Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 20:53:51 … · 2011-12-02 · states the equivalent content of dexpanthenol. Where 3,7-Dimethyl-9-(2,6,6-trimethyl-1- cyclohexen-1-yl)

USP 35 Dietary Supplements / Vitamins 1567

CONTAMINANTS USP Niacinamide RS• MICROBIAL ENUMERATION TESTS ⟨2021⟩: The total aerobic 3-Pyridinecarboxamide.

microbial count does not exceed 3000 cfu/mL, and the C6H6N2O 122.12combined molds and yeasts count does not exceed 300 USP Pyridoxine Hydrochloride RScfu/mL. 3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-,

• MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED hydrochloride.MICROORGANISMS ⟨2022⟩: Meet the requirements of the C8H11NO3 · HCl 205.64tests for absence of Salmonella species, Escherichia coli, USP Racemic Panthenol RSand Staphylococcus aureus Butanamide, 2,4-dihydroxy-N-(3-hydroxypropyl)-3,3-

dimethyl-,(±)-.ADDITIONAL REQUIREMENTS C9H19NO4 205.25• PACKAGING AND STORAGE: Preserve in tight, light-resistant USP Riboflavin RS

containers, under an inert gas or with a minimum of Riboflavine.headspace. C17H20N4O6 376.36

• LABELING:5 The label states that the product is Oil- and USP Sodium Fluoride RSWater-Soluble Vitamins with Minerals Oral Solution. The Sodium fluoride.label states the quantity of each vitamin and mineral in a NaF 41.99given volume of the Oral Solution and, where necessary, USP Thiamine Hydrochloride RSthe chemical form in which a vitamin is present, and Thiazolium, 3-[(4-amino-2-methyl-5-states also the salt form of the mineral used as the source pyrimidinyl)methyl]-5-(2-hydroxyethyl)-4-methyl-,of each element. Where the product contains vitamin E, chloride, monohydrochloride.the label indicates whether it is the d- or dl- form. Where C12H17ClN4OS · HCl 337.27the product is labeled to contain panthenol, the label USP Vitamin A RSstates the equivalent content of dexpanthenol. Where 3,7-Dimethyl-9-(2,6,6-trimethyl-1- cyclohexen-1-yl)more than one assay method is given for a particular 2,4,6,8-nonatetraen-1-ol acetate (vitamin A acetate)vitamin or mineral, the labeling states with which assaymethod the product complies only if Method 1 is notused.

.• USP REFERENCE STANDARDS ⟨11⟩USP Alpha Tocopherol RS Oil- and Water-Soluble Vitamins with2H-1-Benzopyran-6-ol, 3,4-dihydro-2,5,7,8,-tet- Minerals Tabletsramethyl-2-(4,8,12-trimethyltridecyl)-.C29H50O2 430.70

DEFINITIONUSP Biotin RSOil- and Water-Soluble Vitamins with Minerals Tablets con-1H-Thieno[3,4-d]imidazole-4-pentanoic acid, hexahydro-

tain one or more of the following oil-soluble vitamins: Vi-2-oxo-, 3aS-[(3aα,4β,6aα)]-.tamin A, Vitamin D as Ergocalciferol (Vitamin D2) orC10H16N2O3S 244.31Cholecalciferol (Vitamin D3), Vitamin E, Phytonadione (Vi-USP Calcium Pantothenate RStamin K1), and Beta Carotene; one or more of the follow-β-Alanine, N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-,ing water-soluble vitamins: Ascorbic Acid or its equivalentcalcium salt (2:1), (R)-.as Calcium Ascorbate or Sodium Ascorbate, Biotin, Cyano-C18H32CaN2O10 476.53cobalamin, Folic Acid, Niacin or Niacinamide, PantothenicUSP Cholecalciferol RSAcid (as Calcium Pantothenate or Racemic Calcium Panto-9,10-Secocholesta-5,7,10(19)-trien-3-ol, (3β,5Z,7E)-.thenate), Pyridoxine Hydrochloride, Riboflavin, and Thia-C27H44O 384.64mine Hydrochloride or Thiamine Mononitrate; and one orUSP Cyanocobalamin RSmore minerals derived from substances generally recog-Vitamin B12. nized as safe, furnishing one or more of the followingC63H88CoN14O14P 1355.37elements in ionizable form: boron, calcium, chromium,USP Dexpanthenol RScopper, fluorine, iodine, iron, magnesium, manganese,Butanamide, 2,4-dihydroxy-N-(3-hydroxypropyl)-3,3-molybdenum, nickel, phosphorus, potassium, selenium,dimethyl-, (R)-.tin, vanadium, and zinc. Tablets contain NLT 90.0% andC9H19NO4 205.25NMT 165.0% of the labeled amounts of vitamin AUSP Ergocalciferol RS(C20H30O) as retinol or esters of retinol in the form of9,10-Secoergosta-5,7,10 (19),22-tetraen-3-ol,retinyl acetate (C22H32O2) or retinyl palmitate (C36H60O2),(3β,5Z,7E,22E)-.vitamin D as cholecalciferol (C27H44O) or ergocalciferolC28H44O 396.65(C28H44O), vitamin E as alpha tocopherol (C29H50O2) or al-USP Niacin RSpha tocopheryl acetate (C31H52O3) or alpha tocopheryl3-Pyridinecarboxylic acid.acid succinate (C33H54O5), phytonadione (C31H46O2), andC6H5NO2 123.11beta carotene (C40H56); NLT 90.0% and NMT 150.0% of

5 USP Units of activity for vitamins, where such exist or formerly existed, are the labeled amounts of ascorbic acid (C6H8O6) or its saltsequivalent to the corresponding international units, where such formerly as calcium ascorbate (C12H14CaO12 · 2H2O) or sodium as-existed. The USP Unit for Vitamin E has been discontinued. International units

corbate (C6H7NaO6), biotin (C10H16N2O3S), cyanocobala-(IU) for vitamins also have been discontinued; however, the use of IU on thelabels of vitamin products continues. Where articles are labeled in terms of min (C63H88CoN14O14P), folic acid (C19H19N7O6), niacinUnits in addition to the required labeling, the relationship of the USP Units or (C6H5NO2) or niacinamide (C6H6N2O), calcium pantothen-IU to mass is as follows. One USP Vitamin A Unit = 0.3 µg of all-trans-retinol ate (C18H32CaN2O10), pyridoxine hydrochloride(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 (C8H11NO3 · HCl), riboflavin (C17H20N4O6), and thiamineretinol (3.3 USP Vitamin A Units) = 1 retinol equivalent (RE); 1 IU of beta (C12H17ClN4OS) as thiamine hydrochloride or thiaminecarotene = 0.6 µg of all-trans-beta carotene; 1 USP Vitamin D Unit = 0.025 mononitrate; NLT 90.0% and NMT 125.0% of the labeledµg of ergocalciferol or cholecalciferol; and 1 mg of dl-alpha tocopherol = 1.1

amounts of calcium (Ca), copper (Cu), iron (Fe), manga-former USP Vitamin E Units, 1 mg of dl-alpha tocopheryl acetate = 1 formerUSP Vitamin E Unit, 1 mg of dl-alpha tocopheryl acid succinate = 0.89 former nese (Mn), magnesium (Mg), phosphorus (P), potassiumUSP Vitamin E Unit, 1 mg of d-alpha tocopherol = 1.49 former USP Vitamin E (K), and zinc (Zn); and NLT 90.0% and NMT 160.0% ofUnits, and 1 mg of d-alpha tocopheryl acetate = 1.36 former USP Vitamin E the labeled amounts of boron (B), chromium (Cr), fluor-Units, 1 mg of d-alpha tocopheryl acid succinate = 1.21 former USP Vitamin EUnits. In terms of d-alpha tocopherol equivalents, 1 mg of d-alpha tocopheryl ine (F), iodine (I), molybdenum (Mo), nickel (Ni), sele-acetate = 0.91, 1 mg of d-alpha tocopheryl acid succinate = 0.81, 1 mg of dl- nium (Se), tin (Sn), and vanadium (V).alpha tocopherol = 0.74, 1 mg of dl-alpha tocopheryl acetate = 0.67, and 1mg of dl-alpha tocopheryl acid succinate = 0.60.

Official from May 1, 2012Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.

Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 20:53:51 EST 2011

1568 Vitamins / Dietary Supplements USP 35

They may contain other labeled added substances that are CS = concentration of retinyl acetate (C22H32O2)generally recognized as safe, in amounts that are from USP Vitamin A RS in the Standardunobjectionable. solution (µg/mL)

CU = nominal concentration of vitamin A, as retinolSTRENGTH (C20H30O) in the Sample solution (µg/mL)[NOTE—In the following assays, where more than one assay F = factor used to convert retinyl acetate, the estermethod is given for an individual ingredient, the require- form present in USP Vitamin A RS, to retinol,ments may be met by following any one of the specified 0.872methods, the method used being stated in the labeling only [NOTE—The molar responses of retinyl acetate andif Method 1 is not used.] retinyl palmitate are equivalent.]• VITAMIN A, Method 1 Acceptance criteria: 90.0%–165.0% of of the labeled

[NOTE—Where the use of a vitamin A ester (retinyl ace- amount of vitamin A, as retinol (C20H30O)tate or retinyl palmitate) is specified in the following • VITAMIN A, Method 2procedure, use the chemical form present in the formu- [NOTE—Where a vitamin A ester (retinyl acetate or retinyllation. USP Vitamin A RS is retinyl acetate. It is to be palmitate) is indicated in the following procedure, useused where USP Vitamin A RS is specified. Use low-ac- the chemical form present in the formulation. USPtinic glassware throughout this procedure.] Vitamin A RS is retinyl acetate. It is to be used where

Mobile phase: n-Hexane USP Vitamin A RS is specified. Use low-actinic glasswareStandard solution: 15 µg/mL of retinyl acetate from throughout this procedure.]USP Vitamin A RS in n-hexane 3 N methanolic sulfuric acid solution: Cautiously add 9

System suitability stock solution: 15 µg/mL of retinyl mL of sulfuric acid to 80 mL of methanol in a 100-mLpalmitate in n-hexane volumetric flask. Cool, and dilute with methanol to

System suitability solution: Mix equal volumes of Sys- volume.tem suitability stock solution and the Standard solution to Sodium ascorbate–pyrogallol solution: Transfer 10 g ofobtain concentrations of 7.5 µg/mL each of retinyl ace- sodium ascorbate and 5 g of pyrogallol to a 100-mLtate and retinyl palmitate. volumetric flask, and add sufficient water to dissolve.

