1818 / Dietary Supplements USP 36

Standard stock solution 2 (B, Cr, Mo, Ni, Se, Sn, and PERFORMANCE TESTS V): [NOTE—It is only necessary to include the minerals • DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS of interest in the solution.] Using commercially available 〈2040〉: Meet the requirements for Dissolution element standard (single- or multi-element) solutions in • WEIGHT VARIATION OF DIETARY SUPPLEMENTS 〈2091〉: Meet 20% (v/v) hydrochloric acid solution, pipet the the requirements appropriate amount of element standard solution into a volumetric flask, and dilute with 20% (v/v) hydrochloric SPECIFIC TESTS acid solution to obtain a solution with final • MICROBIAL ENUMERATION TESTS—NUTRITIONAL AND DIETARY concentrations of about 200 mg/L of boron, and SUPPLEMENTS 〈2021〉: The total aerobic microbial count 100 mg/L of chromium, molybdenum, nickel, selenium, does not exceed 3000 cfu/g, and the combined molds tin, and vanadium each. and yeasts count does not exceed 300 cfu/g. Standard solutions: Prepare a mixture of Standard • ABSENCE OF SPECIFIED MICROORGANISMS—NUTRITIONAL AND stock solution 1 and Standard stock solution 2, as DIETARY SUPPLEMENTS 〈2022〉: Meet the requirements of required, in Diluent, to prepare a six-point calibration the tests for absence of Salmonella species, Eshcerichia curve to bracket the concentration range of each coli, and Staphylococcus aureus. mineral of interest. Sample solution: Weigh, then transfer 5 Capsules to a ADDITIONAL REQUIREMENTS 250-mL volumetric flask, and heat gently on a hot plate • PACKAGING AND STORAGE: Preserve in tight, light-resistant until the contents begin to release. Cautiously add containers. 25 mL of Stock aqua regia solution in 5-mL increments, • LABELING: The label states that the product is Water- and swirl. Heat, continue to swirl until the Capsules Soluble Vitamins with Minerals Capsules. The label also dissolve into the acid, immediately remove from the states the quantity of each and mineral in terms heat source, and add 150 mL of water. Cool, and dilute of metric units per dosage unit and where necessary the with water to volume. Filter about 30 mL into a chemical form in which a vitamin is present and also centrifuge tube, using a 5-µm pore size nylon syringe states the salt form of the mineral used as the source of filter. If necessary, make any further adjustments using each element. Where more than one assay method is Diluent. given for a particular vitamin or mineral, the labeling Spectrometric conditions states with which assay method the product complies (See Spectrochemistry 〈730〉.) only if Method 1 is not used. Mode: Inductively coupled plasma spectrometry, using • USP REFERENCE STANDARDS 〈11〉 a spectrometer, set to measure the emission of each USP RS mineral of interest at about the corresponding USP Calcium Pantothenate RS USP RS wavelength. [NOTE—The operating conditions may be developed and optimized based on the manufacturer’s USP Dexpanthenol RS recommendation. The wavelengths selected should be USP Folic Acid RS demonstrated experimentally to provide sufficient USP RS specificity, sensitivity, linearity, accuracy, and USP Niacinamide RS precision.] USP Hydrochloride RS System suitability USP RS Sample: System suitability solution USP Sodium Fluoride RS USP Hydrochloride RS [NOTE—Analyze the System suitability solution and obtain the response as directed for Analysis.] Suitability requirements Relative standard deviation: NMT 2.0% Analysis . Samples: Standard solutions and Sample solution Water-Soluble Vitamins with Minerals Determine the emission of each mineral of interest in the Oral Solution Standard solutions and Sample solution with an Inductively coupled plasma system using the Diluent as DEFINITION the blank. Plot the emission of the Standard solutions Water-Soluble Vitamins with Minerals Oral Solution contains versus the concentration, in mg/L, of the minerals of one or more of the following water-soluble vitamins: Cya- interest, and draw the straight line best fitting the nocobalamin, Niacin or Niacinamide, Dexpanthenol or plotted points. From the graph so obtained, determine Panthenol, (as Calcium Pantothenate or the concentration, C, in mg/L, for each mineral of Racemic Calcium Pantothenate), Pyridoxine Hydrochlo- interest in the Sample solution. Calculate the percentage ride, Riboflavin or Riboflavin-5′-Phosphate Sodium, and of the labeled amount for each mineral: Thiamine Hydrochloride or Thiamine Mononitrate; and one or more minerals derived from substances generally Result = C × (V/W) × F × (CW/L) × 100 recognized as safe, furnishing one or more of the follow- ing elements in ionizable form: iodine, iron, magnesium, C = measured concentration of the relevant manganese, and zinc. It contains NLT 90.0% and NMT element in the Sample solution (mg/L) 450.0% of the labeled amount of cyanocobalamin V = volume of the Sample solution (L) (C63H88CoN14O14P); NLT 90.0% and NMT 250.0% of the W = sample weight (mg) labeled amount of thiamine (C12H17ClN4OS) as thiamine F = dilution factor of the Sample solution hydrochloride or thiamine mononitrate; NLT 90.0% and CW = average Capsule weight (mg) NMT 150.0% of the labeled amounts of calcium panto- L = labeled amount of the relevant element/ thenate (C18H32CaN2O10), dexpanthenol (C9H19NO4) or Capsule (mg/Capsule) panthenol (C9H19NO4), niacin (C6H5NO2) or niacinamide Acceptance criteria: 90.0%–125.0% of the labeled (C6H6N2O), pyridoxine hydrochloride (C8H11NO3 · HCl), amount of calcium (Ca), copper (Cu), iron (Fe), and riboflavin (C17H20N4O6) or riboflavin-5′-phosphate so- magnesium (Mg), manganese (Mn), phosphorus (P), dium (C17H20N4NaO9P); NLT 90.0% and NMT 160.0% of potassium (K), and zinc (Zn); and 90.0%–160.0% of the labeled amount of iodine (I); and NLT 90.0% and the labeled amounts of boron (B), chromium (Cr), NMT 125.0% of the labeled amounts of iron (Fe), magne- fluorine (F), iodine (I), molybdenum (Mo), nickel (Ni), sium (Mg), manganese (Mn), and zinc (Zn). selenium (Se), tin (Sn), and vanadium (V) USP 36 Dietary Supplements / Vitamins 1819

STRENGTH Vitamin solution B: 20 mg of p-aminobenzoic acid, [NOTE—In the following assays, where more than one assay 10 mg of calcium pantothenate, 40 mg of pyridoxine method is given for an individual ingredient, the require- hydrochloride, 40 mg of pyridoxal hydrochloride, 8 mg ments may be met by following any one of the specified of dihydrochloride, and 2 mg of folic acid methods, the method used being stated in the labeling in a mixture of water and neutralized (3:1) to only if Method 1 is not used.] make 400 mL. Store, protected from light, in a • CYANOCOBALAMIN refrigerator. [NOTE—Use low-actinic glassware throughout this Basal medium stock solution: Prepare the medium ac- procedure.] cording to the following formula and directions. A de- Standard stock solution: 1.0 µg/mL of cyanocobalamin hydrated mixture containing the same ingredients may from USP Cyanocobalamin RS in 25% alcohol. Store in be used provided that, when constituted as directed in a refrigerator. the labeling, it yields a medium comparable to that ob- Standard solution: Dilute a suitable volume of Standard tained from the formula given herein. stock solution with water to a measured volume such Add the ingredients in the order listed, carefully dissolv- that after the incubation period as described for Analy- ing cystine and tryptophan in the hydrochloric acid sis, the difference in transmittance between the inocu- before adding the next eight solutions. To the resulting lated blank and the 5.0-mL level of the Standard solu- solution add 100 mL of water, and dissolve the dex- tion is NLT that which corresponds to a difference of trose, sodium acetate, and ascorbic acid. Filter, if neces- 1.25 mg in dried cell weight. This concentration usually sary, add the Polysorbate 80 solution, adjust