Oil- and Water-Soluble Vitamins with Minerals Oral Solution
DEFINITION
Oil- and Water-Soluble Vitamins with Minerals Oral Solution contains one or more of the following oil-soluble vitamins: Vitamin A, Vitamin D as Ergocalciferol (Vitamin D2) or Cholecalciferol (Vitamin D3), and Vitamin E; one or more of the following water-soluble vitamins: Ascorbic Acid or its equivalent as Calcium Ascorbate or Sodium Ascorbate, Biotin, Cyanocobalamin, Niacin or Niacinamide, Dexpanthenol or Panthenol, Pantothenic Acid (as Calcium Pantothenate or Racemic Calcium Pantothenate), Pyridoxine Hydrochloride, Riboflavin or Riboflavin-5¢-Phosphate Sodium, and Thiamine Hydrochloride or Thiamine Mononitrate; and one or more minerals derived from substances generally recognized as safe, furnishing one or more of the following elements in ionizable form: chromium, fluorine, iodine, iron, magnesium, manganese, molybdenum, and zinc. It contains NLT 90.0% and NMT 200.0% of the labeled amounts of vitamin A (C20H30O) as retinol or esters of retinol in the form of retinyl acetate (C22H32O2) or retinyl palmitate (C36H60O2), vitamin D as ergocalciferol (C28H44O) or cholecalciferol (C27H44O), vitamin E as alpha tocopherol (C29H50O2) or alpha tocopheryl acetate (C31H52O3) or alpha tocopheryl acid succinate (C33H54O5), ascorbic acid (C6H8O6) or its salts as calcium ascorbate (C12H14CaO12·2H2O) or sodium ascorbate (C6H7NaO6), and thiamine (C12H17 ClN4OS) as thiamine hydrochloride or thiamine mononitrate; NLT 90.0% and NMT 150.0% of the labeled amounts of biotin (C10H16N2O3S), calcium pantothenate (C18H32CaN2O10), dexpanthenol (C9H19NO4) or panthenol (C9H19NO4), niacin (C6H5NO2) or niacinamide (C6H6N2O), pyridoxine hydrochloride (C8H11NO3·HCl), riboflavin (C17H20N4O6) or riboflavin-5¢-phosphate sodium (C17H20N4NaO9P); NLT 90.0% and NMT 450.0% of the labeled amount of cyanocobalamin (C63H88CoN14O14P); NLT 90.0% and NMT 160.0% of the labeled amounts of chromium (Cr), fluorine (F), iodine (I), and molybdenum (Mo); and NLT 90.0% and NMT 125.0% of the labeled amounts of iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn).
STRENGTH
[Note—In the following assays, where more than one assay method is given for an individual ingredient, the requirements may be met by following any one of the specified methods, the method used being stated in the labeling only if Method 1 is not used. ]
•  Vitamin A
[Note—Use low-actinic glassware throughout this procedure. ]
Diluent:  Tetrahydrofuran and acetonitrile (1:1)
Mobile phase:  Methanol, acetonitrile, and n-hexane (46.5:46.5:7.0)
Standard solution:  0.33 mg/mL of retinol (C20H30O) from USP Vitamin A RS in Diluent. [Note—USP Vitamin A RS is retinyl acetate. Use it to analyze Oral Solution that contains vitamin A as retinol, retinyl acetate, or retinyl palmitate. ]
Sample solution:  Transfer an accurately measured volume of Oral Solution, equivalent to 3.3 mg of retinol, to a 500-mL separatory funnel containing 10 mL of water and 20 mL of dehydrated alcohol. Add 150 mL of solvent hexane, insert the stopper, and shake for 1 min. Add another 150 mL of solvent hexane, insert the stopper, shake, and allow the layers to separate. Discard the aqueous layer, and filter the solvent hexane extract through anhydrous sodium sulfate into a 500-mL, round-bottom flask. Evaporate the solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 65. Immediately add 10.0 mL of Diluent, swirl to dissolve the residue, and filter.
Chromatographic system 
Mode:  LC
Detector:  UV 265 nm
Column:  4.6-mm × 50-cm (prepared from two concatenated 4.6-mm × 25-cm columns); packing L1
Column temperature:  40
Flow rate:  1.5 mL/min
Injection size:  20 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Relative standard deviation:  NMT 5.0%
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of the labeled amount of vitamin A as retinol (C20H30O) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × F × 100
rU== peak area of retinol or retinyl ester from the Sample solution
rS== peak area of retinyl acetate from the Standard solution
CS== concentration of retinyl acetate (C22H32O2) in the Standard solution (µg/mL)
CU== nominal concentration of Vitamin A as retinol (C20H30O) in the Sample solution (µg/mL)
F== factor used to convert retinyl acetate, the ester form present in USP Vitamin A RS, to retinol, 0.872
Acceptance criteria:  90.0%–200.0% of the labeled amount of vitamin A as retinol (C20H30O)
•  Cholecalciferol or Ergocalciferol (Vitamin D)
[Note—Where vitamin D (cholecalciferol or ergocalciferol) is specified in the following procedure, use the chemical form present in the formulation and the relevant USP Reference Standard. Use low-actinic glassware throughout this procedure. ]
Diluent and Mobile phase:  Prepare as directed for Vitamin A.
Standard solution:  5 µg/mL of USP Cholecalciferol RS or USP Ergocalciferol RS in Diluent
Sample solution:  Transfer an accurately measured volume of Oral Solution, equivalent to 50 µg of cholecalciferol or ergocalciferol, to a 500-mL separatory funnel containing 10 mL of water and 20 mL of dehydrated alcohol. Add 150.0 mL of solvent hexane, insert the stopper, and shake for 1 min. Add another 150 mL of solvent hexane, insert the stopper, shake, and allow the layers to separate. Discard the aqueous layer. Drain the solvent hexane extract through anhydrous sodium sulfate into a 500-mL, round-bottom flask. Evaporate the solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 65. Immediately add 10.0 mL of Diluent, swirl to dissolve the residue, and filter.
Chromatographic system:  Prepare as directed for Vitamin A.
System suitability 
Sample:  Standard solution
Suitability requirements 
Relative standard deviation:  NMT 5.0%
Analysis 
Samples:  Standard solution and Sample solution
Measure the peak areas of vitamin D. Calculate the percentage of the labeled amount of cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × F × 100
rU== peak areas of cholecalciferol or ergocalciferol from the Sample solution
rS== peak areas of cholecalciferol or ergocalciferol from the Standard solution
CS== concentration of USP Cholecalciferol RS or USP Ergocalciferol RS in the Standard solution (µg/mL)
CU== nominal concentration of cholecalciferol or ergocalciferol in the Sample solution (µg/mL)
F== correction factor to account for the average amount of previtamin D present in the formulation, 1.09
Acceptance criteria:  90.0%–200.0% of the labeled amount of vitamin D as cholecalciferol (C27H44O) or ergocalciferol (C28H44O)
•  Vitamin E
[Note—Use low-actinic glassware throughout this procedure. ]
Diluent:  Acetonitrile and ethyl acetate (1:1)
Potassium hydroxide solution:  Transfer 90 g of potassium hydroxide pellets to a 100-mL volumetric flask containing 60 mL of water. Mix to dissolve, cool, and dilute with water to volume.
Mobile phase:  Methanol, acetonitrile, and n-hexane (46.5:46.5:7.0)
Standard solution:  0.3 mg/mL of USP Alpha Tocopherol RS in Diluent
Sample solution:  Transfer an amount of Oral Solution, equivalent to 1.5 mg of alpha tocopherol, to a 125-mL conical flask fitted with a ground-glass joint, and add 25.0 mL of dehydrated alcohol. Attach a reflux condenser, and reflux in a boiling water bath for 1 min. Cautiously add 3 mL of Potassium hydroxide solution through the condenser, and continue to reflux for 30 min. Remove the flask from the bath, and rinse the condenser with about 15 mL of water. Cool, and transfer with a minimum volume of water to a 250-mL separatory funnel. Rinse the flask with 50 mL of n-hexane, and add the rinsings to the separatory funnel. Insert the stopper, shake vigorously for 1 min, and allow the layers to separate. Drain the aqueous layer into a second 250-mL separatory funnel, and repeat the extraction with 50 mL of n-hexane. Discard the aqueous layer, and combine the hexane extracts. Wash the combined extracts with 25 mL of water, allow the layers to separate, and discard the aqueous layer. Add 3 drops of glacial acetic acid, and repeat the washing procedure two more times. Filter the washed hexane layer through anhydrous sodium sulfate into a 250-mL, round-bottom flask. Rinse the funnel and sodium sulfate with n-hexane, and add the rinsing to the hexane solution in the flask. Evaporate the hexane solution to dryness with the aid of a rotary evaporator over a water bath maintained at about 50. Immediately add 5.0 mL of Diluent, and swirl to dissolve the residue.
Chromatographic system 
Mode:  LC
Detector:  UV 291 nm
Column:  4.6-mm × 25-cm; packing L1
Column temperature:  40
Flow rate:  3.0 mL/min
Injection size:  20 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Relative standard deviation:  NMT 5.0%
Analysis 
Samples:  Standard solution and Sample solution
Measure the peak areas. Calculate the percentage of the labeled amount of vitamin E as alpha tocopherol (C29H50O2) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of alpha tocopherol from the Sample solution
rS== peak area of alpha tocopherol from the Standard solution
CS== concentration of alpha tocopherol in the Standard solution (mg/mL)
CU== nominal concentration of Vitamin E as alpha tocopherol in the Sample solution (mg/mL)
Acceptance criteria:  90.0%–200.0% of the labeled amount of vitamin E.
•  Ascorbic Acid, Method 1: Proceed as directed for Automated Methods of Analysis 16, Assay for Ascorbic Acid.