Sample solution: Finely powder NLT 20 Tablets. Transfer Add 1.7 mL of sulfuric acid, and dilute with water toa portion of the powder, equivalent to 5 Tablets, to a volume.container having a polytef-lined screw cap. Add 10 mL Lecithin solution: 5 mg/mL of lecithin in 2,2,4-of dimethyl sulfoxide and 15 mL of n-hexane, and shake trimethylpentanefor 45 min on a wrist-action shaker in a water bath Mobile phase: n-Hexane and ethyl acetate (99.7:0.3)maintained at 60°. [NOTE—Set up the wrist-action shaker Standard solution: 15 µg/mL of retinyl acetate fromto ensure that the contents of the container are mixed USP Vitamin A RS in 2,2,4-trimethylpentanevigorously and thoroughly.] Centrifuge at 3000 rpm for System suitability stock solution: 15 µg/mL of retinyl10 min, and transfer the hexane layer by means of a palmitate in 2,2,4-trimethylpentanepipet to a 100-mL volumetric flask. Add 15 mL of n- System suitability solution: Mix equal volumes of thehexane to the dimethyl sulfoxide layer, shake thoroughly System suitability stock solution and the Standard solutionfor 5 min, and transfer the hexane layer by means of a to obtain concentrations of 7.5 µg/mL each of retinylpipet to the 100-mL volumetric flask. Repeat this extrac- acetate and retinyl palmitate.tion with three additional 15-mL portions of n-hexane. Sample solution: [NOTE—This preparation is suitable forDilute the extracts in the volumetric flask with n-hexane the determination of vitamin A, vitamin D, and vitaminto volume. Dilute a 10-mL volume of this solution with E, when present in the formulation.] Finely powder NLTn-hexane to obtain a solution with a concentration of 15 20 Tablets. If vitamin D is present in the formulation,µg/mL of vitamin A as retinol (C20H30O). transfer a portion of the powder, equivalent to 30 µg of

Chromatographic system the labeled amount of cholecalciferol or ergocalciferol, to(See Chromatography ⟨621⟩, System Suitability.) a container having a polytef-lined screw cap. If vitamin DMode: LC is not present in the formulation, use a portion of theDetector: UV 325 nm powder, equivalent to 90 mg of the labeled amount ofColumn: 4.6-mm × 15-cm; 3-µm packing L8 vitamin E (as alpha tocopherol, alpha tocopheryl acetate,Flow rate: 1 mL/min or alpha tocopheryl hemisuccinate). If vitamin E is notInjection size: 40 µL present in the formulation, use a portion of the powder,

System suitability equivalent to 2.5 mg of the labeled amount of retinylSample: System suitability solution acetate or retinyl palmitate. Add 0.5 g of sodiumSuitability requirements bicarbonate, 1.5 mL of Lecithin solution, and 12.5 mL of

Resolution: NLT 10 between all-trans-retinyl acetate 2,2,4-trimethylpentane, and disperse on a vortex mixer.and all-trans-retinyl palmitate Add 6 mL of Sodium ascorbate–pyrogallol solution, shake

Relative standard deviation: NMT 3.0% slowly, and allow the solution to degas. ContinueAnalysis shaking until the evolution of gas has ceased, and thenSamples: Standard solution and Sample solution shake for an additional 12 min. Add 6 mL of dimethylMeasure the peak area for all-trans-retinyl acetate from sulfoxide, mix on a vortex mixer to form a suspension,the Standard solution and the peak area for all-trans- and shake for 12 min. Add 6 mL of 3 N methanolicretinyl acetate or all-trans-retinyl palmitate in the chro- sulfuric acid solution, mix on a vortex mixer to form amatogram of the Sample solution. For products con- suspension, and shake for 12 min. Add 12.5 mL of 2,2,4-taining vitamin A acetate or vitamin A palmitate, cal- trimethylpentane, mix on a vortex mixer to form aculate the percentage of the labeled amount of suspension, and shake for 10 min. Centrifuge for 10 minvitamin A, as retinol (C20H30O) in the portion of the to break up the emulsion and to clarify the supernatant.Tablets taken: [NOTE—The supernatant is used for the determination of

vitamin A, and also vitamin D and vitamin E, if present inResult = (rU/rS) × (CS/CU) × F × 100 the formulation.] If necessary, quantitatively dilute a

volume of the supernatant with 2,2,4-trimethylpentanerU = peak area of the all-trans-retinyl ester from the to obtain a concentration close to that of the StandardSample solution solution.rS = peak area of the all-trans-retinyl ester from the Chromatographic systemStandard solution (See Chromatography ⟨621⟩, System Suitability.)




Mode: LC 1.5 mg of retinyl acetate, to a stoppered 125-mL flask.Detector: UV 325 nm Add 5 mL of water, 15 mL of Diluent, and 3 mL ofColumn: 4.6-mm × 25-cm; 5-µm packing L24 Potassium hydroxide solution. Insert the stopper tightly,Flow rate: 1.5 mL/min shake for 15 min over a water bath maintained at 60Injection size: 40 µL ±5°, and cool to room temperature. Add 7 mL of water

System suitability and 25.0 mL of Extraction solvent. Insert the stopperSample: System suitability solution tightly, and shake vigorously for 60 s or longer, ifSuitability requirements necessary, for complete extraction. Rinse the sides of the

Resolution: NLT 8.0 between all-trans-retinyl acetate flask with 60 mL of water, and allow to stand for 10 minand all-trans-retinyl palmitate until the layers separate. [NOTE—Do not shake, because

Relative standard deviation: NMT 3.0% an emulsion may form.] Withdraw a portion of theAnalysis organic layer, and dilute with Extraction solvent to obtainSamples: Standard solution and Sample solution a concentration of 0.34 µg/mL of retinol.Measure the peak area for all-trans-retinyl acetate from Chromatographic systemthe Standard solution and the peak area of all-trans- (See Chromatography ⟨621⟩, System Suitability).retinyl acetate or all-trans-retinyl palmitate from the Mode: LCSample solution. Detector: UV 335 nm

Calculate the percentage of the labeled amount of of Column: 6.2-mm × 8-cm; packing L3vitamin A, as retinol (C20H30O), in the portion of Column temperature: 40°Tablets taken: Flow rate: 4 mL/min

Injection size: 50 µLResult = (rU/rS) × (CS/CU) × F × 100 System suitability

Sample: Standard solutionrU = peak area of the all-trans-retinyl ester from the [NOTE—The relative retention times for 13-cis-retinol

Sample solution and all-trans-retinol are 0.92 and 1.0, respectively.]rS = peak area of the all-trans-retinyl ester from the Suitability requirements

Standard solution Relative standard deviation: NMT 5.0%CS = concentration of retinyl acetate (C22H32O2) Analysis

from USP Vitamin A RS in the Standard Samples: Standard solution and Sample solutionsolution (µg/mL) Measure the peak areas for all-trans-retinol and 13-cis-

CU = nominal concentration of vitamin A, as retinol retinol. Calculate the percentage of the labeled(C20H30O) in the Sample solution (µg/mL) amount of vitamin A, as retinol (C20H30O), in the

F = factor used to convert retinyl acetate, the ester portion of Tablets taken:form present in the USP Vitamin A RS, toretinol, 0.872 Result = (rT1/rT2) × (CS/CU) × F × 100

[NOTE—Account for the initial extraction volume of 26.5mL of 2,2,4-trimethylpentane to calculate the nominal rT1 = sum of the areas of the all-trans-retinol andconcentration. The molar responses of retinyl acetate 13-cis-retinol peaks from the Sample solutionand retinyl palmitate are equivalent.] rT2 = sum of the areas of the all-trans-retinol and

Acceptance criteria: 90.0%–165.0% of the labeled 13-cis-retinol peaks from the Standardamount of vitamin A, as retinol (C20H30O) solution

• VITAMIN A, Method 3 CS = concentration of retinyl acetate (C23H32O2)[NOTE—Where a vitamin A ester (retinyl acetate or retinyl from USP Vitamin A RS in the Standardpalmitate) is indicated in the following procedure, use solution (µg/mL)the chemical form present in the formulation. USP CU = nominal concentration of vitamin A, as retinolVitamin A RS is retinyl acetate. It is to be used where (C20H30O) in the Sample solution (µg/mL)USP Vitamin A RS is specified. Use low-actinic glassware F = factor used to convert retinyl acetate, the esterthroughout this procedure.] form present in USP Vitamin A RS, to retinol,

Extraction solvent: n-Hexane and methylene chloride 0.872(3:1) Acceptance criteria: 90.0%–165.0% of the labeled

Potassium hydroxide solution: 800 mg/mL of amount of vitamin A, as retinol (C20H30O)potassium hydroxide in water. [NOTE—Cautiously add • CHOLECALCIFEROL or ERGOCALCIFEROL (VITAMIN D), Methodpotassium hydroxide in water. Mix, and cool.] 1

Diluent: 10 mg/mL of pyrogallol in alcohol [NOTE—Where vitamin D (cholecalciferol orMobile phase: n-Hexane and isopropyl alcohol (92:8) ergocalciferol) is specified in the following procedure,Standard stock solution: 30 µg/mL of retinyl acetate use the chemical form present in the formulation andfrom USP Vitamin A RS in Diluent. [NOTE—This solution the relevant USP Reference Standard. Use low-actinicmay be stored in a refrigerator for 1 week.] glassware throughout this procedure.]

Standard solution: Dilute a volume of Standard stock Mobile phase: n-Hexane and isopropyl alcohol (99:1)solution with Diluent to obtain a concentration of 1 Standard solution: 2 µg/mL of USP Cholecalciferol RS orµg/mL of USP Vitamin A RS. Transfer 10.0 mL of this USP Ergocalciferol RS in n-hexanesolution to a stoppered 125-mL flask, and add 5 mL of System suitability solution: Heat a volume of thewater, 5 mL of Diluent, and 3 mL of Potassium hydroxide Standard solution at 60° for 1 h to partially isomerizesolution. Insert the stopper tightly, shake for 15 min over vitamin D (cholecalciferol or ergocalciferol) to itsa water bath maintained at 60 ± 5°, and cool to room corresponding precursor.temperature. Add 7 mL of water and 25.0 mL of Sample solution: Transfer NLT 20 mL of the solutionExtraction solvent. Insert the stopper tightly, and shake prepared as directed for Sample solution in Vitamin A,vigorously for 60 s. Rinse the sides of the flask with 60 Method 1 to a suitable container, and evaporate, ifmL of water, and allow to stand for 10 min until the necessary, in vacuum at room temperature to obtain alayers separate. Withdraw a portion of the organic layer concentration of 2 µg/mL of cholecalciferol orfor injection into the chromatograph. This Standard ergocalciferol.solution contains 0.34 µg/mL of retinol. Chromatographic system

Sample solution: Finely powder a counted number of (See Chromatography ⟨621⟩, System Suitability.)Tablets. Transfer a portion of the powder, equivalent to




Mode: LC (C27H44O) or ergocalciferol (C28H44O) in the portion ofDetector: UV 265 nm Tablets taken:Column: 4.6-mm × 15-cm; 3-µm packing L8

Result = (rU/rS) × (CS/CU) × 100Flow rate: 1 mL/minInjection size: 100 µL

rU = peak area of cholecalciferol or ergocalciferolSystem suitabilityfrom the Sample solutionSample: Standard solution and System suitability solution

rS = peak area of cholecalciferol or ergocalciferolSuitability requirementsfrom the Standard solutionResolution: NLT 10 between the vitamin D form

CS = concentration of USP Cholecalciferol RS orpresent and its corresponding precursor, SystemUSP Ergocalciferol RS in the Standard solutionsuitability solution(µg/mL)Relative standard deviation: NMT 3.0%, Standard

CU = nominal concentration of cholecalciferol orsolutionergocalciferol in the Sample solution (µg/mL)Analysis

[NOTE—Account for the initial extraction volume of 26.5Samples: Standard solution and Sample solutionmL of 2,2,4-trimethylpentane to calculate the nominalMeasure the peak areas for vitamin D. Calculate theconcentration.]percentage of the labeled amount of cholecalciferol