with 1 N falls between 0.01 ng/mL and 0.04 ng/mL of Standard sodium hydroxide to a pH of 5.5–6.0, and add Purified solution. Prepare this solution fresh for each assay. Water to make 250 mL. Sample solution: Transfer to an appropriate vessel an accurately measured volume of Oral Solution assumed L-Cystine 0.1 g to contain 1.0 µg of cyanocobalamin for each mL of the Oral Solution taken and 25 mL of an aqueous extracting L-Tryptophan 0.05 g solution prepared just before use to contain, in each 1 N Hydrochloric acid 10 mL 100 mL, 1.29 g of dibasic sodium phosphate, 1.1 g of –guanine–uracil solution 5 mL anhydrous citric acid, and 1.0 g of sodium metabisul- Xanthine solution 5 mL fite. Autoclave the mixture at 121° for 10 min. Allow Vitamin solution A 10 mL any undissolved particles of the extract to settle, and Vitamin solution B 10 mL filter or centrifuge if necessary. Dilute an aliquot of the clear solution with water to obtain a final solution con- Salt solution A 5 mL Salt solution B 5 mL taining activity approximately equivalent to that of the Standard solution. Asparagine solution 5 mL Acid-hydrolyzed casein solution: Mix 100 g of vita- Acid-hydrolyzed casein solution 25 mL min-free casein with 500 mL of 6 N hydrochloric acid, Dextrose, anhydrous 10 g and reflux the mixture for 8–12 h. Remove the hydro- Sodium acetate, anhydrous 5 g chloric acid from the mixture by distillation under re- duced pressure until a thick paste remains. Redissolve Ascorbic acid 1 g the resulting paste in water, adjust the solutiion with Polysorbate 80 solution 5 mL 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated char- Tomato juice preparation: Centrifuge commercially coal, stir for 1 h, and filter. Repeat the treatment with canned tomato juice so that most of the pulp is re- activated charcoal. Store under toluene in a cool place moved. Suspend 5 g/L of analytical filter-aid in the su- pernatant, and filter, with the aid of reduced pressure, at a temperature not below 10°. Filter the solution if a precipitate forms during storage. through a layer of the filter-aid. Repeat, if necessary, until a clear, straw-colored filtrate is obtained. Store Asparagine solution: Dissolve 2.0 g of L-asparagine in water to make 200 mL. Store under toluene in a under toluene in a refrigerator. refrigerator. Culture medium Adenine–guanine–uracil solution: Dissolve 200 mg [NOTE—A dehydrated mixture containing the same ingre- each of adenine sulfate, guanine hydrochloride, and dients may be used provided that, when constituted as uracil, with the aid of heat, in 10 mL of 4 N hydrochlo- directed in the labeling, it yields a medium equivalent ric acid. Cool, and add water to make 200 mL. Store to that obtained from the formula given herein.] under toluene in a refrigerator. Dissolve 0.75 g of yeast extract, 0.75 g of dried peptone, Xanthine solution: Suspend 0.20 g of xanthine in 1.0 g of anhydrous dextrose, and 0.20 g of monobasic potassium phosphate in 60–70 mL of water. Add 10 mL 30–40 mL of water, heat to 70°, add 6.0 mL of 6 N ammonium hydroxide, and stir until the solid is dis- of Tomato juice preparation and 1 mL of Polysorbate 80 solved. Cool, and add water to make 200 mL. Store solution. Adjust with 1 N sodium hydroxide to a pH of under toluene in a refrigerator. 6.8, and add water to make 100 mL. Place 10-mL por- Salt solution A: Dissolve 10 g of monobasic potassium tions of the solution in test tubes, and plug with cot- phosphate and 10 g of dibasic potassium phosphate in ton. Sterilize the tubes and contents in an autoclave at water to make 200 mL, and add 2 drops of hydrochloric 121° for 15 min. Cool as rapidly as possible to avoid acid. Store this solution under toluene. color formation resulting from overheating the medium. Salt solution B: Dissolve 4.0 g of magnesium sulfate, Suspension medium: Dilute a measured volume of Ba- 0.20 g of sodium chloride, 0.20 g of ferrous sulfate, and sal medium stock solution with an equal volume of 0.20 g of manganese sulfate in water to make 200 mL, water. Place 10-mL portions of the diluted medium in and add 2 drops of hydrochloric acid. Store this solu- test tubes. Sterilize, and cool as directed for Culture tion under toluene. medium. Polysorbate 80 solution: 20 g of polysorbate 80 in al- Stock culture of Lactobacillus leichmannii: To 100 mL cohol to make 200 mL. Store in a refrigerator. of Culture medium add 1.0–1.5 g of agar, and heat the Vitamin solution A: 10 mg of riboflavin, 10 mg of thia- mixture on a steam bath, with stirring, until the agar dissolves. Place 10-mL portions of the hot solution in mine hydrochloride, 100 µg of biotin, and 20 mg of ni- acin in 0.02 N acetic acid to make 400 mL. Store under test tubes, cover the tubes, sterilize at 121° for 15 min toluene, protected from light, in a refrigerator. in an autoclave, and allow the tubes to cool in an up- right position. Inoculate three or more of the tubes by 1820 Vitamins / Dietary Supplements USP 36

stab transfer of a pure culture of Lactobacillus leichman- a temperature between 30° and 40°, held constant to nii. [NOTE—Before first using a fresh culture in this as- within ±0.5°, for 16–24 h. say, make NLT 10 successive transfers of the culture in a Terminate growth by heating to a temperature NLT 80° 2-week period.] for 5 min. Cool to room temperature. After agitating its Incubate for 16–24 h at a temperature between 30° and contents, read the transmittance at 530 nm when a 40° held constant to within ±0.5°. Store in a steady state is reached. This steady state is observed a refrigerator. few seconds after agitation when the reading remains Prepare fresh stab cultures at least three times each constant for 30 s or more. Allow approximately the week, and do not use them for preparing the Inoculum same time interval for the reading on each tube. if more than 4 days old. The activity of the microorgan- With the transmittance set at 100% for the uninoculated ism can be increased by daily or twice-daily transfer of blank, read the transmittance of the inoculated blank. If the stab culture to the point where definite turbidity in the difference is greater than 5% or if there is evidence the liquid Inoculum can be observed 2–4 h after inocu- of contamination with a foreign microorganism, disre- lation. A slow-growing culture seldom gives a suitable gard the results of the assay. response curve and may lead to erratic results. With the transmittance set at 100% for the uninoculated Inoculum blank, read the transmittance of each of the remaining [NOTE—A frozen suspension of Lactobacillus leichmannii tubes. Disregard the results of the assay if the slope of may be used as the stock culture, provided it yields an the standard curve indicates a problem with sensitivity. Inoculum comparable to a fresh culture.] Calculation: Prepare a standard concentration-re- Make a transfer of cells from the Stock culture of Lactoba- sponse curve by the following procedure. Test for and cillus leichmannii to two sterile tubes each containing replace any aberrant individual transmittances. For 10 mL of the Culture medium. Incubate these cultures each level of the Standard, calculate the response from for 16–24 h at a temperature between 30° and 40° the sum of the duplicate values of the transmittances held constant to within ± 0.5°. Under aseptic conditions (ΣS) as the difference, y = 2.00 − ΣS. Plot this response centrifuge the cultures, and decant the supernatant. on the ordinate of cross-section paper against the log- Suspend the cells from the culture in 5 mL of sterile arithm of the mL of Standard solution per tube on the Suspension medium, and combine. Using sterile Suspen- abscissa, using for the ordinate either an arithmetic or sion medium, adjust the volume so that a 1-in-20 dilu- a logarithmic scale, whichever gives the better approx- tion in saline TS produces 70% transmittance