Acceptance criteria:  90.0%–200.0% of the labeled amount of ascorbic acid (C6H8O6)
•  Ascorbic Acid, Method 2
Sample solution:  Transfer an accurately measured volume of Oral Solution, equivalent to 80 mg of ascorbic acid, to a conical flask. Add 50 mL of water, 100 mL of 0.1 N sulfuric acid VS, and 15.0 mL of 0.1 N iodine VS. Stir the contents for 30 s, and add 5 mL of starch TS.
Analysis:  Immediately titrate with 0.1 N sodium thiosulfate VS to the disappearance of the color. Each mL of 0.1 N iodine is equivalent to 8.806 mg of ascorbic acid (C6H8O6).
Acceptance criteria:  90.0%–200.0% of the labeled amount of ascorbic acid (C6H8O6)
•  Calcium Ascorbate, Method 1: Proceed as directed for Automated Methods of Analysis 16, Assay for Ascorbic Acid.
Acceptance criteria:  90.0%–200.0% of the labeled amount of calcium ascorbate (C12H14CaO12·2H2O)
•  Calcium Ascorbate, Method 2: Proceed as directed for Ascorbic Acid, Method 2. Each mL of 0.1 N iodine is equivalent to 10.66 mg of C12H14CaO12·2H2O.
Acceptance criteria:  90.0%–200.0% of the labeled amount of calcium ascorbate (C12H14CaO12·2H2O)
•  Sodium Ascorbate, Method 1: Proceed as directed for Automated Methods of Analysis 16, Assay for Ascorbic Acid.
Acceptance criteria:  90.0%–200.0% of the labeled amount of sodium ascorbate (C6H7NaO6)
•  Sodium Ascorbate, Method 2: Proceed as directed for Ascorbic Acid, Method 2. Each mL of 0.1 N iodine is equivalent to 9.905 mg of C6H7NaO6.
Acceptance criteria:  90.0%–200.0% of the labeled amount of sodium ascorbate (C6H7NaO6)
•  Biotin, Method 1
[Note—Use low-actinic glassware throughout this procedure. ]
Dehydrated mixtures yielding formulations similar to the media described herein may be used provided that, when constituted as directed, they have growth-promoting properties equal to or superior to those obtained with the media prepared as described herein.
Standard stock solution:  50 µg/mL of USP Biotin RS in 50% alcohol. Store this solution in a refrigerator.
Standard solution:  On the day of the assay, prepare 0.1 ng/mL of biotin from the Standard stock solution in water.
Sample solution:  Dilute an accurately measured portion of the Oral Solution with water to an assumed concentration of 0.1 ng/mL.
Acid-hydrolyzed casein solution:  Mix 100 g of vitamin-free casein with 500 mL of 6 N hydrochloric acid, and reflux the mixture for 8–12 h. Remove the hydrochloric acid from the mixture by distillation under reduced pressure until a thick paste remains. Redissolve the resulting paste in water, adjust the solution with 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 h, and filter. Repeat the treatment with activated charcoal. Store under toluene in a cool place at a temperature not below 10. Filter the solution if a precipitate forms during storage.
Cystine–tryptophan solution:  Suspend 4.0 g of l-cystine in a solution of 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700–800 mL of water, heat to 70–80, and add dilute hydrochloric acid (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, and add water to make 1000 mL. Store under toluene in a cool place at a temperature not below 10.
Adenine–guanine–uracil solution:  Dissolve 200 mg each of adenine sulfate, guanine hydrochloride, and uracil, with the aid of heat, in 10 mL of 4 N hydrochloric acid, cool, and add water to make 200 mL. Store under toluene in a refrigerator.
Polysorbate 80 solution:  100 mg/mL of polysorbate 80 in alcohol
Calcium pantothenate solution:  10 µg/mL of calcium pantothenate in 50% alcohol. Store in a refrigerator.
Riboflavin–thiamine hydrochloride solution:  20 µg/mL of riboflavin and 10 µg/mL of thiamine hydrochloride in 0.02 N acetic acid. Store under toluene, protected from light, in a refrigerator.
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution:  10 µg/mL of p-aminobenzoic acid, 50 µg/mL of niacin, and 40 µg/mL of pyridoxine hydrochloride in neutralized alcohol and water (1:3). Store in a refrigerator.
Salt solution A:  50 mg/mL of monobasic potassium phosphate and 50 mg/mL of dibasic potassium phosphate in water. Add 5 drops of hydrochloric acid. Store under toluene.
Salt solution B:  20 g of magnesium sulfate, 1 mg/mL of sodium chloride, 1 mg/mL of ferrous sulfate, and 1 mg/mL of manganese sulfate in water. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Basal medium stock solution:  Dissolve the anhydrous dextrose and anhydrous sodium acetate in the solutions previously mixed according to Table 1, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL.
Table 1
Acid-hydrolyzed casein solution 25 mL
Cystine–tryptophan solution 25 mL
Polysorbate 80 solution 0.25 mL
Dextrose, anhydrous 10 g
Sodium acetate, anhydrous 5 g
Adenine–guanine–uracil solution 5 mL
Calcium pantothenate solution 5 mL
Riboflavin–thiamine hydrochloride solution 5 mL
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution 5 mL
Salt solution A 5 mL
Salt solution B 5 mL
Stock culture of Lactobacillus plantarum Dissolve 2.0 g of yeast extract in 100 mL of water; add 500 mg of anhydrous dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g of agar; and heat the mixture on a steam bath, with stirring, until the agar dissolves. Add 10-mL portions of the hot solution to test tubes, close or cover the tubes, sterilize in an autoclave at 121 for 15 min, and allow the tubes to cool in an upright position. Prepare stab cultures in three or more of the tubes, using a pure culture of Lactobacillus plantarum,1 incubating for 16–24 h at a temperature between 30 and 37 held constant to within ± 0.5. Store in a refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for Inoculum if the culture is more than 1 week old.
Culture medium:  To each of a series of test tubes containing 5.0 mL of Basal medium stock solution, add 5.0 mL of water containing 0.5 ng of biotin. Plug the tubes with cotton, sterilize in an autoclave at 121 for 15 min, and cool.
Inoculum:  [Note—A frozen suspension of Lactobacillus plantarum may be used as the stock culture, provided it yields an Inoculum comparable to a fresh culture. ]
Transfer cells from the Stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of Culture medium. Incubate this culture for 16–24 h at a temperature between 30 and 37 held constant to within ± 0.5. The cell suspension so obtained is the Inoculum.
Analysis:  To similar separate test tubes add, in 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 empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL. To similar test tubes add, in duplicate, volumes of the Sample solution corresponding to three or more of the levels specified for the Standard solution, including the levels of 2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL of the Basal medium stock solution and sufficient water to make 10 mL. Place one complete set of Standard and sample tubes together in one tube rack and the duplicate set in a second rack or section of a rack, preferably in random order.
Cover the tubes of both series to prevent contamination, and sterilize in an autoclave at 121 for 5 min. Cool, add 1 drop of Inoculum to each tube, except two of the four tubes containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature between 30 and 37 held constant to within ± 0.5 until, following 16–24 h of incubation, there has been no substantial increase in turbidity in the tubes containing the highest level of Standard during a 2-h period.
Determine the transmittance of the tubes in the following manner. Mix the contents of each tube, and transfer to a spectrophotometer cell. Place the cell in a spectrophotometer that has been set at a specific wavelength from 540 to 660 nm, and read the transmittance when a steady state is reached. This steady state is observed a few seconds after agitation when the reading remains constant for 30 s or more. Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 1.00 for the uninoculated blank, read the transmittance of the inoculated blank. With the transmittance set at 1.00 for the inoculated blank, read the transmittance for each of the remaining tubes. If there is evidence of contamination with a foreign microorganism, disregard the result of the assay.
Calculation:  Prepare a standard concentration-response curve as follows. For each level of the Standard, calculate the response from the sum of the duplicate values of the transmittance (SS) as the difference, y = 2.00 SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
Calculate the response, y = 2.00 SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution corresponding to each of those values of y that fall within the range of lowest and highest points plotted for the standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the Sample solution to obtain the difference, x, for each dosage level. Average the values of x for each of three or more dosage levels to obtain x, which equals the log-relative potency, M¢, of the Sample solution. Determine the quantity, in ng, of biotin (C10H16N2O3S) in the portion of Oral Solution taken:
antilog M = antilog (M¢ + log R)
R== number of ng of biotin assumed to be present in the portion of Oral solution taken
Calculate the percentage of the labeled amount of biotin in the portion of Oral Solution taken:
Result = [(antilog M)/N] × 100
N== nominal amount of biotin, in ng, in the portion of Oral Solution taken
Replication:  Repeat the entire determination at least once, using separately prepared Sample solutions. If the difference between the two log-potencies M is NMT 0.08, their mean, M, is the assayed log-potency of the test material (see Design and Analysis of Biological Assays 111, Confidence Interval and Limits of Potency). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
Acceptance criteria:  90.0%–150.0% of the labeled amount of biotin (C10H16N2O3S)
•  Biotin, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Solution A:  Transfer 800 mL of water and 100 mL of triethylamine to a 1000-mL volumetric flask. Add 80 mL of 85% phosphoric acid, and dilute with water to volume.
Mobile phase:  Transfer 80 mL of acetonitrile and 10 mL of Solution A to a 1000-mL volumetric flask. Dilute with water to volume.
Standard solution:  0.6 µg/mL of USP Biotin RS in water. [Note—A portion of the Standard solution will be used to determine the percent recovery of biotin from the Solid-phase extraction procedure. ]
Sample solution:  0.6 µg/mL of biotin, from the Oral Solution in water. Adjust the solution with either dilute acetic acid or 0.1 N sodium hydroxide to a pH between 6.0 and 7.0.