Acceptance criteria: 90.0%–165.0% of the labeled(C27H44O) or ergocalciferol (C28H44O) in the portion ofamount of vitamin D as cholecalciferol (C27H44O) orTablets taken:ergocalciferol (C28H44O)

• CHOLECALCIFEROL or ERGOCALCIFEROL (VITAMIN D), MethodResult = (rU/rS) × (CS/CU) × F × 1003

rU = peak area of cholecalciferol or ergocalciferol [NOTE—Where vitamin D (cholecalciferol orfrom the Sample solution ergocalciferol) is specified in the following procedure,

rS = peak area of cholecalciferol or ergocalciferol use the chemical form present in the formulation andfrom the Standard solution the relevant USP Reference Standard. Use low-actinic

CS = concentration of USP Cholecalciferol RS or glassware throughout this procedure.]USP Ergocalciferol RS in the Standard solution Diluted acetic acid: Glacial acetic acid solution (1 in 10)(µg/mL) in water

CU = nominal concentration of cholecalciferol or Phenolphthalein solution: 10 mg/mL ofergocalciferol in the Sample solution (µg/mL) phenolphthalein in alcohol

F = correction factor to account for the average Potassium hydroxide solution: Slowly dissolve 14 g ofamount of previtamin D present in the potassium hydroxide in a mixture of 31 mL ofSample solution, 1.09 dehydrated alcohol and 5 mL of water. Prepare fresh

Acceptance criteria: 90.0%–165.0% of the labeled daily.amount of vitamin D as cholecalciferol (C27H44O) or Extraction solvent: Methylene chloride and isopropylergocalciferol (C28H44O) alcohol (99.8:0.2)

• CHOLECALCIFEROL or ERGOCALCIFEROL (VITAMIN D), Method Mobile phase: Acetonitrile and methanol (91:9)2 Standard stock solution: 0.2 mg/mL of USP[NOTE—Where vitamin D (cholecalciferol or Cholecalciferol RS or USP Ergocalciferol RS in dehydratedergocalciferol) is specified in the following procedure, alcohol. [NOTE—Prepare fresh every 4 weeks. Store in ause the chemical form present in the formulation and freezer.]the relevant USP Reference Standard. Use low-actinic Standard solution: [NOTE—Condition the solid-phaseglassware throughout this procedure.] extraction column specified for use in the Standard

3 N methanolic sulfuric acid solution, Sodium solution and the Sample solution by initially washing theascorbate–pyrogallol solution, Lecithin solution, and column with 4.0 mL of a mixture of methylene chlorideSample solution: Proceed as directed in Vitamin A, and isopropyl alcohol (4:1), followed by 5.0 mL ofMethod 2. Extraction solvent. Do not allow the column to dry.]

Mobile phase: n-Hexane and tertiary butyl alcohol Dilute a volume of Standard stock solution with(98.75:1.25) dehydrated alcohol to obtain a concentration of 5

Standard solution: 1 µg/mL of USP Cholecalciferol RS or µg/mL of USP Cholecalciferol RS or USP ErgocalciferolUSP Ergocalciferol RS in 2,2,4-trimethylpentane RS. Prepare this solution fresh daily. Transfer 2.0 mL of

System suitability solution: Heat a volume of the this solution to a stoppered 125-mL flask. Add 15.0 mLStandard solution at 60° for 1 h to partially isomerize of water and 15.0 mL of Potassium hydroxide solution,vitamin D (cholecalciferol or ergocalciferol) to its insert the stopper, and shake for 30 min in a water bathcorresponding precursor. maintained at 60°. Allow to cool to room temperature,

Chromatographic system and transfer the contents of the flask to a 250-mL(See Chromatography ⟨621⟩, System Suitability.) separatory funnel. Add 15.0 mL of water to the flask,Mode: LC insert the stopper, shake vigorously, and transfer thisDetector: UV 265 nm solution to the separatory funnel. Rinse the flask with 60Column: 4.6-mm × 25-cm; 5-µm packing L24 mL of n-hexane, and transfer the rinsing to theFlow rate: 1 mL/min separatory funnel. Insert the stopper, shake vigorously forInjection size: 40 µL 90 s, and allow to stand for 15 min until the layers

System suitability separate. Drain and discard the aqueous layer. Add 15.0Sample: Standard solution and System suitability solution mL of water to the hexane layer in the separatory funnel,Suitability requirements insert the stopper, and shake vigorously. Allow to stand

Resolution: NLT 4.0 between the vitamin D form for 10 min until the layers separate, and discard thepresent and its corresponding precursor, System aqueous layer. Add 1 drop of Phenolphthalein solutionsuitability solution and 15.0 mL of water to the separatory funnel. Add

Relative standard deviation: NMT 3.0%, Standard Diluted acetic acid dropwise, with shaking, until thesolution washing is neutral. Allow to stand for 10 min until the

Analysis layers separate. Drain and discard the aqueous layer.Samples: Standard solution and Sample solution Filter the hexane layer through anhydrous sodium sulfateMeasure the peak areas for vitamin D. Calculate the supported by a small pledget of cotton into a 100-mL,percentage of the labeled amount of cholecalciferol round-bottom flask. Rinse the funnel and sodium sulfate




with a few mL of n-hexane, and collect the rinsings in Sample solution: Transfer NLT 20 mL of the solutionthe same flask. Evaporate the hexane in the flask on a prepared as directed for Sample solution in Vitamin A,rotary evaporator at 50° to dryness. Immediately add 2.0 Method 1 to a suitable container, and evaporate inmL of Extraction solvent to dissolve the residue. Transfer vacuum at room temperature to dryness. Transfer thethis solution to a freshly conditioned solid-phase residue with the aid of methanol to a suitable volumetricextraction column containing silica packing with a flask, and dilute with methanol to volume to obtain asorbent mass to column volume ratio of 500 mg to 2.8 concentration of 2 mg/mL of alpha tocopherol, alphamL or equivalent, rinse the round-bottom flask with 1.0 tocopheryl acetate, or alpha tocopheryl acid succinate.mL of Extraction solvent, and transfer to the column. Chromatographic systemElute the column with 2.0 mL of Extraction solvent, and (See Chromatography ⟨621⟩, System Suitability.)discard this fraction. Elute the column with 7.0 mL of Mode: LCExtraction solvent, and collect the eluate in a suitable Detector: UV 254 nmflask. Place the flask in a warm water bath maintained at Column: 8-mm × 10-cm; 5-µm packing L142°, and evaporate the solvent with the aid of a stream Flow rate: 2 mL/minof nitrogen. Immediately add 2.0 mL of acetonitrile to Injection size: 100 µLthe residue, and use the solution for injection into the System suitabilitychromatograph. Samples: Standard solution and System suitability

Sample solution: Finely powder NLT 20 Tablets. Transfer solutiona portion of the powder, equivalent to 10 µg of [NOTE—The relative retention times for ergocalciferolcholecalciferol or ergocalciferol, to the stoppered 125-mL and alpha tocopheryl acetate are about 0.5 and 1.0,flask, and proceed as directed for the Standard solution, respectively.]beginning with “Add 15.0 mL of water and 15.0 mL of Suitability requirementsPotassium hydroxide solution”. Resolution: NLT 12 between ergocalciferol and alpha

Chromatographic system tocopheryl acetate, System suitability solution(See Chromatography ⟨621⟩, System Suitability.) Tailing factor: Between 0.8 and 1.2, System suitabilityMode: LC solutionDetector: UV 265 nm Relative standard deviation: NMT 3.0%, StandardColumn: 4.6-mm × 25-cm; 5-µm packing L1 solutionColumn temperature: 27° AnalysisFlow rate: 0.7 mL/min Samples: Standard solution and Sample solutionInjection size: 15 µL Measure the peak areas. Calculate the percentage of

System suitability the labeled amount of alpha tocopherol (C29H50O2),Sample: Standard solution alpha tocopheryl acetate (C31H52O3), or alphaSuitability requirements tocopheryl acid succinate (C33H54O5) in the portion of

Relative standard deviation: NMT 4.0% Tablets taken:Analysis

Result = (rU/rS) × (CS/CU) × 100Samples: Standard solution and Sample solutionMeasure the peak areas for vitamin D. Calculate the

rU = peak area of the relevant vitamin E form frompercentage of the labeled amount of cholecalciferolthe Sample solution(C27H44O) or ergocalciferol (C28H44O) in the portion of

rS = peak area of the relevant vitamin E form fromTablets taken:the Standard solution

CS = concentration of the corresponding USPResult = (rU/rS) × (CS/CU) × 100Reference Standard in the Standard solution

rU = peak area of cholecalciferol or ergocalciferol (mg/mL)from the Sample solution CU = nominal concentration of the corresponding

rS = peak area of cholecalciferol or ergocalciferol form of vitamin E in the Sample solutionfrom the Standard solution (mg/mL)

CS = concentration of USP Cholecalciferol RS or Acceptance criteria: 90.0%–165.0% of the labeledUSP Ergocalciferol RS in the Standard solution amount of vitamin E as alpha tocopherol (C29H50O2),(µg/mL) alpha tocopheryl acetate (C31H52O3), or alpha tocopheryl

CU = nominal concentration of cholecalciferol or acid succinate (C33H54O5)ergocalciferol in the Sample solution (µg/mL) • VITAMIN E, Method 2

Acceptance criteria: 90.0%–165.0% of the labeled [NOTE—Where vitamin E (alpha tocopherol, alphaamount of vitamin D as cholecalciferol (C27H44O) or tocopheryl acetate, or alpha tocopheryl acid succinate)ergocalciferol (C28H44O) is specified in the following procedure, use the chemical

• VITAMIN E, Method 1 form present in the formulation and the relevant USP[NOTE—Where vitamin E (alpha tocopherol, alpha Reference Standard. Use low-actinic glasswaretocopheryl acetate, or alpha tocopheryl acid succinate) throughout this procedure.]is specified in the following procedure, use the chemical Mobile phase: Mix 240 mL of methanol with 10 mL ofform present in the formulation and the relevant USP water followed by 0.5 mL of 50% phosphoric acid, andReference Standard. Use low-actinic glassware dilute with acetonitrile to 1000 mL.throughout this procedure.] System suitability solution: 2 mg/mL each of USP Alpha

Solution A: Phosphoric acid solution (1 in 100) in water Tocopherol RS, USP Alpha Tocopheryl Acetate RS, andMobile phase: Methanol and Solution A (19:1) USP Alpha Tocopheryl Acid Succinate RS in methanolSystem suitability solution: Prepare a 0.65-mg/mL Standard solution: 2 mg/mL of USP Alpha Tocopherolsolution of USP Ergocalciferol RS in methanol. Transfer RS, USP Alpha Tocopheryl Acetate RS, or USP Alpha1.0 mL of this solution to a 100-mL volumetric flask Tocopheryl Acid Succinate RS in methanolcontaining 100 mg of USP Alpha Tocopheryl Acetate RS. Sample solution: Proceed as directed for Sample solutionDissolve in 30 mL of methanol, with the aid of in Vitamin A, Method 2. Transfer a volume of thesonication if necessary, and dilute with methanol to supernatant 2,2,4-trimethylpentane to a suitablevolume. Store this solution in a refrigerator. volumetric flask, the volume of the specimen withdrawn

Standard solution: 2 mg/mL of USP Alpha Tocopherol from the 2,2,4-trimethylpentane and the size of theRS, USP Alpha Tocopheryl Acetate RS, or USP Alpha volumetric flask being such that the final concentrationTocopheryl Acid Succinate RS in methanol of the Sample solution is equivalent to that of the




Standard solution. Evaporate nearly to dryness, add to separate, and discard the aqueous layer. Add 3 dropsseveral mL of methanol, and evaporate the remaining of glacial acetic acid, and repeat the washing procedure2,2,4-trimethylpentane. Dilute with methanol to volume. two more times. Filter the washed hexane layer through

Chromatographic system anhydrous sodium sulfate into a 250-mL round-bottom(See Chromatography ⟨621⟩, System Suitability.) flask. Rinse the funnel and sodium sulfate with a few mLMode: LC of n-hexane, and add the rinsing to the hexane solutionDetector: UV 280 nm in the flask. Place the flask in a water bath maintained atColumn: 4.6-mm × 25-cm; 5-µm packing L1 50°, and evaporate the hexane solution with the aid of aFlow rate: 1.5 mL/min rotary evaporator to dryness. Immediately add 25.0 mLInjection size: 25 µL of Diluent, and swirl to dissolve the residue.

