses flask. Dissolve and dilute to volume and calculate the relative standard de- with distilled water and mix. viation as described for system suit- (2) Buffer solution, pH 7.0. Transfer ability tests in the U.S.P. XX General 5.68 grams of sodium phosphate, dibasic
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USP and 3.63 grams of potassium phos- microliters of the sample solution pre- phate monobasic to a 1-liter volu- pared as described in paragraph (d)(2) metric flask. Dissolve and dilute to of this section into the chromatograph volume with distilled water and mix. and repeat the procedure described for (3) Mobile phase. Mix buffer solution, the working standard solution. Allow pH 3.6: acetonitrile (9:1). Filter through an elution time sufficient to obtain a suitable glass fiber filter or equiva- satisfactory separation of the expected lent that is capable of removing partic- components. The elution order is void ulate contamination to 1 micron in di- volume, salicylic acid and cefazolin. ameter. Degas the mobile phase just (f) Calculation. Calculate the prior to its introduction into the chro- micrograms of cefazolin per milligram matograph pumping system. of sample as follows: (4) Internal standard solution. Transfer 1.2 grams of salicylic acid to a 200-mil- Micrograms of × × Ru P s 100 liliter volumetric flask. Dissolve in 10 cefazolin per = × × − milliliters of methyl alcohol, dilute to milligram Rs C u () 100 m volume with buffer solution, pH 7.0, where: and mix. Ru = Area of the cefazolin peak in the chro- (c) Operating conditions. Perform the matogram of the sample (at a retention assay at ambient temperature with a time equal to that observed for the typical flow rate of 2 milliliters per standard) /Area of internal standard peak; minute. Use a detector sensitivity set- Rs = Area of the cefazolin peak in the chro- ting that gives a peak height for the matogram of the cefazolin working working standard that is at least 50 standard/Area of internal standard peak; percent of scale. The minimum be- Ps = Cefazolin activity in the cefazolin work- tween peaks must be no more than 2 ing standard solution in micrograms per millimeters above the initial baseline. milliliter; (d) Preparation of working standard Cu = Milligrams of sample per milliliter of sample solution; and and sample solutions—(1) Working stand- m = Percent moisture content of the sample. ard solution. Place approximately 50 milligrams of cefazolin working stand- [48 FR 33478, July 22, 1983; 48 FR 34947, Aug. 2, 1983] ard, accurately weighed, into a 50-mil- liliter volumetric flask. Dissolve and § 436.343 High-pressure liquid dilute to volume with buffer solution, chromatographic assay for pH 7.0, and mix. Transfer 4.0 milliliters cefuroxime. of this solution to a 200-milliliter volu- (a) Equipment. A suitable high-pres- metric flask, add 5.0 milliliters of in- sure liquid chromatograph equipped ternal standard solution, dilute to vol- with: ume with buffer solution, pH 7.0, and (1) A low dead volume cell 8 to 20 mix. microliters; (2) Sample solution. Place approxi- (2) A light path length of 1 centi- mately 50 milligrams of the sample, ac- meter; curately weighed, into a 50-milliliter (3) A suitable ultraviolet detection volumetric flask. Dissolve and dilute system operating at a wavelength of to volume with buffer solution, pH 7.0, 254 nanometers; and mix. Transfer 4.0 milliliters of this (4) A suitable recorder of at least 25.4 solution to a 200-milliliter volumetric centimeter deflection; flask, add 5.0 milliliters of internal (5) A suitable integrator; and standard solution, dilute to volume (6) A 15-centimeter column having an with buffer solution, pH 7.0, and mix. inside diameter of 4.6 millimeters and (e) Procedure. Using the equipment, packed with hexyl silane chemically mobile phase, and operating conditions bonded to porous silica or ceramic listed in paragraphs (a), (b), and (c) of microparticles, 5 micrometers in diam- this section, inject 10 microliters of the eter. working standard solution prepared as (b) Reagents—(1) Acetate buffer, pH 3.4. directed in paragraph (d)(1) of this sec- Place 50 milliliters of 0.1M sodium ace- tion into the chromatograph. After tate into a 1,000-milliliter volumetric separation of the working standard so- flask and dilute to volume with 0.1M lution has been completed, inject 10 acetic acid. Mix.
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(2) Mobile phase. Mix 0.1M acetate (a) Micrograms of cefuroxime per milli- buffer, pH 3.4:acetonitrile (10:1). Filter gram. Dissolve an accurately weighed the mobile phase through a suitable portion of the sample with sufficient glass fiber filter or equivalent that is distilled water to obtain a stock solu- capable of removing particulate con- tion containing 1.0 milligram of tamination to 1 micron in diameter. cefuroxime per milliliter. Immediately Degas the mobile phase just prior to its transfer 5.0 milliliters of the stock so- introduction into the chromatograph lution to a 100-milliliter volumetric pumping system. flask, add 20.0 milliliters of internal (3) Internal standard solution. Prepare standard solution and dilute to 100 mil- a 1.5 milligram per milliliter solution liliters with distilled water and mix. of orcinol monohydrate in water. Store the solution in a refrigerator and (c) Operating conditions. Perform the use within 6 hours. Using this sample assay at ambient temperature with a solution, proceed as directed in para- typical flow rate of 2.0 milliliters per graph (e) of this section. minute. Use a detector sensitivity set- (b) Milligrams of cefuroxime per con- ting that gives a peak height for the tainer. Reconstitute the sample as di- working standard that is at least 50 rected in the labeling. Then using a percent of scale. suitable hypodermic needle and sy- (d) Preparation of working standard ringe, remove all of the withdrawable and sample solutions—(1) Preparation of contents if it is represented as a single- working standard solution. Dissolve an dose container; or, if the labeling speci- accurately weighed portion of the fies the amount of potency in a given cefuroxime working standard with suf- volume of the resultant preparation, ficient distilled water to obtain a stock remove an accurately measured rep- solution containing 1.0 milligram of resentative portion from each con- cefuroxime per milliliter. Immediately tainer. Dilute the solution thus ob- transfer 5.0 milliliters of the stock so- tained with distilled water to obtain a lution to a 100-milliliter volumetric stock solution of 1.0 milligram per mil- flask, add 20.0 milliliters of internal liliter. Immediately transfer 5.0 milli- standard solution and dilute to 100 mil- liters of the stock solution to a 100-mil- liliters with distilled water and mix. liliter volumetric flask, add 20.0 milli- Store the solution in a refrigerator and liters of internal standard solution and use within 6 hours. dilute to 100 milliliters with distilled (2) Preparation of sample solutions—(i) water and mix. Store the solution in a Product not packaged for dispensing refrigerator and use within 6 hours. (micrograms of cefuroxime per milligram). Using this sample solution, proceed as Dissolve an accurately weighed portion directed in paragraph (e) of this sec- of the sample with sufficient distilled tion. water to obtain a stock solution con- (e) Procedure. Using the equipment, taining 1.0 milligram of cefuroxime per reagents, and operating conditions as milliliter. Immediately transfer 5.0 listed in paragraphs (a), (b), and (c) of milliliters of the stock solution to a this section, inject 10 microliters of the 100-milliliter volumetric flask, add 20.0 working standard solution into the milliliters of internal standard solu- chromatograph. Allow an elution time tion and dilute to 100 milliliters with sufficient to obtain satisfactory sepa- distilled water and mix. Store the solu- ration of the expected components. tion in a refrigerator and use within 6 After separation of the working stand- hours. Using this sample solution, pro- ard solution has been completed, inject ceed as directed in paragraph (e) of this 10 microliters of the sample solution section. prepared as described in paragraph (ii) Product packaged for dispensing. (d)(2)(i) of this section into the chro- Determine both micrograms of matograph and repeat the procedure cefuroxime per milligram of the sample described for the working standard so- and milligrams of cefuroxime per con- lution. If the sample is packaged for tainer. Use separate containers for dispensing, repeat the procedure for preparation of each sample solution as each sample solution prepared as de- described in paragraphs (d)(2)(ii) (a) scribed in paragraphs (d)(2)(ii)(a) and and (b) of this section. (d)(2)(ii)(b) of this section.
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(f) Calculations. (1) Calculate the grams each of the working standard micrograms of cefuroxime per milli- and sample in 5 milliliters of a 50 per- gram of sample as follows: cent aqueous acetone solution. (d) Procedure. Pour the developing Micrograms of × × solvent into the glass trough at the RPu s 100 cefuroxime per = bottom of the chromatography tank. × × − milligram Rs C u ()100 m Cover and seal the tank. Allow it to where: equilibrate for 1 hour. Prepare a plate Ru=Area of the cefuroxime peak in the chro- as follows: On a line 2 centimeters from matogram of the sample (at a retention the base of the plate, and at intervals time equal to that observed for the of 2 centimeters, spot 5 microliters standard)/Area of internal standard peak; each of the sample and working stand- Rs=Area of the cefuroxime peak in the chro- ard solutions. After all spots are thor- matogram of the cefuroxime working standard/Area of internal standard peak; oughly dry, place the plate directly Ps=Cefuroxime activity in the cefuroxime into the glass trough of the chroma- working standard solution in tography tank. Cover and seal the tank micrograms per milliliter; tightly. Allow the solvent front to Cu=Milligrams of sample per milliliter of travel a minimum of 15 centimeters sample solution; and from the starting line. Remove the m=Percent moisture content of the sample. plate from the tank and allow it to air (2) Calculate the cefuroxime content dry. Observe under ultraviolet light of the vial as follows: (254 nanometers). (e) Evaluation. Measure the distance R× P × d Milligrams of = u s the solvent front traveled from the cefuroxime per vial × starting line and the distance the spots Rs 1, 000 are from the starting line. Calculate where: the Rf value by dividing the latter by Ru=Area of the cefuroxime peak in the chro- the former. The sample and standard matogram of the sample (at a retention should have spots of corresponding Rf time equal to that observed for the values. standard)/Area of internal standard peak; Rs=Area of the cefuroxime peak in the chro- [48 FR 38461, Aug. 24, 1983] matogram of the cefuroxime working standard/Area of internal standard peak; § 436.345 High-pressure liquid Ps=Cefuroxime activity in the cefuroxime chromatographic assay for working standard solution in ceftizoxime. micrograms per milliliter; and d= Dilution factor of the sample. (a) Equipment. A suitable high-pres- sure liquid chromatograph equipped [48 FR 38460, Aug. 24, 1983; 48 FR 40704, Sept. with: 9, 1983] (1) A low dead volume cell 8 to 20 § 436.344 Thin layer chromatographic microliters; identity test for cefuroxime. (2) A light path length of 1 centi- (a) Equipment—(1) Chromatography meter; tank. Use a rectangular tank approxi- (3) A suitable ultraviolet detection mately 23×23×9 centimeters, with a system operating at a wavelength of glass solvent trough on the bottom and 254 nanometers; a tight-fitting cover. Line the inside (4) A suitable recorder of at least 25.4 walls of the tank with Whatman #3MM centimeter deflection; chromatographic paper or equivalent. (5) A suitable integrator; and (2) Plates. Use 20 × 20 centimeter thin (6) A 30-centimeter column having an layer chromatography plates coated inside diameter of 4.0 millimeters and with Silica Gel F or equivalent to a packed with octadecyl silane chemi- thickness of 250 microns. cally bonded to porous silica or ce- (b) Developing solvent. Mix chloro- ramic microparticles, 5 to 10 microm- form, methanol, and formic acid in vol- eters in diameter, USP XX. umetric proportions of 90:16:4, respec- (b) Reagents—(1) pH 3.6 buffer solution. tively. Transfer 2.31 grams of sodium phos- (c) Preparation of the spotting solu- phate diabasic dodecahydrate and 1.42 tions. Dissolve approximately 200 milli- grams of citric acid monohydrate to a
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1-liter volumetric flask. Dissolve and ple and milligrams of ceftizoxime per dilute to volume with distilled water. container. Use separate containers for (2) pH 7.0 buffer solution. Transfer preparation of each sample solution as 14.33 grams of sodium phosphate diba- described in paragraphs (e)(2) (i) and sic dodecahydrate and 3.63 grams of po- (ii) of this section. tassium phosphate monobasic to a 1- (i) Micrograms of ceftizoxime per milli- liter volumetric flask. Dissolve and di- gram. Dissolve an accurately weighed lute to volume with distilled water. portion of the sample with sufficient (3) Mobile phase. Mix pH 3.6 buffer so- pH 7.0 buffer solution to obtain a con- lution:acetonitrile (9:1). Filter the mo- centration of 1.0 milligram of bile phase through a suitable glass ceftizoxime per milliliter. Transfer 2.0 fiber filter or equivalent that is capa- milliliters of this solution to a 100-mil- ble of removing particulate contamina- liliter volumetric flask, add 5.0 milli- tion to 1 micron in diameter. Degas the liters of internal standard solution, di- mobile phase just prior to its introduc- lute to volume with pH 7.0 buffer solu- tion into the chromatograph pumping tion and mix. Using this sample solu- system. tion, proceed as directed in paragraph (4) Internal standard solution. Place 1.2 (f) of this section. grams of salicyclic acid in a 200-milli- (ii) Milligrams of ceftizoxime per con- liter volumetric flask. Dissolve in 10 tainer. Reconstitute the sample as di- milliliters of methyl alcohol, dilute to rected in the labeling. Then using a volume with pH 7.0 buffer solution and suitable hypodermic needle and sy- mix. ringe, remove all of the withdrawable (c) Operating conditions. Perform the contents if it is represented as a single- assay at ambient temperature with a dose container; or, if the labeling speci- typical flow rate of 2.0 milliliters per fies the amount of potency is a given minute. Use a detector sensitivity set- volume of the resultant preparation, ting that gives a peak height for the remove an accurately measured rep- working standard that is at least 50 resentative portion from each con- percent of scale. tainer. Further dilute an aliquot of the (d) Preparation of working standard so- solution thus obtained with sufficient lution. Dissolve an accurately weighed pH 7.0 buffer solution to obtain a con- portion of the ceftizoxime working centration of 1.0 milligram per milli- standard with sufficient pH 7.0 buffer liter. Transfer 2.0 milliliters of this so- solution to obtain a solution contain- lution to a 100-milliliter volumetric ing 1,000 micrograms of ceftizoxime ac- flask, add 5.0 milliliters of internal tivity per milliliter. Transfer 2.0 milli- standard solution, dilute to volume liters of this solution to a 100-milliliter with pH 7.0 buffer solution and mix. volumetric flask, add 5.0 milliliters of Using this sample solution, proceed as internal standard solution, dilute to directed in paragraph (f) of this sec- volume with pH 7.0 buffer solution and tion. mix. (f) Procedure. Using the equipment, (e) Preparation of sample solutions—(1) reagents, and operating conditions as Product not packaged for dispensing listed in paragraphs (a), (b), and (c) of (micrograms of ceftizoxime per milligram). this section, inject 10 microliters of the Dissolve an accurately weighed portion working standard solution into the of the sample with sufficient pH 7.0 chromatograph. Allow an elution time buffer solution to obtain a concentra- sufficient to obtain satisfactory sepa- tion of 1.0 milligram per milliliter. ration of the expected components. The Transfer 2.0 milliliters of this solution elution order is void volume, to a 100-milliliter volumetric flask, add ceftizoxime, and internal standard. 5.0 milliliters of internal standard solu- After separation of the working stand- tion, dilute to volume with pH 7.0 buff- ard solution has been completed, inject er solution and mix. Using this sample 10 microliters of the sample solution solution, proceed as directed in para- prepared as described in paragraph graph (f) of this section. (e)(1) of this section into the chro- (2) Product packaged for dispensing. matograph and repeat the procedure Determine both micrograms of described for the working standard so- ceftizoxime per milligram of the sam- lution. If the sample is packaged for
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dispensing, repeat the procedure for (5) An oven or water bath capable of each sample solution prepared as de- maintaining the column at an operat- scribed in paragraphs (e)(2) (i) and (ii) ing temperature of 70° C; of this section. (6) A steel capillary tube, 1 meter in (g) Calculations—(1) Calculate the length, having an inside diameter of micrograms of ceftizoxime per milli- 0.25 millimeter. This tube is inserted gram of sample as follows: between the injection system and the chromatographic column and is equili- Micrograms of R ×P ×100 brated to 70° C; and ceftizoxime per = u s (7) A sample injection valve on which × × − milligram Rs C u ()100 m the loop determines the sample size. where: (b) Columns. The chromatographic column is packed with microparticu- Ru=Area of the ceftizoxime peak in the chro- late (3 to 10 micrometers in diameter) matogram of the sample (at a retention reversed phase packing materials that time equal to that observed for the standard)/Area of internal standard peak; exhibit some degree of polarity such as the hydrocarbon bonded silicas with di- Rs=Area of the ceftizoxime peak in the chro- matogram of the ceftizoxime working methyl, trimethyl, or octyl groups. standard/Area of internal standard peak; Connect a saturation column gravity Ps=Ceftizoxime activity in the ceftizoxime packed with similarly bonded silica working standard solution in particles 40 to 60 microns in diameter micrograms per milliliter; to the inlet of the analytical column. Cu=Milligrams of sample per milliliter of (c) Mobile phase. Mix acetonitrile, sample solution; and water, methanol, and o-phosphoric acid m=Percent moisture content of the sample. (900:525:75:0.075 by volume). Degas by R×P × d passing through a 0.5-micrometer filter Milligrams of ceftizoxime = u s with vacuum and ultrasonicate for no per vial R ×1, 000 less than 2 minutes before use. The mo- s bile phase may be sparged perceptibly where: with helium through a 2-micrometer Ru=Area of the ceftizoxime peak in the chro- metal filter for the duration of the matogram of the sample (at a retention time equal to that observed for the analysis. Adjust the ratio of acetoni- standard)/Area of internal standard peak; trile to aqueous buffer as necessary to Rs=Area of the ceftizoxime peak in the chro- obtain satisfactory retention of the matogram of the ceftizoxime working peaks. standard/Area of internal standard peak; (d) Operating conditions. Perform the Ps=Ceftizoxime activity in the ceftizoxime assay at a constant operating tempera- working standard solution in ture of 70° C with a typical flow rate of micrograms per milliliter; and 2.0 milliliters per minute. Use a detec- d=Dilution factor of the sample. tor sensitivity setting that gives a [48 FR 46270, Oct. 12, 1983; 48 FR 49656, Oct. 27, peak height for the working standard 1983] that is at least 50 percent of scale with a typical chart speed of 2.5 millimeters § 436.346 High-pressure liquid per minute. Obtain chromatograms for chromatographic assay for performance parameters at a chart cyclosporine. speed of not less than 25 millimeters (a) Equipment. A suitable high-pres- per minute to allow a more accurate sure liquid chromatograph equipped measurement of peak geometry. with: (e) Preparation of working standard (1) A suitable pump capable of and sample solutions. Prepare the work- reproducibly delivering a liquid to a ing standard and sample solutions as pressure of 4,500 pounds per square inch directed in the individual monographs and a flow rate of at least 5 milliliters for cyclosporine. per minute; (f) Systems suitability. Equilibrate and (2) A suitable ultraviolet detection condition the column by passage of system operating at a wavelength of about 10 to 15 void volumes of mobile 210 nanometers; phase followed by about 5 injections of (3) A suitable recorder; not less than 10 microliters each of (4) A suitable integrator; working standard solution. Proceed
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with the analysis when the following t=Retention time of solute; and the sub- minimum performance requirements scripts i and j designate two different have been met or exceeded. peaks and where tj is larger than ti; and (1) Capacity ratio factor. Calculate the W=Width of peak at baseline as determined capacity ratio (k) of the cyclosporine by extrapolating the relative straight sides to the baseline. Both t and W must peak as follows: be measured in the same units. Resolution between the cyclosporine t -tm k = peak and any other peak must be at tm least 1.1. where: (g) Procedure. Using the equipment, t=Retention time of solute; and columns, mobile phase, operating con- tm=Retention time of solvent or unretained ditions and the working standard and substance. sample solutions listed in paragraphs The capacity ratio is satisfactory if it (a), (b), (c), (d), and (e) of this section, is not less than 3 or not more than 10. inject 20 microliters of the working (2) Coefficient of variation. The coeffi- standard solution into the chro- cient of variation of at least five rep- matograph. Allow an elution time suf- licate injections is less than 1 percent. ficient to obtain satisfactory separa- (3) Efficiency. Calculate the efficiency tion of expected components. After sep- (n) as follows: aration of the working standard solu- tion has been completed, inject 20 2 microliters of the sample solution into t n = 5. 545 the chromatograph and repeat the pro- cedure described for the working stand- W0. 5 ard solution. where: t=Retention time of solute; and (h) Calculations. Calculate the cyclosporine content of cyclosporine W0.5=Peak width at half height. Both t and W0.5 must be measured in the same units. and its dosage forms as directed in the individual monographs. The efficiency is satisfactory if it is greater than 1,500 theoretical plates [49 FR 22631, May 31, 1984; 49 FR 27489, July when assaying cyclosporine and great- 5, 1984] er than 700 theoretical plates when as- saying finished dosage forms. § 436.347 High-pressure liquid (4) Asymmetry factor. Calculate the chromatographic assay for cefoxitin. asymmetry factor (As) as follows: (a) Equipment. A suitable high-pres- W sure liquid chromatograph equipped = 0. 1 As with: 2f (1) A low dead volume cell 8 to 20 where: microliters; W0.1=Horizontal distance measured from a (2) A light path length of 1 centi- point on the cyclosporine peak ascent 10 meter; percent above the baseline to an inter- (3) A suitable ultraviolet detection cept with the cyclosporine peak descent; and system operating at a wavelength of f=Horizontal distance from point of 10 per- 254 nanometers; cent ascent above the baseline of the (4) A suitable recorder of at least 25.4 cyclosporine peak to point of maximum centimeter deflection; peak height. (5) A suitable integrator; and The asymmetry factor is satisfactory if (6) A 30-centimeter column having an it is not more than 1.5. inside diameter of 4.0 millimeters and (5) Resolution. Calculate the resolu- packed with octadecyl silane chemi- tion (Rs) as follows: cally bonded to porous silica or ce- ramic microparticles, 5 micrometers to 2 (t − t ) 10 micrometers in diameter, U.S.P. XX. R = j i s + (b) Reagents—(1) One percent potas- ()WWi j sium phosphate buffer, pH 6.0. Prepare where: as described in § 436.101(a)(1).