Solid-phase extraction:   [Note—Condition the extraction column specified in this procedure in the following manner. Wash the column with a 2-mL portion of methanol. Equilibrate with a 2-mL portion of water. ] Separately pipet 5.0 mL each of the Sample solution and Standard solution into freshly conditioned solid-phase extraction columns consisting of a mixed-mode packing with a sorbent-mass of 60 mg. [Note—The mixed-mode packing consists of anion-exchange and reversed-phase sorbents. The reverse-phase component is a copolymer of N-vinylpyrrolidone and divinylbenzene. The anion exchange moiety is a trialkylamino group.2 ] Wash the column with 10 mL of 30% (v/v) methanol in water. Apply an appropriate volume (4.9 mL) of 30% (v/v) methanol in 0.1 N hydrochloric acid to the column. Collect the eluate in a 5-mL volumetric flask, containing 100 µL of 40% (w/v) sodium acetate in water, and dilute with 30% (v/v) methanol in 0.1 N hydrochloric acid to volume.
Chromatographic system 
Mode:  LC
Detector:  UV 200 nm
Column:  4.6-mm × 25-cm; packing L1
Flow rate:  2 mL/min
Injection size:  100 µL
System suitability 
Samples:  Standard solution and a portion of the Standard solution that has undergone Solid-phase extraction
Suitability requirements 
Tailing factor:  NMT 1.5, Standard solution
Relative standard deviation:  NMT 2.0%, Standard solution and the Standard solution that has undergone Solid-phase extraction
Recovery:  95%–100%, Standard solution that has undergone Solid-phase extraction
Analysis 
Samples:  Standard solution and Sample solution that have both undergone Solid-phase extraction
Measure the peak areas of biotin. Calculate the percentage of the labeled amount of biotin (C10H16N2O3S) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of biotin from the Sample solution
rS== peak area of biotin from the Standard solution
CS== concentration of USP Biotin RS in the Standard solution (µg/mL)
CU== nominal concentration of biotin in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–150.0% of the labeled amount of biotin (C10H16N2O3S)
•  Cyanocobalamin
[Note—Use low-actinic glassware throughout this procedure. ]
Standard stock solution:  1.0 µg/mL of cyanocobalamin from USP Cyanocobalamin RS in 25% alcohol. Store in a refrigerator.
Standard solution:  Dilute a suitable volume of Standard stock solution with water to a measured volume such that after the incubation period as described for Analysis, the difference in transmittance between the inoculated blank and the 5.0-mL level of the Standard solution is NLT that which corresponds to a difference of 1.25 mg in dried cell weight. This concentration usually falls between 0.01 and 0.04 ng/mL of Standard solution. Prepare this solution fresh for each assay.
Sample solution:  Transfer an accurately measured volume of Oral Solution, assumed to contain 1.0 µg of cyanocobalamin, to an appropriate vessel containing, for each mL of the Oral Solution taken, 25 mL of an aqueous extracting solution prepared just before use to contain, in each 100 mL, 1.29 g of dibasic sodium phosphate, 1.1 g of anhydrous citric acid, and 1.0 g of sodium metabisulfite. Autoclave the mixture at 121 for 10 min. Allow any undissolved particles of the extract to settle, and filter or centrifuge if necessary. Dilute an aliquot of the clear solution with water to obtain a final solution containing vitamin B12 activity approximately equivalent to that of the Standard solution.
Acid-hydrolyzed casein solution:  Prepare as directed in Biotin, Method 1.
Asparagine solution:  Dissolve 2.0 g of l-asparagine in water to make 200 mL. Store under toluene in a refrigerator.
Adenine–guanine–uracil solution:  Prepare as directed for Biotin, Method 1.
Xanthine solution:  Suspend 0.20 g of xanthine in 30–40 mL of water, heat to 70, add 6.0 mL of 6 N ammonium hydroxide, and stir until the solid is dissolved. Cool, and add water to make 200 mL. Store under toluene in a refrigerator.
Salt solution A:  Dissolve 10 g of monobasic potassium phosphate and 10 g of dibasic potassium phosphate in water to make 200 mL, and add 2 drops of hydrochloric acid. Store this solution under toluene.
Salt solution B:  Dissolve 4.0 g of magnesium sulfate, 0.20 g of sodium chloride, 0.20 g of ferrous sulfate, and 0.20 g of manganese sulfate in water to make 200 mL, and add 2 drops of hydrochloric acid. Store this solution under toluene.
Polysorbate 80 solution:  20 g of polysorbate 80 in alcohol to make 200 mL. Store in a refrigerator.
Vitamin solution A:  10 mg of riboflavin, 10 mg of thiamine hydrochloride, 100 µg of biotin, and 20 mg of niacin in 0.02 N acetic acid to make 400 mL. Store under toluene, protected from light, in a refrigerator.
Vitamin solution B:  20 mg of p-aminobenzoic acid, 10 mg of calcium pantothenate, 40 mg of pyridoxine hydrochloride, 40 mg of pyridoxal hydrochloride, 8 mg of pyridoxamine dihydrochloride, and 2 mg of folic acid in a mixture of water and neutralized alcohol (3:1) to make 400 mL. Store, protected from light, in a refrigerator.
Basal medium stock solution:  Prepare the medium according to the following formula and directions. A dehydrated mixture containing the same ingredients may be used provided that, when constituted as directed in the labeling, it yields a medium comparable to that obtained from the formula given herein.
Add the ingredients in the order listed in Table 2, carefully dissolving cystine and tryptophan in the hydrochloric acid before adding the next eight solutions in the resulting solution. Add 100 mL of water, and dissolve dextrose, sodium acetate, and ascorbic acid. Filter, if necessary, add the Polysorbate 80 solution, adjust with 1 N sodium hydroxide to a pH of 5.5–6.0, and add Purified Water to make 250 mL.
Table 2
l-Cystine 0.1 g
l-Tryptophan 0.05 g
1 N Hydrochloric acid 10 mL
Adenine–guanine–uracil solution 5 mL
Xanthine solution 5 mL
Vitamin solution A 10 mL
Vitamin solution B 10 mL
Salt solution A 5 mL
Salt solution B 5 mL
Asparagine solution 5 mL
Acid-hydrolyzed casein solution 25 mL
Dextrose, anhydrous 10 g
Sodium acetate, anhydrous 5 g
Ascorbic acid 1 g
Polysorbate 80 solution 5 mL
Tomato juice preparation:  Centrifuge commercially canned tomato juice so that most of the pulp is removed. Suspend 5 g/L of analytical filter-aid in the supernatant, and filter, with the aid of reduced pressure, through a layer of the filter-aid. Repeat, if necessary, until a clear, straw-colored filtrate is obtained. Store under toluene in a refrigerator.
Culture medium 
[Note—A dehydrated mixture containing the same ingredients may be used provided that, when constituted as directed in the labeling, it yields a medium equivalent to that obtained from the formula given herein. ]
Dissolve 0.75 g of yeast extract, 0.75 g of dried peptone, 1.0 g of anhydrous dextrose, and 0.20 g of monobasic potassium phosphate in 60–70 mL of water. Add 10 mL of Tomato juice preparation and 1 mL of Polysorbate 80 solution. Adjust with 1 N sodium hydroxide to a pH of 6.8, and add water to make 100 mL. Place 10-mL portions of the solution in test tubes, and plug with cotton. Sterilize the tubes and contents in an autoclave at 121 for 15 min. Cool as rapidly as possible to avoid color formation resulting from overheating the medium.
Suspension medium:  Dilute a measured volume of Basal medium stock solution with an equal volume of water. Place 10-mL portions of the diluted medium in test tubes. Sterilize, and cool as directed for Culture medium.
Stock culture of Lactobacillus leichmannii To 100 mL of Culture medium add 1.0–1.5 g of agar, and heat the mixture on a steam bath, with stirring, until the agar dissolves. Place 10-mL portions of the hot solution in test tubes, cover the tubes, sterilize at 121 for 15 min in an autoclave, and allow the tubes to cool in an upright position. Inoculate three or more of the tubes by stab transfer of a pure culture of Lactobacillus leichmannii.3 [Note—Before first using a fresh culture in this assay, make NLT 10 successive transfers of the culture in a 2-week period. ]
Incubate for 16–24 h at a temperature between 30 and 40 held constant to within ±0.5. Store in a refrigerator.
Prepare fresh stab cultures at least three times each week, and do not use them for preparing the Inoculum if more than 4 days old. The activity of the microorganism can be increased by daily or twice-daily transfer of the stab culture to the point where definite turbidity in the liquid Inoculum can be observed 2–4 h after inoculation. A slow-growing culture seldom gives a suitable response curve and may lead to erratic results.
Inoculum:  [Note—A frozen suspension of Lactobacillus leichmannii may be used as the stock culture, provided it yields an Inoculum comparable to a fresh culture. ]
Transfer cells from the Stock culture of Lactobacillus leichmannii to two sterile tubes containing 10 mL of the Culture medium each. Incubate these cultures for 16–24 h at a temperature between 30 and 40 held constant to within ± 0.5. Under aseptic conditions centrifuge the cultures, and decant the supernatant. Suspend the cells from the culture in 5 mL of Suspension medium, and combine. Using Suspension medium, adjust the volume so that a 1-in-20 dilution in saline TS produces 70% transmittance when read on a suitable spectrophotometer that has been set at a wavelength of 530 nm, equipped with a 10-mm cell, and read against saline TS set at 100% transmittance. Prepare a 1-in-400 dilution of the adjusted suspension using sterile Basal medium stock solution. The cell suspension so obtained is the Inoculum. [Note—This dilution may be altered, when necessary, to obtain the desired test response. ]
Calibration of spectrophotometer:  Check the wavelength of the spectrophotometer periodically, using a standard wavelength cell or other suitable device. Before reading any tests, calibrate the spectrophotometer for 0% and 100% transmittance, using water and with the wavelength set at 530 nm.