System suitability Chromatographic systemSamples: Standard solution and System suitability (See Chromatography ⟨621⟩, System Suitability.)solution Mode: LC[NOTE—The relative retention times for alpha tocopheryl Detector: UV 291 nmacid succinate, alpha tocopherol, and alpha tocopheryl Column: 4.6-mm × 25-cm; packing L1acetate are about 0.6, 0.8, and 1.0, respectively.] Column temperature: 40°

Suitability requirements Flow rate: 3 mL/minResolution: NLT 4.0 between alpha tocopheryl acid Injection size: 20 µLsuccinate and alpha tocopherol; NLT 3.0 between System suitabilityalpha tocopherol and alpha tocopheryl acetate, Sample: Standard solutionSystem suitability solution Suitability requirements

Relative standard deviation: NMT 3.0%, Standard Relative standard deviation: NMT 5.0%solution Analysis

Analysis Samples: Standard solution and Sample solutionSamples: Standard solution and Sample solution Measure the peak areas. Calculate the percentage ofMeasure the peak areas. Calculate the percentage of the labeled amount of vitamin E as alpha tocopherolthe labeled amount of alpha tocopherol (C29H50O2), (C29H50O2) in the portion of Tablets taken:alpha tocopheryl acetate (C31H52O3), or alpha

Result = (rU/rS) × (CS/CU) × 100tocopheryl acid succinate (C33H54O5) in the portion ofTablets taken:

rU = peak area of alpha tocopherol from theSample solutionResult = (rU/rS) × (CS/CU) × 100

rS = peak area of alpha tocopherol from therU = peak area of the relevant vitamin E form from Standard solution

the Sample solution CS = concentration of alpha tocopherol in therS = peak area of the relevant vitamin E form from Standard solution (mg/mL)

the Standard solution CU = nominal concentration of vitamin E as alphaCS = concentration of the corresponding USP tocopherol in the Sample solution (mg/mL)

Reference Standard in the Standard solution [NOTE—Calculate the nominal amount of vitamin E as(mg/mL) alpha tocopherol (C29H50O2), by multiplying the

CU = nominal concentration of the corresponding content of alpha tocopheryl acetate (C31H52O3) orform of vitamin E in the Sample solution alpha tocopheryl acid succinate (C33H54O5), in(mg/mL) mg/tablet, by the factors 0.91 or 0.81, respectively.]

[NOTE—Account for the initial extraction volume of 26.5 Acceptance criteria: 90.0%–165.0% of the labeledmL of 2,2,4-trimethylpentane and the dilution factor amount of vitamin E as alpha tocopherol (C29H50O2),to exchange the solvent from 2,2,4-trimethylpentane alpha tocopheryl acetate (C31H52O3), or alpha tocopherylto methanol to calculate the nominal concentration.] acid succinate (C33H54O5)

Acceptance criteria: 90.0%–165.0% of the labeled • PHYTONADIONE, Method 1amount of vitamin E as alpha tocopherol (C29H50O2), [NOTE—Use low-actinic glassware throughout thisalpha tocopheryl acetate (C31H52O3), or alpha tocopheryl procedure.]acid succinate (C33H54O5) Mobile phase: Methanol and water (19:1)

• VITAMIN E, Method 3 Standard stock solution: 200 µg/mL of USPDiluent: Acetonitrile and ethyl acetate (1:1) Phytonadione RS in methanol. Dissolve with the aid ofMobile phase: Methanol, acetonitrile, and n-hexane sonication if necessary.(46.5:46.5:7.0) Standard solution: 20 µg/mL of USP Phytonadione RS

Standard solution: 0.3 mg/mL of USP Alpha Tocopherol from Standard stock solution diluted with methanol.RS in methanol System suitability solution: 0.65 mg/mL of USP Alpha

Sample solution: Finely powder NLT 20 Tablets. Transfer Tocopheryl Acetate RS and 20 µg/mL of USPa portion of the powder, equivalent to 8 mg of alpha Phytonadione RS from Standard stock solution dilutedtocopherol, to a 125-mL flask fitted with a ground-glass with methanol. [NOTE—Dissolve USP Alpha Tocopheryljoint. Add 25.0 mL of water, 25.0 mL of dehydrated Acetate RS in a portion of methanol, add the Standardalcohol, and 3.5 g of potassium hydroxide pellets. Shake stock solution, and then dilute with methanol to volume.]for 1 h in a water bath maintained at 55°. Cool, and Sample solution: Transfer NLT 20 mL of the solutiontransfer with the aid of a minimum volume of water to a prepared as directed for Sample solution in Vitamin A,125-mL separatory funnel. Rinse the flask with 50 mL of Method 1 to a suitable container, and evaporate in an-hexane, and add the rinsing to the separatory funnel. vacuum at room temperature to dryness. Transfer theInsert the stopper, shake vigorously for 60 s, and allow residue with the aid of methanol to a suitable volumetricthe layers to separate. Drain the aqueous layer into a flask, and dilute with methanol to volume to obtain asecond 250-mL separatory funnel, and repeat the concentration of 20 µg/mL of phytonadione.extraction with 50 mL of n-hexane. Discard the aqueous Chromatographic systemlayer, and combine the hexane extracts. Wash the (See Chromatography ⟨621⟩, System Suitability.)combined extracts with 25 mL of water, allow the layers




Mode: LC in a volume of Solvent to obtain a concentration of 1Detector: UV 254 nm µg/mL of phytonadione.Column: 8-mm × 10-cm; 5-µm packing L1 Chromatographic systemFlow rate: 2 mL/min (See Chromatography ⟨621⟩, System Suitability.)Injection size: 100 µL Mode: LC

System suitability Detector: Fluorometric detector set at 320 nm forSamples: Standard solution and System suitability excitation and 420 nm for emissionsolution Column: 4.6-mm × 25-cm; 5-µm, end-capped packing[NOTE—The relative retention times for alpha tocopheryl L1, and a postcolumn reactor constituted with a 4.6-acetate and phytonadione are 0.68 and 1.0, mm × 3-cm PEEK column tightly packed with zincrespectively.] powder. [NOTE—Prepare the postcolumn reactor daily,

Suitability requirements or as necessary, to meet the system suitabilityResolution: NLT 5 between alpha tocopheryl acetate requirements.]and phytonadione, System suitability solution Flow rate: 1 mL/min

Relative standard deviation: NMT 3.0%, Standard Injection size: 25 µLsolution System suitability

Analysis Sample: Standard solutionSamples: Standard solution and Sample solution [NOTE—The relative retention times for the internalMeasure the peak areas. Calculate the percentage of standard and phytonadione are 1.0 and 1.4,the labeled amount of phytonadione (C31H46O2) in the respectively.]portion of Tablets taken: Suitability requirements

Column efficiency: NLT 2500 theoretical plates forResult = (rU/rS) × (CS/CU) × 100 the phytonadione peak

Tailing factor: NMT 1.5 for the phytonadione peakrU = peak area from the Sample solution Relative standard deviation: NMT 3.0%rS = peak area from the Standard solution AnalysisCS = concentration of USP Phytonadione RS in the Samples: Standard solution and Sample solution

Standard solution (µg/mL) Calculate the percentage of the labeled amount ofCU = nominal concentration of phytonadione in the phytonadione (C31H46O2) in the portion of Tablets

Sample solution (µg/mL) taken:Acceptance criteria: 90.0%–165.0% of the labeledamount of phytonadione (C31H46O2) Result = (RU/RS) × (CS/CU) × 100

• PHYTONADIONE, Method 2[NOTE—Use low-actinic glassware throughout this RU = peak response ratio of phytonadione to that ofprocedure.] the internal standard from the Sample

Solvent: Methanol and isopropyl alcohol (19:1) solutionMobile phase: Mix 800 mL of methanol, 200 mL of RS = peak response ratio of phytonadione to that ofmethylene chloride, 0.1 mL of glacial acetic acid, 1.36 g the internal standard from the Standardof zinc chloride, and 0.41 g of sodium acetate. solution

Internal standard solution: 5 µg/mL of menaquinone 4 CS = concentration of USP Phytonadione RS in the(vitamin K2) in Solvent. [NOTE—A concentrated stock Standard solution (µg/mL)solution of menaquinone 4 (100 µg/mL) can be stored CU = nominal concentration of phytonadione in thefor 2 months in a refrigerator.] Sample solution (µg/mL)

Standard stock solution: 5 µg/mL of USP Phytonadione Acceptance criteria: 90.0%–165.0% of labeled amountRS, prepared by dissolving in methylene chloride with of phytonadione (C31H46O2)the aid of sonication, and diluting with Solvent to final • BETA CAROTENEvolume. [NOTE—Use low-actinic glassware throughout this

Standard solution: Transfer 1.0 mL of the Standard stock procedure.]solution and 1.0 mL of the Internal standard solution to a Potassium hydroxide solution: Dissolve 58.8 gsuitable flask and dilute with Solvent to 5 mL. Pass potassium hydroxide in 50 mL of water.through a membrane filter of 0.45-µm or finer pore size. Iodine solution: 0.01 mg/mL of iodine in cyclohexane.