when imation to a straight line. Draw the straight line or read on a suitable spectrophotometer that has been set smooth curve that best fits the plotted points. at a wavelength of 530 nm, equipped with a 10 mm Calculate the response, y = 2.00 − ΣU, adding together cell, and read against saline TS set at 100% transmit- the two transmittances (ΣU) for each level of the Sam- tance. Prepare a 1-in-400 dilution of the adjusted sus- ple solution. Read from the standard curve the loga- pension using sterile Basal medium stock solution. The rithm of the volume of the Standard solution corre- cell suspension so obtained is the Inoculum. sponding to each of those values of y that falls within [NOTE—This dilution may be altered, when necessary, to the range of the lowest and highest points plotted for obtain the desired test response.] the Standard. Subtract from each logarithm so ob- Calibration of spectrophotometer: Check the wave- tained the logarithm of the volume, in mL, of the length of the spectrophotometer periodically, using a Sample solution to obtain the difference, X, for each standard wavelength cell or other suitable device. dosage level. Average the values of X for each of three Before reading any tests, calibrate the spectrophotome- or more dosage levels to obtain X, which equals the ter for 0% and 100% transmittance, using water and log-relative potency, M′, of the Sample solution. Deter- with the wavelength set at 530 nm. mine the quantity, in µg, of cyanocobalamin (C63H88 Analysis: Because of the high sensitivity of the test or- CoN14O14P), in the portion of Oral Solution taken: ganism to minimum amounts of vitamin B12 activity and to traces of many cleansing agents, cleanse meticu- antilog M = antilog (M′ + log R) lously by suitable means, followed preferably by heating at 250° for 2 h, using hard-glass 20-mm × 150-mm test R = µg of cyanocobalamin assumed to be present tubes, and other necessary glassware. in the portion of Oral Solution taken To separate test tubes add, in duplicate, 1.0, 1.5, 2.0, Calculate the percentage of the labeled amount of 3.0, 4.0, and 5.0 mL of the Standard solution. To each cyanocobalamin in the portion of the Oral Solution of these tubes and to four similar empty tubes, add taken: 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL. Result = [(antilog M)/N] x 100 To similar separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Sample solution. To each N = nominal amount of cyanocobalamin in the tube add 5.0 mL of Basal medium stock solution and suf- portion of Oral Solution taken ficient water to make 10 mL. Place one complete set of Replication: Repeat the entire determination at least standard and sample tubes together in one tube rack once, using separately prepared Sample solutions. If the and the duplicate set in a second rack or section of a difference between the two log-potencies M is NMT rack, preferably in random order. 0.08, their mean, M, is the assayed log-potency of the Cover the tubes to prevent bacterial contamination, and test material (see Design and Analysis of Biological sterilize in an autoclave at 121° for 5 min, arranging to Assays 〈111〉, Vitamin B12 Activity). If the two reach this temperature in NMT 10 min by preheating determinations differ by more than 0.08, conduct one the autoclave if necessary. Cool as rapidly as possible to or more additional determinations. From the mean of avoid color formation resulting from overheating the two or more values of M that do not differ by more medium. Take precautions to maintain uniformity of than 0.15, compute the mean potency of the sterilizing and cooling conditions throughout the assay, preparation under assay. because packing the tubes too closely in the autoclave Acceptance criteria: 90.0%–450.0% of the labeled or overloading it may cause variation in the heating amount of cyanocobalamin (C63H88 CoN14O14P) rate. • CALCIUM PANTOTHENATE, Method 1 Aseptically add 0.5 mL of Inoculum to each tube so pre- Mobile phase: Methanol and 0.2 M monobasic sodium pared, except two of the four containing no Standard phosphate (3:97). Adjust with 1.7 M phosphoric acid to solution (the uninoculated blanks). Incubate the tubes at a pH of 3.2 ± 0.1. USP 36 Dietary Supplements / Vitamins 1821

Standard solution: 80 µg/mL of USP Calcium the resulting paste in water, adjust the solution with Pantothenate RS in Mobile phase 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add System suitability solution: 80 µg/mL of USP Racemic water to make 1000 mL. Add 20 g of activated Panthenol RS in Mobile phase. Mix the resulting solution charcoal, stir for 1 h, and filter. Repeat the treatment and Standard solution (1:1). with activated charcoal. Store under toluene in a Sample solution: Equivalent to 80 µg/mL of calcium refrigerator at a temperature not below 10°. Filter the pantothenate from the Oral Solution, in Mobile phase solution if a precipitate forms during storage. Chromatographic system Cystine–tryptophan solution: Suspend 4.0 g of (See Chromatography 〈621〉, System Suitability.) L-cystine in a solution of 1.0 g of L-tryptophan (or 2.0 g Mode: LC of D,L-tryptophan) in 700–800 mL of water, heat to Detector: UV 210 nm 70°–80°, and add dilute hydrochloric acid (1 in 2) Column: 4-mm × 10-cm; packing L1 dropwise, with stirring, until the solids are dissolved. Flow rate: 1 mL/min Cool, and add water to make 1000 mL. Store under Injection size: 20 µL toluene in a refrigerator at a temperature not below System suitability 10°. Samples: Standard solution and System suitability Adenine–guanine–uracil solution: Dissolve 200 mg solution each of adenine sulfate, guanine hydrochloride, and Suitability requirements uracil, with the aid of heat, in 10 mL of 4 N Resolution: NLT 1.5 between panthenol and calcium hydrochloric acid. Cool, and add water to make pantothenate, System suitability solution 200 mL. Store under toluene in a refrigerator. Tailing factor: NMT 2.0 for both the calcium Polysorbate 80 solution: 100 mg/mL of polysorbate 80 pantothenate and the panthenol peaks, System in alcohol suitability solution Riboflavin–thiamine hydrochloride–biotin solution: Relative standard deviation: NMT 2.0%, Standard 20 µg/mL of riboflavin, 10 µg/mL of thiamine solution hydrochloride, and 0.04 µg/mL of biotin in 0.02 N Analysis acetic acid. Store under toluene, protected from light, Samples: Standard solution and Sample solution in a refrigerator. Calculate the percentage of the labeled amount of p-Aminobenzoic acid–niacin–pyridoxine hydrochloride calcium pantothenate (C18H32CaN2O10) in the portion solution: 10 µg/mL of p-aminobenzoic acid, 50 µg/mL of Oral Solution taken: of niacin, and 40 µg/mL of pyridoxine hydrochloride in a mixture of neutralized alcohol and water (1:3). Store Result = (rU/rS) × (CS/CU) × 100 in a refrigerator. Salt solution A: Dissolve 25 g of monobasic potassium rU = peak area for calcium pantothenate from the phosphate and 25 g of dibasic potassium phosphate in Sample solution water to make 500 mL. Add 5 drops of hydrochloric rS = peak area for calcium pantothenate from the acid. Store under toluene. Standard solution Salt solution B: Dissolve 10 g of magnesium sulfate, CS = concentration of USP Calcium Pantothenate 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and RS in the Standard solution (mg/mL) 0.5 g of manganese sulfate in water to make 500 mL. CU = nominal concentration of the Sample solution Add 5 drops of hydrochloric acid. Store under toluene. (mg/mL) Basal medium stock solution Acceptance criteria: 90.0%–150.0% of the labeled amount of calcium pantothenate (C18H32CaN2O10) Acid-hydrolyzed casein solution 25 mL • CALCIUM PANTOTHENATE, Method 2 Standard stock solution: Dissolve 50 mg of USP Cystine–tryptophan solution 25 mL Calcium Pantothenate RS, previously dried and stored in Polysorbate 80 solution 0.25 mL the dark over