Analysis:  Because of the high sensitivity of the test organism to minimum amounts of vitamin B12 activity and to traces of many cleansing agents, cleanse meticulously by suitable means, followed preferably by heating at 250 for 2 h, using hard-glass 20-mm × 150-mm test tubes, and other necessary glassware.
To separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. To each of these tubes and to four similar empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.
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 tube add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL. Place one complete set of Standard and sample tubes together in one tube rack and the duplicate set in a second rack or section of a rack, preferably in random order.
Cover the tubes to prevent bacterial contamination, and sterilize in an autoclave at 121 for 5 min, arranging to reach this temperature in NMT 10 min by preheating the autoclave if necessary. Cool as rapidly as possible to avoid color formation resulting from overheating the medium. Take precautions to maintain uniformity of sterilizing and cooling conditions throughout the assay, because packing the tubes too closely in the autoclave or overloading it may cause variation in the heating rate.
Aseptically add 0.5 mL of Inoculum to each tube so prepared, except two of the four containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature between 30 and 40, held constant to within ± 0.5 for 16–24 h.
Terminate growth by heating to NLT 80 for 5 min. Cool to room temperature. After agitating its contents, read the transmittance at 530 nm when a steady state is reached. This steady state is observed a few seconds after agitation when the reading remains constant for 30 s or more. Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 100% for the uninoculated blank, read the transmittance of the inoculated blank. If the difference is greater than 5% or if there is evidence of contamination with a foreign microorganism, disregard the results of the assay.
With the transmittance set at 100% for the uninoculated blank, read the transmittance of each of the remaining tubes. Disregard the results of the assay if the slope of the standard curve indicates a problem with sensitivity.
Calculation:  Prepare a standard concentration-response curve by the following procedure. Test for and replace any aberrant individual transmittances. For each level of the Standard, calculate the response from the sum of the duplicate values of the transmittances (SS) as the difference, y = 2.00 SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
Calculate the response, y = 2.00 SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution corresponding to each of those values of y that falls within the range of the lowest and highest points plotted for the standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the Sample solution to obtain the difference, x, for each dosage level. Average the values of x for each of three or more dosage levels to obtain x, which equals the log-relative potency, M¢, of the Sample solution. Determine the quantity, in µg, of cyanocobalamin (C63H88CoN14O14P), in the portion of Oral Solution taken:
antilog M = antilog (M¢ + log R)
R== number of µg of cyanocobalamin assumed to be present in the portion of Oral Solution taken
Calculate the percentage of the labeled amount of cyanocobalamin in the portion of the Oral Solution taken:
Result = [(antilog M)/N] x 100
N== nominal amount of cyanocobalamin in the portion of Oral Solution taken
Replication:  Repeat the entire determination at least once, using separately prepared Sample solutions. If the difference between the two log-potencies M is NMT 0.08, their mean, M, is the assayed log-potency of the test material (see Design and Analysis of Biological Assays 111, Vitamin B12 Activity). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
Acceptance criteria:  90.0%–450.0% of the labeled amount of cyanocobalamin (C63H88CoN14O14P)
•  Calcium Pantothenate, Method 1
Mobile phase:  Methanol and 0.2 M monobasic sodium phosphate (3:97). Adjust with 1.7 M phosphoric acid to a pH of 3.2 ± 0.1.
Standard solution:  80 µg/mL of USP Calcium Pantothenate RS in Mobile phase
System suitability solution:  80 µg/mL of USP Racemic Panthenol RS in Mobile phase. Mix the resulting solution and Standard solution (1:1).
Sample solution:  Equivalent to 80 µg/mL of calcium pantothenate from the Oral Solution in Mobile phase
Chromatographic system 
Mode:  LC
Detector:  UV 210 nm
Column:  4.0-mm × 10-cm; packing L1
Flow rate:  1 mL/min
Injection size:  20 µL
System suitability 
Samples:  Standard solution and System suitability solution
Suitability requirements 
Resolution:  NLT 1.5 between panthenol and calcium pantothenate, System suitability solution
Tailing factor:  NMT 2.0 for both the calcium pantothenate and the panthenol peaks, Standard solution
Relative standard deviation:  NMT 2.0%, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Measure the peak areas of calcium pantothenate. Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of calcium pantothenate from the Sample solution
rS== peak area of calcium pantothenate from the Standard solution
CS== concentration of USP Calcium Pantothenate RS in the Standard solution (mg/mL)
CU== nominal concentration of calcium pantothenate in the Sample solution (mg/mL)
Acceptance criteria:  90.0%–150.0% of the labeled amount of calcium pantothenate (C18H32CaN2O10)
•  Calcium Pantothenate, Method 2
Standard stock solution:  Dissolve 50 mg of USP Calcium Pantothenate RS, previously dried and stored in the dark over phosphorus pentoxide and protected from absorption of moisture while weighing, in 500 mL of water in a 1000-mL volumetric flask. Add 10 mL of 0.2 N acetic acid and 100 mL of sodium acetate solution (1 in 60), and dilute with water to volume to obtain a concentration of 50 µg/mL of USP Calcium Pantothenate RS. Store under toluene in a refrigerator.
Standard solution:  On the day of the assay, dilute a volume of Standard stock solution with water to obtain a concentration of 0.01–0.04 µg/mL of calcium pantothenate, the exact concentration being such that the responses obtained as directed for Analysis, 2.0 and 4.0 mL of the Standard solution being used, are within the linear portion of the log-concentration response curve.
Sample solution:  Transfer an accurately measured volume of Oral Solution equivalent to 50 mg of calcium pantothenate to a 1000-mL volumetric flask containing 500 mL of water. Add 10 mL of 0.2 N acetic acid and 100 mL of sodium acetate solution (1 in 60), dilute with water to volume, and filter. Dilute a measured volume of this solution quantitatively, and stepwise if necessary, with water to obtain a solution having about the same concentration as that of the Standard solution.
Acid-hydrolyzed casein solution:  Mix 100 g of vitamin-free casein with 500 mL of 6 N hydrochloric acid, and reflux the mixture for 8–12 h. Remove the hydrochloric acid from the mixture by distillation under reduced pressure until a thick paste remains. Redissolve the resulting paste in water, adjust the solution with 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 h, and filter. Repeat the treatment with activated charcoal. Store under toluene in a cool place at a temperature not below 10. Filter the solution if a precipitate forms during storage.
Cystine–tryptophan solution:  Suspend 4.0 g of l-cystine in a solution of 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700–800 mL of water, heat to 70–80, and add dilute hydrochloric acid (1 in 2) dropwise, with stirring, until the solids are dissolved. Cool, and add water to make 1000 mL. Store under toluene in a cool place at a temperature not below 10.
Adenine–guanine–uracil solution:  Dissolve 200 mg each of adenine sulfate, guanine hydrochloride, and uracil, with the aid of heat, in 10 mL of 4 N hydrochloric acid. Cool, and add water to make 200 mL. Store under toluene in a refrigerator.
Polysorbate 80 solution:  100 mg/mL of polysorbate 80 in alcohol
Riboflavin–thiamine hydrochloride–biotin solution:  20 µg/mL of riboflavin, 10 µg/mL of thiamine hydrochloride, and 0.04 µg/mL of biotin in 0.02 N acetic acid. Store under toluene, protected from light, in a refrigerator.
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution:  10 µg/mL of p-aminobenzoic acid, 50 µg/mL of niacin, and 40 µg/mL of pyridoxine hydrochloride in a mixture of neutralized alcohol and water (1:3). Store in a refrigerator.
Salt solution A:  Dissolve 25 g of monobasic potassium phosphate and 25 g of dibasic potassium phosphate in water to make 500 mL. Add 5 drops of hydrochloric acid. Store under toluene.
Salt solution B:  Dissolve 10 g of magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese sulfate in water to make 500 mL. Add 5 drops of hydrochloric acid. Store under toluene.
Basal medium stock solution:  Dissolve the anhydrous dextrose and anhydrous sodium acetate in the solutions previously mixed according to Table 3, and adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute with water to 250 mL.
Table 3
Acid-hydrolyzed casein solution 25 mL
Cystine–tryptophan solution 25 mL
Polysorbate 80 solution 0.25 mL
Dextrose, anhydrous 10 g
Sodium acetate, anhydrous 5 g
Adenine–guanine–uracil solution 5 mL
Riboflavin–thiamine hydrochloride–biotin solution 5 mL
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution 5 mL
Salt solution A 5 mL
Salt solution B 5 mL
Stock culture of Lactobacillus plantarum Dissolve 2.0 g of yeast extract in 100 mL of water; add 500 mg of anhydrous dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g of agar; and heat the mixture on a steam bath, with stirring, until the agar dissolves. Add 10-mL portions of the hot solution to the test tubes, close or cover the tubes, sterilize in an autoclave at 121 for 15 min, and allow the tubes to cool in an upright position. Prepare stab cultures in three or more of the tubes, using a pure culture of Lactobacillus plantarum1, incubating for 16–24 h at a temperature between 30 and 37 held constant to within ± 0.5. Store in a refrigerator. Prepare a fresh stab of the stock culture every week, and do not use for Inoculum if the culture is more than 1 week old.
Culture medium:  To each of a series of test tubes containing 5.0 mL of Basal medium stock solution, add 5.0 mL of water containing 0.2 µg of calcium pantothenate. Plug the tubes with cotton, sterilize in an autoclave at 121 for 15 min, and cool.