Sample solution: Finely powder NLT 20 Tablets. To a [NOTE—Prepare this solution fresh daily.]centrifuge tube fitted with a cap transfer an amount of Sample solution: Weigh NLT 20 Tablets. Grind thepowder, not exceeding 800 mg and equivalent to an Tablets to a fine powder, and transfer a quantity of theamount of phytonadione not exceeding 50 µg. Add 4 powder, equivalent to 2 mg of beta carotene, to a 500-mL of water. Insert the stopper, and mix using a vortex mL saponification flask. Add 100 mL of alcohol, 6 mL ofmixer until the sample is dispersed. Place the tube in a Potassium hydroxide solution, and a magnetic stirring bar.water bath at 60° for 5 min. Remove from the bath, and Attach an air condenser to the flask, and heat underagain shake or mix using a vortex mixer for 1 min while reflux for 45 min with constant stirring. Cool to roomthe preparation is still hot. Add 8 mL of alcohol, and temperature, add 170 mL of solvent hexane, and stir forswirl the contents to mix. Place the tube in a water bath 30 min. Quantitatively transfer the contents of the flaskat 60° for 5 min. Remove from the bath, and again to a 500-mL separatory funnel with portions of solventshake or mix using a vortex mixer for 2 min while the hexane. Allow the layers to separate for 5–10 min, andpreparation is still hot. Cool to room temperature. Add a transfer the upper organic layer to a 500-mL volumetricvolume of Internal standard solution, equivalent to 1.0 flask. Transfer the lower aqueous layer into themL per each 5 µg of the expected amount of saponification flask, add 170 mL of solvent hexane, andphytonadione in the aliquot taken. Add 20.0 mL of stir for an additional 20 min. Quantitatively transfer thepetroleum ether, and cap the tube tightly. Shake or mix contents of the saponification flask to the separatoryusing a vortex mixer for 15 min to thoroughly mix the funnel with the aid of portions of solvent hexane. Allowcontents. Centrifuge to separate the two layers. Transfer the layers to separate for 10 min. Drain the lowera volume of the top layer of petroleum ether, equivalent aqueous layer, and discard. Transfer the organic layer toto 5–50 µg of the nominal amount of phytonadione, to the volumetric flask containing the previously collectedan appropriate flask. Place the flask in a water bath at organic layer. Rinse the separatory funnel with small35°–45°, and evaporate the solvent under a stream of portions of solvent hexane, and transfer the washings tonitrogen until an oily residue is left. Dissolve the residue the volumetric flask. Dilute the hexane extracts with




solvent hexane to volume, add 3 g of anhydrous sodium Mobile phase: Mix 85 mL of acetonitrile, 1 g of sodiumsulfate, shake, and allow to settle. Quantitatively transfer perchlorate, 1 mL of phosphoric acid, and dilute witha volume of this solution, equivalent to 100 µg of beta water to 1000 mL.carotene, to a 50-mL volumetric flask. Evaporate under a Standard stock solution: 0.333 mg/mL of USP Biotin RSstream of nitrogen to dryness, and immediately add in dimethyl sulfoxidecyclohexane. Add 2 mL of Iodine solution, and heat for Standard solution: 5 µg/mL of USP Biotin RS prepared15 min in a water bath maintained at 65°. Cool rapidly, by diluting the Standard stock solution in waterand dilute with cyclohexane to volume. Sample solution: Finely powder NLT 20 Tablets. Transfer

Instrumental conditions a portion of the powder, equivalent to 1 mg of biotin, to(See Spectrophotometry and Light-Scattering ⟨851⟩.) a 200-mL volumetric flask, add 3 mL of dimethylMode: Vis sulfoxide, and swirl to wet. Place the flask in a waterAnalytical wavelength: 452 nm bath at 60°–70° for 5 min. Sonicate for 5 min, diluteBlank: Cyclohexane with water to volume, and filter.

Analysis Chromatographic systemSample: Sample solution (See Chromatography ⟨621⟩, System Suitability.)Determine the absorbance against the Blank. Calculate Mode: LCthe percentage of the labeled amount of beta carotene Detector: UV 200 nm(C40H56) in the portion of Tablets taken: Column: 4.6-mm × 15-cm; 3-µm packing L7

Flow rate: 1.2 mL/minResult = (AU/F) × (100/CU) Injection size: 100 µL

System suitabilityAU = absorbance of the Sample solution Sample: Standard solutionF = absorptivity of beta carotene at 452 nm, 223 Suitability requirementsCU = nominal concentration of beta carotene in the Relative standard deviation: NMT 3.0%

Sample solution (mg/mL) AnalysisAcceptance criteria: 90.0%–165.0% of the labeled Samples: Standard solution and Sample solutionamount of beta carotene (C40H56) Measure the responses for the biotin peaks. Calculate

• ASCORBIC ACID, Method 1 the percentage of the labeled amount of biotinSample solution: Finely powder NLT 20 Tablets. Transfer (C10H16N2O3S) in the portion of Tablets taken:a portion of the powder, equivalent of 100 mg ofascorbic acid, to a 200-mL volumetric flask, and add 75 Result = (rU/rS) × (CS/CU) × 100mL of metaphosphoric–acetic acids TS. Insert a stopperinto the flask, and shake by mechanical means for 30 rU = peak response from the Sample solutionmin. Dilute with water to volume. Transfer a portion of rS = peak response from the Standard solutionthe solution to a centrifuge tube, and centrifuge until a CS = concentration of USP Biotin RS in the Standardclear supernatant is obtained. Pipet 4.0 mL of this solution (µg/mL)solution into a 50-mL conical flask, and add 5 mL of CU = nominal concentration of biotin in the Samplemetaphosphoric–acetic acids TS. solution (µg/mL)

Analysis: Titrate with standard dichlorophenol–indophe- Acceptance criteria: 90.0%–150.0% of the labelednol solution VS to a rose-pink color that persists for at amount of biotin (C10H16N2O3S)least 5 s. Correct for the volume of dichlorophenol– • BIOTIN, Method 2indophenol solution consumed by a mixture of 5.5 mL [NOTE—Use low-actinic glassware throughout thisof metaphosphoric–acetic acids TS and 15 mL of water. procedure.]From the ascorbic acid equivalent of the standard Dehydrated mixtures yielding formulations similar to thedichlorophenol–indophenol solution, calculate the media described herein may be used provided that,content of ascorbic acid in each Tablet. when constituted as directed, they have growth-

Acceptance criteria: 90.0%–150.0% of the labeled promoting properties equal to or superior to thoseamount of ascorbic acid (C6H8O6) obtained with the media prepared as described herein.

• ASCORBIC ACID, Method 2: Proceed as directed in Standard stock solution: 50 µg/mL of USP Biotin RS inAutomated Methods of Analysis ⟨16⟩, Assay for Ascorbic 50% alcohol. Store this solution in a refrigerator.Acid. Standard solution: 0.1 ng/mL of USP Biotin RS in water,

Acceptance criteria: 90.0%–150.0% of the labeled prepared by dilution of the Standard stock solution withamount of ascorbic acid (C6H8O6) water on the day of the assay.

• CALCIUM ASCORBATE, Method 1: Proceed as directed in Sample solution: Finely powder NLT 30 Tablets. TransferAscorbic Acid, Method 1. a portion of the powder, equivalent to 100 µg of biotin,

Acceptance criteria: 90.0%–150.0% of the labeled to a 200-mL volumetric flask. Add 3 mL of 50% alcohol,amount of calcium ascorbate (C12H14CaO12 · 2H2O) and swirl to wet the contents. Heat the flask in a water

• CALCIUM ASCORBATE, Method 2: Proceed as directed in bath at 60°–70° for 5 min. Sonicate for 5 min, diluteAutomated Methods of Analysis ⟨16⟩, Assay for Ascorbic with 50% alcohol to volume, and filter. Dilute a volumeAcid. of the filtrate, quantitatively and stepwise if necessary,

Acceptance criteria: 90.0%–150.0% of the labeled with water to obtain a solution with a concentration ofamount of calcium ascorbate (C12H14CaO12 · 2H2O) 0.1 ng/mL.

• SODIUM ASCORBATE, Method 1: Proceed as directed in Acid-hydrolyzed casein solution: Mix 100 g of vitamin-Ascorbic Acid, Method 1. free casein with 500 mL of 6 N hydrochloric acid, and

Acceptance criteria: 90.0%–150.0% of the labeled reflux the mixture for 8–12 h. Remove the hydrochloricamount of sodium ascorbate (C6H7NaO6) acid from the mixture by distillation under reduced

• SODIUM ASCORBATE, Method 2: Proceed as directed in pressure until a thick paste remains. Redissolve theAutomated Methods of Analysis ⟨16⟩, Assay for Ascorbic resulting paste in water, adjust the solution with 1 NAcid. sodium hydroxide to a pH of 3.5 ±0.1, and add water to

Acceptance criteria: 90.0%–150.0% of the labeled make 1000 mL. Add 20 g of activated charcoal, stir for 1amount of sodium ascorbate (C6H7NaO6) h, and filter. Repeat the treatment with activated

• BIOTIN, Method 1 charcoal. Store under toluene in a cool place at a[NOTE—Use low-actinic glassware throughout this temperature not below 10°. Filter the solution if aprocedure.] precipitate forms during storage.




Cystine–tryptophan solution: Suspend 4.0 g of L- Culture medium: To each of a series of test tubescystine in a solution of 1.0 g of L-tryptophan (or 2.0 g of containing 5.0 mL of Basal medium stock solution add 5.0D,L-tryptophan) in 700–800 mL of water, heat to mL of water containing 0.5 ng of biotin. Plug the tubes70°–80°, and add dilute hydrochloric acid (1 in 2) with cotton, sterilize in an autoclave at 121° for 15 min,dropwise, with stirring, until the solids are dissolved. and cool.Cool, and add water to make 1000 mL. Store under Inoculum: [NOTE—A frozen suspension of Lactobacillustoluene in a cool place at a temperature not below 10°. plantarum may be used as the stock culture, provided it

Adenine–guanine–uracil solution: Dissolve 200 mg yields an inoculum comparable to a fresh culture.] Makeeach of adenine sulfate, guanine hydrochloride, and a transfer of cells from the Stock culture of Lactobacillusuracil, with the aid of heat, in 10 mL of 4 N hydrochloric plantarum to a sterile tube containing 10 mL of cultureacid. Cool, and add water to make 200 mL. Store under medium. Incubate this culture for 16–24 h at atoluene in a refrigerator. temperature between 30° and 37° held constant to

Polysorbate 80 solution: 100 mg/mL of polysorbate 80 within ±0.5°. The cell suspension so obtained is thein alcohol Inoculum.