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(2) Mobile phase. Mix distilled (6) A 30-centimeter column having an water:glacial acetic acid:acetonitrile inside diameter of 4.0 millimeters and (800:10:190). Filter the mobile phase packed wth octadecyl silane chemi- through a suitable glass fiber filter or cally bonded to porous silica or ce- equivalent that is capable of removing ramic microparticles, 5 micrometers to particulate contamination to 1 micron 10 micrometers in diameter, U.S.P. XX. in diameter. Degas the mobile phase A particular column used for analysis just prior to its introduction into the of ceforanide should not be used for the chromatograph pumping system. analysis of other drugs.— (c) Operating conditions. Perform the (b) Mobile phase. Mix 18.0 milliliters assay at ambient temperature with a of 10 percent aqueous typical flow rate of 1.0 milliliter per tetrabutylammonium hydroxide and minute. Use a detector sensitivity set- 8.56 milliliters of 11N potassium hy- ting that gives a peak height for the droxide. Add the mixture to approxi- working standard that is at least 50 mately 700 milliliters of distilled percent of scale. The minimum be- water. Add 200 milliliters of reagent tween peaks must be no more than 2 grade methanol. Adjust the pH of the millimeters above the baseline. mixture to pH 7.0 with concentrated (d) Preparation of working standard phosphoric acid and dilute to 1,000 mil- and sample solutions. Use the working liliters with distilled water. Prepare standard and sample solutions prepared fresh daily. Filter the mobile phase as described in the individual mono- through a suitable glass fiber filter or graphs for the drug being tested. equivalent which is capable of remov- (e) Procedure. Using the equipment, ing particulate contamination to 1 mi- reagents, and operating conditions as cron in diameter. Degas the mobile described in paragraphs (a), (b), and (c) phase just prior to its introduction of this section, inject 10 microliters of into the chromatograph pumping sys- the working standard solution into the tem. chromatograph. Allow an elution time sufficient to obtain separation of the (c) Operating conditions. Perform the expected components. After separation assay at ambient temperature with a of the working standard solution has typical flow rate of 1 milliliter per been completed, inject 10 microliters of minute. Use a detector sensitivity set- the sample solution into the chro- ting that gives a peak height for the matograph and repeat the procedure working standard that is at least 50 described for the working standard so- percent of scale. lution. (d) Preparation of working standard (f) Calculations. Calculate the and sample solutions—(1) Preparation of cefoxitin content as described in the working standard solution. Prepare a so- individual monographs for the drug lution containing 1,000 micrograms of being tested. ceforanide activity per milliliter in mobile phase. Inject working standard [49 FR 47827, Dec. 7, 1984] solution within 5 minutes after dissolu- § 436.348 High-pressure liquid tion. chromatographic assay for (2) Preparation of sample solution. Pre- ceforanide. pare the sample solution as directed in (a) Equipment. A suitable high-pres- the individual monograph for the drug sure liquid chromatograph equipped being tested. Inject sample solution with: within 5 minutes after dissolution. (1) A low dead volume cell 8 to 20 (e) Procedure. Use the equipment, mo- microliters; bile phase, operating conditions, and (2) A light path length of 1 centi- working standard and sample solutions meter; described in paragraphs (a), (b), (c), and (3) A suitable ultraviolet detection (d) of this section, and proceed as di- system operating at a wavelength of rected in paragraph (e)(1) of this sec- 254 nanometers; tion. (4) A suitable recorder of at least (1) System suitability test. Equilibrate 25.4-centimeter deflection; and condition the column by passage of (5) A suitable integrator; and about 10 to 15 void volumes of mobile
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phase followed by three replicate injec- (1) A suitable pump capable of tions of 10 microliters each of the reproducibly delivering a liquid to a working standard solution. Allow an pressure of 5,000 pounds per square elution time sufficient to obtain satis- inch; factory separation of expected compo- (2) A suitable ultraviolet detection nents after each injection. Record the system operating at a wavelength of peak responses and calculate the tail- 254 nanometers; ing factor, efficiency of the column, co- (3) A suitable recorder; efficient of variation, and capacity fac- (4) A suitable integrator; and tor as described for system suitability (5) A 25-centimeter column having an tests in the U.S.P. XX General Chapter inside diameter of 4.6 millimeters and 621 chromatography. Proceed as di- packed with octadecyl silane chemi- rected in paragraph (e)(2) of this sec- cally bonded to porous silica or ce- tion if the following minimum per- ramic microparticles, 5 micrometers to formance requirements have been met: 10 micrometers in diameter, U.S.P. XX. (i) Tailing factor. The tailing factor is (b) Reagents—(1) 2,4- satisfactory if it is not more than 1.2; Dinitrofluorobenzene solution. Weigh (ii) Efficiency of the column. The effi- accurately approximately 760 milli- ciency of the column is satisfactory if grams of 2,4-dinitrofluorobenzene into it is greater than 1,900 theoretical a 50-milliliter volumetric flask. Dis- plates; solve and dilute to volume with abso- (iii) Coefficient of variation. The coef- lute ethyl alcohol. ficient of variation of at least three (2) Tris (hydroxymethyl) replicate injections is satisfactory if it aminomethane (THAM) solution. is not more than 1.5 percent; and Weigh accurately approximately 1.44 (iv) Capacity factor. The capacity fac- grams of THAM into a 100-milliliter tor is satisfactory if it is not less than volumetric flask. Dissolve and dilute 1.8 and not more than 5. to volume with distilled water. If the minimum performance require- (c) Mobile phase. Mix methanol and ments are not met, adjustments must water (62:38), and adjust to pH 3.0 with be made to the system to obtain satis- glacial acetic acid. factory operation before proceeding as (d) Operating conditions. Perform the described in paragraph (e)(2) of this assay at ambient temperature with a section. typical flow rate of 1.5 milliliters per (2) Determination of the chromatogram. minute. Use a detector sensitivity set- Inject 10 microliters of the working ting that gives a peak height for the standard solution into the chro- standard that is at least 50 percent of matograph. Allow an elution time suf- scale with a typical chart speed of 0.2 ficient to obtain satisfactory separa- inch per minute. tion of the expected components. After (e) Preparation of standard and sample separation of the working standard so- solutions—(1) Preparation of standard so- lution has been completed, inject 10 lution. Weigh accurately approximately microliters of the sample solution into 36 milligrams of L-lysine used as the the chromatograph and repeat the pro- standard into a 100-milliliter volu- cedure described for the working stand- metric flask. Dissolve and dilute to ard solution. volume with distilled water. Transfer (f) Calculations. Calculate the 2.0 milliliters of the L-lysine solution ceforanide content as directed in the into a 10-milliliter volumetric flask, individual monograph for the drug add 2.0 milliliters of THAM solution being tested. and 3.0 milliliters of 2,4- [49 FR 25846, June 25, 1984; 49 FR 34347, Aug. dinitrofluorobenzene solution. Cap 30, 1984] tightly and mix well. Place the flask in a 50° C water bath for 30 minutes. Re- § 436.349 High-pressure liquid move from water bath, allow the flask chromatographic assay for L-lysine to cool to room temperature, and di- in ceforanide for injection. lute to volume with methanol. Mix (a) Equipment. A suitable high-pres- well. sure liquid chromatograph equipped (2) Preparation of sample solution. with: Weigh accurately approximately 150
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milligrams of the sample, ceforanide (v) Capacity factor. The capacity fac- for injection, into a 100-milliliter volu- tor is satisfactory if it is not less than metric flask. Dissolve and dilute to 4 and not more than 6. volume with distilled water. Transfer If the minimum performance require- 2.0 milliliters of the sample solution ments are not met, adjustments must into a 10-milliliter volumetric flask, be made to the system to obtain satis- add 2.0 milliliters of THAM solution factory operation before proceeding as and 3.0 milliliters of 2,4- described in paragraph (f)(2) of this sec- dinitrofluorobenzene solution. Cap tion. tightly and mix well. Place the flask in (2) Determination of the chromatogram. a 50° C water bath for 30 minutes. Re- Inject 20 microliters of the standard so- move from water bath, allow the flask lution into the chromatograph. Allow to cool to room temperature, and di- an elution time sufficient to obtain lute to volume with methanol. Mix satisfactory separation of the expected well. components. After separation of the (f) Procedure. Use the equipment, re- standard solution is completed, inject agents, mobile phase, operating condi- 20 microliters of the sample solution tions, and standard and sample solu- into the chromatograph and repeat the tions described in paragraphs (a), (b), procedure described for the standard (c), (d), and (e) of this section, and pro- solution. ceed as directed in paragraph (f)(1) of (g) Calculations. Calculate the percent this section. of L-lysine per milligram of ceforanide (1) System suitability test. Equilibrate for injection as follows: and condition the column by passage of about 10 to 15 void volumes of mobile AP× Percent of L -lysine = u s phase followed by three replicate injec- × × tions of 20 microliters each of the ACs u 10 standard solution. Allow an elution where:
time sufficient to obtain satisfactory Au = Area of the L-lysine peak in the chro- separation of the expected components matogram of the sample (at a retention after each injection. Record the peak time equal to that observed for the responses and calculate the resolution standard); factor, tailing factor, efficiency of the As = Area of the L-lysine peak in the chro- matogram of the L-lysine standard: column, coefficient of variation, and Ps = L-lysine content in the L-lysine stand- capacity factor as described for system ard solution in micrograms per milli- suitability tests in the U.S.P. XX Gen- liter; and
eral Chapter 621 chromatography. Pro- Cu = Milligrams of sample per milliliter of ceed as directed in paragraph (f)(2) of sample solution. this section if the following minimum [49 FR 25846, June 25, 1984; 49 FR 34347, Aug. performance requirements have been 30, 1984; 49 FR 40006, Oct. 12, 1984] met: (i) Resolution factor. The resolution § 436.350 High-performance liquid factor between the peak for derivatized chromatographic assay for L-lysine and from the peak for the cefonicid. dinitrofluorobenzene derivatizing rea- (a) Apparatus. A suitable high-per- gent is satisfactory if it is not less formance liquid chromatograph than 4.5; equipped with: (ii) Tailing factor. The tailing factor (1) A suitable detection system speci- is satisfactory if it is not more than fied in the monograph for the drug 1.3; being tested; (iii) Efficiency of the column. The effi- (2) A suitable recording device of at ciency of the column is satisfactory if least 25-centimeter deflection; it is greater than 1,500 theoretical (3) A suitable chromatographic data plates; managing system; and (iv) Coefficient of variation. The coeffi- (4) An analytical column, 3 to 30 cen- cient of variation of at least three rep- timeters long, packed with a material licate injections is satisfactory if it is as defined in the monograph for the not more than 1.5 percent; and drug being tested; and if specified in
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that monograph, the inlet of this col- n=Efficiency, as number of theoretical plates umn may be connected to a guard col- for column; umn 3 to 5 centimeters in length, tR=Retention time of solute; and packed with the same material of 40 to wh=Peak width at half-height. 60 micrometers particle size. (3) Resolution factor. Calculate the (b) Procedure. Perform the assay and resolution factor (R), between calculate the drug content using the desacetyl cefonicid and cefonicid, as temperature, instrumental conditions, follows: and calculations specified in the mono- graph for the drug being tested with a 2(t− t ) flow rate not to exceed 2.0 milliliters R = 2 1 per minute. Use a detector sensitivity + w w2 setting that gives a peak height for the 1 working standard that is at least 50 where: percent of scale with typical chart t1=Retention time of desacetyl cefonicid; speed of not less than 2.5 millimeters t2=Retention time of cefonicid; and w and w =Widths of the bases of the cor- per minute. Use the apparatus de- 1 2 responding peaks obtained by extrapolat- scribed in paragraph (a) of this section; ing the relatively straight sides of the and the reagents and working standard peaks to the baseline. and sample solutions described in the monograph for the drug being tested. (4) Coefficient of variation (relative Equilibrate and condition the column standard deviation). Calculate the coef- by passage of 10 to 15 void volumes of ficient of variation (SR in percent) as mobile phase followed by five replicate follows: injections of the same volume (between 1/ 2 10 and 20 microliters) of the working n − 2 standard solution. Allow an operating ∑(XXi ) 100 = time sufficiently long to obtain satis- S = i 1 factory separation and elution of the R X N −1 expected components after each injec- tion. Record the peak responses and calculate the prescribed system suit- where: ability requirements as follows: X¯ is the mean of N individual measurements (c) System suitability test. Using the of Xi. apparatus and procedure described in If the complete operating system this section, test the chromatographic meets the system suitability require- system for assay as follows: ments of the monograph for the drug (1) Tailing factor. Calculate the tail- being tested, proceed as described in ing factor (T), from distances measured paragraph (b) of this section, using the along the horizontal line at 5 percent sample solution in lieu of the working of the peak height above the baseline, standard solution. as follows: [49 FR 34347, Aug. 30, 1984, as amended at 49 FR 44460, Nov. 7, 1984; 50 FR 29209, July 18, W0.05 T = 1985] 2 f where: § 436.351 High-performance liquid chromatographic assay for W0.05 = Width of peak at 5 percent height; and f = Horizontal distance from point of ascent amoxicillin and clavulanic acid. to a point coincident with maximum (a) Apparatus. A suitable high-per- peak height. formance liquid chromatograph (2) Efficiency of the column. Calculate equipped with: the number of theoretical plates (n) of (1) A suitable detection system speci- the column as follows: fied in the monograph for the drug being tested; 2 t (2) A suitable recording device of at n = 5. 545 R least 25-centimeter deflection; Wh (3) A suitable chromatographic data where: managing system; and
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(4) An analytical column, 10 to 30 (2) Efficiency of the column. Calculate centimeters long, packed with a mate- the number of theoretical plates (n) of rial as defined in the monograph for the column as follows: the drug being tested; and if specified 2 in that monograph, the inlet of this t column may be connected to a guard n = 5. 545 R column, 3 to 5 centimeters in length, Wh packed with the same material of 40 to where: 60 micrometers particle size. n=Efficiency, as number of theoretical plates (b) Procedure. Perform the assay and for column; calculate the drug content using the tR=Retention time of amoxicillin or temperature, instrumental conditions, clavulanic acid peaks; and and calculations specified in the mono- wh=Corresponding peak width at half-height. graph for the drug being tested with a flow rate not to exceed 2.0 milliliters (3) Resolution factor. Calculate the per minute. Use a detector sensitivity resolution factor (R) as follows: setting that gives a peak height for the 2(t− t ) working standard that is at least 50 R = 2 1 percent of scale with typical chart + speed of not less than 2.5 millimeters w1 w2 per minute. Use the apparatus de- where:
scribed in paragraph (a) of this section; t1=Retention time of amoxicillin peak; and the reagents and working standard t2=Retention time of clavulanic acid peak; and sample solutions described in the and monograph for the drug being tested. w1 and w2=Widths of the bases of the cor- Equilibrate and condition the column responding peaks obtained by extrapolat- by passage of 10 to 15 void volumes of ing the relatively straight sides of the mobile phase followed by five replicate peaks to the baseline. injections of the same volume (between (4) Coefficient of variation (Relative 10 and 20 microliters) of the working standard deviation). Calculate the coef- standard solution. Allow an operating ficient of variation (SR in percent) as time sufficiently long to obtain satis- follows: factory separation and elution of the expected components after each injec- 1/ 2 n 2 tion. The retention times for ∑(XX− ) amoxicillin and clavulanic acid are i 100 i=1 about 2.1 and 1.0 minutes, respectively, S = R X N −1 under these prescribed conditions. Record the peak responses and cal- culate the prescribed system suit- where: ability requirements as follows: ¯ (c) System suitability test. Using the X is the mean of N individual measurements of X . apparatus and procedure described in i this section, test the chromatographic If the complete operating system system for assay as follows: meets the system suitability require- (1) Tailing factors for the amoxicillin ments of the monograph for the drug and clavulanic acid peaks. Calculate the being tested, proceed as described in tailing factors (T), from distances paragraph (b) of this section, using the measured along the horizontal line at 5 sample solution in lieu of the working percent of the peak height above the standard solution. baseline, as follows: [49 FR 39671, Oct. 10, 1984] W T = 0. 05 § 436.352 High-performance liquid chromatographic assay for deter- 2 f mining clavam-2-carboxylate con- where: tent in clavulanate potassium. W0.05=Width of peak at 5 percent height; and f=Horizontal distance from point of ascent to (a) Apparatus. A suitable high-per- a point coincident with maximum peak formance liquid chromatograph height. equipped with:
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(1) A suitable detection system speci- (c) System suitability test. Using the fied in the monograph for the drug apparatus and procedure described in being tested; this section, test the chromatographic (2) A suitable recording device of at system for assay as follows: least 25-centimeter deflection; (1) Tailing factor. Calculate the tail- (3) A suitable chromatographic data ing factor (T), from distances measured managing system; and along the horizontal line at 5 percent (4) An analytical column, approxi- of the peak height above the baseline, mately 30 centimeters in length, as follows: packed with a material as defined in the monograph for the drug being test- W ed. T = 0. 05 (b) Procedure. Perform the assay and 2 f calculate the drug content using the temperature, instrumental conditions, where: and calculations specified in the mono- W0.05=Width of peak at 5 percent height; and graph for the drug being tested with a f=Horizontal distance from point of ascent to a point coincident with maximum peak flow rate not to exceed 0.5 milliliter height. per minute. Use a detector sensitivity setting that gives a peak height for the (2) Efficiency of the column. Calculate working standard that is at least 50 the number of theoretical plates (n) of percent of scale with typical chart the column as follows: speed of not less than 2.5 millimeters 2 per minute. Use the apparatus de- t scribed in paragraph (a) of this section; n = 5. 545 R and the mobile phase and working Wh standard and sample solutions de- where: scribed in the monograph for the drug n=Efficiency, as number of theoretical plates being tested. Equilibrate and condition for column; the column by passage of 10 to 15 void tR=Retention time of clavam-2-carboxylic volumes of mobile phase followed by acid peak; and
five replicate injections of the same wh=Corresponding peak width at half-height. volume (between 10 and 20 microliters) of the working standard solution. (3) Resolution factor. Calculate the Allow an operating time sufficiently resolution factor (R) as follows: long to obtain satisfactory separation 2(t− t ) and elution of the expected components R = 2 1 after each injection. The retention + times for clavam-2-carboxylic acid and w1 w2 clavulanic acid are about 10 and 14 where: minutes, respectively, under these pre- t1=Retention time of clavam-2-carboxylic scribed conditions. The sample solution acid peak; should be injected at least in duplicate t2=Retention time of clavulanic acid peak; and an average should be taken. For and each such series of samples injected, w1 and w2=Widths of the bases of the cor- two injections of standard should be responding peaks obtained by extrapolat- made, one before and one after the ing the relatively straight sides of the peaks to the baseline. sample series, and an average should be taken. Record the peak responses and (4) Coefficient of variation (Relative calculate the prescribed system suit- standard deviation). Calculate the coef- ability requirements as follows: ficient of variation (SR
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n 1/ 2 − 2 ∑(XXi ) 100 = i=1 SR X N −1
where: monograph for the drug being tested. X¯ is the mean of N individual measurements Equilibrate and condition the column of Xi by passage of 10 to 15 void volumes of If the complete operating system mobile phase followed by five replicate meets the system suitability require- injections of the same volume (between ments of the monograph for the drug 10 and 20 microliters) of the working being tested, proceed as described in standard solution. Allow an operating paragraph (b) of this section, using the time sufficiently long to obtain satis- sample solution in lieu of the working factory separation and elution of the standard solution. expected components after each injec- tion. Record the peak responses and [49 FR 39671, Oct. 10, 1984] calculate the prescribed system suit- § 436.353 High-performance liquid ability requirements as described for chromatographic assay for the system suitability test in para- amdinocillin. graph (c) of this section. (a) Apparatus. A suitable high-per- (c) System suitability test. Using the formance liquid chromatograph apparatus and procedure described in equipped with: this section, test the chromatographic (1) A suitable detection system speci- system for assay as follows: fied in the monograph for the drug (1) Tailing factor. Calculate the tail- being tested; ing factor (T), from distances measured (2) A suitable recording device of at along the horizontal line at 5 percent least 25-centimeter deflection; of the peak height above the baseline, (3) A suitable chromatographic data as follows: managing system; and (4) An analytical column, 3 to 30 cen- W0. 05 timeters long, packed with a material T = as defined in the monograph for the 2 f drug being tested; and if specified in where: that monograph, the inlet of this col- W0.05=Width of peak at 5 percent height; and umn may be connected to a guard col- f=Horizontal distance from point of ascent to umn, 3 to 5 centimeters in length, a point coincident with maximum peak packed with the same material of 40 to height. 60 micrometers particle size. (2) Efficiency of the column. Calculate (b) Procedure. Perform the assay and the number of theoretical plates (n) of calculate the drug content using the the column by either of the following temperature, instrumental conditions, formulas: and calculations specified in the mono- graph for the drug being tested with a 2 t flow rate not to exceed 2.0 milliliters n = 5. 545 R ; or per minute. Use a detector sensitivity Wh setting that gives a peak height for the where: working standard that is at least 50 n=Efficiency, as number of theoretical plates percent of scale with typical chart for column; speed of not less than 2.5 millimeters tR=Retention time of solute; per minute. Use the apparatus de- wh=Peak width at half-height; and scribed in paragraph (a) of this section; W=Width of the base of the peak obtained by and the reagents and working standard extrapolating the relatively straight and sample solutions described in the sides of the peak to the baseline.