phosphorus pentoxide and protected Dextrose, anhydrous 10 g from absorption of moisture while weighing, in 500 mL Sodium acetate, anhydrous 5 g of water in a 1000-mL volumetric flask. Add 10 mL of Adenine–guanine–uracil solution 5 mL 0.2 N acetic acid and 100 mL of sodium acetate solution (1 in 60), and dilute with water to volume to Riboflavin–thiamine hydrochloride–biotin solution 5 mL obtain a concentration of 50 µg/mL of USP Calcium p-Aminobenzoic acid–niacin–pyridoxine 5 mL Pantothenate RS. Store under toluene in a refrigerator. hydrochloride solution Standard solution: On the day of the assay, dilute a Salt solution A 5 mL volume of Standard stock solution with water to obtain a Salt solution B 5 mL concentration of 0.01–0.04 µg/mL of calcium pantothenate, the exact concentration being such that Dissolve the anhydrous dextrose and anhydrous sodium the responses obtained as directed for Analysis, 2.0 and acetate in the solutions previously mixed, and adjust 4.0 mL of the Standard solution being used, are within with 1 N sodium hydroxide to a pH of 6.8. Dilute with the linear portion of the log-concentration response water to 250 mL. curve. Stock culture of Lactobacillus plantarum: Dissolve Sample solution: Transfer an accurately measured 2.0 g of yeast extract in 100 mL of water; add 500 mg volume of Oral Solution equivalent to 50 mg of calcium of anhydrous dextrose, 500 mg of anhydrous sodium pantothenate to a 1000-mL volumetric flask containing acetate, and 1.5 g of agar; and heat the mixture on a 500 mL of water. Add 10 mL of 0.2 N acetic acid and steam bath, with stirring, until the agar dissolves. Add 100 mL of sodium acetate solution (1 in 60), dilute with 10-mL portions of the hot solution to the test tubes, water to volume, and filter. Dilute a measured volume close or cover the tubes, sterilize in an autoclave at of this solution quantitatively, and stepwise if necessary, 121° for 15 min, and allow the tubes to cool in an with water to obtain a solution having about the same upright position. Prepare stab cultures in three or more concentration as that of the Standard solution. of the tubes, using a pure culture of Lactobacillus Acid-hydrolyzed casein solution: Mix 100 g of plantarum, incubating for 1–24 h at a temperature vitamin-free casein with 500 mL of 6 N hydrochloric between 30° and 37° held constant to within ±0.5°. acid, and reflux the mixture for 8–12 h. Remove the Store in a refrigerator. Prepare a fresh stab of the stock hydrochloric acid from the mixture by distillation under culture every week, and do not use for Inoculum if the reduced pressure until a thick paste remains. Redissolve culture is more than 1 week old. 1822 Vitamins / Dietary Supplements USP 36

Culture medium: To each of a series of test tubes or more dosage levels to obtain X, which equals the containing 5.0 mL of Basal medium stock solution, add log-relative potency, M′, of the Sample solution. 5.0 mL of water containing 0.2 µg of calcium Determine the quantity, in mg, of USP Calcium pantothenate. Plug the tubes with cotton, sterilize in an Pantothenate RS corresponding to the calcium autoclave at 121° for 15 min, and cool. pantothenate (C18H32CaN2O10) in the portion of Oral Inoculum Solution taken: [NOTE—A frozen suspension of Lactobacillus plantarum may be used as the stock culture, provided it yields an antilog M = antilog (M′ + log R) Inoculum comparable to a fresh culture.] Make a transfer of cells from the Stock culture of R = mg of calcium pantothenate assumed to be Lactobacillus plantarum to a sterile tube containing present in the portion of Oral Solution taken 10 mL of Culture medium. Incubate this culture for Calculate the percentage of the labeled amount of 16–24 h at a temperature between 30° and 37° held calcium pantothenate (C18H32CaN2O10) in the portion constant to within ±0.5°. The cell suspension so of Oral Solution taken: obtained is the Inoculum. Analysis: To similar separate test tubes add, in Result = [(antilog M)/N] × 100 duplicate, 1.0 and/or 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. To each tube and to four similar N = nominal amount of calcium pantothenate in empty tubes, add 5.0 mL of Basal medium stock solution the portion of Oral Solution (mg) and sufficient water to make 10 mL. Replication: Repeat the entire determination at least To similar separate test tubes add, in duplicate, volumes once, using separately prepared Sample solutions. If the of the Sample solution corresponding to three or more difference between the two log-potencies M is NMT of the levels specified for the Standard solution, 0.08, their mean, M, is the assayed log-potency of the including the levels of 2.0, 3.0, and 4.0 mL. To each test material (see Design and Analysis of Biological tube add 5.0 mL of the Basal medium stock solution and Assays 〈111〉, The Confidence Interval and Limits of sufficient water to make 10 mL. Place one complete set Potency). If the two determinations differ by more than of standard and sample tubes together in one tube rack 0.08, conduct one or more additional determinations. and the duplicate set in a second rack or section of a From the mean of two or more values of M that do rack, preferably in random order. not differ by more than 0.15, compute the mean Cover the tubes of both series to prevent contamination, potency of the preparation under assay. and sterilize in an autoclave at 121° for 5 min. Cool, Acceptance criteria: 90.0%–150.0% of the labeled add 1 drop of Inoculum to each tube, except two of the amount of calcium pantothenate (C18H32CaN2O10) four tubes containing no Standard solution (the • DEXPANTHENOL OR PANTHENOL, Method 1 uninoculated blanks). Incubate the tubes at a Mobile phase and Chromatographic system: Proceed temperature between 30° and 37°, held constant to as directed in the assay for Calcium Pantothenate, within ±0.5° until, following 16–24 h of incubation, Method 1. there has been no substantial increase in turbidity in Standard solution: 80 µg/mL of USP Dexpanthenol RS the tubes containing the highest level of Standard or USP Racemic Panthenol RS in Mobile phase. [NOTE— during a 2-h period. Use USP Dexpanthenol RS to analyze Oral Solution that Determine the transmittance of the tubes in the contains dexpanthenol and use USP Racemic Panthenol following manner. Mix the contents of each tube, and RS to analyze Oral Solution that contains panthenol.] transfer to an optical container if necessary. Read the System suitability solution: 80 µg/mL of USP Calcium transmittance between 540 and 660 nm when a steady Pantothenate RS in Mobile phase. Mix the resulting state is reached. This steady state is observed a few solution and Standard solution (1:1). seconds after agitation when the galvanometer reading Sample solution: Equivalent to 80 µg/mL of remains constant for 30 s or more. Allow approximately dexpanthenol or panthenol from Oral Solution in Mobile the same time interval for the reading on each tube. phase With the transmittance set at 1.00 for the uninoculated Analysis blank, read the transmittance of the inoculated blank. Samples: Standard solution and Sample solution With the transmittance set at 1.00 for the inoculated Calculate the percentage of the labeled amount of blank, read the transmittance for each of the remaining dexpanthenol or panthenol (C9H19NO4) in the portion tubes. If there is evidence of contamination with a of Oral Solution taken: foreign microorganism, disregard the result of the Result = (rU/rS) × (CS/CU) × 100 assay. Calculation: Prepare a standard concentration- rU = peak area for dexpanthenol or panthenol from response curve as follows. For each level of the the Sample solution Standard, calculate the response from the sum of the rS = peak area for dexpanthenol or panthenol from duplicate values of the transmittance (ΣS) as the the Standard solution difference, y = 2.00 S. Plot this response on the − Σ CS = concentration of USP Dexpanthenol RS or USP ordinate of cross-section paper against the logarithm Racemic Panthenol RS in the Standard of the mL of Standard solution per tube on the solution (mg/mL) abscissa, using for the ordinate either an arithmetic or CU = concentration of the Sample solution (mg/mL) a logarithmic scale, whichever gives the better Acceptance criteria: 90.0%–150.0% of the labeled approximation to a straight line. Draw the straight line amount of dexpanthenol or panthenol (C9H19NO4) or smooth curve that best fits the plotted points. • DEXPANTHENOL OR PANTHENOL, Method 2 Calculate the response, y = 2.00 − ΣU, adding together [NOTE—The following procedure is applicable also to the the two transmittances (ΣU) for each level of the determination of the dextrorotatory component of Sample solution. Read from the standard curve the racemic panthenol in preparations containing logarithm of the volume of the Standard solution panthenol.] corresponding to each of those values of y that fall Dehydrated mixtures yielding formulations similar to the within the range of the lowest and highest points media described herein may be used provided that, plotted for the Standard. Subtract from each logarithm when constituted as directed, they have growth- so obtained the logarithm of the volume, in mL, of the promoting properties equal to or superior to those Sample solution to obtain the difference, X, for each obtained with the media prepared as described herein. dosage level. Average the values of X for each of three USP 36 Dietary Supplements / Vitamins 1823

Standard stock solution: 800 µg/mL of USP Adenine–guanine–uracil solution 5 mL Dexpanthenol RS or 1600 µg/mL of USP Racemic Riboflavin–thiamine hydrochloride–biotin solution 5 mL Panthenol RS in water. Store in a refrigerator, protected p-Aminobenzoic acid–niacin–pyridoxine 5 mL from light, and use within 30 days. [NOTE—Use USP hydrochloride solution Dexpanthenol RS to analyze Oral Solution that contains dexpanthenol and use USP Racemic Panthenol RS to Salt solution 1 5 mL analyze Oral Solution that contains panthenol.] Salt solution 2 5 mL Standard solution: On the day of the assay, prepare Pyridoxal–calcium pantothenate solution 5 mL 1.2 µg/mL of dexpanthenol or 2.4 µg/mL of racemic Polysorbate 40–oleic acid solution 5 mL panthenol from Standard stock solution with water. Sample solution: 1.2 µg/mL of dexpanthenol or Dissolve anhydrous dextrose and sodium acetate in the 2.4 µg/mL of panthenol from Oral Solution in water solutions previously mixed, and adjust with 1 N sodium Acid-hydrolyzed casein solution: Mix 100 g of hydroxide to a pH of 6.8. Finally, dilute with water to vitamin-free casein with 500 mL of 6 N hydrochloric 250 mL, and mix. acid, and reflux the mixture for 8–12 h. Remove the Double-strength modified pantothenate medium: hydrochloric acid from the mixture by distillation under Prepare as directed under Modified pantothenate reduced pressure until a thick paste remains. Redissolve medium, but make the final dilution to 125 mL instead the resulting paste in about 500 mL of water, adjust the of 250 mL. Prepare fresh. solution with 1 N sodium hydroxide to a pH of 3.5 ± Stock culture of Pediococcus acidilactici: Dissolve in 0.1, and add water to make 1000 mL. Add 20 g of 800 mL of water, with the aid of heat, 6.0 g of activated charcoal, stir for 1 h, and filter. Repeat the peptone, 4.0 g of pancreatic digest of casein, 3.0 g of treatment with activated charcoal. Store under toluene yeast extract, 1.5 g of beef extract, 1.0 g of dextrose, in a refrigerator at a temperature not below 10°. Filter and 15.0 g of agar. Adjust with 0.1 N sodium the solution if a precipitate forms during storage. hydroxide or 0.1 N hydrochloric acid to a pH between Cystine–tryptophan solution: Suspend 4.0 g of 6.5 and 6.6, dilute with water to 1000 mL, and mix. L-cystine in a solution of 1.0 g of L-tryptophan (or 2.0 g Add 10-mL portions of the solution to culture tubes, of D,L-tryptophan) in 700 to 800 mL of water, heat to place caps on the tubes, and sterilize in an autoclave at 75 ± 5°, and add 6 M hydrochloric acid dropwise, with 121° for 15 min. Cool on a slant, and store in a stirring, until the solids are dissolved. Cool, add water refrigerator. Prepare a stock culture of Pediococcus to make 1000 mL, and mix. Store under toluene in a acidilactici1 on a slant of this medium. Incubate at 35° refrigerator at a temperature not below 10°. for 20–24 h, and store in a refrigerator. Maintain the Adenine–guanine–uracil solution: Dissolve 200 mg stock culture by monthly transfer onto fresh slants. each of adenine sulfate, guanine hydrochloride, and Inoculum: Inoculate three 250-mL portions of Modified uracil, with the aid of heat, in 10 mL of 4 N pantothenate medium from a stock culture slant, and hydrochloric acid. Cool, add water to make 200 mL, incubate at 35° for 20–24 h. Centrifuge the suspension and mix. Store under toluene in a refrigerator. from the combined portions, and wash the cells with Polysorbate 80 solution: 100 mg/mL of polysorbate 80 Modified pantothenate medium. Resuspend the cells in in alcohol sufficient Modified pantothenate medium so that a 1-in- Riboflavin–thiamine hydrochloride–biotin solution: 50 dilution, when tested in a 13-mm diameter test Prepare a solution of riboflavin, thiamine hydrochloride, tube, gives 80% light transmission at 530 nm. Transfer and biotin in 0.02 N acetic acid containing 20 µg/mL of 1.2-mL portions of this stock suspension to glass riboflavin, 10 µg/mL of thiamine hydrochloride, and ampuls, seal, freeze in liquid nitrogen, and store in a 0.04 µg/mL of biotin. Store under toluene, protected freezer. On the day of the assay, allow the ampuls to from light, in a refrigerator. reach room temperature, mix the contents, and dilute p-Aminobenzoic acid–niacin–pyridoxine hydrochloride 1 mL of thawed culture with sterile saline TS to 150 mL. solution: Prepare a solution in neutral 25% alcohol [NOTE—This dilution may be altered when necessary to containing 10 µg/mL of p-aminobenzoic acid, 50 µg/mL obtain the desired test response.] of niacin, and 40 µg/mL of pyridoxine hydrochloride. Analysis: Prepare in triplicate a series of eight culture Store in a refrigerator. tubes by adding the following quantities of water to the Salt solution 1: Dissolve 25 g of monobasic potassium tubes within a set: 5.0, 4.5, 4.0, 3.5, 3.0, 2.0, 1.0, and phosphate and 25 g of dibasic potassium phosphate in 0.0 mL. To these same tubes, and in the same order, water to make 500 mL. Add 5 drops of hydrochloric add 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the acid, and mix. Store under toluene. Standard solution. Salt solution 2: Dissolve 10 g of magnesium sulfate, Prepare in duplicate a series of five culture tubes by 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and adding the following quantities of water to the tubes 0.5 g of manganese sulfate in water to make 500 mL. within a set: 4.0, 3.5, 3.0, 2.0, and 1.0 mL. To these Add 5 drops of hydrochloric acid, and mix. Store under same tubes, and in the same order, add 1.0, 1.5, 2.0, toluene. 3.0, and 4.0 mL of the Sample solution. Pyridoxal–calcium pantothenate solution: Dissolve Add 5.0 mL of Double-strength modified pantothenate 40 mg of pyridoxal hydrochloride and 375 µg of medium to each tube, and mix. Cover the tubes with calcium pantothenate in 10% alcohol to make 200 mL, metal caps, and sterilize in an autoclave at 121° for 5 and mix. Store in a refrigerator, and use within 30 days. min. Cool to room temperature in a chilled water bath, Polysorbate 40–oleic acid solution: Dissolve 25 g of and inoculate each tube with 0.5 mL of the Inoculum. polysorbate 40 and 0.25 g of oleic acid in 20% alcohol Allow to incubate at 37° for 16 h. Terminate growth by to make 500 mL, and mix. Store in a refrigerator, and heating to a temperature not below 80°, such as by use within 30 days. steaming at atmospheric pressure in a sterilizer for 5 to Modified pantothenate medium 10 min. Cool, and concomitantly determine the percentage transmittance of the suspensions, in cells of equal path length, on a suitable spectrophotometer, at Acid-hydrolyzed casein solution 25 mL a wavelength of 530 nm. Cystine–tryptophan solution 25 mL Calculation: Draw a dose-response curve on arithmetic Polysorbate 80 solution 0.25 mL graph paper by plotting the average response, in Dextrose, anhydrous 10 g percent transmittance, for each set of tubes of the Sodium acetate, anhydrous 5 g 1ATCC No. 8042 is suitable. 1824 Vitamins / Dietary Supplements USP 36