Calcium pantothenate solution: 10 µg/mL of calcium Analysispantothenate in 50% alcohol. Store in a refrigerator. Samples: Standard solution and Sample solution

Riboflavin–thiamine hydrochloride solution: 20 µg/mL To similar separate test tubes add, in duplicate, 1.0 and/of riboflavin and 10 µg/mL of thiamine hydrochloride in or 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard0.02 N acetic acid. Store under toluene, protected from solution. To each tube and to four similar empty tubeslight, in a refrigerator. add 5.0 mL of Basal medium stock solution and sufficient

p-Aminobenzoic acid–niacin–pyridoxine hydrochloride water to make 10 mL.solution: 10 µg/mL of p-aminobenzoic acid, 50 µg/mL To similar test tubes add, in duplicate, volumes of theof niacin, and 40 µg/mL of pyridoxine hydrochloride in a Sample solution corresponding to three or more of themixture of neutralized alcohol and water (1:3). Store in a levels specified for the Standard solution, including therefrigerator. levels of 2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL

Salt solution A: 25 g of monobasic potassium phosphate of the Basal medium stock solution and sufficient waterand 25 g of dibasic potassium phosphate in water to to make 10 mL. Place one complete set of Standardmake 500 mL. Add 5 drops of hydrochloric acid. Store and sample tubes together in one tube rack and theunder toluene. duplicate set in a second rack or section of a rack,

Salt solution B: 10 g of magnesium sulfate, 0.5 g of preferably in random order.sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of Cover the tubes of both series to prevent contamination,manganese sulfate in water to make 500 mL. Add 5 and sterilize in an autoclave at 121° for 5 min. Cool,drops of hydrochloric acid, and mix. Store under and add 1 drop of Inoculum to each tube, except twotoluene. of the four tubes containing no Standard solution (the

Basal medium stock solution: Dissolve the anhydrous uninoculated blanks). Incubate the tubes at adextrose and anhydrous sodium acetate in the solutions temperature between 30° and 37° held constant topreviously mixed according to Table 1, and adjust with within ±0.5° until, following 16–24 h of incubation,1 N sodium hydroxide to a pH of 6.8. Dilute with water there has been no substantial increase in turbidity into 250 mL. the tubes containing the highest level of Standard

during a 2-h period.Determine the transmittance of the tubes in theTable 1following manner. Mix the contents of each tube, and

Acid-hydrolyzed casein solution 25 mL transfer to a spectrophotometer cell. Place the cell in aspectrophotometer that has been set at a specificCystine–tryptophan solution 25 mLwavelength from 540 to 660 nm, and read thePolysorbate 80 solution 0.25 mLtransmittance when a steady state is reached. ThisDextrose, anhydrous 10 g steady state is observed a few seconds after agitation

Sodium acetate, anhydrous 5 g when the galvanometer reading remains constant forAdenine–guanine–uracil solution 5 mL 30 s or more. Allow approximately the same timeCalcium pantothenate solution 5 mL interval for the reading on each tube.

With the transmittance set at 1.00 for the uninoculatedRiboflavin–thiamine hydrochloride solution 5 mLblank, read the transmittance of the inoculated blank.p-Aminobenzoic acid–niacin–pyridoxine 5 mLWith the transmittance set at 1.00 for the inoculatedhydrochloride solutionblank, read the transmittance for each of the remainingSalt solution A 5 mL tubes. If there is evidence of contamination with a

Salt solution B 5 mL foreign microorganism, disregard the result of theassay.Stock culture of Lactobacillus plantarum: Dissolve 2.0 Calculation: Prepare a standard concentration-responseg of yeast extract in 100 mL of water. Add 500 mg of curve as follows. For each level of the Standard, calculateanhydrous dextrose, 500 mg of anhydrous sodium the response from the sum of the duplicate values of theacetate, and 1.5 g of agar, and heat the mixture on a transmittance (ΣS) as the difference, y = 2.00 − ΣS. Plotsteam bath, with stirring, until the agar dissolves. Add this response on the ordinate of cross-section paper10-mL portions of the hot solution to test tubes, close or against the logarithm of the mL of Standard solution percover the tubes, sterilize in an autoclave at 121° for 15 tube on the abscissa, using for the ordinate either anmin, and allow the tubes to cool in an upright position. arithmetic or a logarithmic scale, whichever gives thePrepare stab cultures in three or more of the tubes, better approximation to a straight line. Draw the straightusing a pure culture of Lactobacillus plantarum,1 line or smooth curve that best fits the plotted points.incubating for 16–24 h at a temperature between 30° Calculate the response, y = 2.00 − ΣU, adding togetherand 37° held constant to within ±0.5°. Store in a the two transmittances for each level of the Samplerefrigerator. Prepare a fresh stab of the stock culture solution (ΣU). Read from the standard curve theevery week, and do not use for Inoculum if the culture is logarithm of the volume of the Standard solutionmore than 1 week old. corresponding to each of those values of y that fall

1 ATCC No. 8014 is suitable. This strain was formerly known as Lactobacillus within the range of lowest and highest points plottedarabinosus 17-5. 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 Chromatographic systemdosage level. Average the values of X for each of three (See Chromatography ⟨621⟩, System Suitability.)or more dosage levels to obtain X, which equals the Mode: LClog-relative potency, M′, of the Sample solution. Detector: UV 200 nmDetermine the quantity, in µg, of biotin in the portion Column: 4.6-mm × 25-cm; packing L1of Tablets taken: Flow rate: 2 mL/min

Injection size: 100 µLantilog M = antilog (M′ + log R) System suitability

Samples: Standard solution and portion of StandardR = number of µg of biotin that was assumed to solution that has undergone Solid-phase extraction

be present in the portion of the Tablets Suitability requirementstaken Tailing factor: NMT 1.5, Standard solution

Calculate the percentage of the labeled amount of biotin Relative standard deviation: NMT 2.0%, Standard(C10H16N2O3S) in the portion of the Tablets taken solution and the Standard solution that has undergone

Solid-phase extractionResult = [(antilog M)/N] × 100 Recovery: 95%–100%, Standard solution that has

undergone Solid-phase extractionN = nominal amount of biotin in the portion of Analysisthe Tablets taken (µg) Samples: Standard solution and Sample solution thatReplication: Repeat the entire determination at least have both undergone Solid-phase extractiononce, using separately prepared Sample solutions. If the Measure the responses for the biotin peak. Calculatedifference between the two log-potencies M is NMT the percentage of the labeled amount of biotin0.08, their mean, M, is the assayed log-potency of the (C10H16N2O3S) in the portion of Tablets taken:test material (see Design and Analysis of Biological Assays⟨111⟩, The Confidence Interval and Limits of Potency). If Result = (rU/rS) × (CS/CU) × 100the two determinations differ by more than 0.08,conduct one or more additional determinations. From rU = peak response from the Sample solutionthe mean of two or more values of M that do not differ rS = peak response from the Standard solutionby more than 0.15, compute the mean potency of the CS = concentration of USP Biotin RS in the Standardpreparation under assay. solution (µg/mL)

Acceptance criteria: 90.0%–150.0% of the labeled CU = nominal concentration of biotin in the Sampleamount of biotin (C10H16N2O3S) solution (µg/mL)

• BIOTIN, Method 3 Acceptance criteria: 90.0%–150.0% of the labeled[NOTE—Use low-actinic glassware throughout this amount of biotin (C10H16N2O3S)procedure.] • CYANOCOBALAMIN, Method 1

Solution A: Transfer 800 mL of water and 100 mL of [NOTE—Use low-actinic glassware throughout thistriethylamine to a 1000-mL volumetric flask. Add 80 mL procedure.]of 85% phosphoric acid, and dilute with water to Mobile phase: Methanol and water (7:13)volume. Standard stock solution: 10 µg/mL of USP

Mobile phase: Transfer 80 mL of acetonitrile and 10 mL Cyanocobalamin RS in water. [NOTE—Store this stockof Solution A to a 1000-mL volumetric flask. Dilute with solution in a dark place, and discard after 1 week.]water to volume. Standard solution: 1 µg/mL of USP Cyanocobalamin RS

Standard solution: 0.6 µg/mL of USP Biotin RS in water. from Standard stock solution diluted with water[NOTE—A portion of the Standard solution will be used to Sample solution: Finely powder NLT 30 Tablets. Transferdetermine the percent recovery of biotin from the Solid- a portion of the powder, equivalent to 100 µg ofphase extraction procedure.] cyanocobalamin, to a 250-mL flask. Quantitatively add

Sample solution: Finely powder NLT 20 Tablets. Transfer 100.0 mL of water, and carefully extract for 2 min. Filteran amount of powdered Tablets to a volumetric flask to 10 mL of the extract, and use the filtrate.obtain a concentration of 0.6 µg/mL of biotin. Add Chromatographic systemwater up to 80% of the flask capacity, sonicate for (See Chromatography ⟨621⟩, System Suitability.)30–40 min, with occasional mixing, to dissolve. Dilute Mode: LCwith water to volume, and filter. Adjust the pH of the Detector: 550 nmsolution with either dilute acetic acid or 0.1 N sodium Column: 4.6-mm × 15-cm; 5-µm packing L1hydroxide to 6.0–7.0. Flow rate: 0.5 mL/min

Solid-phase extraction: [NOTE—Condition the extraction Injection size: 200 µLcolumn specified in this procedure in the following System suitabilitymanner. Wash the column with a 2-mL portion of Sample: Standard solutionmethanol. Equilibrate with a 2-mL portion of water.] Suitability requirementsSeparately pipet 5.0 mL of the Sample solution and Relative standard deviation: NMT 3.0%Standard solution into freshly conditioned solid-phase Analysisextraction columns consisting of a mixed-mode packing Samples: Standard solution and Sample solutionwith a sorbent-mass of 60 mg. [NOTE—The mixed-mode Measure the peak responses for cyanocobalamin.packing consists of anion-exchange and reversed-phase Calculate the percentage of the labeled amount ofsorbents. The reverse-phase component is a polymer of cyanocobalamin (C63H88CoN14O14P) in the portion ofcopolymer N-vinylpyrrolidone and divinylbenzene. The Tablets taken:anion exchange moiety is a trialkylamino group.2] Washthe column with 10 mL of 30% methanol in water. Result = (rU/rS) × (CS/CU) × 100Apply an appropriate volume (4.9 mL) of 30% methanolin 0.1 N hydrochloric acid to the column. Collect the rU = peak response from the Sample solutioneluate in a 5-mL volumetric flask, containing 100 µL of rS = peak response from the Standard solution40% (w/v) sodium acetate in water, and dilute with 30% CS = concentration of USP Cyanocobalamin RS inmethanol in 0.1 N hydrochloric acid to volume. the Standard solution (µg/mL)

CU = nominal concentration of cyanocobalamin in2 A suitable cartridge is the Waters, Oasis MAX Vac RC cartridge, particle size the Sample solution (µg/mL)30 µm, part 186000371.




Acceptance criteria: 90.0%–150.0% of the labeled acid. Filter, if necessary. Add the Polysorbate 80 solution,amount of cyanocobalamin (C63H88CoN14O14P) adjust with 1 N sodium hydroxide to a pH of between

• CYANOCOBALAMIN, Method 2 5.5 and 6.0, and add Purified Water to make 250 mL.[NOTE—Use low-actinic glassware throughout thisprocedure.] Table 2

Standard stock solution: 1.0 µg/mL of USPL-Cystine 0.1 gCyanocobalamin RS in 25% alcohol. Store in a

refrigerator. L-Tryptophan 0.05 gStandard solution: Dilute a suitable volume of Standard 1 N hydrochloric acid 10 mLstock solution with water to a measured volume such that Adenine–guanine–uracil solution 5 mLafter the incubation period as described in the Analysis,

Xanthine solution 5 mLthe difference in transmittance between the inoculatedVitamin solution A 10 mLblank and the 5.0-mL level of the Standard solution isVitamin solution B 10 mLNLT that which corresponds to a difference of 1.25 mg

in dried cell weight. This concentration usually falls Salt solution A 5 mLbetween 0.01 and 0.04 ng/mL of Standard solution. Salt solution B 5 mLPrepare this solution fresh for each assay. Asparagine solution 5 mLSample solution: Finely powder NLT 20 Tablets. Transfer