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(3) Resolution factor. Calculate the per minute, Use a detector sensitivity resolution factor (R) as follows: setting that gives a peak height for the working standard that is at least 50 − 2(tRj t Ri ) percent of scale. Use the apparatus de- R = scribed in paragraph (a) of this section; + wi w j and also, use the system suitability re- where: quirements, reagents, working stand- tRj=Retention time for a solute eluting after ard, test and sample solutions, and cal- i (tRj is larger than tRi); culations as directed in the individual tRi=Retention time for any solute; monograph for the drug being tested. wi=Width of peak at baseline for any solute; Equilibrate and condition the column and by passage of 10 to 15 void volumes of wj=Width of peak at baseline for any solute mobile phase followed by five replicate eluting after i. injections of 20 microliters each of the (4) Coefficient of variation (relative test solution. Allow an operating time standard deviation). Calculate the coef- sufficiently long to obtain satisfactory ficient of variation (SR separation and elution of the expected components after each injection. n 1/ 2 Record the peak responses and cal- 2 − culate the prescribed system suit- ∑(XXi ) 100 ability requirements as described for = i=1 SR the system suitability test in para- X N −1 graph (c) of this section. (c) System suitability test. Using the apparatus and procedure described in where: this section, test the chromatographic X¯ is the mean of N individual measurements system for assay as follows: of Xi. (1) Capacity factor. Calculate the ca- If the complete operating system pacity factor (k) as follows: meets the system suitability require- − ments of the monograph for the drug tRM t being tested, proceed as described in k = paragraph (b) of this section, using the tM sample solution in lieu of the working where: standard solution. tR=Retention time of solute; and tM=Retention time of solvent or unretained [50 FR 7764, Feb. 26, 1985; 50 FR 10220, Mar. 14, substance. 1985; 50 FR 18243, Apr. 30, 1985] (2) Resolution. Calculate the resolu- § 436.354 High-performance liquid tion (R) as follows: chromatographic assay for ceftriaxone. 2(t− t ) R = Rj Ri (a) Apparatus. A suitable high-per- + formance liquid chromatograph wi w j equipped with: where: (1) A suitable detection system speci- tRj=Retention time for a solute eluting after fied in the monograph for the drug i (tRj is larger than tRi); being tested; tRi=Retention time for any solute; wi=Width of peak at baseline for any solute; (2) A suitable recording device of at and least 25-centimeter deflection; wj=Width of peak at baseline for any solute (3) A suitable chromatographic data eluting after i. managing system; and (3) Asymmetry factor. Calculate the (4) An analytical column, 3 to 30 cen- asymmetry factor (A timeters long, packed with a material s as defined in the monograph for the a+b drug being tested. = As (b) Procedure. Perform the assay at 2a the temperature specified in the mono- where: graph for the drug being tested with a a=Horizontal distance from point of ascent flow rate not to exceed 2.0 milliliters to point of maximum peak height; and
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b=Horizontal distance from point of maxi- § 436.355 High-performance liquid mum peak height to point of descent. chromatographic assay for (4) Efficiency of the column. Calculate ticarcillin-clavulanic acid. the efficiency of the column (reduced (a) Equipment. A suitable high-per- formance liquid chromatograph plate height) (hr (i) equipped with: (1) A suitable detection system speci- 2 fied in the monograph for the drug being testing; tR n = 5.545 ; (2) A suitable recording device of at Wh least 25-centimeter deflection; (3) A suitable chromatographic data (ii) managing system; and (4) An analytical column, 10 to 30 L h = ; and centimeters long, packed with a mate- rial as defined in the monograph for n the drug being tested; and if specified (iii) in that monograph, the inlet of this column may be connected to a guard = h column, 3 to 5 centimeters in length, hr . d packed with the same material of 40 to p 60 micrometers particle size. where: (b) Procedure. Perform the assay and n=Efficiency, as number of theoretical plates calculate the drug content using the for column; temperature, instrumental conditions,
tR=Retention time of solute; and calculations specified in the mono- wh=Peak width at half-height; graph for the drug being tested with a h=Efficiency, as height equivalent to one flow rate not to exceed 2.0 milliliters theoretical plate; per minute. Use a detector sensitivity L=Length of column; and setting that gives a peak height for the dp=Average diameter of particle in column. working standard that is at least 50 (5) Coefficient of variation (relative percent of scale with typical chart speed of not less than 2.5 millimeters standard deviation). Calculate the coef- per minute. Use the equipment de- ficient of variation (S R scribed in paragraph (a) of this section; and the reagents and working standard n 1/ 2 2 and sample solutions described in the ( − ) ∑ XXi monograph for the drug being tested. 100 = i=1 Equilibrate and condition the column SR X N −1 by passage of 10 to 15 void volumes of mobile phase followed by five replicate injections of the same volume (between where: 10 and 20 microliters) of the working X¯ is the mean of N individual measurements standard solution. Allow an operating time sufficiently long to obtain satis- of Xi. factory separation and elution of the The complete operating system is ac- expected components after each injec- ceptable for assay if it meets the sys- tion. The clavulanic acid peak is sharp tem suitability requirements of the and the chromatograms of standard monograph for the drug being tested. If and sample solutions show baseline the complete operating system is ac- separations between it and any neigh- ceptable, proceed as described in para- boring peaks. The retention times for graph (b) of this section using the sam- clavulanic acid and ticarcillin are ap- ple solution in lieu of the test solution. proximately 3 minutes and 14 minutes, Calculate the drug content as described respectively. Record the peak re- in the individual monograph for the sponses and calculate the prescribed drug being tested. system suitability requirements de- scribed for the system suitability test [50 FR 9999, Mar. 13, 1985] in paragraph (c) of this section.
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(c) System suitability test. Using the 1/ 2 equipment and procedure described in n − 2 this section, test the chromatographic ∑(XXi ) 100 system for assay as follows: = i=1 SR (1) Tailing factors for the ticarcillin and X N −1 clavulanic acid peaks. Calculate the tailing factors (T), from distances measured along the horizontal line at 5 where: percent of the peak height above the X¯ is the mean of N individual measurements baseline, as follows: of Xi. W If the complete operating system T = 0. 05 meets the system suitability require- 2 f ments of the monograph for the drug being tested, proceed as described in where: paragraph (b) of this section, using the W =Width of peak at 5 percent height; and 0.05 sample solution in lieu of the working f=Horizontal distance from point of ascent to a point coincident with maximum peak standard solution. height. [50 FR 33517, Aug. 20, 1985; 50 FR 43384, Oct. 25, 1985] (2) Efficiency of the column. Calculate the number of theoretical plates (n) of § 436.356 High-performance liquid the column as follows: chromatographic assay for ceftazidime. 2 t (a) Equipment. A suitable high-per- n = 5.545 R formance liquid chromatograph Wh equipped with: (1) A suitable detection system speci- where: fied in the monograph for the drug n=Efficiency, as number of theoretical plates for column; being tested; (2) A suitable recording device of at tR=Retention time of ticarcillin or clavulanic acid peaks; and least 25-centimeter deflection;
wh=Corresponding peak width at half-height. (3) A suitable chromatographic data managing system; and (3) Resolution factor. Calculate the (4) An analytical column, 3 to 30 cen- resolution factor (R) as follows timeters long, packed with a material 2(t− t ) as defined in the monograph for the R = 2 1 drug being tested; and if specified in w+ w that monograph, the inlet of this col- 1 2 umn may be connected to a guard col- where: umn, 3 to 5 centimeters in length, t1=Retention time of clavulanic acid peak; packed with the same material of 40 to t2=Retention time of ticarcillin peak; and 60 micrometers particle size. w and w =Widths of the bases of the cor- 1 2 (b) Procedure. Perform the assay and responding peaks obtained by extrapolat- ing the relatively straight sides of the calculate the drug content using the peaks to the baseline. temperature, instrumental conditions, flow rate, and calculations specified in When using the method to assay the monograph for the drug being test- clavulanic acid alone, the resolution ed. Use a detector sensitivity setting factor is not applicable. that gives a peak height for the work- (4) Coefficient of variation (Relative ing standard that is at least 50 percent standard deviation). Calculate the coef- of scale with typical chart speed of not ficient of variation (SR less than 2.5 millimeters per minute.
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Use the equipment described in para- 2(t− t ) graph (a) of this section. Use the re- R = Rj Ri agents, working standard solution, and w+ w sample solution described in the mono- i j graph for the drug being tested. Equili- where: brate and condition the column by pas- tRj=Retention time of a solute eluting after sage of 10 to 15 void volumes of mobile i(tRj is larger than tRi); t =Retention time of any solute; phase followed by five replicate injec- Ri wi=Width of peak at baseline measured by tions of the same volume (between 10 extrapolating the relatively straight and 20 microliters) of the working sides to the baseline of any solute; and standard solution for the system suit- wj=Width of peak at baseline measured by ability test. Allow an operating time extrapolating the relatively straight sufficiently long to obtain satisfactory sides to the baseline of any solute eluting after i. separation and elution of the expected components after each injection. (4) Coefficient of variation (relative Record the peak responses and cal- standard deviation). Calculate the coef- culate the prescribed system suit- ficient of variation (SR in percent) as ability requirements described for the follows: system suitability test in paragraph (c) n 1/ 2 of this section. 2 − (c) System suitability test. Select the ∑(XXi ) 100 system suitability requirements speci- = i=1 SR fied in the monograph for the drug X N −1 being tested. Then, using the equip- ment and procedure described in this section, test the chromatographic sys- where: tem for assay as follows: X¯ is the mean of N individual measurements (1) Tailing factor. Calculate the tail- of Xi. ing factor (T), from distances measured If the complete operating system along the horizontal line at 5 percent meets the system suitability require- of the peak height above the baseline, ments of the monograph for the drug as follows: being tested, proceed as described in paragraph (b) of this section, except al- W0. 05 ternate injections of the working T = standard solution with injections of 2 f the sample solution. where: [50 FR 48397, Nov. 25, 1985] W0.05=Width of peak at 5 percent height; and f=Horizontal distance from point of ascent to § 436.357 Atomic absorption test for so- a point coincident with maximum peak dium carbonate content. height. (a) Equipment. A suitable atomic ab- (2) Efficiency of the column. Calculate sorbance spectrophotometer equipped the number of theoretical plates (n) of with: the column as follows: (1) A suitable sodium hollow-cathode discharge lamp; 2 (2) An oxidizing air-acetylene flame; tR (3) A nebulizer-burner system; n =5 . 545 (4) An optical dispersing device capa- wh ble of isolating a resonance line of so- where: dium from other wavelengths produced n=Efficiency, as number of theoretical plates by the emission source; and for column; (5) A suitable radiation detector.
tR=Retention time of solute; and (b) Ionization buffer. Dissolve and di- wh=Peak width at half-height. lute 19.07 grams of potassium chloride in distilled water to 1,000 milliliters. (3) Resolution. Calculate the resolu- (c) Preparation of reference standard tion (R) as follows: and sample solutions—(1) Reference
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standard solution. Accurately weigh ap- (1) A suitable detection system speci- proximately 140 milligrams of sodium fied in the monograph for the drug chloride, which has been previously being tested; dried for 40 to 50 minutes at a tempera- (2) A suitable recording device of at ture of 500 to 650 ° C. Dissolve and di- least 25-centimeter deflection; lute with sufficient distilled water to (3) A suitable chromatographic data obtain a stock solution containing 5.5 managing system; and micrograms of sodium per milliliter. (4) An analytical column, 15 to 25 Mix 10 milliliters of the stock solution centimeters long, packed with a mate- with 10 milliliters of ionization buffer rial as defined in the monograph for and dilute the mixture with distilled the drug being tested; and if specified water to obtain a solution containing in that monograph, the inlet of this 0.55 microgram of sodium per milli- column may be connected to a guard liter. column, 3 to 5 centimeters in length, (2) Sample solution. Dilute the stock packed with the same material of 40 to sample solution, prepared as directed 60 micrometers particle size. in the monograph for the drug being (b) Procedure. Perform the assay and tested, with distilled water to obtain a calculate the pyridine content using solution containing 5.5 micrograms of the temperature, instrumental condi- sodium per milliliter (estimated). Mix tions, flow rate, and calculations speci- 10 milliliters of this solution with 10 fied in the monograph for the drug milliliters of ionization buffer and di- being tested. Use a detector sensitivity lute the mixture with distilled water to setting that gives a peak height for the obtain a solution containing 0.55 working standard that is at least 25 microgram of sodium per milliliter (es- percent of scale with typical chart timated). speed of not less than 2.5 millimeters (3) Procedure. Determine the atomic per minute. Use the equipment de- absorbance of the reference standard scribed in paragraph (a) of this section. and sample solutions at a wavelength Use the reagents, working standard so- of 589 nanometers, using the atomic ab- lution, and sample solution described sorbance spectrophotometer and a rea- in the monograph for the drug being gent blank prepared by diluting 10 mil- tested. Equilibrate and condition the liliters of ionization buffer to 100 milli- column by passage of 10 to 15 void vol- liters with distilled water. umes of mobile phase followed for the (d) Calculations. Calculate the percent system suitability test by five rep- sodium carbonate (S) as follows: licate injections of the same volume AP× × 2. 304 (between 10 and 20 microliters) of the Percent sodium= u s system suitability test solution. Allow carbonate A ×C ×10 an operating time sufficiently long to s u obtain satisfactory separation and where: elution of the expected components Au=Absorbance of sodium in the sample solu- after each injection. Record the peak tion; responses and calculate the prescribed As=Absorbance of sodium in the reference standard solution; system suitability requirements de- scribed for the system suitability test Ps=Sodium concentration in the reference standard solution in micrograms per mil- in paragraph (c) of this section. liliter; and (c) System suitability test. Select the Cu=Milligrams of sample per milliliter of system suitability requirements speci- sample solution. fied in the monograph for the drug [50 FR 48398, Nov. 25, 1985, as amended at 54 being tested. Then, using the equip- FR 20785, May 15, 1989] ment and procedure described in this section, test the chromatographic sys- § 436.358 High-performance liquid tem for assay as follows: chromatographic assay for pyri- (1) Tailing factor. Calculate the tail- dine. ing factor for the pyridine peak (T), (a) Equipment. A suitable high-per- from distances measured along the hor- formance liquid chromatograph izontal line at 5 percent of the peak equipped with: height above the baseline, as follows:
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(2) A suitable recording device of at W0. 05 least 25-centimeter deflection; T = (3) A suitable chromatographic data 2 f managing system; and where: (4) An analytical column, 50 centi- W0.05=Width of peak at 5 percent height; and meters long and 9 millimeters internal f=Horizontal distance from point of ascent to diameter, packed with a material as a point coincident with maximum peak defined in the monograph for the drug height. being tested. (2) Resolution. Calculate the resolu- (b) Procedure. Perform the assay and tion (R) as follows: calculate the high molecular weight polymer content using the tempera- − 2(tRj t Ri ) ture, instrumental conditions, and cal- R = culations specified in the monograph + wi w j for the drug being tested. Use a detec- tor sensitivity setting that gives a where: peak height for the working standard tRj=Retention time of t-butyl ceftazidime; t =Retention time of pyridine; that is at least 10 percent of scale with Ri a typical chart speed of not less than wi=Width of pyridine peak at the baseline measured by extrapolating the relatively 2.5 millimeters per minute. Use the straight sides to the baseline; and equipment described in paragraph (a)
wj=Width of t-butyl ceftazidime peak at the of this section. Use the reagents, work- baseline measured by extrapolating the ing standard solution, and sample solu- relatively straight sides to the baseline. tion described in the monograph for (3) Coefficient of variation (relative the drug being tested. Equilibrate and standard deviation). Calculate the coef- condition the column by passage of mo- ficient of variation for the pyridine bile phase for not less than 18 hours, peak (S in percent ) as follows: removing any voids that may form at R the top of the column, followed by five replicate injections of the same volume n 1/ 2 − 2 (100 microliters) of the blue dextran ∑(XXi ) 100 system suitability test solution. Allow = i=1 an operating time sufficiently long to SR X N −1 obtain satisfactory separation and elution of the expected components after each injection. Record the peak where: responses and calculate the prescribed X¯ is the mean of N individual measurements system suitability requirements de- of Xi. scribed for the system suitability test in paragraph (c) of this section. If the complete operating system (c) System suitability test. Select the meets the system suitability require- system suitability requirements speci- ments of the monograph for the drug fied in the monograph for the drug being tested, proceed as described in being tested. Then, using the equip- paragraph (b) of this section, except al- ment and procedure described in this ternate injections of the working section, test the chromatographic sys- standard solution with injections of tem for assay as follows: the sample solution. (1) Tailing factor. Calculate the tail- [50 FR 48398, Nov. 25, 1985; 50 FR 53308, Dec. ing factor (T), from distances measured 31, 1985] along the horizontal line at 5 percent of the peak height above the baseline, § 436.360 Gel permeation as follows: chromatographic assay for high mo- lecular weight polymer. W0. 05 (a) Equipment. A suitable gel perme- T = ation chromatograph equipped with. 2 f (1) A suitable detection system speci- where: fied in the monograph for the drug W0.05=Width of peak at 5 percent height; and being tested; f=Horizontal distance from point of ascent to
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a point coincident with maximum peak drug being tested; and if specified in height. that monograph, the inlet of this col- (2) Efficiency of the column. Calculate umn may be connected to a guard col- the number of theoretical plates (n) of umn, 3 to 5 centimeters in length, the column as follows: packed with the same material of 40 to 60 micrometers particle size. 2 (b) Procedure. Perform the assay and tR calculate the drug content using the n = 5.545 temperature, instrumental conditions, wh flow rate, and calculations specified in where: the monograph for the drug being test- n=Efficiency, as number of theoretical plates ed. Use a detector sensitivity setting for column; that gives a peak height for the work- tR=Retention time of solute; and ing standard that is at least 50 percent wh=Peak width at half-height. of scale with typical chart speed of not (3) Coefficient of variation (relative less than 2.5 millimeters per minute. standard deviation). Calculate the coef- Use the equipment described in para- ficient of variation (SR in percent) as graph (a) of this section. Use the re- follows: agents, working standard solution, and sample solution described in the mono- n 1/ 2 graph for the drug being tested. Equili- 2 − brate and condition the column by pas- ∑(XXi ) 100 sage of 10 to 15 void volumes of mobile = i=1 SR phase followed by five replicate injec- X N −1 tions of the same volume (between 10 and 20 microliters) of the working standard solution. Allow an operating where: time sufficiently long to obtain satis- X¯ is the mean of N individual measurements factory separation and elution of the of Xi. expected components after each injec- If the complete operating system tion. Record the peak responses and meets the system suitability require- calculate the prescribed system suit- ments of the monograph for the drug ability requirements described for the being tested, proceed as described in system suitability test in paragraph (c) paragraph (b) of this section, using two of this section. injections of the same volume (100 (c) System suitability test. Select the microliters) of the working standard system suitability requirements speci- solution followed by one injection of fied in the monograph for the drug the same volume (100 microliters) of being tested. Then, using the equip- the sample solution. ment and procedure described in this [50 FR 48398, Nov. 25, 1985; 51 FR 1367, Jan. 13, section, test the chromatographic sys- 1986] tem for assay as follows: (1) Tailing factor. Calculate the tail- § 436.361 High-performance liquid ing factor (T), from distances measured chromatographic assay for along the horizontal line at 5 percent aztreonam. of the peak height above the baseline, (a) Equipment. A suitable high-per- as follows: formance liquid chromatograph equipped with: W0. 05 (1) A suitable detection system speci- T = fied in the monograph for the drug 2 f being tested; where: (2) A suitable recording device of at W0.05=Width of peak at 5 percent height; and least 25-centimeter deflection; f=Horizontal distance from point of ascent to (3) A suitable chromatographic data a point coincident with maximum peak managing system; and height. (4) An analytical column, 3 to 30 cen- (2) Efficiency of the column. Calculate timeters long, packed with a material the number of theoretical plates (n) of as defined in the monograph for the the column as follows:
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the bottom and a tight-fitting cover for 2 the top. t n = 5. 545 R (2) Plates. Use a 20- by 20-centimeter W precoated silica gel 60 F–254 thin-layer h chromatography plate. Before using, where: place the plate in an unlined develop- n=Efficiency, as number of theoretical plates ing chamber containing approximately for column; 100 milliliters of anhydrous methanol tR=Retention time of solute; and and allow the solvent front to travel to wh=Peak width at half-height. the top of the plate, marking the direc- (3) Resolution. Calculate the resolu- tion of travel. Remove the plate and tion (R) as follows: allow to drip dry. Store in a dry place. (b) Reagents—(1) Developing solvent. 2(t− t ) Mix 15 milliliters of chloroform and 85 R = Rj Ri milliliters of anhydrous methanol. Use + fresh developing solvent for each test. wi w j (2) Spray solution. Dissolve 150 milli- where: grams of xanthydrol in a mixture of 7.5 tRj=Retention time of a solute eluting after i milliliters of glacial acetic acid and (tRj is larger than tRi); 92.5 milliliters of 37 percent hydro- tRi=Retention time of any solute; chloric acid. wi=Width of peak at baseline of any solute; (c) Preparation of spotting solutions— and (1) Sample solution. Prepare a solution wj=Width of peak at baseline of any solute eluting after i. of the sample in anhydrous methanol to contain 10 milligrams per milliliter. (4) Coefficient of variation (relative standard deviation). Calculate the coef- NOTE: It is advisable to prepare the sample and standard solutions immediately before ficient of variation (SR in percent ) as spotting to minimize the possibility of deg- follows: radation in solution.)