standard curve against the standard level concentra- • PYRIDOXINE HYDROCHLORIDE tions. The curve is drawn by connecting each adjacent Diluent, Mobile phase, and Chromatographic system: pair of points with a straight line. From this standard Proceed as directed in the assay for Niacin or curve, determine by interpolation the potency, in Niacinamide. terms of dexpanthenol, of each tube containing Standard solution: Equivalent to 24 µg/mL of USP portions of the Sample solution. Divide the potency of Pyridoxine Hydrochloride RS in Diluent each tube by the amount of the Sample solution added Sample solution: Equivalent to 24 µg/mL of pyridoxine to it, to obtain the individual responses. Calculate the hydrochloride from Oral Solution in Diluent mean response by averaging the individual responses Analysis that vary from their mean by NMT 15%, using NLT Samples: Standard solution and Sample solution half the total number of tubes. Calculate the potency Calculate the percentage of the labeled amount of of the portion of the material taken for assay, by pyridoxine hydrochloride (C8H11NO3 · HCl) in the multiplying the mean response by the appropriate portion of Oral Solution taken: dilution factor. Calculate the percentage of the labeled amount of dexpanthenol or panthenol in the portion Result = (rU/rS) × (CS/CU) × 100 of Oral Solution taken: rU = peak area for pyridoxine hydrochloride from Result = (P/N) × 100 the Sample solution rS = peak area for pyridoxine hydrochloride from P = calculated potency of dexpanthenol or the Standard solution panthenol in the portion of Oral Solution CS = concentration of USP Pyridoxine Hydrochloride taken (mg) RS in the Standard solution (mg/mL) N = nominal amount of dexpanthenol or CU = nominal concentration of pyridoxine panthenol in the portion of Oral Solution hydrochloride in the Sample solution taken (mg) (mg/mL) Acceptance criteria: 90.0%–150.0% of the labeled Acceptance criteria: 90.0%–150.0% of the labeled amount of dexpanthenol or panthenol (C9H19NO4) amount of pyridoxine hydrochloride (C8H11NO3 · HCl) • NIACIN OR NIACINAMIDE • RIBOFLAVIN OR RIBOFLAVIN-5′-PHOSPHATE SODIUM, Method 1 [NOTE—Use low-actinic glassware throughout this [NOTE—Riboflavin-5′-phosphate sodium is quantitated procedure.] against USP Riboflavin RS in this procedure. In the Diluent: 25 mg/mL of edetate disodium in water chromatogram of the Sample solution, the riboflavin-5′- Mobile phase: Methanol, glacial acetic acid, phosphate peak is the only peak measured for triethylamine, and 0.008 M sodium 1-hexanesulfonate calculation.] (350:15:0.4:1634.6) Diluent, Mobile phase, and Chromatographic system: Standard solution: 0.10 mg/mL of USP Niacin RS or Proceed as directed in the assay for Niacin or USP Niacinamide RS in Diluent Niacinamide. [NOTE—Use USP Niacin RS for Oral Solution that contains Standard solution: Equivalent to 8 µg/mL of USP niacin and use USP Niacinamide RS for Oral Solution Riboflavin RS in Diluent, by heating if necessary that contains niacinamide.] Sample solution: Equivalent to 8 µg/mL of riboflavin Sample solution: Dilute an accurately measured from the Oral Solution in Diluent volume of Oral Solution with Diluent to obtain a Analysis solution with a concentration of 0.1 mg/mL of niacin or Samples: Standard solution and Sample solution niacinamide. [NOTE—The relative retention times for riboflavin-5′- Chromatographic system phosphate and riboflavin are about 0.18 and 1.0, (See Chromatography 〈621〉, System Suitability.) respectively.] Mode: LC Calculate the percentage of the labeled amount of Detector: UV 270 nm riboflavin (C17H20N4O6) in the portion of Oral Solution Column: 4.6-mm × 25-cm; packing L7 taken: Flow rate: 2 mL/min Injection size: 5 µL Result = (rU/rS) × (CS/CU) × F × 100 System suitability Sample: Standard solution rU = peak area for riboflavin-5′-phosphate from the Suitability requirements Sample solution Relative standard deviation: NMT 2.0% rS = peak area for riboflavin from the Standard Analysis solution Samples: Standard solution and Sample solution CS = concentration of USP Riboflavin RS in the Calculate the percentage of the labeled amount of Standard solution (mg/mL) niacin (C6H5NO2) or niacinamide (C6H6N2O) in the CU = nominal concentration of riboflavin in the portion of Oral Solution taken: Sample solution (mg/mL) F = factor for converting the response obtained Result = (rU/rS) × (CS/CU) × 100 for riboflavin-5′-phosphate to riboflavin, 1.493 [NOTE—Riboflavin Phosphate Sodium rU = peak area for niacin or niacinamide from the is a mixture of isomeric monophosphates Sample solution and diphosphates containing an average rS = peak area for niacin or niacinamide from the amount of 67% of riboflavin-5′- Standard solution monophosphate, which separates in this CS = concentration of USP Niacin RS or USP chromatographic system. The factor 1.493 Niacinamide RS in the Standard solution assumes 67% of riboflavin-5′- (mg/mL) monophosphate.] CU = nominal concentration of niacin or Acceptance criteria: 90.0%–150.0% of the labeled niacinamide in the Sample solution (mg/mL) amount of riboflavin (C17H20N4O6) Acceptance criteria: 90.0%–150.0% of the labeled amount of niacin (C6H5NO2) or niacinamide (C6H6N2O) USP 36 Dietary Supplements / Vitamins 1825

• RIBOFLAVIN OR RIBOFLAVIN-5′-PHOSPHATE SODIUM, Method 2 Standard stock solution: 1.3 mg/mL of potassium [NOTE—Use low-actinic glassware throughout this iodide in Mobile phase. This solution has a procedure.] concentration of 1 mg/mL of iodide. Solvent blank: 1 N hydrochloric acid, 2.5 M sodium Standard solution: 2.5 µg/mL of iodide from Standard acetate, and water (1:2:97) stock solution in Mobile phase Riboflavin stock solution: 0.16 mg/mL of USP System suitability solution: Transfer 0.13 g of Riboflavin RS in 1 N hydrochloric acid, 2.5 M sodium potassium iodide and 0.5 g of potassium iodate to a acetate, and water (1:2:97). Mix the resulting solution 100-mL volumetric flask. Dissolve in Mobile phase, using and water (1:9). sonication if necessary, dilute with Mobile phase to Standard solution: Riboflavin stock solution, 1 N volume, and mix. Transfer 1.0 mL of this solution to a hydrochloric acid, 2.5 M sodium acetate, and water 100-mL volumetric flask, dilute with Mobile phase to (1:1:2:96) volume, and mix. Transfer 25.0 mL of this solution to a Sample solution: Transfer equivalent to 0.8 mg of 100-mL volumetric flask, and dilute with Mobile phase riboflavin from the Oral Solution to a 100-mL to volume. volumetric flask, and dilute with water to volume. Mix Sample solution: Dilute an accurately measured the resulting solution, 1 N hydrochloric acid, 2.5 M volume of Oral Solution to obtain a solution having a sodium acetate, and water (2:1:2:95). concentration of 2.5 µg/mL of iodine in Mobile phase Spectrometric conditions Chromatographic system (See Spectrophotometry and Light-Scattering 〈851〉). (See Chromatography 〈621〉, System Suitability.) Mode: Fluorescence Mode: LC Analytical wavelength Detector: UV 225 nm Excitation: 440 nm Column: 4.6-mm × 15-cm; packing L1 Emission: 530 nm Flow rate: 1.5 mL/min Blank: Solvent