Inoculum:  [Note—A frozen suspension of Lactobacillus plantarum may be used as the stock culture, provided it yields an Inoculum comparable to a fresh culture. ]
Transfer cells from the Stock culture of Lactobacillus plantarum to a sterile tube containing 10 mL of Culture medium. Incubate this culture for 16–24 h at a temperature between 30 and 37 held constant to within ± 0.5. The cell suspension so obtained is the Inoculum.
Analysis:  To similar separate test tubes add, in 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 empty tubes, add 5.0 mL of Basal medium stock solution and sufficient water to make 10 mL.
To similar separate test tubes add, in duplicate, volumes of the Sample solution corresponding to three or more of the levels specified for the Standard solution, including the levels of 2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL of the Basal medium stock solution and sufficient water to make 10 mL. Place one complete set of Standard and sample tubes together in one tube rack and the duplicate set in a second rack or section of a rack, preferably in random order.
Cover the tubes of both series to prevent contamination, and sterilize in an autoclave at 121 for 5 min. Cool, add 1 drop of Inoculum to each tube, except two of the four tubes containing no Standard solution (the uninoculated blanks). Incubate the tubes at a temperature between 30 and 37, held constant to within ± 0.5 until, following 16–24 h of incubation, there has been no substantial increase in turbidity in the tubes containing the highest level of Standard during a 2-h period.
Determine the transmittance of the tubes in the following manner. Mix the contents of each tube, and transfer to an optical container if necessary. Read the transmittance between 540 and 660 nm when a steady state is reached. This steady state is observed a few seconds after agitation when the galvanometer reading remains constant for 30 s or more. Allow approximately the same time interval for the reading on each tube.
With the transmittance set at 1.00 for the uninoculated blank, read the transmittance of the inoculated blank. With the transmittance set at 1.00 for the inoculated blank, read the transmittance for each of the remaining tubes. If there is evidence of contamination with a foreign microorganism, disregard the result of the assay.
Calculation:  Prepare a standard concentration-response curve as follows. For each level of the Standard, calculate the response from the sum of the duplicate values of the transmittance (SS) as the difference, y = 2.00 SS. Plot this response on the ordinate of cross-section paper against the logarithm of the mL of Standard solution per tube on the abscissa, using for the ordinate either an arithmetic or a logarithmic scale, whichever gives the better approximation to a straight line. Draw the straight line or smooth curve that best fits the plotted points.
Calculate the response, y = 2.00 SU, adding together the two transmittances (SU) for each level of the Sample solution. Read from the standard curve the logarithm of the volume of the Standard solution corresponding to each of those values of y that fall within the range of the lowest and highest points plotted for the Standard. Subtract from each logarithm so obtained the logarithm of the volume, in mL, of the Sample solution to obtain the difference, x, for each dosage level. Average the values of x for each of three or more dosage levels to obtain x, which equals the log-relative potency, M¢, of the Sample solution. Determine the quantity, in mg, of USP Calcium Pantothenate RS corresponding to the calcium pantothenate (C18H32CaN2O10) in the portion of Oral Solution taken:
antilog M = antilog (M¢ + log R)
R== number of mg of calcium pantothenate assumed to be present in the portion of Oral Solution taken
Calculate the percentage of the labeled amount of calcium pantothenate (C18H32CaN2O10) in the portion of Oral Solution taken:
Result = [(antilog M)/N] × 100
N== nominal amount of calcium pantothenate in the portion of Oral Solution (mg)
Replication:  Repeat the entire determination at least once, using separately prepared Sample solutions. If the difference between the two log-potencies M is NMT 0.08, their mean, M, is the assayed log-potency of the test material (see Design and Analysis of Biological Assays 111, Confidence Interval and Limits of Potency). If the two determinations differ by more than 0.08, conduct one or more additional determinations. From the mean of two or more values of M that do not differ by more than 0.15, compute the mean potency of the preparation under assay.
Acceptance criteria:  90.0%–150.0% of the labeled amount of calcium pantothenate (C18H32CaN2O10)
•  Dexpanthenol or Panthenol, Method 1
Mobile phase and Chromatographic system:  Proceed as directed in the assay for Calcium Pantothenate, Method 1.
Standard solution:  80 µg/mL of USP Dexpanthenol RS or USP Racemic Panthenol RS in Mobile phase. [Note—Use USP Dexpanthenol RS to analyze Oral Solution that contains dexpanthenol and USP Racemic Panthenol RS to analyze Oral Solution that contains panthenol. ]
System suitability solution:  80 µg/mL of USP Calcium Pantothenate RS in Mobile phase. Mix the resulting solution and Standard solution (1:1).
Sample solution:  Equivalent to 80 µg/mL of dexpanthenol or panthenol from the Oral Solution in Mobile phase
Analysis 
Samples:  Standard solution and Sample solution
Measure the peak areas of panthenol. Calculate the percentage of the labeled amount of dexpanthenol or panthenol (C9H19NO4) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of dexpanthenol or panthenol from the Sample solution
rS== peak area of dexpanthenol or panthenol from the Standard solution
CS== concentration of USP Dexpanthenol RS or USP Racemic Panthenol RS in the Standard solution (mg/mL)
CU== nominal concentration of panthenol or dexpanthenol in the Sample solution (mg/mL)
Acceptance criteria:  90.0%–150.0% of the labeled amount of dexpanthenol or panthenol (C9H19NO4)
•  Dexpanthenol or Panthenol, Method 2
[Note—The following procedure is applicable also to the determination of the dextrorotatory component of racemic panthenol in preparations containing panthenol. ]
Dehydrated mixtures yielding formulations similar to the media described herein may be used provided that, when constituted as directed, they have growth-promoting properties equal to or superior to those obtained with the media prepared as described herein.
Standard stock solution:  800 µg/mL of USP Dexpanthenol RS or 1600 µg/mL of USP Racemic Pantenol RS in water. Store in a refrigerator, protected from light, and use within 30 days. [Note—Use USP Dexpanthenol RS to analyze Oral Solution that contains dexpanthenol and USP Racemic Panthenol RS to analyze Oral Solution that contains panthenol. ]
Standard solution:  On the day of the assay, prepare 1.2 µg/mL of dexpanthenol or 2.4 µg/mL of racemic panthenol from the Standard stock solution with water.
Sample solution:  1.2 µg/mL of dexpanthenol or 2.4 µg/mL of panthenol from the Oral Solution in water
Acid-hydrolyzed casein solution:  Mix 100 g of vitamin-free casein with 500 mL of dilute hydrochloric acid (1 in 2), and reflux the mixture for 8–12 h. Remove the hydrochloric acid from the mixture by distillation under reduced pressure until a thick paste remains. Redissolve the resulting paste in about 500 mL of water, adjust the solution with 1 N sodium hydroxide to a pH of 3.5 ± 0.1, and add water to make 1000 mL. Add 20 g of activated charcoal, stir for 1 h, and filter. Repeat the treatment with activated charcoal. Store under toluene in a cool place at a temperature not below 10. Filter the solution if a precipitate forms during storage.
Cystine–tryptophan solution:  Suspend 4.0 g of l-cystine in a solution of 1.0 g of l-tryptophan (or 2.0 g of d,l-tryptophan) in 700–800 mL of water, heat to 75 ± 5, and add 6 M hydrochloric acid solution dropwise, with stirring, until the solids are dissolved. Cool, add water to make 1000 mL, and mix. Store under toluene in a cool place at a temperature not below 10.
Adenine–guanine–uracil solution:  Dissolve 200 mg each of adenine sulfate, guanine hydrochloride, and uracil, with the aid of heat, in 10 mL of 4 N hydrochloric acid. Cool, add water to make 200 mL, and mix. Store under toluene in a refrigerator.
Polysorbate 80 solution:  100 mg/mL of polysorbate 80 in alcohol
Riboflavin–thiamine hydrochloride–biotin solution:  Prepare a solution of riboflavin, thiamine hydrochloride, and biotin in 0.02 N acetic acid containing 20 µg/mL of riboflavin, 10 µg/mL of thiamine hydrochloride, and 0.04 µg/mL of biotin. Store under toluene, protected from light, in a refrigerator.
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution:  Prepare a solution in neutral 25% alcohol containing 10 µg/mL of p-aminobenzoic acid, 50 µg/mL of niacin, and 40 µg/mL of pyridoxine hydrochloride. Store in a refrigerator.
Salt solution A:  Dissolve 25 g of monobasic potassium phosphate and 25 g of dibasic potassium phosphate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Salt solution B:  Dissolve 10 g of magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese sulfate in water to make 500 mL. Add 5 drops of hydrochloric acid, and mix. Store under toluene.
Pyridoxal–calcium pantothenate solution:  Dissolve 40 mg of pyridoxal hydrochloride and 375 µg of calcium pantothenate in 10% alcohol to make 200 mL, and mix. Store in a refrigerator, and use within 30 days.
Polysorbate 40–oleic acid solution:  Dissolve 25 g of polysorbate 40 and 0.25 g of oleic acid in 20% alcohol to make 500 mL, and mix. Store in a refrigerator, and use within 30 days.
Modified pantothenate medium:  Dissolve anhydrous dextrose and sodium acetate in the solutions previously mixed according to Table 4, adjust with 1 N sodium hydroxide to a pH of 6.8, dilute with water to 250 mL, and mix.
Table 4
Acid-hydrolyzed casein solution 25 mL
Cystine–tryptophan solution 25 mL
Polysorbate 80 solution 0.25 mL
Dextrose, anhydrous 10 g
Sodium acetate, anhydrous 5 g
Adenine–guanine–uracil solution 5 mL
Riboflavin–thiamine hydrochloride–biotin solution 5 mL
p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solution 5 mL
Salt solution A 5 mL
Salt solution B 5 mL
Pyridoxal–calcium pantothenate solution 5 mL
Polysorbate 40–oleic acid solution 5 mL
Double-strength modified pantothenate medium:  Prepare as directed under Modified pantothenate medium, but make the final dilution to 125 mL instead of 250 mL. Prepare fresh.