1/ 2 (2) Standard solution. Prepare a solu- n − 2 tion of erythromycin base reference ∑(XXi ) 100 standard in anhydrous methanol to = i=1 contain 1 milligram per milliliter. SR X N −1 Weigh 99.5, 99.0, and 97.0 milligrams of erythromycin estolate (propionyl erythromycin lauryl sulfate) reference where: standard and transfer to separate 10- X¯ is the mean of N individual measurements milliliter volumetric flasks. To these of Xi. flasks add 0.5, 1.0, and 3.0 milliliters, respectively, of the 1-milligram-per- If the complete operating system milliliter solution of erythromycin meets the system suitability require- base reference standard and dilute to ments of the monograph for the drug volume with anhydrous methanol. being tested, proceed as described in These solutions contain, respectively, paragraph (b) of this section, except al- 0.5 percent, 1.0 percent, and 3.0 percent ternate injections of the working erythromycin base in erythromycin standard solution with injections of estolate. Prepare a solution of erythro- the sample solution. mycin estolate reference standard in [52 FR 4611, Feb. 13, 1987; 52 FR 8550, Mar. 18, anhydrous methanol to contain 10 mil- 1987] ligrams per milliliter. Prepare a solu- tion of erythromycin base reference § 436.362 Thin-layer chromatographic standard in anhydrous methanol to test for free erythromycin content contain 0.1 milligram per milliliter. in erythromycin estolate bulk. (d) Procedure. Pour 100 milliliters of (a) Equipment—(1) Chromatography developing solvent into the glass tank. A rectangular tank approxi- trough on the bottom of the unlined mately 23 centimeters long, 23 centi- chromatography tank. Cover and seal meters high, and 9 centimeters wide, the tank. Allow it to equilibrate while equipped with a glass solvent trough in the plate is being prepared. Prepare a
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plate as follows: On a line 2.0 centi- essary to repeat the test using a dif- meters from the base of the thin-layer ferent set of standards. plate, apply 1.0 microliter of each of the following solutions: [53 FR 1919, Jan. 25, 1988] (1) 10-milligrams-per-milliliter solu- § 436.363 High-performance liquid tion of erythromycin estolate reference chromatographic assay for standard, equivalent to 10 micrograms cefmenoxime. of erythromycin estolate; (2) 0.5 percent base-in-estolate solu- (a) Apparatus. A suitable high-per- tion, equivalent to 0.05 microgram of formance liquid chromatograph base and 9.95 micrograms of estolate; equipped with: (3) 1.0 percent base-in-estolate solu- (1) A suitable detection system speci- tion, equivalent to 0.10 microgram of fied in the monograph for the drug base and 9.90 micrograms of estolate; being tested; (4) 3.0 percent base-in-estolate solu- (2) A suitable recording device of at tion, equivalent to 0.30 microgram of least 18-centimeter deflection; base and 9.70 micrograms of estolate; (3) A suitable chromatographic data (5) 0.1-milligram-per-milliliter solu- managing system; and tion of erythromycin base reference (4) An analytical column, 3 to 30 cen- standard, equivalent to 0.1 microgram timeters long, packed with a material of erythromycin base; and as defined in the monograph for the (6) Sample solution, equivalent to 10 drug being tested; and if specified in micrograms of erythromycin estolate. that monograph, the inlet of this col- Allow the spots to dry. Place the plate umn may be connected to a guard col- directly in the chromatograph tank. umn, 3 to 5 centimeters in length, Cover and seal the tank. Allow the sol- packed with the same material of 30 to vent front to travel a distance of 7 cen- 60 micrometers particle size. timeters (about 27 minutes). Remove (b) Procedure. Perform the assay and the plate from the tank, and allow it to calculate the drug content using the air dry under a hood. With the plate temperature, instrumental conditions, still under the hood, spray uniformly and calculations specified in the mono- with the spray solution. Heat the graph for the drug being tested with a sprayed plate in an oven at 100 °C for 5 flow rate not to exceed 2.0 milliliters minutes. (CAUTION: Avoid exposure to per minute. Use a detector sensitivity the acid fumes while removing the setting that gives a peak height for the plate from the oven.) working standard that is at least 50 (e) Evaluation. Erythromycin base percent of scale with typical chart and erythromycin estolate appear as speed of not less than 2.5 millimeters reddish-violet spots on the sprayed and per minute. Use the apparatus de- heated plate. Better visualization of scribed in paragraph (a) of this section; the erythromycin base spots may be and the reagents and working standard gained by viewing the plate under long- and sample solutions described in the wavelength (366 nanometers) ultra- monograph for the drug being tested. violet light, erythromycin base appear- Equilibrate and condition the column ing as dark spots on a yellow-green flu- by passage of 10 to 15 void volumes of orescent background. Erythromycin mobile phase followed by 5 replicate in- base has an Rfvalue of about 0.3. Eryth- jections of the same volume (between romycin estolate has an Rf value of 10 and 20 microliters) of the working about 0.7. Compare the size and inten- standard solution. Allow an operating sity of any erythromycin base spots in time sufficiently long to obtain satis- the sample lane with the erythromycin factory separation and elution of the base spots in the erythromycin base expected components after each injec- reference standard lane and in the 0.5 tion. Record the peak responses and percent, 1.0 percent, and 3.0 percent calculate the prescribed system suit- base-in-estolate lanes, and report the ability requirements described for the percentage of erythromycin base (free system suitability test in paragraph (c) erythromycin) in the sample. For a of this section. more accurate determination of free (c) System suitability test. Using the erythromycin content, it may be nec- apparatus and procedure described in
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this section, test the chromatographic ments of the monograph for the drug system for assay as follows: being tested, proceed as described in (1) Tailing factor. Calculate the tail- paragraph (b) of this section, using the ing factor (T), from distances measured sample solution in lieu of the working standard solution. along the horizontal line at 5 percent of the peak height above the baseline, [53 FR 13401, Apr. 25, 1988; 53 FR 19368, May as follows: 27, 1988] W § 436.364 Atomic absorption test for so- T = 0. 05 dium carbonate content of cefmenoxime hydrochloride for in- 2 f jection. where: (a) Apparatus. A suitable atomic ab- W0.05=Width of peak at 5 percent height; and f=Horizontal distance from point of ascent to sorbance spectrophotometer equipped a point coincident with maximum peak with: height. (1) A suitable sodium hollow-cathode (2) Efficiency of the column. Calculate discharge lamp; the number of theoretical plates (n) of (2) An oxidizing air-acetylene flame; the column as follows: (3) A nebulizer-burner system; (4) An optical dispersing device capa- 2 ble of isolating a resonance line of so- t dium from other wavelengths produced n = 5. 545 R by the emission source; and Wh (5) A suitable radiation detector. where: (b) Reagents. Ionization buffer: Dis- n=Efficiency, as number of theoretical plates solve 19.07 grams of potassium chloride for column; in distilled water and dilute to 1,000 tR=Retention time of solute; and milliliters. wh=Peak width at half-height. (c) Preparation of reference standard (3) Resolution. Calculate the resolu- and sample solutions—(1) Reference tion (R) as follows: standard solution. Accurately weigh ap- proximately 140 milligrams of sodium 2()t− t chloride which has been previously R = RJ Ri dired for 40 to 50 minutes at a tempera- + ture of 500 to 650 ° C. Dissolve and di- wi w J lute with sufficient distilled water to where: t =Retention time of a solute eluting after i obtain a stock solution containing 5.5 RJ micrograms of sodium per milliliter. (tRJ is larger than tRi); tRi=Retention time of any solute; Mix 10 milliliters of the stock solution wi=Width of peak at baseline of any solute; with 10 milliliters of ionization buffer and and dilute the mixture with distilled wJ=Width of peak at baseline of any solute water to obtain a solution containing eluting after i. 0.55 microgram of sodium per milli- (4) Coefficient of variation (Relative liter. standard deviation). (2) Sample solution. Dilute the sample Calculate the coefficient of variation solution used in § 442.222(b)(1)(ii)(B)(1) (SR) in percent) as follows: of this chapter, with sufficient distilled water to obtain a stock solution con- 1 taining 5.5 micrograms of sodium per N 2 ()XX− 2 milliliter (estimated). Mix 10 milli- ∑ i liters of the stock solution with 10 mil- 100 i=1 liliters of ionization buffer and dilute SR = X N −1 the mixture with distilled water to ob- tain a solution containing 0.55 microgram of sodium per milliliter (es- where: timated). X is the mean of N individual measurements (3) Procedure. Determine the atomic of Xi. If the complete operating system absorbance of the reference standard meets the system suitability require- and sample solutions at a wavelength
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of 589 nanometers, using the atomic ab- liliters of ionization buffer to 100 milli- sorbance spectrophotometer and a rea- liters with distilled water. gent blank prepared by diluting 10 mil- (d) Calculations. Calculate the percent sodium carbonate as follows:
A× P ×100 × 0. 9068 × d Percent sodium carbonate = u s × ACs u
where: solution to points 2 and 3. After all the Au=Absorbance of sodium in the sample solu- spots are thoroughly dry, place the tion; plate into the trough in the bottom of As=Absorbance of sodium in the reference the tank. Cover and tightly seal the standard solution; tank. Allow the solvent front to travel Ps=Milligrams of sodium chloride per milli- liter of the reference standard solution; about 7 centimeters from the starting Cu=Milligrams of sample per milliliter of line. Remove the plate from the tank sample solution; and and air dry. d= Dilution factor of the sample. (e) Evaluation. Measure the distance [53 FR 13401, Apr. 25, 1988] the solvent front traveled from the starting line, and the distance the red § 436.365 Thin layer chromatographic spots are from the starting line. Divide identity test for rifampin. the latter by the former to calculate (a) Equipment—(1) Chromatography the Rf value. tank. Use a rectangular tank approxi- [54 FR 38375, Sept. 18, 1989; 54 FR 42886, Oct. mately 23×23×9 centimeters, with a 18, 1989] glass solvent trough on the bottom and a tight-fitting cover, lined with § 436.366 High-performance liquid Whatman #3MM chromatographic chromatography assay for deter- paper or equivalent. mining chromatographic purity of (2) Plates. Use 20×20 centimeter thin vancomycin. layer chromatography plates coated (a) Apparatus. A suitable high-per- with silica gel 60 F–254 or equivalent to formance liquid chromatograph a thickness of 250 microns. equipped with: (b) Developing solvent. Mix chloroform (1) A suitable ultraviolet detection and methanol in volumetric propor- system operating at a wavelength of tions of 90:10, respectively. 254 nanometers or preferably 280 (c) Spotting solutions—(1) Preparation nanometers; of working standard solution. Dissolve (2) A suitable recording device of at approximately 50 milligrams of least 25-centimeter deflection; rifampin working standard in 5 milli- (3) A suitable chromatographic data liters of chloroform. managing system; and (2) Preparation of sample solution. Dis- (4) A 25-centimeter analytical col- solve the contents of a sample vial in umn having an inside diameter of 4.6 60 milliliters of chloroform. millimeters and packed with octadecyl (d) Procedure. Pour the developing silane chemically bonded to porous solvent into the glass trough on the silica or ceramic microparticles; 5 mi- bottom of the tank and onto the paper crometers in diameter. lining the walls of the tank. Cover and (b) Reagents—(1) 0.2 percent seal the tank. Allow it to equilibrate. triethylammonium phosphate buffer. To Prepare a plate as follows: On a line 2.5 2,000 milliliters of distilled water, ei- centimeters from the base of the thin ther add 4 milliliters of triethylamine layer chromatography plate and at in- or 4 grams of triethylammonium chlo- tervals of 2.0 centimeters, spot 3 micro- ride. Adjust the pH to 3.2 with phos- liters of the working standard solution phoric acid. to points 1 and 3. When these spots are (2) Sample solvents. (i) Vancomycin dry, apply 3 microliters of the sample hydrochloride: Mobile Phase A.
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(ii) Vancomycin base: 5 milliliters vancomycin B peak; the second Mobile Phase A; add 0.1N HCl dropwise desamido isomer elutes during the gra- with swirling until sample dissolves: dient ramp and is used to demonstrate dilute to volume with Mobile Phase A. the effective performance of this stage. (c) Mobile Phases—(1) Mobile Phase A. (2) Sample preparation. In a volu- Add 70 milliliters of acetonitrile and 10 metric flask either dissolve a rep- milliliters of tetrahydrofuran to 920 resentative sample or dilute a rep- milliliters of 0.2 percent resentative portion with sample sol- triethylammonium phosphate buffer vent to give a sample preparation con- and mix well. Filter the mobile phase taining approximately 10 milligrams through a suitable glass fiber filter or per milliliter. Pipet 2 milliliters of this equivalent that is capable of removing sample solution into a separate 50-mil- particulate contamination to 1 micron liliter volumetric flask and dilute to in diameter. Degas the mobile phase, volume with sample solvent to give a briefly, just prior to its introduction diluted sample preparation containing into the chromatographic pumping sys- approximately 0.4 milligram per milli- tem. liter. (2) Mobile Phase B. Add 290 milliliters (f) Procedure. Optimize of acetonitrile and 10 milliliters of chromatographic conditions under tetrahydofuran to 700 milliliters of 0.2 isocratic conditions by equilibrating percent triethylammonium phosphate buffer and mix well. Filter the mobile the system while pumping 100 percent phase through a suitable glass fiber fil- mobile phase A through the column. ter or equivalent that is capable of re- Inject 20 microliters of the resolution moving particulate contamination to 1 solution onto the column and record micron in diameter. Degas the mobile the chromatogram. Adjust the acetoni- phase, briefly, just prior to its intro- trile concentration of mobile phase A duction into the chromatographic as needed to provide a retention time pumping system. for vancomycin B of 7.5 to 10.5 minutes. (d) Operating conditions. Perform the Use the resolution solution to perform assay at ambient temperature with a the system suitability tests. The typical flow rate of about 2.0 milliliters elution order is resolution compound 1, per minute. Use a detector sensitivity vancomycin B, resolution compound 2. setting that gives a peak height for the Return the system to the initial gra- main peak (Vancomycin B) that is at dient operating conditions. Separately least 50 percent of scale. The run time inject 20 milliliters of each diluted (0.4 is 30 minutes per injection and the gra- milligram per milliliter) and con- dient conditions are as follows: (0, 12, centrated (10 milligrams per milliliter) 12.5, 8, 0, 2) sample solution onto the column and record each chromatogram. Mobile Mobile Time (min- phase A phase B (g) System suitability test. Using the utes) (per- (per- Gradient condition resolution solution described in para- cent) cent) graph (e)(1) of this section, test the 0 ...... 100 0 Initial conditions. performance of the chromatographic 12 ...... 100 0 Isocratic region. system as follows: 20 ...... 0 100 Linear ramp. 22 ...... 0 100 Isocratic region. (1) Asymmetry factor. Calculate the 23 ...... 100 0 Return to initial. asymmetry factor (A), measured at a 30 ...... 100 0 Reequilibration. point that is 10 percent of the vancomycin B peak height from the (e) Preparation of resolution and sam- baseline, as follows: ple solutions—(1) Resolution solution. Prepare a solution of vancomycin hy- a+b drochloride reference standard in water A = containing 0.5 milligram per milliliter. s 2a Heat at 65 °C for 24 hours and allow to where: cool. This procedure generates two a=Horizontal distance from point of ascent desamido-vancomycin isomers. The to point of maximum peak height; and first desamido isomer elutes during the b=Horizontal distance from point of maxi- isocratic period and before the mum peak height to point of descent.
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The asymmetry factor (Ar) is satisfactory paragraph (f) of this section. Alternate if it is not less than 0.8 and not more than chromatographic conditions are acceptable 1.8. provided that the system suitability param- eters are met. However, the sample prepara- (2) Efficiency of the column. From the tion described in paragraph (e)(2) of this sec- number of theoretical plates (n) cal- tion should not be changed. culated as described in § 436.216(c)(2) (h) Calculations. (1) Calculate the per- calculate the reduced plate height (hr) for the vancomycin B peak as follows: centage of vancomycin B in the speci- men as follows: (L)(10,000) h = Percentage of A r =B ×100 percent (n)() dp vancomycin B ATotal where: L=Length of the column in centimeters; where: n=Number of theoretical plates; and AB=Area of the vancomycin B peak in the dp=Average diameter of the particles in the dilute (0.4 milligram per milliliter) sample column in micrometers. solution; and ATotal=Area of the vancomycin B peak in The absolute efficiency (hr) is satisfactory if it is not more than 40 for the vancomycin B the dilute (0.4 milligram per milliliter) solu- peak in the resolution solution. tion+[Area of the total related substances peaks (exclude the area of the vancomycin B (3) Resolution. The resolution (R) be- peak) in the concentrated solution (10 milli- tween the vancomycin B peak and the grams per milliliter) divided by 25]. peak for resolution compound 1 is not (2) Calculate the percentage of each less than 3.0. Resolution compound 2 is other peak as follows: eluted between 3 and 6 minutes after the start of the period when the per- Percentage []A centage of mobile phase B is increasing of related =i/25 ×100 percent from 0 percent to 100 percent. substance (i ) A (4) Coefficient of variation (relative Total standard deviation). The coefficient of where: variation (S in percent) of five rep- Ai=Area of any given peak, other than the R main peak in the concentrated solution (10 licate injections of the resolution solu- milligrams per milliliter); and tion is calculated as described in ATotal=Area of the vancomycin B peak in § 436.216(c)(4) is satisfactory if it is not the dilute (0.4 milligram per milliliter) solu- more than 2.0 percent. tion+[Area of the total related substances (5) Capacity factor (k). Calculate the peaks (exclude the area of the vancomycin B capacity factor (k) for vancomycin B as peak) in the concentrated solution (10 milli- follows: grams per milliliter) divided by 25]. [54 FR 20383, May 11, 1989; 54 FR 25849, June t− t 20, 1989] k = r m tm § 436.367 Thin-layer chromatographic identity test for cephalexin hydro- where: chloride. tr=Retention time of solute; and tm=Retention time of solvent or unretained (a) Equipment—(1) Chromatography substance, calculated as follows: tank. Use a rectangular tank approxi- mately 23 × 23 × 9 centimeters, with a 2 glass solvent trough in the bottom and = (31416 . )(DL )( )(0.75) t m a tight-fitting cover. Line the inside 4F walls of the tank with Whatman #3 MM where: chromatographic paper or equivalent. D=Column diameter in centimeters; (2) Plates. Use 20 × 20 centimeter thin L=Column length in centimeters; layer chromatographic plates coated 0.75=Average total column porosity; and F=Flow rate in milliliters per minute. with silica gel 60F–254 or equivalent to a thickness of 250 microns. The capacity factor (k) for vancomycin B is satisfactory if it is not less than 2.6 and not (b) Developing solvent. Mix more than 3.3. ethylacetate, acetonitrile, water and When the system suitability requirements glacial acetic acid in volumetric pro- have been met, then proceed as described in portions of 42:14:18:14, respectively.
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(c) Preparation of the spotting solu- (3) Detection reagent. Iodine vapor. tions. Prepare a solution of the sample (c) Assay solutions—(1) Reference containing 25 milligrams per milliliter standard solution. Dissolve 50 milli- of cephalexin hydrochloride in water. grams of cefprozil (Z) reference stand- Prepare a solution of cephalexin ard in 10 milliliters of diluent. monohydrate reference material at a (2) Sample solution. Place an amount concentration of 25 milligrams per mil- of sample containing approximately 50 liliter. Add water and 0.1N hydrochloric milligrams of cefprozil in a 20-milli- acid in a dropwise mode until the ma- liter glass stoppered vial. Add 10 milli- terial is completely dissolved. liters of diluent. Shake for 5 minutes (d) Procedure. Pour the developing and allow the solids to settle. solvent into the glass trough at the (d) Procedure. Pour a suitable quan- bottom of the chromatography tank. tity of the developing solvent into a Cover and seal the tank. Allow it to glass, chromatographic tank lined with equilibrate for 1 hour. Prepare a plate filter paper and allow to equilibrate for as follows: On a line 2 centimeters from 1 hour. On a line 2 centimeters from the base of the plate, and at intervals the bottom edge of the plate, spot 10 of 2 centimeters, spot approximately 5 microliters each of the reference solu- microliters of the standard solution to tion and sample solution. Draw a line points 1 and 3 and approximately 5 indicating the distance to which the microliters of the sample solution to developing solvent must travel at a point 2. After all spots are thoroughly point 12 centimeters from the bottom dry, place the plate directly into the edge of the plate. Place the plate in the glass trough of the chromatography tank and allow the solvent to migrate tank. Cover and seal the tank. Allow to the finishing line. Remove the plate the solvent front to travel approxi- and air dry in a fume hood. Place the mately 15 centimeters from the start- dried plate in a chamber containing io- ing line. Remove the plate from the dine vapors. tank and allow it to air dry. (e) Evaluation. Measure the distance (e) Evaluation. View the dry plate the solvent front traveled from the under ultraviolet light (254 starting line, and the distance the nanometers). Measure the distance the spots are from the starting line. Divide solvent front traveled from the start- the latter by the former to calculate ing line and the distance the spots are the Rf value. The identity test is posi- from the starting line. Calculate the Rf tive if the sample solution produces a value by dividing the latter by the yellow spot at the same Rf value and former. The sample and standard has the same appearance as the spot should have spots of corresponding Rf obtained for the reference solution. values of approximately 0.35. The Rf value for cefprozil (Z) is ap- proximately 0.45. Cefprozil (E), has an [54 FR 48860, Nov. 28, 1989; 54 FR 51816, Dec. 18, 1989] Rf value of approximately 0.47.Cefprozil (Z) is ‘‘absent’’ if the above test is per- § 436.368 Thin layer chromatographic formed and no spots, which correspond identity test for cefprozil. to those from the reference solution, (a) Equipment—(1) Chromatography are obtained for the sample. tank. Use a glass rectangular tank ap- [58 FR 26660, May 4, 1993] proximately 23 x 23 x 9 centimeters lined with filter paper and equipped § 436.369 Thin layer chromatography with a tight-fitting cover. test for free N-isobutylpiperidone (2) Plates. Use 20 x 20 centimeter thin content in rifabutin. layer chromatography plates coated (a) Equipment—(1) Chromatography with silica gel GF to a thickness of 250 tank. A rectangular tank, approxi- microns. mately 23 X 23 X 9 centimeters, with a (b) Reagents—(1) Diluent. Mix 0.1N glass solvent trough on the bottom and HCl and acetone in volumetric propor- a tight-fitting cover. tions of 1:4. (2) Iodine vapor chamber. A rectangu- (2) Developing solvent. Mix n-butanol, lar tank, approximately 23 X 23 X 9 glacial acetic acid and water in volu- centimeters, with a suitable cover, con- metric proportions of 60:20:20. taining iodine crystals.