Acid-hydrolyzed casein solution 25 mLa portion of the powdered Tablets, equivalent to 1.0 µgDextrose, anhydrous 10 gof cyanocobalamin, to an appropriate vessel containing,Sodium acetate, anhydrous 5 gfor each g of powdered Tablets taken, 25 mL of an

aqueous extracting solution prepared just before use to Ascorbic acid 1 gcontain, in each 100 mL, 1.29 g of dibasic sodium Polysorbate 80 solution 5 mLphosphate, 1.1 g of anhydrous citric acid, and 1.0 g ofsodium metabisulfite. Autoclave the mixture at 121° for Tomato juice preparation: Centrifuge commercially10 min. Allow any undissolved particles of the extract to canned tomato juice so that most of the pulp issettle, and filter or centrifuge if necessary. Dilute an removed. Suspend 5 g/L of analytical filter aid in thealiquot of the clear solution with water to obtain a final supernatant, and pass, with the aid of reduced pressure,solution containing vitamin B12 activity equivalent to the through a layer of the filter aid. Repeat, if necessary,nominal activity of the Standard solution. until a clear, straw-colored filtrate is obtained. Store

Acid-hydrolyzed casein solution: Prepare as directed in under toluene in a refrigerator.Biotin, Method 2. Culture medium: [NOTE—A dehydrated mixture

Asparagine solution: Dissolve 2.0 g of L-asparagine in containing the same ingredients may be used providedwater to make 200 mL. Store under toluene in a that, when constituted as directed in the labeling, itrefrigerator. yields a medium equivalent to that obtained from the

Adenine–guanine–uracil solution: Prepare as directed in formula given herein.] Dissolve 0.75 g of yeast extract,Biotin, Method 2. 0.75 g of dried peptone, 1.0 g of anhydrous dextrose,

Xanthine solution: Suspend 0.20 g of xanthine in and 0.20 g of monobasic potassium phosphate in 60–7030–40 mL of water, heat to 70°, add 6.0 mL of 6 N mL of water. Add 10 mL of Tomato juice preparation andammonium hydroxide, and stir until the solid is 1 mL of Polysorbate 80 solution. Adjust with 1 N sodiumdissolved. Cool, and add water to make 200 mL. Store hydroxide to a pH of 6.8, and add water to make 100under toluene in a refrigerator. mL. Place 10-mL portions of the solution in test tubes,

Salt solution A: Dissolve 10 g of monobasic potassium and plug with cotton. Sterilize the tubes and contents inphosphate and 10 g of dibasic potassium phosphate in an autoclave at 121° for 15 min. Cool as rapidly aswater to make 200 mL, and add 2 drops of hydrochloric possible to avoid color formation resulting fromacid. Store this solution under toluene. overheating the medium.

Salt solution B: Dissolve 4.0 g of magnesium sulfate, Suspension medium: Dilute a measured volume of Basal0.20 g of sodium chloride, 0.20 g of ferrous sulfate, and medium stock solution with an equal volume of water.0.20 g of manganese sulfate in water to make 200 mL, Place 10-mL portions of the diluted medium in testand add 2 drops of hydrochloric acid. Store this solution tubes. Sterilize, and cool as directed for Culture medium.under toluene. Stock culture of Lactobacillus leichmannii: To 100 mL

Polysorbate 80 solution: 20 g of polysorbate 80 in of Culture medium add 1.0–1.5 g of agar, and heat thealcohol to make 200 mL. Store in a refrigerator. mixture on a steam bath, with stirring, until the agar

Vitamin solution A: 10 mg of riboflavin, 10 mg of dissolves. Place 10-mL portions of the hot solution in testthiamine hydrochloride, 100 µg of biotin, and 20 mg of tubes, cover the tubes, sterilize at 121° for 15 min in anniacin in 0.02 N acetic acid to make 400 mL. Store autoclave, and allow the tubes to cool in an uprightunder toluene, protected from light, in a refrigerator. position. Inoculate three or more of the tubes by stab

Vitamin solution B: 20 mg of p-aminobenzoic acid, 10 transfer of a pure culture of Lactobacillus leichmannii.3 mg of calcium pantothenate, 40 mg of pyridoxine [NOTE—Before first using a fresh culture in this assay,hydrochloride, 40 mg of pyridoxal hydrochloride, 8 mg make NLT 10 successive transfers of the culture in a 2-of pyridoxamine dihydrochloride, and 2 mg of folic acid week period.]in a mixture of water and neutralized alcohol (3:1) to Incubate for 16–24 h at a temperature between 30° andmake 400 mL. Store, protected from light, in a 40° held constant to within ±0.5°. Store in arefrigerator. refrigerator.

Basal medium stock solution: Prepare the medium Prepare fresh stab cultures at least three times eachaccording to the following formula and directions. A week, and do not use them for preparing the Inoculumdehydrated mixture containing the same ingredients if more than 4 days old. The activity of themay be used provided that, when constituted as directed microorganism can be increased by daily or twice-dailyin the labeling, it yields a medium comparable to that transfer of the stab culture, to the point where definiteobtained from the formula given herein. turbidity in the liquid Inoculum can be observed 2–4 hAdd the ingredients in the order listed in Table 2, after inoculation. A slow-growing culture seldom gives acarefully dissolving cystine and tryptophan in the suitable response curve and may lead to erratic results.hydrochloric acid before adding the next eight solutions

3 Pure cultures of Lactobacillus leichmannii may be obtained as No. 7830 fromto the resulting solution. Add 100 mL of water, and ATCC, 10801 University Blvd., Manassas, VA 20110-2209.dissolve the dextrose, sodium acetate, and ascorbic




Inoculum: [NOTE—A frozen suspension of Lactobacillus tubes. Disregard the results of the assay if the slope ofleichmannii may be used as the stock culture, provided it the standard curve indicates a problem with sensitivity.yields an Inoculum comparable to a fresh culture.] Make Calculation: Prepare a standard concentration-responsea transfer of cells from the Stock culture of Lactobacillus curve by the following procedure. Test for and replaceleichmannii to two sterile tubes containing 10 mL of the any aberrant individual transmittances. For each level ofCulture medium each. Incubate these cultures for 16–24 the Standard, calculate the response from the sum ofh at a temperature between 30° and 40° held constant the duplicate values of the transmittances (ΣS) as theto within ±0.5°. Under aseptic conditions centrifuge the difference, y = 2.00 − ΣS. Plot this response on thecultures, and decant the supernatant. Suspend the cells ordinate of cross-section paper against the logarithm offrom the culture in 5 mL of sterile Suspension medium, the mL of Standard solution per tube on the abscissa,and combine. Using sterile Suspension medium, adjust the using for the ordinate either an arithmetic or avolume so that a 1-in-20 dilution in saline TS produces logarithmic scale, whichever gives the better70% transmittance when read on a suitable approximation to a straight line. Draw the straight linespectrophotometer that has been set at a wavelength of or smooth curve that best fits the plotted points.530 nm, equipped with a 10-mm cell, and read against Calculate the response, y= 2.00 − ΣU, adding togethersaline TS set at 100% transmittance. Prepare a 1-in-400 the two transmittances for each level of the Sampledilution of the adjusted suspension using sterile Basal solution (ΣU). Read from the standard curve themedium stock solution. The cell suspension so obtained is logarithm of the volume of the Standard solutionthe Inoculum. [NOTE—This dilution may be altered, when corresponding to each of those values of y that fallsnecessary, to obtain the desired test response.] within the range of the lowest and highest points

Calibration of spectrophotometer: Check the plotted for the Standard. Subtract from each logarithmwavelength of the spectrophotometer periodically, using so obtained the logarithm of the volume, in mL, of thea standard wavelength cell or other suitable device. Sample solution to obtain the difference, X, for eachBefore reading any tests, calibrate the spectrophotometer dosage level. Average the values of X for each of threefor 0% and 100% transmittance, using water and with or more dosage levels to obtain X, which equals thethe wavelength set at 530 nm. log-relative potency, M′, of the Sample solution.

Analysis Determine the quantity, in µg, of cyanocobalamin inSamples: Standard solution and Sample solution the portion of Tablets taken:Because of the high sensitivity of the test organism to

antilog M = antilog (M′ + log R)minute amounts of vitamin B12 activity and to traces ofmany cleansing agents, cleanse meticulously by suitable

R = number of µg of cyanocobalamin that wasmeans, followed preferably by heating at 250° for 2 h,assumed to be present in the portion ofusing hard-glass 20-mm × 150-mm test tubes, andTablets takenother necessary glassware.

Calculate the percentage of the labeled amount ofTo separate test tubes add, in duplicate, 1.0, 1.5, 2.0,cyanocobalamin in the portion of the Tablets taken:3.0, 4.0, and 5.0 mL of the Standard solution. To each

of these tubes and to four similar empty tubes add 5.0Result = [(antilog M)/N] × 100mL of Basal medium stock solution and sufficient water

to make 10 mL. N = nominal amount of cyanocobalamin in theTo similar separate test tubes add, in duplicate, 1.0, 1.5, portion of the Tablets taken (µg)2.0, 3.0, and 4.0 mL of the Sample solution. To each Replication: Repeat the entire determination at leasttube add 5.0 mL of Basal medium stock solution and once, using separately prepared Sample solutions. If thesufficient water to make 10 mL. Place one complete set difference between the two log-potencies M is NMTof standard and sample tubes together in one tube rack 0.08, their mean, M, is the assayed log-potency of theand the duplicate set in a second rack or section of a test material (see Vitamin B12 Activity in Design andrack, preferably in random order. Analysis of Biological Assays ⟨111⟩, The ConfidenceCover the tubes to prevent bacterial contamination, and Interval and Limits of Potency). If the two determinationssterilize in an autoclave at 121° for 5 min, arranging to differ by more than 0.08, conduct one or morereach this temperature in NMT 10 min by preheating additional determinations. From the mean of two orthe autoclave if necessary. Cool as rapidly as possible to more values of M that do not differ by more than 0.15,avoid color formation resulting from overheating the compute the mean potency of the preparation undermedium. Take precautions to maintain uniformity of assay.sterilizing and cooling conditions throughout the assay, Acceptance criteria: 90.0%–150.0% of the labeledbecause packing the tubes too closely in the autoclave amount of cyanocobalamin (C63H88CoN14O14P)or overloading it may cause variation in the heating • FOLIC ACID, Method 1rate. [NOTE—Use low-actinic glassware throughout thisAseptically add 0.5 mL of Inoculum to each tube so procedure.]prepared, except two of the four containing no Reagent A: 25% solution of tetrabutylammoniumStandard solution (the uninoculated blanks). Incubate hydroxide in methanolthe tubes at a temperature between 30° and 40°, held Reagent B: Transfer 5.0 g of pentetic acid to a 50-mLconstant to within ±0.5°, for 16–24 h. volumetric flask. Using sonication if necessary, dissolve inTerminate growth by heating to a temperature NLT 80° and dilute with 1 N sodium hydroxide to volume.for 5 min. Cool to room temperature. After agitating its Mobile phase: 2 g of monobasic potassium phosphatecontents, and read the transmittance at 530 nm when in 650 mL of water. Add 12.0 mL of Reagent A, 7.0 mLa steady state is reached. This steady state is observed a of 3 N phosphoric acid, and 240 mL of methanol. Coolfew seconds after agitation when the reading remains to room temperature, adjust with phosphoric acid orconstant for 30 s or more. Allow approximately the ammonia TS to a pH of 7.0, dilute with water to 1000same time interval for the reading on each tube. mL, and filter. Recheck the pH before use. [NOTE—TheWith the transmittance set at 100% for the uninoculated methanol and water content may be varied (betweenblank, read the transmittance of the inoculated blank. If 1% and 3%) by adding water or methanol to thethe difference is greater than 5% or if there is evidence prepared Mobile phase to obtain baseline separation ofof contamination with a foreign microorganism, folic acid and the internal standard. The pH may bedisregard the results of the assay. increased up to 7.15 to obtain better separation.]With the transmittance set at 100% for the uninoculatedblank, read the transmittance of each of the remaining