Stock culture of Pediococcus acidilactici Dissolve 6.0 g of peptone, 4.0 g of pancreatic digest of casein, 3.0 g of yeast extract, 1.5 g of beef extract, 1.0 g of dextrose, and 15.0 g of agar in 800 mL of water, with the aid of heat. Adjust with 0.1 N sodium hydroxide or 0.1 N hydrochloric acid to a pH between 6.5 and 6.6, dilute with water to 1000 mL, and mix. Add 10-mL portions of the solution to culture tubes, place caps on the tubes, and sterilize in an autoclave at 121 for 15 min. Cool on a slant, and store in a refrigerator. Prepare a stock culture of Pediococcus acidilactici4 on a slant of this medium. Incubate at 35 for 20–24 h, and store in a refrigerator. Maintain the stock culture by monthly transfer onto fresh slants.
Inoculum:  Inoculate three 250-mL portions of sterile Modified pantothenate medium from a stock culture slant, and incubate at 35 for 20–24 h. Centrifuge the suspension from the combined portions, and wash the cells with sterile Modified pantothenate medium. Resuspend the cells in sufficient sterile Modified pantothenate medium so that a 1-in-50 dilution, when tested in a 13-mm diameter test tube, gives 80% light transmission at 530 nm. Transfer 1.2-mL portions of this stock suspension to sterile glass ampuls, seal, freeze in liquid nitrogen, and store in a freezer. On the day of the assay, allow the ampuls to reach room temperature, mix the contents, and dilute 1 mL of thawed culture with sterile saline TS to 150 mL.
[Note—This dilution may be altered when necessary to obtain the desired test response. ]
Analysis:  Prepare in triplicate a series of eight culture tubes by adding the following quantities of water to the tubes within a set: 5.0, 4.5, 4.0, 3.5, 3.0, 2.0, 1.0, and 0.0 mL. To these same tubes, and in the same order, add 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution.
Prepare in duplicate a series of five culture tubes by adding the following quantities of water to the tubes within a set: 4.0, 3.5, 3.0, 2.0, and 1.0 mL. To these same tubes, and in the same order, add 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Sample solution.
Add 5.0 mL of Double-strength modified pantothenate medium to each tube, and mix. Cover the tubes with metal caps, and sterilize in an autoclave at 121 for 5 min. Cool to room temperature in a chilled water bath, and inoculate each tube with 0.5 mL of the Inoculum. Allow to incubate at 37 for 16 h. Terminate growth by heating to a temperature not below 80, such as by steaming at atmospheric pressure in a sterilizer for 5 to 10 min. Cool, and concomitantly determine the percentage transmittance of the suspensions, in cells of equal pathlength, on a suitable spectrophotometer, at a wavelength of 530 nm.
Calculation:  Draw a dose-response curve on arithmetic graph paper by plotting the average response, in percent transmittance, for each set of tubes of the standard curve against the standard level concentrations. The curve is drawn by connecting each adjacent pair of points with a straight line. From this standard curve, determine by interpolation the potency, of each tube containing portions of the Sample solution. Divide the potency of each tube by the amount of the Sample solution added to it, to obtain the individual responses. Calculate the mean response by averaging the individual responses that vary from their mean by NMT 15%, using NLT half the total number of tubes. Calculate the potency of the portion of the Oral Solution taken for assay, by multiplying the mean response by the appropriate dilution factor. Calculate the percentage of the labeled amount of dexpanthenol or panthenol in the portion of Oral Solution taken:
Result = (P/N) × 100
P== calculated potency of dexpanthenol or panthenol in the portion of Oral Solution taken (mg)
N== nominal amount of dexpanthenol or panthenol in the portion of Oral Solution taken (mg)
Acceptance criteria:  90.0%–150.0% of the labeled amount of dexpanthenol or panthenol (C9H19NO4)
•  Niacin or Niacinamide
[Note—Use low-actinic glassware throughout this procedure. ]
Diluent:  25 mg/mL of edetate disodium in water
Mobile phase:  Mix 350 mL of methanol, 15.0 mL of glacial acetic acid, 0.4 mL of triethylamine, and dilute with 0.008 M sodium 1-hexanesulfonate to 2000 mL.
Standard solution:  0.10 mg/mL of USP Niacin RS or USP Niacinamide RS in Diluent. [Note—Use USP Niacin RS for Oral Solution that contains niacin and USP Niacinamide RS for Oral Solution that contains niacinamide. ]
Sample solution:  Dilute an accurately measured volume of Oral Solution with Diluent to obtain a solution with a concentration of 0.1 mg/mL of niacin or niacinamide.
Chromatographic system 
Mode:  LC
Detector:  UV 270 nm
Column:  4.6-mm × 25-cm; packing L7
Flow rate:  2 mL/min
Injection size:  5 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of the labeled amount of niacin (C6H5NO2) or niacinamide (C6H6N2O) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of niacin or niacinamide from the Sample solution
rS== peak area of niacin or niacinamide from the Standard solution
CS== concentration of USP Niacin RS or USP Niacinamide RS in the Standard solution (mg/mL)
CU== nominal concentration of niacin or niacinamide in the Sample solution (mg/mL)
Acceptance criteria:  90.0%–150.0% the labeled amount of niacin (C6H5NO2) or niacinamide (C6H6N2O)
•  Pyridoxine Hydrochloride
Diluent, Mobile phase, and Chromatographic system:  Proceed as directed for Niacin or Niacinamide.
Standard solution:  24 µg/mL of USP Pyridoxine Hydrochloride RS in Diluent
Sample solution:  Equivalent to 24 µg/mL of Pyridoxine Hydrochloride from Oral Solution in Diluent
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of pyridoxine hydrochloride from the Sample solution
rS== peak area of pyridoxine hydrochloride from the Standard solution
CS== concentration of USP Pyridoxine Hydrochloride RS in the Standard solution (mg/mL)
CU== nominal concentration of pyridoxine hydrochloride in the Sample solution (mg/mL)
Acceptance criteria:  90.0%–150.0% of the labeled amount of pyridoxine hydrochloride (C8H11NO3·HCl)
•  Riboflavin-5¢-Phosphate Sodium, Method 1
[Note—Riboflavin-5¢-phosphate sodium is quantitated against USP Riboflavin RS in this procedure. In the chromatogram of the Sample solution, the riboflavin-5¢-phosphate peak is the only peak measured for calculation. ]
Diluent, Mobile phase, and Chromatographic system:  Proceed as directed for Niacin or Niacinamide.
Standard solution:  8 µg/mL of USP Riboflavin RS in Diluent, by heating if necessary.
Sample solution:  Equivalent to 8 µg/mL of Riboflavin from the Oral Solution in Diluent
Analysis 
Samples:  Standard solution and Sample solution
[Note—The relative retention times for riboflavin-5¢-phosphate and riboflavin are about 0.18 and 1.0, respectively. ]
Calculate the percentage of the labeled amount of riboflavin (C17H20N4O6) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × F × 100
rU== peak area of riboflavin-5¢-phosphate from the Sample solution
rS== peak area of riboflavin from the Standard solution
CS== concentration of USP Riboflavin RS in the Standard solution (mg/mL)
CU== nominal concentration of riboflavin in the Sample solution (mg/mL)
F== factor for converting the response obtained for riboflavin-5¢-phosphate to riboflavin, 1.493 [Note—Riboflavin phosphate sodium is a mixture of isomeric monophosphates and diphosphates containing an average amount of 67% of riboflavin-5¢-monophosphate, which separates in this chromatographic system. The factor 1.493 assumes 67% of riboflavin-5¢-monophosphate. ]
Acceptance criteria:  90.0%–150.0% of the labeled amount of riboflavin (C17H20N4O6)
•  Riboflavin or Riboflavin-5¢-Phosphate Sodium, Method 2
[Note—Use low-actinic glassware throughout this procedure. ]
Solvent blank:  1 N hydrochloric acid, 2.5 M sodium acetate, and water (1:2:97)
Standard stock solution:  Transfer 0.16 mg/mL of USP Riboflavin RS in 1 N hydrochloric acid, 2.5 M sodium acetate, and water (1:2:97). Mix the resulting solution and water (1:9).
Standard solution:  Transfer 5.0 mL of the Standard stock solution to a 500-mL volumetric flask, add 5.0 mL of 1 N hydrochloric acid, 10.0 mL of 2.5 M sodium acetate, and dilute with water to volume.
Sample solution:  Transfer an accurately measured volume of Oral Solution, equivalent to 0.8 mg of riboflavin to a 100-mL volumetric flask, and dilute with water to volume. Transfer 10.0 mL of the resulting solution to a 500-mL volumetric flask, add 5.0 mL of 1 N hydrochloric acid, 10.0 mL of 2.5 M sodium acetate, and dilute with water to volume.
Spectrometric conditions 
Mode:  Fluorescence
Analytical wavelength 
Excitation:  440 nm
Emission:  530 nm
Blank:  Solvent blank
Analysis 
Samples:  Standard solution and Sample solution
Determine the maximum fluorescence intensities, IS and IU, of the Standard solution and the Sample solution, respectively. Calculate the percentage of the labeled amount of riboflavin (C17H20N4O6) in the portion of Oral Solution taken:
Result= (IU/IS) × (CS/CU) × 100
IU== fluorescence intensity from the Sample solution
IS== fluorescence intensity from the Standard solution
CS== concentration of USP Riboflavin RS in the Standard solution (mg/mL)
CU== nominal concentration of riboflavin in the Sample solution (mg/mL)
Acceptance criteria:  90.0%–150.0% of the labeled amount of riboflavin (C17H20N4O6)
•  Thiamine
Diluent, Mobile phase, and Chromatographic system:  Proceed as directed for Niacin or Niacinamide.