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(3) Plates. Use 20 X 20 centimeter thin the Rf value by dividing the latter by layer chromatography plates coated the former. N-isobutylpiperidone has with silica gel 60F 254 or equivalent to an Rf value of about 0.3. Rifabutin has a thickness of 250 microns. an Rf value of about 0.1. Compare the (b) Reagents—(1) Developing solvent. size and intensity of any N- Mix petroleum ether (b.p. 60 to 80 ° C) isobutylpiperidone spots in the sample and acetone in volumetric proportions lane with the N-isobutylpiperidone of 100:30, respectively. spots in the standard lanes, and report (2) Spray solution. Prepare a 1 percent the percentage of N-isobutylpiperidone solution of soluble starch in water in the sample. (containing 0.01 percent mercuric io- [59 FR 40806, Aug. 10, 1994] dide). (c) Preparation of spotting solutions— § 436.370 Spectrophotometric identity (1) Sample solution. Prepare a solution test for rifabutin capsules. of the rifabutin sample in 1:1 chloro- (a) Equipment. A suitable spectro- form/methanol to contain 10 milli- photometer capable of recording the grams per milliliter. ultraviolet spectrum in the 200 to 400 (2) Standard solution. Prepare a solu- nanometer range, using suitable quartz tion of N-isobutylpiperidone standard cells of 1 centimeter pathlength. in 1:1 chloroform/methanol to contain 1 (b) Preparation of working standard milligram per milliliter. Transfer and sample solution—(1) Working stand- aliquots of 0.5, 1.0, 2.0, 5.0, and 10.0 mil- ard solution. Suspend approximately 200 liliters into separate 100-milliliter vol- milligrams of rifabutin working stand- umetric flasks and dilute to volume ard in 20 milliliters of methanol and with 1:1 chloroform/methanol. These sonicate for approximately 5 minutes. solutions contain, respectively, the Filter the resulting solution through a equivalent of 0.05, 0.1, 0.2, 0.5, and 1.0 suitable 0.5 micrometer filter. Transfer percent of N-isobutylpiperidone. a 2-milliliter aliquot of the filtered so- (d) Procedure. Pour 100 milliliters of lution to a 100-milliliter volumetric developing solvent into the glass flask and fill to volume with methanol. trough on the bottom of the unlined Further dilute with methanol to obtain chromatography tank. Cover and seal a solution containing 20 micrograms of the tank. Allow it to equilibrate while rifabutin activity per milliliter. the plate is being prepared. Prepare a (2) Sample solution. Empty and com- plate as follows: on a line 2.0 centi- bine the contents of five capsules. Sus- meters from the base of the thin layer pend a quantity of the capsule contents chromatography plate, and at intervals equivalent to 200 milligrams of of 2.0 centimeters, apply 10 microliters rifabutin in 20 milliliters of methanol. of each of the standard solutions and Sonicate for about 5 minutes and then the sample solution prepared as di- filter through an appropriate 0.5 mi- rected above. After the spots are thor- crometer filter. Transfer a 2-milliliter oughly dry, place the plate into the aliquot to a 100-milliliter volumetric trough in the bottom of the tank. flask and dilute to volume with meth- Cover and tightly seal the tank, allow anol. Further dilute with methanol to the solvent front to travel about 15 obtain a solution containing 20 centimeters from the starting line and micrograms of rifabutin activity per then remove the plate from the tank. milliliter (estimated). Air dry the plate. Warm the iodine (c) Procedure. Using a suitable spec- vapor chamber to vaporize the iodine trophotometer equipped with 1.0 centi- crystals and place the dry plate in the meter cells and methanol as the blank, iodine vapor chamber until the spots determine the absorbance spectra of are visible (usually about 5 minutes). the working standard and sample solu- Remove the plate from the iodine tions over the ultraviolet range of 250 vapor chamber and spray with 1 per- to 300 nanometers. cent starch solution. (d) Evaluation. Compare the spectrum (e) Evaluation. Measure the distance of the sample to that of the working the solvent front traveled from the standard. The identity of the rifabutin starting line and the distance the spots capsules is confirmed by quantitative are from the starting line. Calculate comparison of the two spectra with an
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absorbance maximum being observed Subpart H—Tests for Specific at about 275 nanometers. Antibiotic Dosage Forms [59 FR 40807, Aug. 10, 1994] § 436.500 Penicillin in oil and wax. Subpart G—Chemical Tests for (a) Potency. Proceed as directed in Nonantibiotic Active Ingredients § 440.80a(b)(1) of this chapter except paragraph (b)(1)(ix) thereof and, in lieu § 436.400 Thin layer chromatographic of the directions in § 440.80a(b)(1)(iv) of identity test for this chapter prepare sample as follows: iodochlorhydroxyquin. Liquefy the sample by warming, thor- (a) Equipment—(1) Chromatography oughly mix, and withdraw 1.0 milliliter tank. A rectangular tank, approxi- using a sterile syringe equipped with mately 9 × 9 × 3.5 inches with a glass an 18-gauge needle. Transfer to a sepa- solvent trough on the bottom. ratory funnel containing approxi- (2) Plates. Use 20 × 20 centimeter thin mately 50 milliliters of peroxide-free layer chromatography plates coated ether. Shake the separatory funnel vig- with Silica Gel G or equivalent to a orously to bring about complete mix- thickness of 250 microns. ing of the material with the ether. (b) Developing solvent. Mix benzene Shake with a 25-milliliter portion of 1 and methanol in volumetric propor- percent phosphate buffer at pH 6.0. Re- tions of 90:10. move the buffer layer and repeat the (c) Preparation of spotting solutions— extraction with three 25-milliliter (1) Sample solution. Use the sample solu- quantites of buffer. Combine the ex- tion prepared as described in the sec- tracts and make the proper estimated tion for the particular product to be dilutions in 1 percent phosphate buffer tested. at pH 6.0. The sample may also be pre- (2) Reference solution. Prepare a solu- pared by transferring aseptically 1.0 tion containing 0.5 milligram of milliliter of the penicillin in oil and iodochlorhydroxyquin U.S.P. reference wax to a blending jar containing 100 standard per milliliter in acetone. milliliters of 1 percent phosphate buff- (d) Procedure. Pour developing sol- er at pH 6.0. Using a high-speed blend- vent into the glass trough on the bot- er, blend this mixture for 1 minute and tom of the chromatography tank. then make the proper estimated dilu- Cover and seal the tank. Allow it to tions in 1 percent phosphate buffer at equilibrate for 1 hour. Spot a plate as pH 6.0. If the label represents the po- follows: Apply approximately 10 micro- tency of the penicillin in oil and wax as liters each of the sample solution and 200,000 units per milliliter or less, it is of the reference solution on a line 2.0 satisfactory if it is 85 percent or more centimeters from the base of the silica of the potency so represented; if rep- gel plate and at intervals of not less resented as more than 200,000 units per than 2.0 centimeters. After all spots milliliter, it is satisfactory if it is 90 are thoroughly dry, place the silica gel percent or more of the potency so rep- plate directly into the glass trough of resented. the chromatography tank. Cover and (b) Sterility. Proceed as directed in reseal the tank. Allow the solvent § 436.20, using the method described in front to travel about 15 centimeters paragraph (e)(2) of that section, except from the starting line, remove the using medium B in lieu of medium A. plate from the tank, and allow to air (c) Moisture—(1) Reagents—(i) dry. Examine under a strong source of KarlFischer reagent. Preserve the rea- ultraviolet light. The sample and gent in glass-stoppered bottles and use standard are visible as dark spots. from an all glass automatic burette, (e) Evaluation. Measure the distance protecting the solution from the mois- the solvent front traveled from the ture in the air. starting line and the distance the spots (ii) Water-methanol solution. Use are from the starting line. Calculate methanol containing approximately 1 the Rf value by dividing the latter by mg. of water per milliliter. Store the the former. The sample and standard solution in a glass bottle attached to should have spots of corresponding Rf an automatic burette and protect from values (0.55 to 0.60). moisture in the air at all times.
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(2) Standardization of Karl Fischer rea- (d) Measurement of penicillin particle gent. Add a known volume of the Karl size. Vigorously shake the container to Fischer reagent to a suitable titrating obtain an even suspension of the peni- vessel which has been previously dried cillin particles and immediately with- at 105° C. and cooled in a desiccator. In- draw therefrom approximately 0.5 mil- troduce a mechanical stirrer and two liliter of the drug into a clean, dry, tu- platinum electrodes which are con- berculin syringe using a dry 18-gauge nected to a suitable electrometric ap- needle. Discard approximately the first paratus for measurement of the end- 5 drops of the mixture extruded from point. Start the stirrer and titrate with the needle and then extrude approxi- the water-methanol solution until the mately 1 minim of the remaining mix- endpoint is reached. Calculate the mil- ture into a test tube containing 3 to 4 liliters of Karl Fischer reagent equiva- milliliters of light mineral oil. Thor- lent to each milliliter of water-meth- oughly mix the contents of the tube anol. Add an accurately weighed quan- and by means of a bacteriological loop tity of water (approximately 50 milli- (2 millimeters inside diameter, 22 grams) to a dry titrating vessel, add an gauge wire), immediately place one excess of the Karl Fischer reagent and loopful of the suspension on each ruled back titrate with the water-methanol chamber of a bright line hemocytom- solution as above. Calculate the milli- eter. (It is not necessary to use a cover slip.) Confirm by means of the low grams of water equivalent to each mil- power objective of the microscope the liliter of the Karl Fischer reagent. even distribution of particles over the Standardize the Karl Fischer reagent ruled areas of both chambers and re- in this manner daily. peat with another loopful of the sus- W pension if even dispersion is not ob- e = tained. Use a magnification of 430 or V− V f 440 diameters and a calibrated ocular 1 2 micrometer to measure the penicillin where: particles. For the purpose of measure- e=milligrams of water equivalent to 1 ml. ment and calculation, the predominant Karl Fischer reagent. type of crystals observed shall be con- w=weight of water in milligrams. sidered to represent the type of crys- v1=volume of Karl Fischer reagent used. tals present and the thickness and den- v2=volume of methanol used. sity of all particles shall be considered f=volume ratio of Karl Fischer reagent to constant. Center a large penicillin par- water-methanol solution. ticle in the microscopic field; measure (3) Procedure. Transfer 1.0 milliliter the particle and all other particles in of the penicillin in oil and wax to a dry the field and repeat this operation on titrating vessel, add 10 milliliters of other fields until at least 200 particles dry chloroform and an excess of the are measured. Particles of less than 5 Karl Fischer reagent and back titrate microns in length are disregarded. The with the water-methanol solution until grouping of the particles by length, the the endpoint is reached. Transfer 10 midpoint, the ratio of the midpoints, milliliters of the dry chloroform used and the square of the ratio of the to a dry titrating vessel, add an excess midpoints for each group are tabulated of Karl Fischer reagent, and titrate below: with the water-methanol as above. Cal- Length Mid- Ratio culate the milliliters of Karl Fischer Group in mi- of mid- (Ratio) 2 reagent equivalent to 10 milliliters of crons point points chloroform. 1 ...... 5±14 9.5 1.00 1.00 2 ...... 15±29 22.0 2.31 5.34 ()V− V f − b× e ×100 3 ...... 30±49 39.5 4.16 17.31 Percent = 1 2 moisture 4 ...... 50±69 59.5 6.26 39.19 s ×100 5 ...... 70±99 84.5 8.89 79.03 where: 6 ...... 100±149 124.5 13.10 171.61 b=milliliters Karl Fischer reagent equiva- 7 ...... 150±199 174.5 18.36 337.09 lent to 10 ml. of chloroform. 8 ...... 200±249 224.5 23.63 558.38 9 ...... 250±300 275.0 28.95 838.10 s=volume of the sample in milliliters.
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Calculate the percent particles in each (3) Colorimetric determination of pro- group from the total number measured. caine penicillin in the solution for assay. Determine the percent relative weight Transfer an aliquot of the solution for for each group as follows: assay to a 50-milliliter volumetric flask. Determine the quantity of pro- Plate type particles. Relative weight= (ratio) 2×% of total particles in group. caine penicillin in this solution by the following method: Relativeweight ×100 (i) Reagents—(a) Sodium nitrite solu- % relativeweight = tion. Dissolve 0.1 gram of sodium ni- Totalrelativeweight trite in 100 milliliters of distilled Rod-shaped particles. In the case of rod- water. Prepare fresh solution every shaped particles measure the width as well week and store under refrigeration. as the length. (b) Ammonium sulfamate solution. Dis- Relative weight=ratio × average width × % of solve 0.5 gram of ammonium sulfamate total particles in group in 100 milliliters of distilled water and store under refrigeration. Relative weight ×100 %relativeweight= (c) N-(1-naphthyl)-ethylenediamine so- Total relative weight lution. Dissolve 0.1 gram of N-(1- naphthyl) ethylenediamine When examined by the method de- dihydrochloride in 100 milliliters of dis- scribed in this section not less than 50 tilled water. Prepare fresh solutions percent of the total relative weight of every week and store under refrigera- the penicillin in the drug consists of tion. penicillin having a particle size of not (d) Standard procaine solution. Prepare less than 50 microns in length. a standard solution containing 27.55 milligrams of procaine hydrochloride § 436.503 Procaine penicillin and U.S.P. in a liter of distilled water (each buffered crystalline penicillin for milliliter of the standard solution is aqueous injection. equivalent to 60 units of procaine peni- (a) Total potency (except in single-dose cillin). container), sterility, moisture, pyrogens, (ii) Standards. Transfer, respectively, toxicity, pH. Proceed as directed in 1.0, 2.0, 3.0, 4.0, and 5.0 milliliters of the § 440.274b(b) of this chapter. standard solution and 5.0 milliliters of (b) Buffered crystalline penicillin con- distilled water to each of six 50-milli- tent—(1) Preparation of the solution for liter volumetric flasks. Add 4.0, 3.0, 2.0, assay. Add the indicated amount of dis- and 1.0 milliliters of water to the first tilled water to the contents of a vial of four flasks, respectively, to give each a the sample, and shake well. Withdraw volume to 5.0 milliliters. one dose of the suspension with a hypo- (iii) Procedure. To each flask for the dermic syringe and place in a 10-milli- standards and the solution for assay liter volumetric flask. Add 20-percent add 0.5 milliliter of 4 N HCl, 1.0 milli- sodium sulfate solution almost to the liter of the sodium nitrite solution, 1.0 mark, centrifuge sufficiently to see the milliliter of the ammonium sulfamate, meniscus, make to volume with 20-per- and 1.0 milliliter of the N-(1-naphthyl)- cent sodium sulfate solution, shake ethylenediamine solution. Mix and well, and centrifuge to obtain a clear wait two minutes after each addition. or reasonably clear solution. Dilute a Make each flask to volume of 50 milli- 5.0-milliliter aliquot of this clear solu- liters with distilled water. Determine tion with 1-percent phosphate buffer, the absorbency of the colored solutions pH 6.0, to give a solution for assay of at 550 Mµ in a suitable photo electric approximately 2,000 units per milli- colorimeter. The instrument is bal- liter. anced so that the zero concentration (2) Iodometric assay for total penicillin reads zero absorbency. Plot the stand- in the solution for assay. Determine the ard curve on coordinate graph paper. quantity of penicillin in the solution Obtain the procaine penicillin content for assay by the iodometric assay pro- of the solution for assay directly from cedure described in § 440.80a(b)(5)(iv)(a) the point on the standard curve cor- of this chapter. responding to its absorbency.
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(4) The content of buffered crys- of that paragraph. Its content of peni- talline pencillin in one dose of the cillin is satisfactory if it contains not product is calculated as follows: less than 85 percent of the number of units it is represented to contain. A=(B¥C)F, (2) Bacitracin content. Proceed as di- where: rected in § 448.510a(b)(1) of this chapter, A=buffered crystalline penicillin content of except that sufficient penicillinase is the product. added to the sample under test to com- B=total number of units of penicillin per pletely inactivate the penicillin milliliter as determined in paragraph present. Its content of bacitracin is (b)(2) of this section. C=number of units of procaine penicillin satisfactory if it contains not less than per milliliter as determined in 85 percent of the number of units it is paragaph (b)(3) of this section. represented to contain. F=appropriate dilution factor depending on (b) Moisture. Proceed as directed in the dilution made in the preparation of § 436.201. the solution for assay. [39 FR 18944, May 30, 1974, as amended at 40 The content of buffered crystalline FR 13497, Mar. 27, 1975] penicillin in the batch is satisfactory when determined by the method de- § 436.505 Penicillin-streptomycin-baci- scribed in this paragraph if it is not tracin ointment; penicillin-dihydro- less than 85 percent of that which it is streptomycin-bacitracin ointment; penicillin-streptomycin-bacitracin represented to contain. methylene disalicylate ointment; (c) Procaine penicillin. The procaine penicillin-dihydrostreptomycin-bac- penicillin content of the batch is the itracin methylene disalicylate oint- difference between the total potency ment. determined by the method described in (a) Potency—(1) Content of penicillin, paragraph (a) or (d) of this section and streptomycin, and dihydrostreptomycin. the content of the buffered crystalline Proceed as directed in § 536.501(a) of penicillin determined by the method this chapter. described in paragraph (b) of this sec- (2) Bacitracin content. Proceed as di- tion. The procaine penicillin content of rected in § 448.510a(b)(1) of this chapter, the batch is satisfactory when deter- except that: mined by the method described in this (i) Sufficient penicillinase is added to paragraph if it is not less than 85 per- the sample under test to completely in- cent of that which it is represented to activate the penicillin present. contain. (ii) Use as the test organism the (d) Total potency of a one-dose con- streptomycin dihydrostreptomycin re- tainer. Wash out the material remain- sistant strain of either Micrococcus ing in the 10-milliliter volumetric flask flavus (ATCC 10240A) 1 or Sarcina referred to in paragraph (b)(1) of this subflava (ATCC 7468/d), 1 grown and section with 1-percent phosphate buff- maintained in media containing 500 er, pH 6.0. Dilute to give a concentra- micrograms of streptomycin or dihy- tion of approximately 2,000 units per drostreptomycin per milliliter of milliliter, and assay by the iodometric media, or calculate from the quantity method described in § 440.80a of streptomycin or dihydrostrepto- (b)(5)(iv)(a) of this chapter. Obtain the mycin found, using the method pre- total potency by adding the number of scribed by paragraph (a)(1) of this sec- units found in this solution (units per tion, the quantity that would be milliliter × volume) to the number of present when the sample is diluted to units found (units per milliliter × vol- contain one unit of bacitracin (labeled ume) in the solution assayed in accord- potency) per milliliter. Prepare the ance with paragraph (b)(2) of this sec- bacitracin standard curve by adding tion. the calculated quantity of strepto- mycin or dihydrostreptomycin to each § 436.504 Penicillin-bacitracin oint- concentration of bacitracin used for ment. (a) Potency—(1) Penicillin content. 1 Available from: American Type Culture Proceed as directed in § 540.380a(b)(1) of Collection, 12301 Parklawn Drive, Rockville, this chapter, except the last sentence MD 20852.
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the curve. Use this standard curve to to make 10 milliliters. Shake well and calculate the bacitracin content of the centrifuge to obtain a clear, or reason- sample. ably clear, solution. Dilute a 5.0-milli- (3) Bacitracin methylene disalicylate liter aliquot to 50 milliliters with buff- content. Proceed as directed in para- er and proceed as directed in graph (a)(2) of this section, except pre- § 440.80a(b)(1) of this chapter to deter- pare the sample as follows: Place a rep- mine the number of units per milliliter resentative portion of the sample (usu- of this solution, and from this value ally approximately 1 gram, accurately calculate the number of units per mil- weighed) or the entire contents of a liliter of the drug. The content of single-dose container in blending jar, buffered crystalline penicillin is satis- add 99 milliliters of a 2.0-percent aque- factory if it is not less than 85 percent ous solution of sodium bicarbonate and of that which it is represented to con- 1 milliliter of a 10-percent aqueous so- tain. lution of polysorbate 80 and blend for 3 (c) Benzathine penicillin G content. minutes in a high-speed blender. Allow The benzathine penicillin G content of the foam to subside, remove an aliquot the batch is the difference between the of the solution, and dilute to 1 unit per total potency as described in paragraph milliliter with 1.0-percent phosphate (a) or (d) of this section and the con- buffer, pH 6.0. tent of buffered crystalline penicillin (b) Moisture. Proceed as directed in determined by the method prescribed § 436.201. in paragraph (b) of this section. The [39 FR 18944, May 30, 1974, as amended at 40 content of benzathine penicillin G is FR 13497, Mar. 27, 1975] satisfactory if it is not less than 85 per- cent of that which it is represented to § 436.506 Benzathine penicillin G and contain. buffered crystalline penicillin for (d) Total potency of a single-dose con- aqueous injection. tainer. Add sufficient distilled water to (a) Total potency (except in single-dose the material remaining in the 10-milli- containers). Proceed as directed in liter volumetric flask referred to in § 440.80a(b)(1) of this chapter, except if paragraph (b) of this section to bring the bioassay method is used prepare the volume back to 10 milliliters and the sample by diluting 1.0 milliliter of determine the number of units per mil- the drug suspension with sufficient di- liliter of this suspension. If the methyl formamide, formamide, or iodometric method is used, 2.0-milli- methyl alcohol to dissolve the liter aliquots are placed in 50-milliliter benzathine penicillin. Make to 100 mil- volumetric flasks (one blank and one liliters with buffer. Shake well and di- to be inactivated). Obtain the total po- lute to 1.0 unit per milliliter. If the tency by adding the number of units iodometric method is used, proceed as found in the 10-milliliter volumetric directed in § 440.55a(b) of this chapter, flask to one-half the content of except in preparing the blank solution buffered crystalline penicillin found in dilute 1.0 milliliter of the drug suspen- paragraph (b) of this section. sion to 250 milliliters with 1-percent (e) Sterility. Proceed as directed in phosphate buffer at pH 6.0. In preparing § 436.20 using the method described in the solution for inactivation dissolve paragraph (e)(2) of that section, except 1.0 milliliter of the drug suspension in use medium C in lieu of medium A, and approximately 20 milliliters of 0.5 N medium F in lieu of medium E. During NaOH. Allow to stand for 15 minutes. the period of incubation, shake the Dilute to 250 milliliters with distilled tubes at least once daily. water. Pipette a 2.0-milliliter aliquot (f) Moisture. Proceed as directed in into a 125-milliliter glass-stoppered Er- § 440.74a(b)(5) of this chapter. lenmeyer flask and add 2.0 milliliters (g) Pyrogens. Proceed as directed in 1.2 N HCl and 10 milliliters 0.01 N io- § 436.500. dine. (h) Toxicity. Proceed as directed in (b) Buffered crystalline penicillin con- § 440.55a(b)(3) of this chapter. tent. Place 1.0 milliliter of the drug sus- (i) pH. Proceed as directed in pension in a 10-milliliter volumetric § 440.80a(b)(5)(ii) of this chapter, using flask and add 20 percent sodium sulfate the suspension resulting when the
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product is reconstituted as directed in caine penicillin per milliliter. Deter- the labeling. mine the procaine penicillin content by the colorimetric procedure described in § 436.507 Benzathine - procaine - § 436.503(b)(3). The content of procaine buffered crystalline penicillins for penicillin is satisfactory if it contains aqueous injection. not less than 85 percent of the number (a) Potency—(1) Total potency. Pro- of units that it is represented to con- ceed as directed in § 440.80a(b)(1) of this tain. chapter, except if the bioassay method (3) Buffered crystalline penicillin con- is used prepare the sample by diluting tent—(i) Preparation of the solution for one dose of the drug suspension with assay. (a) Add the indicated amount of sufficient dimethyl formamide or form- distilled water to the contents of a vial amide or methyl alcohol to dissolve of the sample, and shake well. With- the benzathine penicillin G. Make to draw one dose of the suspension with a 100 milliliters with 1-percent phosphate hypodermic syringe and place in a 10- buffer, pH 6.0. Shake well, and dilute to milliliter volumetric flask. Add 20-per- 1.0 unit per milliliter with buffer. If the cent sodium sulfate solution almost to iodometric method of assay is used, the mark, centrifuge sufficiently to see add the indicated amount of distilled the meniscus, make to volume with 20- water to the contents of a vial of the percent sodium sulfate solution, shake sample, shake well, and proceed as fol- well, and centrifuge to obtain a clear lows (except for single-dose contain- or reasonably clear solution; or ers): (b) If the original product contains (i) Using a standardized hypodermic more than 600,000 units, place it in a 50- syringe, withdraw one dose and dilute milliliter volumetric flask, add 20-per- with 1-percent phosphate buffer, pH 6.0, cent sodium sulfate to the mark, shake to give a concentration of approxi- well, place a 10-milliliter portion in a mately 2,000 units per milliliter. Use 2.0 centrifuge tube, and centrifuge to ob- milliliters of this suspension as the tain a reasonably clear solution. blank in the iodometric assay proce- (c) Dilute a 5.0-milliliter aliquot of dure described in § 440.80a(b)(5)(iv)(a) of the clear solution obtained in para- this chapter. graph (a) (3)(i) (a) or (b) of this section (ii) Using a standardized hypodermic with 1-percent phosphate buffer, pH 6.0, syringe, withdraw another dose, place to give a solution for assay of approxi- in a flask, and add 20 milliliters of 0.5 mately 2,000 units per milliliter. N NaOH for each 300,000 units of (ii) Iodometric assay for total penicillin benzathine penicillin, mix well, being in the solution for assay. Determine the sure that all penicillin is in solution, total quantity of penicillin in the solu- and allow to stand for 15 minutes. Add tion for assay by the iodometric assay 1 milliliter of 1.2 N HCl for each 2 milli- procedure described in § 440.80a liters of 0.5 N NaOH, mix, and dilute (b)(5)(iv)(a) of this chapter. with distilled water to the same vol- (iii) Colorimetric determination of pro- ume as was used in paragraph (a)(1)(i) caine penicillin in the solution for assay. of this section. Place 2.0 milliliters in a Proceed as directed in § 436.503 (b)(3). 125-milliliter glass-stoppered Erlen- The content of procaine penicillin in meyer flask, add 10 milliliters of 0.01 N the batch is satisfactory if it is not less iodine, allow to stand for 15 minutes, than 85 percent of that which it is rep- and titrate with 0.01 N sodium resented to contain. thiosulfate as directed in the (iv) The buffered crystalline penicil- iodometric assay procedure in § 440.80a lin in one dose of the product is cal- (b)(5)(iv)(a) of this chapter. The total culated as follows: potency of the batch is satisfactory if it contains not less than 85 percent of A=(B¥C)F, that which it is represented to contain. where: (2) Procaine penicillin content (except A=the buffered crystalline penicillin con- for single-dose containers). Make suit- tent of the product. able dilutions of the solution prepared B=the number of units of penicillin per in paragraph (a)(1)(ii) of this section to milliliter as determined in paragraph obtain approximately 60 units of pro- (a)(3)(ii) of this section.