blank Injection size: 30 µL Analysis System suitability Samples: Standard solution and Sample solution Samples: Standard solution and System suitability Determine the maximum fluorescence intensities, IS and solution IU, of the Standard solution and the Sample solution, [NOTE—The relative retention times for iodate and respectively. Calculate the percentage of the labeled iodide are about 0.32 and 1.0, respectively.] amount of riboflavin (C17H20N4O6) in the portion of Oral Suitability requirements Solution taken: Resolution: NLT 2.5 between iodate and iodide, System suitability solution Result = (IU/IS) × (CS/CU) × 100 Relative standard deviation: NMT 2.0% for the iodide peak, Standard solution IU = fluorescence value from the Sample solution Analysis IS = fluorescence value from the Standard solution Samples: Standard solution and Sample solution CS = concentration of USP Riboflavin RS in the Calculate the percentage of the labeled amount of Standard solution (mg/mL) iodine (I) in the portion of Oral Solution taken: CU = nominal concentration of riboflavin in the Sample solution (mg/mL) Result = (rU/rS) × (CS/CU) × 100 Acceptance criteria: 90.0%–150.0% of the labeled amount of riboflavin (C17H20N4O6) rU = peak area for iodide from the Sample solution • THIAMINE rS = peak area for iodide from the Standard solution Diluent, Mobile phase, and Chromatographic system: CS = concentration of iodide in the Standard Proceed as directed in the assay for Niacin or solution (µg/mL) Niacinamide. CU = nominal concentration of iodine in the Sample Standard solution: 24 µg/mL of USP Thiamine solution (µg/mL) Hydrochloride RS in Diluent Acceptance criteria: 90.0%–160.0% of the labeled Sample solution: Equivalent to 24 µg/mL of thiamine amount of iodine (I) hydrochloride from the Oral Solution in Diluent • IODIDE, Method 2: Proceed as directed under Automated Analysis Methods of Analysis 〈16〉, Assay for Iodide. Samples: Standard solution and Sample solution • IRON For products containing thiamine hydrochloride, Iron standard stock solution: Transfer 100 mg of iron calculate the percentage of the labeled amount of powder to a 1000-mL volumetric flask, dissolve in 6 N thiamine hydrochloride (C12H17ClN4OS · HCl) in the hydrochloric acid, and dilute with water to volume. portion of Oral Solution taken: Standard solutions: To separate 100-mL volumetric flasks, transfer 2.0, 4.0, 5.0, 6.0, and 8.0 mL of Iron Result = (rU/rS) × (CS/CU) × 100 standard stock solution. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain rU = peak area for thiamine from the Sample concentrations of 2.0, 4.0, 5.0, 6.0, and 8.0 µg/mL of solution iron. rS = peak area for thiamine from the Standard Sample solution: 6 µg/mL of iron from Oral Solution in solution 0.125 N hydrochloric acid CS = concentration of USP Thiamine Hydrochloride Spectrometric conditions RS in the Standard solution (mg/mL) (See Spectrophotometry and Light-Scattering 〈851〉.) CU = nominal concentration of thiamine Mode: Atomic absorption spectrophotometer hydrochloride in the Sample solution Lamp: Iron hollow-cathode (mg/mL) Flame: Air–acetylene Acceptance criteria: 90.0%–250.0% of the labeled Analytical wavelength: 248.3 nm amount of thiamine hydrochloride (C12H17ClN4OS · HCl) Blank: 0.125 N hydrochloric acid • IODIDE, Method 1 Analysis Mobile phase: Dissolve 5.15 g of tetrabutylammonium Samples: Standard solutions and Sample solution bromide in 320 mL of acetonitrile. Dilute with water to Determine the absorbances of the solutions, using the 2000 mL. Blank. Plot the absorbances of the Standard solutions versus concentration, in µg/mL, of iron, and draw the 1826 Vitamins / Dietary Supplements USP 36

straight line best fitting the five plotted points. From Spectrometric conditions the graph so obtained, determine the concentration, (See Spectrophotometry and Light-Scattering 〈851〉.) C, in µg/mL, of iron in the Sample solution. Calculate Mode: Atomic absorption spectrophotometer the percentage of the labeled amount of iron (Fe) in Lamp: Manganese hollow-cathode the portion of Oral Solution taken: Flame: Air–acetylene Analytical wavelength: 279.5 nm Result = (C/CU) × 100 Blank: 0.125 N hydrochloric acid Analysis C = concentration of iron in the Sample solution Samples: Standard solutions and Sample solution from the graph (µg/mL) Determine the absorbances of the solutions, using the CU = nominal concentration of iron in the Sample Blank. Plot the absorbances of the Standard solutions solution (µg/mL) versus concentration, in µg/mL, of manganese, and Acceptance criteria: 90.0%–125.0% of the labeled draw the straight line best fitting the five plotted amount of iron (Fe) points. From the graph so obtained, determine the • MAGNESIUM concentration, C, in µg/mL, of manganese in the Magnesium standard solution: Transfer 1.00 g of Sample solution. Calculate the percentage of the magnesium ribbon to a 1000-mL volumetric flask. labeled amount of manganese (Mn) in the portion of Dissolve in 50 mL of 6 N hydrochloric acid, and dilute Oral Solution taken: with water to volume. Standard stock solution: 20 µg/mL of magnesium Result = (C/CU) × 100 from Magnesium standard solution in 0.125 N hydrochloric acid C = concentration of manganese in the Sample Standard solutions: To separate 100-mL volumetric solution from the graph (µg/mL) flasks transfer 5.0, 7.5, 10.0, 12.5, and 15.0 mL of CU = nominal concentration of manganese in the Standard stock solution. Dilute with 0.125 N Sample solution (µg/mL) hydrochloric acid to volume to obtain concentrations of Acceptance criteria: 90.0%–125.0% labeled amount of 1.0, 1.5, 2.0, 2.5, and 3.0 µg/mL of magnesium. manganese (Mn) Sample solution: 2.5 µg/mL of magnesium from Oral • ZINC Solution in 0.125 N hydrochloric acid Zinc standard solution: Transfer 311 mg of zinc oxide Spectrometric conditions to a 250-mL volumetric flask, and add 80 mL of 6 N (See Spectrophotometry and Light-Scattering 〈851〉.) hydrochloric acid, warming if necessary to dissolve. Mode: Atomic absorption spectrophotometer Cool, dilute with water to volume, and mix to obtain a Lamp: Magnesium hollow-cathode solution having a known concentration of 1000 µg of Flame: Air–acetylene zinc/mL. Analytical wavelength: 285.2 nm Standard stock solution: 50 µg/mL of zinc from Zinc Blank: 0.125 N hydrochloric acid standard solution in 0.125 N hydrochloric acid Analysis Standard solutions: Transfer 1.0, 2.0, 3.0, 4.0, and Samples: Standard solutions and Sample solution 5.0 mL of Standard stock solution to separate 100-mL Determine the absorbances of the solutions, using the volumetric flasks. Dilute the contents of each flask with Blank. Plot the absorbances of the Standard solutions 0.125 N hydrochloric acid to volume to obtain versus concentration, in µg/mL, of magnesium, and concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of draw the straight line best fitting the five plotted zinc. points. From the graph so obtained, determine the Sample solution: Dilute an accurately measured concentration, C, in µg/mL, of magnesium in the volume of Oral Solution to obtain a solution having a Sample solution. Calculate the percentage of the labeled concentration of 1 µg/mL of zinc in 0.125 N amount of magnesium (Mg) in the portion of Oral hydrochloric acid Solution taken: Spectrometric conditions (See Spectrophotometry and Light-Scattering 〈851〉.) Result = (C/CU) × 100 Mode: Atomic absorption spectrophotometer Lamp: Zinc hollow-cathode C = concentration of magnesium in the Sample Flame: Air–acetylene solution from the graph (µg/mL) Analytical wavelength: 213.8 nm CU = nominal concentration of magnesium in the Blank: 0.125 N hydrochloric acid Sample solution (µg/mL) Analysis Acceptance criteria: 90.0%–125.0% of the labeled Samples: Standard solutions and Sample solution amount of magnesium (Mg) Determine the absorbances of the solutions, using