Standard solution:  24 µg/mL of USP Thiamine Hydrochloride RS in Diluent
Sample solution:  Equivalent to 24 µg/mL of thiamine hydrochloride or thiamine mononitrate from Oral Solution in Diluent
Analysis 
Samples:  Standard solution and Sample solution
Measure the areas of the major peaks. Calculate the percentage of the labeled amount of thiamine hydrochloride (C12H17ClN4OS·HCl) or thiamine mononitrate (C12H17N5O4S) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × F × 100
rU== peak area of thiamine from the Sample solution
rS== peak area of thiamine from the Standard solution
CS== concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL)
CU== nominal concentration of thiamine hydrochloride or thiamine mononitrate in the Sample solution (mg/mL)
F== 1 (for products containing thiamine hydrochloride) and 0.97 (for products containing thiamine mononitrate)
Acceptance criteria:  90.0%–200.0% of the labeled amount of thiamine hydrochloride (C12H17ClN4OS·HCl) or thiamine mononitrate (C12H17N5O4S)
[Note—Commercially available atomic absorption standard solutions for the minerals, where applicable, may be used where preparation of a standard stock solution is described in the following assays. Use deionized water where water is specified. Where atomic absorption spectrophotometry is specified in the assay, the Standard solutions and the Sample solution may be diluted quantitatively with the solvent specified, if necessary, to yield solutions of suitable concentrations adaptable to the linear or working range of the instrument. ]
•  Chromium
Chromium standard solution:  1000 µg/mL of chromium from potassium dichromate, previously dried at 120 for 4 h in water. Store in a polyethylene bottle.
Standard stock solution:  10 µg/mL of chromium from Chromium standard solution diluted with 6 N hydrochloric acid and water (1 in 20)
Standard solutions:  Transfer 10.0 mL and 20.0 mL of the Standard stock solution to separate 100-mL volumetric flasks, and transfer 15.0 mL and 20.0 mL of the Standard stock solution to separate 50-mL volumetric flasks. Dilute the contents of each of the four flasks with 0.125 N hydrochloric acid to volume to obtain concentrations of 1.0, 2.0, 3.0, and 4.0 µg/mL of chromium.
Sample solution:  Dilute an accurately measured volume of Oral Solution to obtain a solution equivalent to 1 µg/mL of chromium in 0.125 N hydrochloric acid.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometer
Lamp:  Chromium hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  357.9 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions, against the blank. Plot the absorbances of the Standard solutions versus concentration, in µg/mL, of chromium, and draw the straight line best fitting the four plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of chromium in the Sample solution. Calculate the percentage of the labeled amount of chromium (Cr) in the portion of Oral Solution taken:
Result = (C/CU) × 100
C== concentration of chromium in the Standard solution (µg/mL)
CU== nominal concentration of chromium in the Sample solution, (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of chromium (Cr)
•  Fluoride
[Note—Use plastic containers throughout this procedure. ]
Ascorbic acid solution:  70 mg/mL of ascorbic acid in water
Mobile phase:  Alcohol, water, and 1 N sulfuric acid (50:449:1)
Standard stock solution:  220 µg/mL of USP Sodium Fluoride RS in water. This solution contains 100 µg/mL of fluoride.
Standard solution:  Transfer 5.0 mL of Standard stock solution to a 100-mL volumetric flask. Add 2 mL of Ascorbic acid solution, 10 mL of alcohol, and about 70 mL of water, and mix. Adjust with 1 N sodium hydroxide to a pH of 4.25 ± 0.05. Dilute with water to volume, and mix to obtain 5 µg/mL of fluoride solution.
Sample solution:  Transfer an accurately measured volume of the Oral Solution, equivalent to 0.5 mg of fluoride, to a 100-mL volumetric flask. Add 1 drop of hydrochloric acid, 10 mL of alcohol, and about 75 mL of water, and mix. Adjust with 1 N sodium hydroxide to a pH of 4.25 ± 0.05. Dilute with water to volume.
Chromatographic system 
Mode:  LC
Detector:  Conductivity
Guard column:  4.6-mm × 3-cm; packing L17
Analytical column:  7.8-mm × 30-cm; packing L17
Flow rate:  0.6 mL/min
Injection size:  100 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Relative standard deviation:  NMT 2.0%
Analysis 
Samples:  Standard solution and Sample solution
Measure the peak areas of fluoride. Calculate the percentage of the labeled amount of fluorine (F) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of fluoride from the Sample solution
rS== peak area of fluoride from the Standard solution
CS== concentration of fluoride in the Standard solution (µg/mL)
CU== nominal concentration of fluorine in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of fluorine (F)
•  Iodide, Method 1
Mobile phase:  Dissolve 5.15 g of tetrabutylammonium bromide in 320 mL of acetonitrile. Dilute with water to 2000 mL.
Standard stock solution:  1.3 mg/mL of potassium iodide in Mobile phase. This solution has a concentration of 1 mg/mL of iodide.
Standard solution:  2.5 µg/mL of iodide from the Standard stock solution in Mobile phase
System suitability solution:  Transfer 0.13 g of potassium iodide and 0.5 g of potassium iodate to a 100-mL volumetric flask. Dissolve in Mobile phase, using sonication if necessary, dilute with Mobile phase to volume, and mix. Transfer 1.0 mL of this solution to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix. Transfer 25.0 mL of this solution to a 100-mL volumetric flask, and dilute with Mobile phase to volume.
Sample solution:  Dilute an accurately measured volume of the Oral Solution to obtain a solution with a concentration of 2.5 µg/mL of iodine in Mobile phase
Chromatographic system 
Mode:  LC
Detector:  UV 225 nm
Column:  4.6-mm × 15-cm; packing L1
Flow rate:  1.5 mL/min
Injection size:  30 µL
System suitability 
Samples:  Standard solution and System suitability solution
[Note—The relative retention times for iodate and iodide are about 0.32 and 1.0, respectively. ]
Suitability requirements 
Resolution:  NLT 2.5 between iodate and iodide, System suitability solution
Relative standard deviation:  NMT 2.0% for the iodide peak, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of the labeled amount of iodine (I) in the portion of Oral Solution taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of iodide from the Sample solution
rS== peak area of iodide from the Standard solution
CS== concentration of iodide in the Standard solution (µg/mL)
CU== nominal concentration of iodine in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of iodine (I)
•  Iodide, Method 2: Proceed as directed for Automated Methods of Analysis 16, Assay for Iodide.
•  Iron
Iron standard stock solution:  Transfer 100 mg of iron powder to a 1000-mL volumetric flask, dissolve in 6 N hydrochloric acid, and dilute with water to volume.
Standard solutions:  To separate 100-mL volumetric flasks, transfer 2.0, 4.0, 5.0, 6.0, and 8.0 mL of Iron standard stock solution. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain concentrations of 2.0, 4.0, 5.0, 6.0, and 8.0 µg/mL of iron.
Sample solution:  6 µg/mL of iron from the Oral Solution in 0.125 N hydrochloric acid
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometer
Lamp:  Iron hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  248.3 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions, against the blank. Plot the absorbances of the Standard solutions versus concentration, in µg/mL, of iron, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of iron in the Sample solution. Calculate the percentage of the labeled amount of iron (Fe) in the portion of Oral Solution taken:
Result = (C/CU) × 100
C== concentration of iron in the Sample solution from the graph (µg/mL)
CU== nominal concentration of iron in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of iron (Fe)
•  Magnesium
Magnesium standard solution:  Transfer 1.00 g of magnesium ribbon to a 1000-mL volumetric flask. Dissolve in 50 mL of 6 N hydrochloric acid, and dilute with water to volume.
Standard stock solution:  20 µg/mL of magnesium from Magnesium standard solution in 0.125 N hydrochloric acid
Standard solutions:  To separate 100-mL volumetric flasks transfer 5.0, 7.5, 10.0, 12.5, and 15.0 mL of Standard stock solution. Dilute with 0.125 N hydrochloric acid to volume to obtain concentrations of 1.0, 1.5, 2.0, 2.5, and 3.0 µg/mL of magnesium.
Sample solution:  2.5 µg/mL of magnesium from the Oral Solution in 0.125 N hydrochloric acid
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometer
Lamp:  Magnesium hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  285.2 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions, against the Blank. Plot the absorbances of the Standard solutions versus concentration, in µg/mL, of magnesium, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of magnesium in the Sample solution. Calculate the percentage of the labeled amount of magnesium (Mg) in the portion of Oral Solution taken:
Result = (C/CU) × 100
C== concentration of magnesium in the Sample solution from the graph (µg/mL)
CU== nominal concentration of magnesium in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of magnesium (Mg)
•  Manganese
Manganese standard solution:  Transfer 1.0 g of manganese to a 1000-mL volumetric flask. Dissolve in 20 mL of nitric acid, and dilute with 6 N hydrochloric acid to volume.
Standard stock solution:  50 µg/mL of manganese from the Manganese standard stock solution in 0.125 N hydrochloric acid
Standard solutions:  To separate 100-mL volumetric 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 0.125 N hydrochloric acid to volume to obtain solutions having known concentrations of 0.5, 0.75, 1.0, 1.5, and 2.0 µg/mL of manganese.