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C=the number of units of procaine penicil- found (units per milliliter ×volume) in lin per milliliter as determined in para- the solution for assay in paragraph graph (a)(3)(iii) of this section. (a)(3)(ii) of this section. F=the appropriate dilution factor depend- ing on the dilutions made in the prepa- (6) Procaine penicillin content of a sin- ration of the solution for assay. gle-dose container. Make suitable dilu- tions of the NaOH-inactivated solution The content of buffered crystalline prepared in paragraph (a)(5) of this sec- penicillin is satisfactory if the batch tion to obtain approximately 60 units contains 85 percent of the number of of procaine penicillin per milliliter. De- units per milliliter that it is rep- termine the procaine penicillin content resented to contain. (units per milliliter× volume) of this (4) Benzathine penicillin content. The solution by the colorimetric procedure sum of the procaine penicillin content described under § 436.503(b)(3). To this determined under paragraph (a)(2) or value add per procaine penicillin con- (6) of this section and the buffered tent (unit per milliliter×volume) of the crystalline penicillin content deter- solution for assay, as found in para- mined under paragraph (a)(3) of this graph (a)(3)(iii) of this section, to ob- section, subtracted from the total po- tain the procaine penicillin content of tency determined in paragraph (a)(1) or the one-dose container. The content of (5) of this section, represents the procaine penicillin in the batch is sat- benzathine penicillin G content. The isfactory if it is not less than 85 per- benzathine penicillin G content is sat- cent of that which it is represented to isfactory if it is not less than 85 per- contain. cent of the number of units that it is (b) Sterility. Proceed as directed in represented to contain. § 436.20, using the method described in (5) Total potency of a single-dose con- paragraph (e)(2) of that section, except Wash out the material remain- tainer. use medium C in lieu of medium A, and ing in the volumetric flask referred to medium F in lieu of medium E. During in paragraph (a)(3)(i)(a) of this section, the period of incubation, shake the or combine the contents remaining in tubes at least once daily. the 50-milliliter volumetric flask and in the centrifuge tube referred to in (c) Pyrogens. Proceed as directed in paragraph (a)(3)(i)(b) of this section. § 440.55a(b)(4) of this chapter. Dissolve the material by adding 10 mil- (d) Toxicity. Proceed as directed in liliters of 1 N NaOH for each 300,000 § 440.55a(b)(3) of this chapter. units of benzathine penicillin and allow (e) Moisture. Proceed as directed in to stand 15 minutes. Add 1 milliliter of § 440.74a(b)(5) of this chapter. 1.2 N HCl for each milliliter of 1 N (f) pH. Proceed as directed in NaOH and then dilute with distilled § 440.80a(b)(5)(ii) of this chapter, using water to give a concentration of ap- the suspension resulting when the proximately 2,000 units per milliliter. product is reconstituted as directed in Place 2.0 milliliters in a 125-milliliter the labeling. glass-stoppered Erlenmeyer flask, add 10 milliliters of 0.01 N iodine, allow to § 436.508 Penicillin - bacitracin - neo- stand for 15 minutes, and then titrate mycin ointment; penicillin-baci- tracin-neomycin in oil. with 0.01 N sodium thiosulfate as di- rected in § 440.80a (b)(5)(iv)(a) of this (a) Potency—(1) Penicillin content; bac- chapter. For the blank determination itracin content. Proceed as directed in prepare a separate sample as directed § 436.504(a). in paragraph (a)(3)(i) (a) or (b) of this (2) Neomycin content. Proceed as di- section and in the first sentence of this rected in § 448.510d(b)(1)(ii) of this chap- paragraph (a)(5), then dilute with 1 per- ter, except that sufficient penicillinase cent phosphate buffer, pH 6.0, to give a is added to the sample under test to concentration of approximately 2,000 completely inactivate the penicillin units per milliliter. The total potency present. Its content of neomycin is sat- of the one-dose container is equal to isfactory if it contains not less than 85 the sum of the number of units found percent of the number of milligrams in this assay (units per milli- per gram that it is represented to con- liter×volume) and the number of units tain.
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(b) Moisture. Proceed as directed in cient penicillinase at 37° C. for 30 min- § 436.201. utes. Make the proper estimated dilu- tions in 10-percent potassium phos- § 436.509 Procaine penicillin-strepto- phate buffer (pH 6.0) to give a con- mycin-polymyxin in oil; procaine centration of 10 units per milliliter (es- penicillin-dihydrostreptomycin- polymyxin in oil; procaine penicil- timated). lin-streptomycin-polymyxin oint- (b) Blending. Place a convenient-sized ment; procaine penicillin - dihydro- representative quantity of the sample streptomycin - polymyxin ointment. in a blending jar containing 1.0 milli- (a) Potency—(1) Penicillin content. liter of polysorbate 80 and sufficient 1- Proceed as directed in § 540.380a(b)(1) of percent phosphate buffer (pH 6.0) to this chapter. Its content of penicillin is give a final volume of 200 milliliters. If satisfactory if it contains not less than the sample consists of substantially 85 percent of the number of units per more than 1 gram, use sufficient buffer milliliter or per gram that it is rep- to give a final volume of 500 milliliters. resented to contain. If the concentration of polymyxin in (2) Streptomycin content. Proceed as the blend is less than 200 units per mil- directed in § 544.373(b)(1)(i) of this chap- liliter, 10-percent phosphate buffer (pH ter, except inactivate the penicillin in 6.0) should be used in lieu of 1-percent the combined extractives with suffi- phosphate buffer (pH 6.0). Using a high- cient penicillinase at 37° C. for 30 min- speed blender, blend the mixture for 2 utes. Its content of streptomycin is minutes. Inactivate the penicillin with satisfactory if it contains not less than sufficient penicillinase at 37° C. for 30 85 percent of the number of milligrams minutes and make the proper esti- per milliliter or per gram that it is rep- mated dilutions in 10-percent phos- resented to contain. phate buffer (pH 6.0) to give a con- (3) Dihydrostreptomycin content. Pro- centration of 10 units per milliliter (es- ceed as directed in paragraph (a)(2) of timated). this section, using the dihydrostrepto- (ii) The standard curve is prepared in mycin working standard as a standard the following concentrations: 6.4, 8.0, of comparison. Its content of dihydro- 10.0, 12.5, and 15.6 units per milliliter in streptomycin is satisfactory if it con- 10-percent potassium phosphate buffer, tains not less than 85 percent of the pH 6.0. The 10 units per milliliter con- number of milligrams per milliliter or centration is used as the reference per gram that it is represented to con- point. Its content of polymyxin is sat- tain. isfactory if it contains not less than 85 (4) Polymyxin content. Proceed as di- percent of the number of units per mil- rected in § 444.170a(b)(2)(i) of this chap- liliter or per gram that it is rep- ter, with the following exceptions: (i) In lieu of the directions for the resented to contain. preparation of the sample described in (b) Moisture. Proceed as directed in § 444.170a(b)(2)(i)(g) of this chapter, pre- § 436.201. pare the sample by one of the following [39 FR 18944, May 30, 1974, as amended at 40 techniques: FR 13497, Mar. 27, 1975; 41 FR 10886, Mar. 15, (a) Extraction. Place a convenient- 1976] sized representative quantity of the sample in a separatory funnel contain- § 436.510 Penicillin-streptomycin- ing approximately 50 milliliters of per- erythromycin ointment; penicillin- oxide-free ether. Shake the sample and dihydro-streptomycin-erythromycin ether until homogeneous. Add 25 milli- ointment. liters of 10-percent potassium phos- (a) Potency—(1) Penicillin content. Ob- phate buffer (pH 6.0) and shake. Re- tain the weight of the content of a sy- move the buffer layer and repeat the ringe by weighing before and after extraction with 25-milliliter portions ejecting the content into a beaker. Stir of buffer at least three times and any until homogeneous. Remove a rep- additional times that may be necessary resentative sample (usually approxi- to insure complete extraction of the mately 1.0 gram, accurately weighed) antibiotic. Combine the extractives. and place in a separatory funnel con- Inactivate the penicillin with suffi- taining 50 milliliters of peroxide-free
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ether. Add 20 milliliters of 0.1 M potas- lute with buffer to give an erythro- sium phosphate buffer (pH 8.0) and mycin content of 1.0 microgram per shake. Remove the buffer layer and re- milliliter (estimated). Its content of peat the extraction with three addi- erythromycin is satisfactory if it con- tional 20-milliliter portions of the buff- tains not less than 85 percent of the er. Place the buffer solution in a sec- number of milligrams that it is pre- ond separatory funnel and wash with sented to contain. three 30-milliliter portions of ether. (b) Moisture. Proceed as directed in Discard the ether washes. Remove an § 436.500(c). aliquot of the buffer solution and pro- [39 FR 18944, May 30, 1974, as amended at 40 ceed as directed in § 440.80a(b) (1) of this FR 13497, Mar. 27, 1975] chapter, except § 440.80a(b)(1)(iv) and (ix) of this chapter. If the iodometric § 436.511 Penicillin-streptomycin-baci- chemical assay is used, proceed as di- tracin methylene disalicylate-neo- rected in § 440.80a(b)(5)(iv)(a) of this mycin ointment; penicillin-dihydro- chapter, except prepare the sample as streptomycin-bacitracin methylene directed in § 536.501(a)(1) of this chap- disalicylate-neomycin ointment. ter. Its content of penicillin is satisfac- (a) Potency—(1) Penicillin content. tory if it contains not less than 85 per- Proceed as directed in § 540.380a(b)(1) of cent of the number of units that it is this chapter. Its penicillin content is represented to contain. satisfactory if it contains not less than (2) Streptomycin content. Using an ali- 85 percent of the number of units that quot of the buffer solution prepared as it is represented to contain. directed in paragraph (a)(1) of this sec- (2) Streptomycin content. Proceed as tion, proceed as directed in § 444.70a directed in § 436.105 of this chapter. Its (b)(1) through (9) of this chapter, ex- content of streptomycin is satisfactory cept add sufficient penicillinase to if it contains not less than 85 percent completely inactivate the penicillin of the number of milligrams that it is present. Its content of streptomycin is represented to contain. satisfactory if it contains not less than (3) Dihydrostreptomycin content. Pro- 85 percent of the number of milligrams ceed as directed in § 436.105 of this chap- that it is represented to contain. ter. Its content of dihydrostreptomycin (3) Dihydrostreptomycin content. Pro- is satisfactory if it contains not less ceed as directed in paragraph (a)(2) of than 85 percent of the number of milli- this section, using the dihydrostrepto- grams that it is represented to contain. mycin working standard as the stand- (4) Bacitracin methylene disalicylate ard of comparison. Its content of dihy- content. Proceed as directed in drostreptomycin is satisfactory if it § 436.505(a)(3). Its potency is satisfac- contains not less than 85 percent of the tory if it contains not less than 85 per- number of milligrams that it is rep- cent of the equivalent number of units resented to contain. of bacitracin that it is represented to (4) Erythromycin content. Proceed as contain. directed in § 444.570b(b)(1)(i)(b) of this (5) Neomycin content. Proceed as di- chapter, except prepare the sample as rected in § 436.105 of this chapter. Its follows: Place a representative sample content of neomycin is satisfactory if (usually approximately 1.0 gram, accu- it contains not less than 85 percent of rately weighed), in a glass blending jar the number of milligrams that it is containing 99 milliliters of 0.1 M potas- represented to contain. sium phosphate buffer, pH 8.0, and 1 (b) Moisture. Proceed as directed in milliliter of polysorbate 80. Using a § 436.201. high-speed blender, blend for 2 to 3 [39 FR 18944, May 30, 1974, as amended at 40 minutes. Add 100 milliliters of 0.1 M po- FR 13497, Mar. 27, 1975] tassium phosphate buffer, pH 8.0, and blend for an additional 2 to 3 minutes. § 436.512 Procaine penicillin G- Prepare an intermediate dilution by di- novobiocin-neomycin-dihydro- luting an aliquot of the filtrate with 0.1 streptomycin in oil. M potassium phosphate buffer (pH 8.0), (a) Potency—(1) Penicillin G content. and add sufficient penicillinase to inac- Proceed as directed in § 440.180d tivate the penicillin. Then further di- (b)(1)(i)(a) of this chapter, using the
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novobiocin-resistant strain of Staphy- sample in a blending jar, add 1.0 milli- lococcus aureus (ATCC 12692),1 except liter of polysorbate 80 and a quantity prepare the sample as follows: Place of 0.1M potassium phosphate buffer, pH the equivalent of one dose of sample in 8.0, sufficient to make a total of 500 a blending jar, add 1.0 milliliter of milliliters. Blend for 5 minutes with a polysorbate 80 and a quantity of 1 per- high-speed blender. To an aliquot, add cent potassium phosphate buffer, pH sufficient penicillinase to inactivate 6.0, sufficient to make a total of 500 the penicillin, further dilute with 0.1M milliliters. Blend for 5 minutes with a potassium phosphate buffer, pH 8.0, to high-speed blender and make appro- give a final concentration of 1.0 priate dilutions, using 1 percent potas- microgram neomycin per milliliter (es- sium phosphate buffer, pH 6.0. Its con- timated), and allow to stand for 1⁄2-hour tent of penicillin G is satisfactory if it at 37° C. before filling the plates. contains not less than 85 percent of the (ii) Aseptically add to the seed agar number of units that it is represented used for this assay, at the time the to contain. bacterial suspension is added, a slurry (2) Novobiocin content. Proceed as di- of Dowex 1–X8, Cl type 200–400 mesh, to rected in § 440.180d(b)(3)(i), with the fol- make a total concentration of 1 per- lowing exceptions: cent. Prepare the slurry by adding 50 (i) Prepare the sample as follows: grams of the resin to 30 milliliters of Place the equivalent of one dose of distilled water and sterilize for 15 min- sample in a blending jar, add 1.0 milli- utes at 15 pounds pressure. Mix the liter of polysorbate 80 and a quantity slurry thoroughly before adding. Its of 0.1M potassium phosphate buffer, pH content of neomycin is satisfactory if 8.0, sufficient to make a total of 500 it contains not less than 85 percent of milliliters. Blend for 5 minutes with a the number of milligrams that it is high-speed blender. To an aliquot, add represented to contain. sufficient penicillinase to inactivate (4) Dihydrostreptomycin content. Pro- the penicillin, further dilute with 10 ceed as directed in § 436.105 except pre- percent potassium phosphate buffer, pH pare the sample by placing the equiva- 6.0 (solution 6) to give a final con- lent of one dose in a blender, add 1.0 centration of 0.5 microgram novobiocin milliliter of polysorbate 80 and a quan- per milliliter (estimated), and allow to tity of 0.1M potassium phosphate buff- 1 ° stand for ⁄2-hour at 37 C. before filling er, pH 8.0, sufficient to make a total of the plates. 500 milliliters. Blend for 5 minutes with (ii) Aseptically add to the seed agar a high-speed blender. To an aliquot, used for this assay, at the time the add sufficient penicillinase to inac- bacterial suspension is added, a slurry tivate the penicillin, further dilute of Dowex 50 WX–4, Na+ type 200–400 with 0.1M potassium phosphate buffer, mesh, sufficient to make a total con- pH 8.0, to give a final concentration of centration of 2 percent. Prepare the 1.0 microgram dihydrostreptomycin per slurry by adding 50 grams of the resin milliliter (estimated), and allow to to 30 milliliters of distilled water and stand for 1⁄2-hour at 37° C. before filling sterilize for 15 minutes at 15 pounds the plates. Its content of dihydro- pressure. Mix the slurry thoroughly be- streptomycin is satisfactory if it con- fore adding. Its content of novobiocin tains not less than 85 percent of the is satisfactory if it contains not less number of milligrams that it is rep- than 85 percent of the number of milli- resented to contain. grams that it is represented to contain. (b) Moisture. Proceed as directed in (3) Neomycin content. Proceed as di- § 436.500(c). rected in § 436.517(b)(1) of this chapter, using the Staphylococcus epidermidis [39 FR 18944, May 30, 1974, as amended at 41 (ATCC 12228) 1 procedure, except: FR 10886, Mar. 15, 1976] (i) Prepare the sample as follows: Place the equivalent of one dose of § 436.513 Chlortetracycline troches; tetracycline hydrochloride troches.
1 Available from: American Type Culture (a) Potency. If it is tetracycline hy- Collection, 12301 Parklawn Drive, Rockville, drochloride proceed as directed in MD 20852. § 446.81a(b)(1) of this chapter and if it is
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chlortetracycline hydrochloride tro- meyer flask, held in a horizontal posi- ches proceed as directed in tion. Add 500 milliliters of 0.1 N HCl § 446.10a(b)(1) of this chapter, except and shake to dissolve the contents. Im- § 446.10a(b)(1)(x), and in lieu of the di- mediately remove aliquots of this solu- rections in § 446.10a(b)(1)(iv) and tion and, using 0.1 M potassium phos- (viii)(c) of this chapter prepare the phate buffer, pH 4.5, for further dilu- sample as follows: Place 12 troches in a tions, proceed as directed in glass blending jar containing 500 milli- § 446.10a(b)(1) of this chapter if it is liters of 0.01N HCl. Using a high-speed chlortetracycline hydrochloride pow- blender, blend for 3 to 5 minutes and der or § 446.81a(b)(1) of this chapter if it then make the proper estimated dilu- is tetracycline hydrochloride powder. tions in the buffer solution. The aver- Calculate the average total amount of age potency of the troches is satisfac- antibiotic expelled from the contain- tory if they contain not less than 85 ers. The total potency is satisfactory if percent of the number of milligrams it contains not less than 85 percent of they are represented to contain. the number of milligrams of chlor- (b) Moisture. Proceed as directed in tetracycline hydrochloride or tetra- § 440.80a(b)(5)(i) of this chapter. cycline hydrochloride that it is rep- resented to contain. § 436.514 Chlortetracycline hydro- (b) Moisture. Proceed as directed in chloride powder topical; tetra- § 440.80a(b)(5)(i) of this chapter, except cycline hydrochloride powder topi- if it is packaged with inert gases pro- cal. ceed as directed in § 536.513(c) of this (a) Potency—(1) Dry powder. Using a chapter. 3.0-gram sample or the entire contents of the immediate container for each de- [39 FR 18944, May 30, 1974, as amended at 40 FR 13497, Mar. 27, 1975] termination, prepare the sample as fol- lows: Using a high-speed blender, blend § 436.515 Capsules tetracycline and a 3.0-gram sample in a glass blending oleandomycin phosphate; capsules jar containing 500 milliliters of 0.01 N tetracycline and troleandomycin; HCl (use 0.1 N HCl if it is tetracycline), capsules tetracycline hydrochloride or reconstitute in the immediate con- and oleandomycin phosphate; cap- tainer as directed in the labeling of the sules tetracycline hydrochloride drug. Transfer an appropriate aliquot and troleandomycin. of 1.0 milliliter to 5.0 milliliters to a (a) Potency—(1) Tetracycline or tetra- 100-milliliter volumetric flask and cycline hydrochloride content by turbi- make to mark with 0.01 N HCl (use 0.1 dimetric assay—(i) Test culture and N HCl if it is tetracycline). Withdraw media. Maintain the test organism an aliquot from the volumetric flask, Escherichia coli (ATCC 10536) 1 on the and if it is chlortetracycline hydro- agar described in § 440.80a(b)(1) (ii)(a) of chloride dilute to 0.06 µ g. per milli- this chapter. For use in the assay, pre- liter, using 0.1 M potassium phosphate pare a suspension of the organism buffer, pH 4.5, and proceed as directed every 2 weeks, as follows: Transfer the in § 446.10a(b)(1) of this chapter. If it is organism to a fresh agar slant and in- tetracycline hydrochloride, dilute to cubate at 37°C. overnight. Wash the 0.24 µ g. per milliliter, using 0.1 M po- growth from the slant with the aid of 2 tassium phosphate buffer, pH 4.5, and milliliters of sterile distilled water and proceed as directed in § 446.81a(b)(1) of sterile glass beads into a Roux bottle this chapter. The average potency is containing 300 milliliters of the main- satisfactory if it contains not less than tenance medium. Incubate overnight at 85 percent of the number of milligrams 37° C. and then harvest the growth with of chlortetracycline hydrochloride or 50 milliliters of sterile distilled water tetracycline hydrocloride per gram or and sterile glass beads. Standardize per immediate container that it is rep- this suspension by determining the di- resented to contain. lution that will permit 40-percent light (2) Powder packaged with inert gases. Spray, as directed in the labeling, the 1 Available from: American Type Culture entire contents of each container to be Collection, 12301 Parklawn Drive, Rockville, tested into a separate 2-liter Erlen- MD 20852.