the • MANGANESE Blank. Plot the absorbances of the Standard solutions Manganese standard stock solution: Transfer 1.0 g of versus concentration, in µg/mL, of zinc, and draw the manganese to a 1000-mL volumetric flask. Dissolve in straight line best fitting the five plotted points. From 20 mL of nitric acid, and dilute with 6 N hydrochloric the graph so obtained, determine the concentration, acid to volume. C, in µg/mL, of zinc in the Sample solution. Calculate Standard stock solution: 50 µg/mL of manganese from the percentage of the labeled amount of zinc (Zn) in Manganese standard stock solution in 0.125 N the portion of Oral Solution taken: hydrochloric acid Standard solutions: To separate 100-mL volumetric Result = (C/CU) × 100 flasks transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of Standard stock solution. Dilute the contents of each flask with C = concentration of zinc in the Sample solution 0.125 N hydrochloric acid to volume to obtain from the graph (µg/mL) solutions having known concentrations of 0.5, 0.75, CU = nominal concentration of zinc in the Sample 1.0, 1.5, and 2.0 µg/mL of manganese. solution (µg/mL) Sample solution: Dilute an accurately measured Acceptance criteria: 90.0%–125.0% of the labeled volume of the Oral Solution to obtain a solution having amount of zinc (Zn) a concentration of 1.5 µg/mL of manganese in 0.125 N hydrochloric acid USP 36 Dietary Supplements / Vitamins 1827

OTHER COMPONENTS (Zn); and NLT 90.0% and NMT 160.0% of the labeled • ALCOHOL DETERMINATION, Method 1 〈611〉 (if present): amounts of boron (B), chromium (Cr), fluorine (F), iodine 90.0%–120.0% of the labeled amount of C2H5OH (I), molybdenum (Mo), nickel (Ni), selenium (Se), tin (Sn), and vanadium (V). CONTAMINANTS They do not contain any form of Beta Carotene or Vitamin • MICROBIAL ENUMERATION TEST—NUTRITIONAL AND DIETARY A, D, E, or K. They may contain other labeled added sub- SUPPLEMENTS 〈2021〉: The total aerobic microbial count stances that are generally recognized as safe, in amounts does not exceed 3000 cfu/mL, and the combined molds that are unobjectionable. and yeasts count does not exceed 300 cfu/mL. • MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED STRENGTH MICROORGANISMS—NUTRITIONAL AND DIETARY SUPPLEMENTS [NOTE—In the following assays, where more than one assay 〈2022〉: Meets the requirements of the tests for absence method is given for an individual ingredient, the require- of Salmonella species, Escherichia coli, and Staphylococcus ments may be met by following any one of the specified aureus. methods, the method used being stated in the labeling only if Method 1 is not used.] ADDITIONAL REQUIREMENTS • ASCORBIC ACID, Method 1 • PACKAGING AND STORAGE: Preserve in tight, light-resistant [NOTE—Finely powder NLT 20 Tablets.] containers, under an inert gas or with a minimum of Sample solution: Transfer a portion of powdered Tab- headspace. lets, equivalent to a nominal amount of 100 mg of as- • LABELING: The label states that the product is Water- corbic acid, to a 200-mL volumetric flask, and add Soluble Vitamins with Minerals Oral Solution. The label 75 mL of metaphosphoric–acetic acids TS. Insert a stop- states the quantity of each vitamin and mineral present per into the flask, and shake by mechanical means for in terms of metric units in a given volume of the Oral 30 min. Dilute with water to volume. Transfer a portion Solution and, where necessary, the chemical form in of the solution to a centrifuge tube, and centrifuge until which a vitamin is present, and also states the salt form a clear supernatant is obtained. Pipet 4.0 mL of this so- of the mineral used as the source of each element. lution into a 50-mL conical flask, and add 5 mL of Where products are labeled to contain panthenol, the metaphosphoric–acetic acids TS. label states the equivalent content of dexpanthenol. Analysis: Titrate with standard dichlorophenol–indophe- Where more than one assay method is given for a nol solution VS to a rose-pink color that persists for at particular vitamin or mineral, the labeling states with least 5 s. Correct for the volume of dichlorophe- which assay method the product complies only if Method nol–indophenol solution consumed by a mixture of 1 is not used. 5.5 mL of metaphosphoric–acetic acids TS and 15 mL of • USP REFERENCE STANDARDS 〈11〉 water. From the ascorbic acid equivalent of the stan- USP Calcium Pantothenate RS dard dichlorophenol– USP Cyanocobalamin RS indophenol solution, calculate the content of ascorbic USP Dexpanthenol RS acid in each Tablet. USP Niacin RS Acceptance criteria: 90.0%–150.0% of the labeled USP Niacinamide RS amount of ascorbic acid (C6H8O6) USP Pyridoxine Hydrochloride RS • ASCORBIC ACID, Method 2: Proceed as directed in Auto- USP Racemic Panthenol RS mated Methods of Analysis 〈16〉, Assay for Ascorbic Acid. USP Riboflavin RS Acceptance criteria: 90.0%–150.0% of the labeled USP Thiamine Hydrochloride RS amount of ascorbic acid (C6H8O6) • CALCIUM ASCORBATE, Method 1: Proceed as directed in the assay for Ascorbic Acid, Method 1. Acceptance criteria: 90.0%–150.0% of the labeled . amount of calcium ascorbate (C12H14CaO12 · 2H2O) Water-Soluble Vitamins with Minerals • CALCIUM ASCORBATE, Method 2: Proceed as directed in Au- Tablets tomated Methods of Analysis 〈16〉, Assay for Ascorbic Acid. Acceptance criteria: 90.0%–150.0% of the labeled DEFINITION amount of calcium ascorbate (C12H14CaO12 · 2H2O) Water-Soluble Vitamins with Minerals Tablets contain one or • SODIUM ASCORBATE, Method 1: Proceed as directed in the more of the following water-soluble vitamins: Ascorbic assay for Ascorbic Acid, Method 1. Acid or its equivalent as Calcium Ascorbate or Sodium Acceptance criteria: 90.0%–150.0% of the labeled Ascorbate, Biotin, Cyanocobalamin, Folic Acid, Niacin or amount of sodium ascorbate (C6H7NaO6) Niacinamide, Pantothenic Acid (as Calcium Pantothenate • SODIUM ASCORBATE, Method 2: Proceed as directed in Au- or Racemic Calcium Pantothenate), Pyridoxine Hydrochlo- tomated Methods of Analysis 〈16〉, Assay for Ascorbic Acid. ride, Riboflavin, and Thiamine Hydrochloride or Thiamine Acceptance criteria: 90.0%–150.0% of the labeled Mononitrate; and one or more minerals derived from sub- amount of sodium ascorbate (C6H7NaO6) stances generally recognized as safe, furnishing one or • BIOTIN, Method 1 more of the following elements in ionizable form: boron, [NOTE—Use low-actinic glassware throughout this calcium, chromium, copper, fluorine, iodine, iron, magne- procedure.] sium, manganese, molybdenum, nickel, phosphorus, po- Mobile phase: Mix 85 mL of acetonitrile, 1 g of sodium tassium, selenium, tin, vanadium, and zinc. Tablets con- perchlorate, and 1 mL of phosphoric acid, and dilute tain NLT 90.0% and NMT 150.0% of the labeled amounts with water to 1000 mL. of ascorbic acid (C6H8O6) or its salts as calcium ascorbate Standard stock solution: 0.333 mg/mL of USP Biotin (C12H14CaO12 · 2H2O) or sodium ascorbate (C6H7NaO6), bi- RS in otin (C10H16N2O3S), cyanocobalamin (C63H88CoN14O14P), Standard solution: 5 µg/mL of USP Biotin RS prepared folic acid (C19H19N7O6), calcium pantothenate by diluting the Standard stock solution in water (C18H32CaN2O10), niacin (C6H5NO2) or niacinamide Sample solution: Finely powder NLT 20 Tablets. Trans- (C6H6N2O), pyridoxine hydrochloride (C8H11NO3 · HCl), ri- fer a portion of powder, equivalent to a nominal boflavin (C17H20N4O6), and thiamine (C12H17ClN4OS) as amount of 1 mg of biotin, to a 200-mL volumetric flask, thiamine hydrochloride or thiamine mononitrate; NLT add 3 mL of dimethyl sulfoxide, and swirl to wet. Place 90.0% and NMT 125.0% of the labeled amounts of cal- the flask in a water bath at 60°–70° for 5 min. Sonicate cium (Ca), copper (Cu), iron (Fe), magnesium (Mg), man- for 5 min, dilute with water to volume, and filter. ganese (Mn), phosphorus (P), potassium (K), and zinc