Sample solution:  Dilute an accurately measured volume of the Oral Solution to obtain 1.5 µg/mL of manganese in 0.125 N hydrochloric acid.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometer
Lamp:  Manganese hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  279.5 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions, against the Blank. Plot the absorbances of the Standard solutions versus concentration, in µg/mL, of manganese, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of manganese in the Sample solution. Calculate the percentage of the labeled amount of manganese (Mn) in the portion of Oral Solution taken:
Result = (C/CU) × 100
C== concentration of manganese in the Sample solution from the graph (µg/mL)
CU== nominal concentration of manganese in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of manganese (Mn)
•  Molybdenum
Molybdenum standard solution:  Transfer 1.0 g of molybdenum wire to a 1000-mL volumetric flask, and dissolve in 50 mL of nitric acid, warming if necessary. Dilute with water to volume.
Standard stock solution:  100 µg/mL of molybdenum from the Molybdenum standard solution in water
Standard solutions:  To separate 100-mL volumetric flasks transfer 0.5, 1.0, 1.5, 2.0 and 3.0 mL of the Standard stock solution. Add 0.125 N hydrochloric acid to volume, and mix to obtain the solutions having known concentrations of about 0.5, 1.0, 1.5, 2.0, and 3.0 µg/mL of molybdenum.
Sample solution:  Dilute an accurately measured volume of the Oral Solution to obtain 1 µg/mL of molybdenum from the Oral Solution in 0.125 N hydrochloric acid.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometer
Lamp:  Molybdenum hollow-cathode
Flame:  Nitrous oxide–acetylene
Analytical wavelength:  313.3 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions, against the Blank. Plot the absorbances of the Standard solutions versus concentration, in µg/mL, of molybdenum, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of molybdenum in the Sample solution. Calculate the percentage of the labeled amount of molybdenum (Mo) in the portion of Oral Solution taken:
Result = (C/CU) × 100
C== concentration of molybdenum in the Sample solution from the graph (µg/mL)
CU== nominal concentration of molybdenum in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–160.0% of the labeled amount of molybdenum (Mo)
•  Zinc
Zinc standard solution:  Transfer 311 mg of zinc oxide to a 250-mL volumetric flask, and add 80 mL of 6 N hydrochloric acid, warming if necessary to dissolve. Cool, dilute with water to volume, and mix to obtain a solution having a known concentration of 1000 µg/mL of zinc.
Standard stock solution:  50 µg/mL of zinc from the Zinc standard solution in 0.125 N hydrochloric acid
Standard solutions:  Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL of Standard stock solution to separate 100-mL volumetric flasks. Dilute the contents of each flask with 0.125 N hydrochloric acid to volume to obtain concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of zinc.
Sample solution:  Dilute an accurately measured volume of the Oral Solution to obtain 1 µg/mL of zinc in 0.125 N hydrochloric acid.
Spectrometric conditions 
Mode:  Atomic absorption spectrophotometer
Lamp:  Zinc hollow-cathode
Flame:  Air–acetylene
Analytical wavelength:  213.8 nm
Blank:  0.125 N hydrochloric acid
Analysis 
Samples:  Standard solutions and Sample solution
Determine the absorbances of the solutions, against the Blank. Plot the absorbances of the Standard solutions versus concentration, in µg/mL, of zinc, and draw the straight line best fitting the five plotted points. From the graph so obtained, determine the concentration, C, in µg/mL, of zinc in the Sample solution. Calculate the percentage of the labeled amount of zinc (Zn) in the portion of Oral Solution taken:
Result = (C/CU) × 100
C== concentration of zinc in the Sample solution from the graph (µg/mL)
CU== nominal concentration of zinc in the Sample solution (µg/mL)
Acceptance criteria:  90.0%–125.0% of the labeled amount of zinc (Zn)
OTHER COMPONENTS
•  Alcohol Determination, Method I 611 (if present): 90.0%–120.0% of the labeled amount of C2H5OH
CONTAMINANTS
•  Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 3000 cfu/mL, and the combined molds and yeasts count does not exceed 300 cfu/mL.
•  Microbiological Procedures for Absence of Specified Microorganisms 2022: Meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers, under an inert gas or with a minimum of headspace.
•  Labeling:5 The label states that the product is Oil- and Water-Soluble Vitamins with Minerals Oral Solution. The label states the quantity of each vitamin and mineral in a given volume of the Oral Solution and, where necessary, the chemical form in which a vitamin is present, and states also the salt form of the mineral used as the source of each element. Where the product contains vitamin E, the label indicates whether it is the d- or dl- form. Where the product is labeled to contain panthenol, the label states the equivalent content of dexpanthenol. Where more than one assay method is given for a particular vitamin or mineral, the labeling states with which assay method the product complies only if Method 1 is not used.
•  USP Reference Standards 11
USP Alpha Tocopherol RS Click to View Structure
2H-1-Benzopyran-6-ol, 3,4-dihydro-2,5,7,8,-tetramethyl-2-(4,8,12-trimethyltridecyl)-.
    C29H50O2        430.70
USP Biotin RS Click to View Structure
1H-Thieno[3,4-d]imidazole-4-pentanoic acid, hexahydro-2-oxo-, 3aS-[(3a,4,6a)]-.
    C10H16N2O3S        244.31
USP Calcium Pantothenate RS Click to View Structure
-Alanine, N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-, calcium salt (2:1), (R)-.
    C18H32CaN2O10        476.53
USP Cholecalciferol RS Click to View Structure
9,10-Secocholesta-5,7,10(19)-trien-3-ol, (3,5Z,7E)-.
    C27H44O        384.64
USP Cyanocobalamin RS Click to View Structure
Vitamin B12.
    C63H88CoN14O14P        1355.37
USP Dexpanthenol RS Click to View Structure
Butanamide, 2,4-dihydroxy-N-(3-hydroxypropyl)-3,3-dimethyl-, (R)-.
    C9H19NO4        205.25
USP Ergocalciferol RS Click to View Structure
9,10-Secoergosta-5,7,10 (19),22-tetraen-3-ol, (3,5Z,7E,22E)-.
    C28H44O        396.65
USP Niacin RS Click to View Structure
3-Pyridinecarboxylic acid.
    C6H5NO2        123.11
USP Niacinamide RS Click to View Structure
3-Pyridinecarboxamide.
    C6H6N2O        122.12
USP Pyridoxine Hydrochloride RS Click to View Structure
3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-, hydrochloride.
    C8H11NO3·HCl        205.64
USP Racemic Panthenol RS Click to View Structure
Butanamide, 2,4-dihydroxy-N-(3-hydroxypropyl)-3,3-dimethyl-,(±)-.
    C9H19NO4        205.25
USP Riboflavin RS Click to View Structure
Riboflavine.
    C17H20N4O6        376.36
USP Sodium Fluoride RS
Sodium fluoride.
    NaF        41.99
USP Thiamine Hydrochloride RS Click to View Structure
Thiazolium, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methyl-, chloride, monohydrochloride.
    C12H17ClN4OS·HCl        337.27
USP Vitamin A RS Click to View Structure
3,7-Dimethyl-9-(2,6,6-trimethyl-1- cyclohexen-1-yl) 2,4,6,8-nonatetraen-1-ol acetate (vitamin A acetate)

1   ATCC No. 8014 is suitable. This strain was formerly known as Lactobacillus arabinosus 17-5.
2  A suitable cartridge is the Waters, Oasis MAX Vac RC cartridge, particle size 30 µm, part 186000371.
3   Pure culture of Lactobacillus leichmannii (listed as Lactobacillus delbrueckii) may be obtained as No. 7030 from ATCC, 10801 University Blvd., Manassas, VA 20110-2209 (www.atcc.org).
4  ATCC No. 8042 is suitable.
5  USP Units of activity for vitamins, where such exist or formerly existed, are equivalent to the corresponding international units, where such formerly existed. The USP Unit for Vitamin E has been discontinued. International units (IU) for vitamins also have been discontinued; however, the use of IU on the labels of vitamin products continues. Where articles are labeled in terms of Units in addition to the required labeling, the relationship of the USP Units or IU to mass is as follows. One USP Vitamin A Unit = 0.3 µg of all-trans-retinol (vitamin A alcohol) or 0.344 µg of all-trans-retinyl acetate (vitamin A acetate) or 0.55 µg of all-trans-retinyl palmitate (vitamin A palmitate), and 1 µg of retinol (3.3 USP Vitamin A Units) = 1 retinol equivalent (RE); 1 IU of beta carotene = 0.6 µg of all-trans-beta carotene; 1 USP Vitamin D Unit = 0.025 µg of ergocalciferol or cholecalciferol; and 1 mg of dl-alpha tocopherol = 1.1 former USP Vitamin E Units, 1 mg of dl-alpha tocopheryl acetate = 1 former USP Vitamin E Unit, 1 mg of dl-alpha tocopheryl acid succinate = 0.89 former USP Vitamin E Unit, 1 mg of d-alpha tocopherol = 1.49 former USP Vitamin E Units, and 1 mg of d-alpha tocopheryl acetate = 1.36 former USP Vitamin E Units, 1 mg of d-alpha tocopheryl acid succinate = 1.21 former USP Vitamin E Units. In terms of d-alpha tocopherol equivalents, 1 mg of d-alpha tocopheryl acetate = 0.91, 1 mg of d-alpha tocopheryl acid succinate = 0.81, 1 mg of dl-alpha tocopherol = 0.74, 1 mg of dl-alpha tocopheryl acetate = 0.67, and 1 mg of dl-alpha tocopheryl acid succinate = 0.60.
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Natalia Davydova
Scientific Liaison
1-301-816-8328
(DS2010) Monographs - Dietary Supplements
2021 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
2022 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
(GCM2010) General Chapters - Microbiology
Reference Standards RS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 1554
Pharmacopeial Forum: Volume No. 32(5) Page 1475