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transmission in a photoelectric col- through the points, either by inspec- orimeter using a 650-millimicron filter tion or by means of the following equa- and an 18-millimeter diameter test tions: tube as an absorption cell. Prepare the daily inoculum by adding 10 milliliters L=(3a+2b+c¥e)/5, of that dilution to each liter of nutri- H=(3e+2d+c¥a)/5, ent broth, prepared as directed in where: § 440.80a (b)(1)(ii)(c) of this chapter, L=absorbance value for the lowest con- needed for the test. centration of the standard curve. H=absorbance value for the highest con- (ii) Working standard and solutions. centration of the standard curve. Dissolve an appropriate amount of the a, b, c, d, e=average absorbance values for working standard in sufficient 0.1 N each concentration of the standard HCl to give a concentration of 1,000 curve. micrograms per milliliter. This stock Plot the values obtained for L and H solution may be kept in the refrig- and connect the points with a straight erator for 1 week. Make daily dilutions line. Average the absorbance values for of the stock solution with 0.1 M potas- the sample and read the tetracycline or sium phosphate buffer (pH 4.5) to ob- tetracycline hydrochloride concentra- tain concentrations of 0.146, 0.187, 0.240, tion from the standard curve. Multiply 0.308, and 0.395 micrograms per milli- the concentration by appropriate dilu- liter. Add 1.0 milliliter of each such tion factors to obtain the tetracycline concentration to each of three 16 milli- or tetracycline hydrochloride content meters x 125 millimeters test tubes. of the sample. Its potency is satisfac- (iii) Preparation of sample. Dissolve tory if it contains the equivalent of not the contents of a representative num- less than 85 percent of the number of ber of capsules in sufficient 0.1 N HCl milligrams of tetracycline hydro- to give a stock solution of convenient chloride that it is represented to con- concentration. Further dilute the tain. stock solution with 0.1 M potassium (2) Oleandomycin content. (i) If phosphate buffer (pH 4.5) to obtain a oleandomycin phosphate is used, pro- final concentration of 0.24 microgram ceed as directed in paragraph (c)(1) of per milliliter (estimated). Add 1.0-mil- this section, except prepare the sample liliter of this dilution to each of three as follows: Dissolve the contents of a 16 millimeters x 125 millimeters test representative number of capsules in tubes. sufficient 0.1 M potassium phosphate (iv) Procedure. To each of the 16 milli- buffer (pH 8.0) to give a stock solution meters x 125 millimeters test tubes pre- of convenient concentration. Further pared in paragraph (a)(1)(ii) and (iii) of dilute with 0.1 M potassium phosphate this section, add 9.0 milliliters of the buffer (pH 8.0) to obtain a final con- inoculated nutrient broth described in centration of 5.0 µ g. of oleandomycin paragraph (a)(1)(i) of this section and activity per milliliter (estimated). place immediately in a 37° C. water (ii) If troleandomycin is used, pro- bath for 3 to 4 hours. After incubation, ceed as follows: Dissolve the contents add 0.5 milliliter of a 12-percent form- of a representative number of capsules aldehyde solution to each tube and in chloroform to give a stock solution read the absorbance values in a suit- of 1.0 milligram of oleandomycin activ- able photoelectric colorimeter using a ity per milliliter. Transfer 30 milli- wavelength of 530 millimicrons. Set the liters of the chloroform solution to a instrument at zero absorbance with glass-stoppered test tube (200 millime- clear uninoculated broth prepared as ters x 22 millimeters) and add 20 milli- described in § 440.80a(b)(1)(ii)(c) of this liters of 1 N sodium hydroxide. Shake chapter. for 1 minute and centrifuge briefly to (v) Estimation of potency. Plot the av- aid in the separation of the layers. erage values for each concentration of With the aid of a syringe and needle, the standard on arithmetic graph paper remove and discard the aqueous layer. with absorbance values on the ordinate Repeat the washing procedure with two and tetracycline or tetracycline hydro- more 20-milliter portions of 1 N sodium chloride concentrations on the ab- hydroxide solution. Filter the chloro- scissa. Construct the best straightline form layer through a pledget of cotton.
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Dilute an aliquot of this solution with iological saline solution onto a large chloroform to give a solution contain- agar surface such as that provided by a ing approximately 25 µ g. of Roux bottle containing 300 milliliters oleandomycin per milliliter. Transfer a of the agar described in paragraph 5.0 milliliter aliquot to a 40 milliliter (c)(1)(ii)(a) of this section. Spread the glass-stoppered centrifuge tube, dilute suspension of organisms over the entire to 20 milliliters, with chloroform, and agar surface with the aid of sterile determine the oleandomycin content glass beads. Incubate for 4 hours at 32° as directed in paragraph (d)(1)(i) of this C. and then wash the resulting growth section. from the agar surface with about 30 Its content of oleandomycin is satisfac- milliliters of sterile physiological sa- tory if it contains not less than 85 per- line solution. Standardize the suspen- cent of the number of milligrams that sion by determining the dilution that it is represented to contain. will give 80-percent light transmission, (b) Moisture. Proceed as directed in using a suitable photoelectric colorim- § 440.80a(b)(5)(1) of this chapter. eter with a 650-millimicron filter and (c) Oleandomycin phosphate used in an 18-millimeter-diameter test tube as making the capsules—(1) Potency—(i) an absorption cell. Run test plates to Cylinders (cups). Used cylinders de- determine the quantity of the diluted scribed in § 440.80a(b)(1)(i) of this chap- suspension (usually 1.5 milliliters) that ter. should be added to each 100 milliliters (ii) Culture media. (a) Use the nutri- of agar to give clear, sharp zones of in- ent agar described in § 440.80a hibition of appropriate size. (b)(1)(ii)(a) of this chapter for the seed (vi) Preparation of plates. Add 21 milli- layer and base layer, except that its pH liters of the agar prepared as described after sterilization is 7.8 to 8.0. in paragraph (c)(1)(ii)(a) of this section (b) Use the nutrient agar described in to each Petri dish (20 millimeters × 100 § 440.80a(b)(1)(ii)(a) of this chapter for millimeters). Distribute the agar even- maintaining the test organism. ly in the plates and allow it to harden. (iii) Working standard. Dissolve a Use the plates the same day they are suitable weighed quantity (usually 25 prepared. Melt a sufficient amount of milligrams or less) of the working the agar described in paragraph standard (obtained from the Food and (c)(1)(ii)(a) of this section, cool to 48° Drug Administration) in 2 milliliters of C., add the proper amount of the test ethanol, then add sufficient 0.1 M po- organism as described in paragraph tassium phosphate buffer, pH 8.0, to (c)(1)(v) of this section and mix thor- give a concentration of 1,000 oughly. Add 4 milliliters of this inocu- micrograms of oleandomycin base per lated agar to each Petri dish. Distrib- milliliter. This stock solution may be ute the agar evenly in the plates, cover kept in the refrigerator for 3 days. with porcelain covers glazed on the (iv) Preparation of sample. Dissolve outside, and allow to harden. After the the sample in sufficient 0.1 M potas- agar has hardened, place 6 cylinders on sium phosphate buffer (pH 8.0) to give a the agar surface so that they are at ap- convenient stock solution. Further di- proximately 60° intervals on a 2.8-centi- lute in 0.1 M potassium phosphate buff- meter radius. er (pH 8.0) to give a final concentration (vii) Standard curve. Prepare the daily of 5.0 micrograms per milliliter (esti- standard curve by further diluting the mated). 1,000 micrograms per milliliter stock (v) Preparation of test organism. The solution in 0.1 M potassium phosphate test organism is Staphylococcus buffer (pH 8.0) to obtain concentrations epidermidis (ATCC 12228) 1 which is of 3.2, 4.0, 5.0, 6.25 and 7.80 micrograms maintained on slants or agar described per milliliter. Use 3 plates for the de- under paragraph (c)(1)(ii)(a) of this sec- termination of each point on the curve, tion. Wash the organism from the agar except the 5.0 micrograms per milli- slant with 3 milliliters of sterile phys- liter concentration, a total of 12 plates. On each of 3 plates fill 3 cylinders with 1 Available from: American Type Culture the 5.0 micrograms per milliliter stand- Collection, 12301 Parklawn Drive, Rockville, ard, and the other 3 cylinders with the MD 20852. concentration under test. Thus, there
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will be 36 five-microgram determina- with the standard 5.0 micrograms per tions and 9 determinations for each of milliliter solution and 3 cylinders with the other points on the curve. After in- the 5.0 micrograms per milliliter (esti- cubation, read the diameters of the cir- mated) sample, alternating standard cles of inhibition in the plates. Average and sample. Incubate all plates, includ- the readings of the 5.0 micrograms per ing those containing the standard milliliter concentration and the read- curve, at 32° C.–35° C. overnight, and ings of the point tested for each set of measure the diameter of each circle of 3 plates and average also all 36 readings inhibition. To estimate the potency of of the 5.0 micrograms per milliliter the sample, average the zone readings concentration. The average of the 36 of the standard and the zone readings readings of the 5.0 micrograms per mil- of the sample on the 3 plates used. If liliter concentration is the correction the sample gives a larger zone size than point for the curve. Correct the aver- the average of the standard, add the age value obtained for each point to difference between them to the 5.0 the figure it would be if the 5.0 micrograms per milliliter zone on the micrograms per milliliter reading for standard curve. If the average sample that set of 3 plates were the same as the correction point. Thus, if in cor- value is lower than the standard value, recting the 4.0-microgram concentra- subtract the difference between them tion, the average of the 36 readings of from the 5.0 micrograms per milliliter the 5.0-microgram concentration were value on the curve. From the standard 20.0 millimeters, and the average of the curve, read the potencies corresponding 5.0-microgram concentration of this set to these corrected values of zone sizes. of 3 plates were 19.8 millimeters, the (2) Toxicity. Proceed as directed in correction would be +0.2 millimeter. If § 440.80a(b)(4) of this chapter, except use the average reading of the 4.0- physiological salt solution as the dilu- microgram concentration of these ent, and inject 0.5 milliliter of a solu- same 3 plates were 19.0 millimeters, the tion containing 8 milligrams per milli- corrected value would be 19.2 millime- liter. ters. Plot these corrected values, in- (3) Moisture. Proceed as directed in cluding the average of the 5.0 § 440.80(b)(5)(i) of this chapter. micrograms per milliliter concentra- (4) pH. Proceed as directed in tion, on 2-cycle semilog paper, using § 440.80a(b)(5)(ii) of this chapter, using a the concentration in micrograms per solution containing 100 milligrams per milliliter as the ordinate (the loga- milliliter. rithmic scale) and the diameter of the (5) Crystallinity. Proceed as directed zone of inhibition as the abscissa. Draw in § 440.80a(b)(5)(iii) of this chapter. the standard curve through these (d) Troleandomycin used in making the points, either by inspection or by capsules—(1) Potency—(i) Chemical meth- means of the following equations: od—(a) Reagents and equipment. (1) L=(3a+2b+c¥e)/5, Methyl orange reagent: Shake 0.5 M H=(3e+2d+c¥a)/5, boric acid solution for about 12 hours (to insure saturation) with an excess of where: L=corrected zone diameter for the lowest methyl orange indicators. An alter- concentration of the standard curve, native method is to heat the mixture H=corrected zone diameter for the highest to about 50° C. and shake for about an concentration of the standard curve, hour. Then allow to cool. Filter the c=average zone diameter for 36 readings of saturated dye solution and wash three the 5.0 micrograms per milliliter stand- times with chloroform. Store the dye ard. a, b, d, e=corrected average values for the solution over chloroform. 3.2, 4.0, 6.25, and 7.81 micrograms per (2) Acid-alcohol solution: Add 2 milli- milliliter standard solutions, respec- liters of concentrated sulfuric acid to tively. 98 milliliters of absolute methyl alco- Plot the values obtained for L and H hol. and connect with a straight line. (3) Glycerin: Reagent grade. (viii) Assay. Use 3 plates for each (4) Centrifuge tubes: 40 milliliters, sample. Fill 3 cylinders on each plate glass-stoppered.
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(b) Procedure. Prepare a chloroform milligrams or less) of the solution containing 50.0 milligrams ac- troleandomycin working standard (ob- tivity of standard oleandomycin base tained from the Food and Drug Admin- in 200 milliliters of solution. Transfer istration) in sufficient 80 percent iso- 10.0 milliliters of the solution to a 100- propyl alcohol-water solution to give a milliliter volumetric flask and dilute concentration of 1,000 micrograms per to volume with chloroform. Transfer milliliter (estimated). Use the solution 2.0, 4.0, 6.0, and 8.0 milliliters of this so- the day that it is prepared. lution to glass-stoppered centrifuge (c) In lieu of the directions in para- tubes (40-milliliter size) and dilute to a graph (c)(1)(iv) of this section, dissolve total volume of 20.0 milliliters each the sample in sufficient 80 percent iso- with chloroform. To the 20.0 milliliters propyl alcohol-water solution to give a of the solution present in each (40-mil- convenient stock solution. Further di- liliter size) centrifuge tube add 0.2 mil- lute in 0.2 M potassium phosphate buff- liliter of glacial acetic acid, 0.20 milli- er, pH 10.5 (35 grams of dipotassium liter of glycerin, and 0.40 milliliter of phosphate plus 2 milliliters of 10 methyl orange reagent. Shake for 5 N minutes and centrifuge for 3 minutes. NaOH, q.s. to 1 liter), to give a final Immediately transfer to another tube a concentration of 15 micrograms per 10.0-milliliter aliquot from the chloro- milliliter (estimated). form (lower) layer. Care must be exer- (d) In lieu of the directions in para- cised to see that no portion of the dye- graph (c)(1)(vi) of this section, use the glycerin-phase is included with the agar described in paragraph (d)(1)(ii)(a) chloroform aliquot. Add 1.0 milliliter of this section for both layers. Use the of acid-alcohol solution to this chloro- plates as soon after seeding as is prac- form aliquot, mix well, and read the tical. If they are not to be used shortly absorbancy at 535 mµ, using a 1-centi- after seeding, then they should be re- meter cell and a suitable photometer frigerated until ready for use. and using chloroform, similarly treat- (e) In lieu of the directions for pre- ed, as a blank. Prepare a standard paring the standard curve in paragraph curve, plotting the absorbance values (c)(1)(vii) of this section, prepare the of the standard solutions against the standard curve by diluting the stock concentration expressed in micrograms solution in 0.2 M potassium phosphate per aliquot. Accurately weigh the sam- buffer, pH 10.5, to give concentrations ple to be tested to give 50 milligrams of 9.6, 12.0, 15.0, 18.8, and 23.4 (estimated) of oleandomycin activity, micrograms per milliliter. The 15.0 dissolve in chloroform, and make to 200 micrograms per milliliter is the ref- milliliters with chloroform. Transfer erence concentration. 10.0 milliliters to a 100-milliliter volu- (f) In lieu of the directions in para- metric flask and make to volume with graph (c)(1)(viii) of this section, incu- chloroform. Transfer 5.0 milliliters to a bate the plates at 37° C. overnight. The glass-stoppered centrifuge tube and concentration of the sample and stand- proceed as above. Determine the po- tency of the sample from the standard ard being tested is 15.0 micrograms per curve. milliliter. (ii) Microbiological assay. Proceed as (2) Toxicity. Administer orally, by directed in paragraph (c)(1) of this sec- means of a cannula or other suitable tion, except: device, to each of five mice within the (a) In lieu of the directions in para- weight range of 18 grams to 25 grams, graph (c)(1)(ii)(a) of this section, use 0.5 milliliter of a suspension containing the nutrient agar described in 200 milligrams per milliliter in normal § 440.80a(b)(1)(ii)(a) of this chapter for saline solution. If no animal dies with- the seed and base layers, except add 2.0 in 48 hours, the sample is nontoxic. If milliliters of polysorbate 80 to each 100 one or more animals die within 48 milliliters of agar. Its pH after steri- hours, repeat the test, using for each lization is 7.8 to 8.0. test five or more previously unused (b) In lieu of the directions in para- mice weighing 20 grams (±0.5 gram) graph (c)(1)(iii) of this section, dissolve each; if the total deaths within 48 a suitable weighed quantity (usually 25 hours is no greater than 10 percent of
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the total number of animals tested, in- (6) Acetyl determination—(i) Apparatus cluding the original test, the sample is and reagents. (a) One three-necked nontoxic. Pyrex flask of approximately 45 milli- (3) Moisture. Proceed as directed in liters capacity, pear-shaped with T- § 440.80a(b)(5)(i) of this chapter. joints, agar inlet tube, glass-stoppered (4) pH. Proceed as directed in funnel, glass condenser, and bubble § 440.80a(b)(5)(ii) of this chapter, using a counter. saturated aqueous-ethanol (1:1) solu- (b) 50-milliliter Pyrex Erlenmeyer tion prepared by adding 100 milligrams flask. per milliliter. (5) Paper chromatograph method—(i) (c) 10-milliliter burette, calibrated in Apparatus and reagents—(a) 0.02 milliliter. Chromatographic chamber (cylinder (d) Anhydrous methanol, reagent glass-stoppered museum jar 11.5 inches grade. × 3.5 inches). (e) 2 N sodium hydroxide solution. (b) Chromatographic paper (8 inches × (f) Sulfuric acid solution prepared by 8 inches Whatman No. 1). adding 100 milliliters of concentrated (c) 0.1 N hydrochloric acid. H2SO4 to 200 milliliters of water. (d) Resolving solvent: Butyl acetate, (g) 1 N barium chloride solution. benzene, nitromethane, pyridine (5:5:5:1 (h) Phenolphthalein solution (1 per- by volume). cent in ethanol). (e) Spray reagent: 15 grams antimony trichloride per 100 milliliters of chloro- (i) Water-pumped nitrogen. form. (j) NaOH solution, 0.015 N. (ii) Procedure. Dissolve the sample in (ii) Procedure. Weight accurately (to chloroform to give a solution contain- 0.01 milligram) approximately 30 milli- ing 10 milligrams to 20 milligrams per grams of the sample into the three- milliliter. Prepare a sheet of necked acetyl flask. Add 2.0 milliliters chromatographic paper by drawing a of methanol to dissolve the sample, line of origin parallel to and 1 inch then add slowly with gentle swirling, from the edge of the paper. Wet the 1.0 milliliter of NaOH solution. Connect paper thoroughly with the 0.1 N hydro- the gas inlet tube with bubble counter chloric acid and blot it firmly between attached, and adjust nitrogen flow to sheets of absorbent paper. Starting 2 about two bubbles a second. Put glass- inches in from the edges and at 1-inch stoppered funnel in centerneck of ace- intervals, apply 3 microliters to 5 tyl flask and put about 5 milliliters of microliters of the sample solutions to H O in the funnel. Add a boiling chip to the starting line. Allow a few minutes 2 the solution and attach condenser in for the paper to dry partially. While the paper is still damp, form a cylinder the refluxing position with water cool- by bringing the outer edges together, ing. Adjust burner flame under acetyl allowing about 1-inch overlap, and se- flask to reflux solution gently. Reflux cure with a paper clip. Stand the paper for 30 minutes. Cool assembly slightly in the chromatographic chamber, then rinse down condenser (still in which has been filled to a depth of 1⁄2- reflux position) with a few milliliters inch with the resolving solvent. After of H2O. Reassemble condenser to the the solvent front rises to a height of 4 distillation position and add water inches to 5 inches above the origin, re- through the funnel to make a total of move the paper from the tank and hang approximately 5 milliliters of H2O it up to air dry. Spray the dried paper added to acetyl flask. Adjust burner with the antimony trichloride reagent. flame so that about 5 milliliters of H2O Hang the paper in a 100° C. oven for 3 and methanol is distilled over in ap- minutes. A purple spot becomes visible proximately 10 minutes. Discard this for trioleandomycin at an Rf value of distillate. Cool acetyl flask slightly. about 0.85. The approximate Rf values Acidify solution in flask by adding 1 for diacetyloleandomycin, milliliter of the sulfuric acid solution monoacetyloleandomycin, and through the funnel. Adjust burner oleandomycin are, respectively, 0.72, flame and distill over approximately 20 0.27, and 0.13.
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milliliters of distillate into an Erlen- § 436.517(b)(1)(iii) is used to prepare the meyer flask in about 20 minutes, add- standard curve, by further diluting ing water through the funnel as nec- with pH 8.0 buffer to final concentra- essary. It is important to keep the liq- tions of 0.64, 0.80, 1.0, 1.25, and 1.56 µ g. uid volume in the acetyl flask around 2 per milliliter. The 1.0 µ g per milliliter milliliters to 3 milliliters in order to solution is the reference concentration. obtain a quantitative recovery of the In lieu of the method described in this acetic acid. Collect a second fraction of subparagraph, the neomycin content distillate, about 10 milliliters in vol- may also be determined as follows. ume. As the second fraction is distill- Using the aqueous solution described, ing, process the first fraction. Heat the prepare the sample and proceed as di- first reaction and boil gently about 20 rected in § 436.517(b)(1), except use seconds. Add a few drops of BaCL2 solu- Staphylococcus aureus (American Type tion to check if any sulfate was dis- Culture Collection 12715) 1 as the test tilled over. If the sulfate is present, organism, which is grown and main- discard and repeat the whole deter- tained on agar containing 100 µ g. of mination. If the sulfate is absent im- tetracycline hydrochloride per milli- mediately titrate the solution with the liter of agar. Its neomycin content is 0.015 N NaOH solution to a faint pink satisfactory if it contains not less than endpoint, using one drop of phenol- 85 percent of the number of milligrams phthalein solution as the indicator. Re- that it is represented to contain. peat the above procedure with the sec- (2) Tetracycline hydrochloride-neomy- ond fraction. If the second fraction re- cin sulfate powder—(i) Tetracycline hy- quires less than 0.10 milliliter of the drochloride content. Prepare the sample 0.015 N NaOH solution and all the ace- as directed in § 436.514(a)(2). Use an ap- tic acid has been distilled over, the de- propriate aliquot and proceed as di- termination is completed. If greater rected in § 446.81a(b)(1) of this chapter. than this, collect a third fraction of ap- Its tetracycline hydrochloride content proximately 10 milliliters and titrate is satisfactory if it contains not less this as before. Total volumes of NaOH than 85 percent of the number of milli- used and calculate results as follows: grams that it is represented to contain. (Milliliters of NaOH × N NaOH × 0.043 × 100)/ (ii) Neomycin content. Use an appro- Weight sample in grams=Percent acetyl. priate aliquot of the solution prepared (7) Crystallinity. Proceed as directed in paragraph (a)(2)(i) of this section in § 440.80a(b)(5)(iii) of this chapter. and proceed as directed in paragraph (a)(1)(ii) of this section. Its neomycin § 436.516 Tetracycline-neomycin com- content is satisfactory if it contains plex powder topical; tetracycline not less than 85 percent of the number hydrochloride-neomycin sulfate of milligrams that it is represented to powder topical. contain. (a) Potency—(1) Tetracycline-neomycin (b) Sterility. Thoroughly cleanse with complex powder—(i) Tetracycline content. a suitable disinfectant the value (do Proceed as directed in § 436.514(a)(2), ex- not flame) of each container to be test- cept use water in lieu of 0.1 N HCl for ed. Into each of two empty, sterile Er- dissolving the sample. Its tetracycline lenmeyer flasks stoppered with a cot- content is satisfactory if it contains ton plug, spray quantitites sufficient not less than 85 percent of the equiva- to yield a residue of approximately the lent number of milligrams of tetra- equivalent of 50 milligrams from 10 cycline hydrochloride that it is rep- separate cans by removing the plug resented to contain. temporarily and using aseptic tech- (ii) Neomycin content. Using 0.1 M po- nique while spraying; allow propellant tassium phosphate buffer, pH 8.0, dilute to evaporate, add 250 milliliters to 500 an appropriate aliquot of the aqueous milliliters of diluting fluid B in lieu of solution, prepared as directed in para- diluting fluid A, and swirl the flasks to graph (a)(1) of this section, to a final dissolve the contents. Then proceed as concentration of 1 µ g. per milliliter (estimated), and proceed as directed in 1 Available from: American Type Culture § 436.515(c)(1), except that the neomycin Collection, 12301 Parklawn Drive, Rockville, standard stock solution described MD 20852.
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directed in § 436.20 of this chapter using (2) Moisture. Proceed as directed in the method described in paragraph § 440.80a(b)(5)(i) of this chapter. (e)(1) of that section. (3) Disintegration time. Proceed as di- (c) Moisture. Proceed as directed in rected in § 440.180a(b)(3). § 536.513(c) of this chapter. (b) Neomycin used in making the tab- (d) Tetracycline-neomycin complex used lets—(1) Potency—(i) Cylinders (cups). in making the drug—(1) Potency—(i) Tet- Use cylinders described under racycline content. Dissolve the sample § 440.80a(b)(1)(i) of this chapter. to be tested in sufficient water to give (ii) Culture medium. Use the medium a convenient stock solution. Using an described in § 440.80a(b)(1) (ii)(a) of this appropriate aliquot, proceed as di- chapter for both the base and seed lay- rected in § 446.81a(b)(1). ers, except its pH after sterilization is (ii) Neomycin content. Using an ali- 7.8 to 8.0. quot of the stock solution prepared as (iii) Working standard. Dry the work- directed in paragraph (d)(1)(i) of this ing standard (obtained from the U.S.P. paragraph, proceed as directed in para- Reference Standards Committee, 46 graph (a)(2) of this section, except the Park Avenue, New York 16, N.Y.) for 3 last sentence of that subparagraph. hours at 60° C. and a pressure of 5 milli- (2) Toxicity. Proceed as directed in meters or less and weigh out a suffi- § 440.80a(b)(4) of this chapter using 0.5 cient quantity to make a convenient milliliter of a solution prepared by di- stock solution by diluting with a 0.1 M luting the sample with physiological potassium phosphate buffer, pH 7.8 to sodium chloride solution to contain 200 8.0. The stock solution, when stored at µ g. of neomycin per milliliter (esti- a temperature of approximately 15° C., mated). or less, may be used for a period not ex- (3) pH. Using a 1-percent aqueous so- ceeding 1 month. lution, proceed as directed in (iv) Standard curve. Using the stock § 440.80a(b)(5)(ii) of this chapter. solution, prepare a daily standard curve as directed in § 444.70a(b)(1)(iv) of [39 FR 18944, May 30, 1974, as amended at 40 FR 13497, Mar. 27, 1975] this chapter, using solutions of the ne- omycin working standard in 0.1M po- § 436.517 Bacitracin-neomycin tablets; tassium phosphate buffer, pH 8.0, in zinc bacitracin-neomycin tablets; concentrations of 6.4, 8.0, 10.0, 12.5, and bacitracin methylene disalicylate- 15.6 micrograms per milliliter if the neomycin tablets. test organism Staphylococcus aureus (a) Tablets—(1) Potency—(i) Bacitracin, (ATCC 6538P), 1 or in concentrations of zinc bacitracin, or bacitracin methylene 0.64, 0.80, 1.0, 1.25, and 1.56 micrograms disalicylate content. Proceed as directed per milliliter if the test organism is in § 448.110a(b)(1). Its content of baci- Staphylococcus epidermidis (ATCC tracin, zinc bacitracin, or bacitracin 12228).1 The 10.0 micrograms per milli- methylene disalicylate is satisfactory liter and the 1.0 microgram per milli- if it contains not less than 85 percent liter concentrations are used as the ref- of the number of units per tablet that erence points. it is represented to contain. (v) Preparation of test organism. The (ii) Neomycin content. Place 5 tablets test organism is Staphylococcus aureus in a blending jar and add thereto 200 (ATCC 6538P), 1 which is maintained on milliliters of a 500-milliliter quantity agar described in § 440.80a(b)(1)(ii)(a) of of 0.10-percent phosphate buffer pH 8.0. this chapter. From a stock slant inocu- After blending for 1 minute with a late a Roux bottle containing this high-speed blender, add the remainder same agar and incubate for 24 hours at of the buffer. Blend again for 1 minute 32° C.–35° C. Wash the resulting growth and make the proper estimated dilu- from the agar surface with about 50 tions in the buffer and proceed as di- milliliters of sterile sodium chloride rected in paragraph (b)(1) of this sec- solution. Standardize this suspension tion. Its content of neomycin is satis- by determining the dilution that will factory if it contains not less than 85 permit 80 percent light transmission percent of the number of milligrams of through a filter at 6500 Angstrom units activity that it is represented to con- tain. 1 See footnote 1 to § 436.516.
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in a photoelectric colorimeter. The agent such as sulfuric acid or silica gel. suspension may be used for 2 weeks if Replace the stopper and place the it is stored under refrigera- weighing bottle in a desiccator over a tion.Staphylococcus epidermidis (ATCC desiccating agent such as phosphorous 12228), 1 which is maintained on agar as pentoxide or silica gel, allow to cool to described in § 440.80a(b)(1)(ii)(a) of this room temperature, and reweigh. Cal- chapter, may also be used as the test culate the percent loss. organism. From a stock slant, inocu- (4) pH. Proceed as directed in late a Roux bottle containing this me- § 440.80a(b)(5)(ii) of this chapter, using a dium and incubate for 24 hours at 32° solution containing 33 milligrams per C.–35° C. Wash the resulting growth milliliter. from the agar surface, using approxi- mately 30 milliliters of sterile sodium § 436.542 Acid resistance/dissolution chloride solution. Standardize the sus- test for enteric-coated erythro- pension by determining the dilution mycin pellets. that will permit 80 percent light trans- (a) Equipment. Use Apparatus 1 as de- mission through a filter of 6500 Ang- scribed in the United States Pharma- strom units in a photoelectric colorim- copeia XX dissolution test. eter. The suspension may be stored for (b) Immersion fluids. All immersion 2 weeks under refrigeration. fluids may be degassed by heating im- (vi) Preparation of plates. Using the mediately prior to use. agar described in subdivision (ii) of this (1) Acid resistance medium. Use 0.06N subparagraph and approximately a 0.5 hydrochloric acid, pH 1.2. percent inoculum of the suspension de- (2) Dissolution medium. Dissolve 6.8 scribed in paragraph (b)(1)(v) of this grams of monobasic potassium phos- section, prepare the plates as directed phate in 250 milliliters of water. Add in § 440.80a(b)(1)(v) of this chapter. 109 milliliters of 0.2N sodium hydroxide (vii) Assay. Dissolve volumetrically and 400 milliliters of water and adjust in 0.1 M potassium phosphate buffer, the resulting solution with 0.2N sodium pH 7.8 to 8.0, the sample to be tested to hydroxide to a pH of 6.8±0.1. Dilute to 1 make a convenient stock solution. Fur- liter. ther dilute volumetrically this solution (c) Procedure. Warm the immersion with 0.1 M potassium phosphate buffer, fluids to a temperature of 37° ±5.0° C. pH 7.8 to 8.0, to a final concentration of Place the contents of one capsule into 10.0 micrograms (estimated) per milli- the basket. Lower the basket into 900 liter, if the test organism is milliliters of acid resistance medium Staphylococcusaureus or 1.0 microgram contained in the beaker. Ensure that per milliliter (estimated) if the test or- all air is displaced from the immersed ganism is Staphylococcus epidermidis. basket and that the pellets remain in (2) Toxicity. Proceed as directed in the basket. Rotate the basket at the § 440.80a(b)(4) of this chapter, using 0.5 speed of 50 revolutions per minute for milliliter of a solution prepared by di- an accurately timed period of 1 hour. luting the sample to approximately 200 Remove the basket from the fluid and micrograms per milliliter with physio- immediately lower the basket into 900 logical salt solution. milliliters of dissolution medium con- (3) Moisture. In an atmosphere of tained in the beaker. Again ensure that about 10 percent relative humidity, all air is displaced from the immersed transfer about 100 milligrams of the basket and that the pellets remain in finely powdered sample to a tared the basket. Rotate the basket at 50 rev- weighing bottle equipped with ground- olutions per minute for an accurately glass top and stopper. Weigh the bottle timed dissolution period of 45 minutes. and place it in a vacuum oven, tilting Withdraw a 25-milliliter sample of the the stopper on its side so that there is dissolution medium from a point mid- no closure during the drying period. way between the stirring shaft and the Dry at a temperature of 60° C. and a wall of the vessel and approximately pressure of 5 millimeters of mercury or midway in depth. Filter the sample less for 3 hours. At the end of the dry- through a Whatman 541 filter paper or ing period fill the vacuum oven with equivalent, discarding the first 2 milli- air dried by passing it through a drying liters. Assay for erythromycin using
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the filtrate as the test solution as di- proximately 345 nanometers. Deter- rected in § 436.105. Repeat the test on mine the exact position of the absorp- five additional capsules. tion peak for the particular instrument (d) Evaluation. Use the interpretation used. described in the United States Pharma- (4) Calculations. Determine the total copeia XX dissolution test. amount of doxycycline dissolved as fol- [46 FR 16678, Mar. 13, 1981, as amended at 50 lows: FR 47213, Nov. 15, 1985; 52 FR 35912, Sept. 24, 1987; 54 FR 41824, Oct. 12, 1989] Milligrams of A× c × d × 900 * doxycycline = u § 436.543 Acid resistance test for pel- dissolved As let-filled doxycycline hyclate cap- where: sules. Au=Absorbance of sample; (a) Equipment. Use Apparatus 1 as de- As=Absorbance of standard; scribed in the United States Pharma- c=Concentration of working standard in copeia XXI dissolution test. milligrams; and (b) Acid resistance medium. Use 0.06N d=Dilution factor of sample withdrawn hydrochloric acid, pH 1.2. May be from beaker. degassed by heating immediately prior *If more than 15 milliliters of dissolution medium is removed, correct for the vol- to use. ume removed. (c) Procedure. Warm the acid resist- ance medium to a temperature of 37±2.0 (e) Evaluation. The pellet-filled cap- ° C. Place the contents of one pellet- sule passes the test if no more than 50 filled capsule into the basket. Lower percent of the drug is dissolved at 20 the basket into a beaker containing 900 minutes. If one pellet-filled capsule milliliters of acid resistance medium. fails to meet this requirement, repeat Ensure that all air is displaced from the test on six additional pellet-filled the immersed basket and that the con- capsules. No more than 2 pellet-filled tents of the pellet-filled capsule remain capsules in 12 may exceed 50 percent of in the basket. the drug dissolved at 20 minutes. Rotate the basket at the speed of 50 [50 FR 41679, Oct. 15, 1985; 50 FR 45603, Nov. 1, revolutions per minute for an accu- 1985] rately timed period of 20 minutes. Withdraw a 5-milliliter sample of the § 436.544 Dissolution test for pellet- acid resistance medium from a point filled doxycycline hyclate capsules. midway between the stirring shaft and (a) Equipment. Use Apparatus 1 as de- the wall of the vessel and approxi- scribed in the United States Pharma- mately midway in depth (this is the copeia XXI dissolution test. sample solution). Assay the sample so- (b) Dissolution medium. Prepare the lution for doxycycline as described in dissolution medium as follows: Dis- paragraph (d) of this section. Repeat solve 10.21 grams of potassium the test on five additional pellet-filled biphthalate and 1.4 grams of sodium capsules. hydroxide in approximately 950 milli- (d) Doxycycline content—(1) Prepara- liters of distilled water and adjust the tion of working standard solution. Dis- pH to 5.5 using 1M sodium hydroxide solve an accurately weighed portion of solution. Dilute with distilled water to doxycycline hyclate working standard 1,000 milliliters. with 0.1M hydrochloric acid to obtain a (c) Procedure. Proceed as directed in concentration of 0.01 milligram per the United States Pharmacopeia XXI milliliter. dissolution test. Ensure that all air is (2) Preparation of sample solution. Di- displaced from the immersed basket lute the 5-milliliter sample portion to and that the contents of the pellet- 25 milliliters with 0.1M hydrochloric filled capsule remain in the basket. Ro- acid. tate the basket at the speed of 50 revo- (3) Procedure. Using a suitable spec- lutions per minute for an accurately trophotometer and 0.1M hydrochloric timed period of 30 minutes. Withdraw a acid as the blank, determine the ab- 5-milliliter sample of the dissolution sorbance of each standard and sample medium from a point midway between solution at the absorbance peak at ap- the stirring shaft and the wall of the
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vessel and approximately midway in medium. Rotate the paddle at the depth (this is the sample solution). speed of 50 revolutions per minute for Assay the sample solution for an accurately timed period of 1 hour. doxycycline as described in paragraph Withdraw a 50-milliliter sample of the (d) of this section. Repeat the test on dissolution medium from a point mid- five additional pellet-filled capsules. way between the stirring shaft and the (d) Doxycycline content—(1) Prepara- wall of the vessel and approximately tion of working standard solution. Dis- midway in depth. Filter the sample solve an accurately weighed portion of through a Whatman No. 1 filter paper doxycycline hyclate working standard or equivalent, discarding the first 5.0 with 0.1M hydrochloric acid to obtain a milliliters. Assay for dissolved erythro- concentration of 0.01 milligram per mycin as directed in paragraph (d) of milliliter. this section using the filtrate as the (2) Preparation of sample solution. Di- sample solution. Repeat the test on lute the 5-milliliter sample portion to five additional tablets. 25 milliliters with 0.1M hydrochloric (d) Arsenomolybdate colorimetric assay acid. for dissolved erythromycin—(1) Appara- (3) Procedure. Using a suitable spec- tus. Automatic analyzer consisting of trophotometer and 0.1M hydrochloric (i) a liquid sampler, (ii) a proportioning acid as the blank, determine the ab- pump, (iii) suitable spectrophotometers sorbance of each standard and sample equipped with matched flow cells and solution at the absorbance peak at ap- analysis capability at 660 nanometers, proximately 345 nanometers. Deter- (iv) a means of recording mine the exact position of the absorp- spectrophotometric readings, and (v) a tion peak for the particular instrument manifold consisting of the components used. illustrated in the diagram in paragraph (4) Calculations. Determine the total (d)(4) of this section. amount of doxycycline dissolved as fol- (2) Reagents—(i) Arsenomolybdate solu- lows: tions—(a) Stock solution. Dissolve 100 grams of ammonium molybdate in ap- Milligrams of × × × proximately 1,700 milliliters of water Au c d 900 * doxycycline = contained in a 2-liter volumetric flask. dissolved As Insert an inert plastic coated stirring where: bar into the flask, and begin mixing. Au=Absorbance of sample; While mixing, slowly add 84 milliliters As=Absorbance of standard; of sulfuric acid (temperature of solu- c=Concentration of working standard in tion should not exceed 50 °C). Dissolve milligrams; and 12 grams of sodium arsenate in 100 mil- d=Dilution factor of sample withdrawn from beaker. liliters of water, and add to the solu- tion in the flask. Remove the stirring *If more than 15 milliliters of dissolution medium is removed, correct for the vol- bar, dilute with water to volume, and ume removed. mix. Store in an amber bottle for 24 hours before using. (This solution (e) Evaluation. Use the dissolution ac- should not be allowed to come into ceptance table and interpretation in contact with rubber.) the United States Pharmacopeia XXI. (b) Working solution. Dilute 1 part of [50 FR 41679, Oct. 15, 1985] stock solution with 2 parts of water, and mix. This solution is freshly pre- § 436.545 Acid resistance test for eryth- pared on the day of use. romycin particles in tablets. (ii) Acetate buffer, pH 4.8. Dissolve 133 (a) Equipment. Use Apparatus 2 as de- grams of ACS grade sodium acetate scribed in the United States Pharma- crystals in about 3.5 liters of water. copeia XXI dissolution test. Adjust the pH to 4.8±0.1 with glacial (b) Acid resistance medium. Use 0.1N acetic acid. Dilute with water to 4,000 hydrochloric acid, 500 milliliters. milliliters, and mix. (c) Procedure. Warm the immersion (iii) 9N Sulfuric acid. Place a 2-liter fluid to a temperature of 37±0.5 °C. volumetric flask containing an inert Place one tablet into a vessel contain- plastic coated magnetic stirring bar ing 500 milliliters of acid resistance and about 1,500 milliliters of water in
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an ice bath, and begin mixing. While and 160 micrograms of erythromycin mixing, cautiously add 300 milliliters per milliliter, respectively. of sulfuric acid. Allow the solution to (4) Procedure. Use the working stand- cool. Remove the stirring bar, dilute ard solutions prepared as described in with water to volume, and mix. paragraph (d)(3) of this section. The ar- (3) Preparation of working standard so- rangement of the apparatus and flow of lutions—(i) Working standard stock solu- the samples and reagents are shown in tion. Accurately weigh approximately the manifold diagram set forth follow- 400 milligrams of USP Erythromycin ing this paragraph. The sampler rate is Reference Standard, previously dried usually 60 per hour, but may be varied. at 60 °C for 3 hours under vacuum (pres- Establish a steady state by pumping sure of 5 millimeters of mercury or less), and transfer to a 100-milliliter reagents until the record trace be- volumetric flask. Dissolve and dilute comes constant. Place cups containing with acetate buffer, pH 4.8 to volume, the four concentrations of working and mix. standard solutions in the sampler fol- (ii) Working standard solutions. Pipet lowed by no more than 12 cups of sam- 5, 10, 15, and 20 milliliters of the stand- ple solutions. Then place four more ard stock solution into separate 500- cups containing the four concentra- milliliter volumetric flasks, add ace- tions of working standard solutions in tate buffer, pH 4.8 to volume, and mix. the sampler. Repeat the sequence The approximate concentrations of above for additional samples by brack- these solutions (before adjusting for eting standards around no more than 12 the standard potency) are 40, 80, 120, sample solutions at a time.
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(5) System suitability test. Perform a ards. The system is suitable for cal- linear regression analysis of absorb- culation if the beginning baseline and ance versus concentration in the ending baseline after assaying a se- micrograms per milliliter of the stand- ries of standard and sample solutions
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does not vary by more than 2 percent (6) Calculations. (i) Calculate the con- transmittance, and the correlation co- centration of each standard curve solu- efficient for each standard curve is tion in micrograms of erythromycin greater than 0.995. per milliliter as follows:
Milligrams of Potency of working Concentration of × Milliliters of working standard (micrograms standard each standard curve solution (micrograms = standard per milligram) × stock solution of erythromycin 100 500 per milliliter) (ii) Calculate the percent of labeled amount of erythromycin released in 60 min- utes as follows: Percent of labeled 500 100 Micrograms of amount of erythromycin = × × erythromycin erythromycin content released in 60 minutes 1,000 per milliliter of tablet
[51 FR 37721, Oct. 24, 1986] 440.71 Penicillin V. 440.73 Penicillin V potassium. 440.74a Sterile penicillin G procaine. PART 440—PENICILLIN ANTIBIOTIC 440.80 Penicillin G potassium. DRUGS 440.80a Sterile penicillin G potassium. 440.81a Sterile penicillin G sodium. Subpart A—Bulk Drugs 440.83a Sterile piperacillin sodium. 440.90a Sterile ticarcillin disodium. Sec. 440.91 Ticarcillin monosodium 440.1a Sterile azlocillin sodium. monohydrate. 440.2a Sterile amdinocillin. 440.3 Amoxicillin trihydrate. Subpart B—Oral Dosage Forms 440.5 Ampicillin. 440.7 Ampicillin trihydrate. 440.103 Amoxicillin oral dosage forms. 440.7a Sterile ampicillin trihydrate. 440.103a Amoxicillin trihydrate capsules. 440.8 Bacampicillin hydrochloride. 440.103b Amoxicillin trihydrate for oral sus- 440.9a Sterile ampicillin sodium. pension. 440.10 Benzylpenicilloyl-polylysine con- 440.103c Amoxicillin trihydrate chewable centrate. tablets. 440.11 Carbenicillin indanyl sodium. 440.103d Amoxicillin trihydrate and 440.13a Sterile carbenicillin disodium. clavulanate potassium tablets. 440.15 Cloxacillin sodium monohydrate. 440.103e Amoxicillin trihydrate and 440.17 Cyclacillin. clavulanate potassium for oral suspen- 440.19 Dicloxacillin sodium monohydrate. sion. 440.19a Sterile dicloxacillin sodium 440.103f Amoxicillin trihydrate-clavulanate monohydrate. potassium chewable tablets. 440.25 Hetacillin. 440.105 Ampicillin oral dosage forms. 440.29 Hetacillin potassium. 440.105a Ampicillin tablets. 440.29a Sterile hetacillin potassium. 440.105b Ampicillin chewable tablets. 440.36a Sterile methicillin sodium 440.105c Ampicillin capsules. monohydrate. 440.105d Ampicillin for oral suspension. 440.37a Sterile mezlocillin sodium 440.107 Ampicillin trihydrate oral dosage monohydrate. forms. 440.41 Nafcillin sodium monohydrate. 440.107a Ampicillin trihydrate chewable 440.41a Sterile nafcillin sodium tablets. monohydrate. 440.107b Ampicillin trihydrate capsules. 440.49 Oxacillin sodium monohydrate. 440.107c Ampicillin trihydrate for oral sus- 440.49a Sterile oxacillin sodium pension. monohydrate. 440.107d Ampicillin trihydrate-probenecid 440.55a Sterile penicillin G benzathine. for oral suspension.
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