Internal standard solution: Transfer 40 mg of Chromatographic systemmethylparaben to a 1000-mL volumetric flask, and add (See Chromatography ⟨621⟩, System Suitability.)220 mL of methanol to dissolve. Dissolve 2.0 g of Mode: LCmonobasic potassium phosphate in 300 mL of water in a Detector: UV 270 nmseparate beaker, quantitatively transfer this solution to Column: 4.6-mm × 25-cm; packing L7the flask containing the methylparaben solution, and add Column temperature: 50°an additional 300 mL of water. Add 19 mL of Reagent A, Flow rate: 2 mL/min7 mL of 3 N phosphoric acid, and 30 mL of Reagent B. Injection size: 5 µLAdjust with ammonia TS to a pH of 9.8, bubble nitrogen System suitabilitythrough the solution for 30 min, dilute with water to Sample: Standard solutionvolume, and mix. Suitability requirements

Standard solution: 0.016 mg/mL of USP Folic Acid RS in Relative standard deviation: NMT 2.0%Internal standard solution Analysis

Sample solution: Finely powder NLT 30 Tablets. Transfer Samples: Standard solution and Sample solutiona portion of powder, equivalent to 0.4 mg of folic acid, Measure the areas of the major peaks. Calculate theto a 50-mL amber-colored centrifuge tube. Add 25.0 mL percentage of the labeled amount of folic acidof Internal standard solution, shake by mechanical means (C19H19N7O6) in the portion of Tablets taken:for 10 min, and centrifuge. Filter a portion of the clear

Result = (rU/rS) × (CS/CU) × 100supernatant, and use the filtrate.Chromatographic system

rU = peak area of folic acid from the Sample(See Chromatography ⟨621⟩, System Suitability.)solutionMode: LC

rS = peak area of folic acid from the StandardDetector: UV 280 nmsolutionColumn: 3.9-mm × 30-cm; packing L1

CS = concentration of USP Folic Acid RS in theFlow rate: 1 mL/minStandard solution (µg/mL)Injection size: 15 µL

CU = nominal concentration of folic acid in theSystem suitabilitySample solution (µg/mL)Sample: Standard solution

Acceptance criteria: 90.0%–150.0% of the labeled[NOTE—The relative retention times for folic acid andamount of folic acid (C19H19N7O6)methylparaben are about 0.8 and 1.0, respectively.]

• CALCIUM PANTOTHENATE, Method 1Suitability requirementsMobile phase: Phosphoric acid and water (1:1000)Relative standard deviation: NMT 3.0%Internal standard solution: 80 mg of p-hydroxybenzoicAnalysisacid in 3 mL of alcohol. Add 50 mL of water and 7.1 gSamples: Standard solution and Sample solutionof dibasic sodium phosphate, and dilute with water toMeasure the peak areas for folic acid and1000 mL. Adjust with phosphoric acid to a pH of 6.7.methylparaben. Calculate the percentage of the

Standard solution: 0.6 mg/mL of USP Calciumlabeled amount of folic acid (C19H19N7O6) in thePantothenate RS in Internal standard solutionportion of Tablets taken:

Sample solution: Finely powder NLT 30 Tablets. Transfera portion of the powder, equivalent to 15 mg of calciumResult = (RU/RS) × (CS/CU) × 100pantothenate, to a centrifuge tube. Add 25.0 mL of the

RU = peak area ratio of folic acid to methylparaben Internal standard solution, and shake vigorously for 10from the Sample solution min. Centrifuge, filter, and use the clear filtrate.

RS = peak area ratio of folic acid to methylparaben Chromatographic systemfrom the Standard solution (See Chromatography ⟨621⟩, System Suitability.)

CS = concentration of USP Folic Acid RS in the Mode: LCStandard solution (µg/mL) Detector: UV 210 nm

CU = nominal concentration of folic acid in the Column: 3.9-mm × 15-cm; packing L1Sample solution (µg/mL) Flow rate: 1.5 mL/min

Acceptance criteria: 90.0%–150.0% of the labeled Injection size: 10 µLamount of folic acid (C19H19N7O6) System suitability

• FOLIC ACID, Method 2 Sample: Standard solution[NOTE—Use low-actinic glassware throughout this [NOTE—The relative retention times for calciumprocedure.] pantothenate and p-hydroxybenzoic acid are about

Reagent: Dissolve 7.5 g of edetate disodium, with 0.5 and 1.0, respectively.]stirring, in 500 mL of water containing 10 mL of Suitability requirementsammonium hydroxide. Relative standard deviation: NMT 3.0%

Diluent: 60 µg/mL of ammonium hydroxide AnalysisMobile phase: Transfer 0.4 mL of triethylamine, 15 mL Samples: Standard solution and Sample solutionof glacial acetic acid, and 350 mL of methanol to a Measure the peak responses for calcium pantothenate2000-mL volumetric flask, and dilute with 0.008 M and the internal standard. Calculate the percentage ofsodium 1-hexanesulfonate to volume. the labeled amount of calcium pantothenate

Standard stock solution: 60 µg/mL of USP Folic Acid RS (C18H32CaN2O10) in the portion of Tablets taken:in Diluent. Prepare this solution fresh daily.

Result = (RU/RS) × (CS/CU) × 100Standard solution: Mix 5.0 mL of Standard stock solutionwith 10.0 mL of methanol and 35.0 mL of Reagent, and

RU = peak response ratio of calcium pantothenateshake for 15 min in a water bath maintained at 60°, andto p-hydroxybenzoic acid from the Samplecool. Filter, discarding the first few mL of the filtrate.solutionSample solution: Transfer a portion of finely powdered

RS = peak response ratio of calcium pantothenateTablets, equivalent to 0.3 mg of folic acid, to a 125-mLto p-hydroxybenzoic acid from the Standardstoppered flask. Add 10.0 mL of methanol and 35.0 mLsolutionof Reagent. Shake for 15 min in a water bath maintained

CS = concentration of USP Calcium Pantothenateat 60°, and cool. Filter, discarding the first few mL of theRS in the Standard solution (mg/mL)filtrate.




CU = nominal concentration of calcium Basal medium stock solution: Dissolve the anhydrouspantothenate in the Sample solution (mg/mL) dextrose and anhydrous sodium acetate in the solutions

Acceptance criteria: 90.0%–150.0% of the labeled previously mixed according to Table 3, and adjust withamount of calcium pantothenate (C18H32CaN2O10) 1 N sodium hydroxide to a pH of 6.8. Dilute with water

• CALCIUM PANTOTHENATE, Method 2 to 250 mL.Standard stock solution: Dissolve 50 mg of USPCalcium Pantothenate RS, previously dried and stored in Table 3the dark over phosphorus pentoxide and protected from

Acid-hydrolyzed casein solution 25 mLabsorption of moisture while weighing, in 500 mL ofwater in a 1000-mL volumetric flask. Add 10 mL of 0.2 Cystine–tryptophan solution 25 mLN acetic acid and 100 mL of sodium acetate solution (1 Polysorbate 80 solution 0.25 mLin 60), and dilute with water to volume, to obtain a Dextrose, anhydrous 10 gconcentration of 50 µg/mL of USP Calcium Pantothenate

Sodium acetate, anhydrous 5 gRS. Store under toluene in a refrigerator.Adenine–guanine–uracil solution 5 mLStandard solution: On the day of the assay, dilute aRiboflavin–thiamine hydrochloride–biotin solution 5 mLvolume of Standard stock solution with water to obtain a

concentration of 0.01–0.04 µg/mL of calcium p-Aminobenzoic acid–niacin–pyridoxine 5 mLpantothenate, the exact concentration being such that hydrochloride solutionthe responses obtained as directed in the Analysis, 2.0 Salt solution A 5 mLand 4.0 mL of the Standard solution being used, are Salt solution B 5 mLwithin the linear portion of the log-concentrationresponse curve. Stock culture of Lactobacillus plantarum: Dissolve 2.0

Sample solution: Finely powder NLT 30 Tablets. Transfer g of yeast extract in 100 mL of water; add 500 mg ofa portion of the powder, equivalent to 50 mg of calcium anhydrous dextrose, 500 mg of anhydrous sodiumpantothenate, to a 1000-mL volumetric flask containing acetate, and 1.5 g of agar; and heat the mixture on a500 mL of water. Add 10 mL of 0.2 N acetic acid and steam bath, with stirring, until the agar dissolves. Add100 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 volume of this close or cover the tubes, sterilize in an autoclave at 121°solution to obtain a solution having approximately the for 15 min, and allow the tubes to cool in an uprightsame concentration as that of the Standard solution. position. Prepare stab cultures in three or more of the

Acid-hydrolyzed casein solution: Mix 100 g of vitamin- tubes, using a pure culture of Lactobacillus plantarum4 free casein with 500 mL of 6 N hydrochloric acid, and incubating for 16–24 h at a temperature between 30°reflux the mixture for 8–12 h. Remove the hydrochloric and 37° held constant to within ±0.5°. Store in aacid from the mixture by distillation under reduced refrigerator. Prepare a fresh stab of the stock culturepressure until a thick paste remains. Redissolve the every week, and do not use for Inoculum if the culture isresulting paste in water, adjust the solution with 1 N more than 1 week old.sodium hydroxide to a pH of 3.5 ±0.1, and add water to Culture medium: To each of a series of test tubesmake 1000 mL. Add 20 g of activated charcoal, stir for 1 containing 5.0 mL of Basal medium stock solution add 5.0h, and filter. Repeat the treatment with activated mL of water containing 0.2 µg of calcium pantothenate.charcoal. Store under toluene in a cool place at a Plug the tubes with cotton, sterilize in an autoclave attemperature not below 10°. Filter the solution if a 121° for 15 min, and cool.precipitate forms during storage. Inoculum: [NOTE—A frozen suspension of Lactobacillus

Cystine–tryptophan solution: Suspend 4.0 g of L- plantarum may be used as the stock culture, provided itcystine in a solution of 1.0 g of L-tryptophan (or 2.0 g of yields an Inoculum comparable to a fresh culture.]D,L-tryptophan) in 700–800 mL of water, heat to Make a transfer of cells from the Stock culture of70°–80°, and add dilute hydrochloric acid (1 in 2) Lactobacillus plantarum to a sterile tube containing 10dropwise, with stirring, until the solids are dissolved. mL of Culture medium. Incubate this culture for 16–24 hCool, and add water to make 1000 mL. Store under at a temperature between 30° and 37° held constant totoluene in a cool place at a temperature not below 10°. within ±0.5°. The cell suspension so obtained is the

Adenine–guanine–uracil solution: Dissolve 200 mg Inoculum.each of adenine sulfate, guanine hydrochloride, and Analysisuracil, with the aid of heat, in 10 mL of 4 N hydrochloric Samples: Standard solution and Sample solutionacid. Cool, and add water to make 200 mL. Store under To similar separate test tubes add, in duplicate, 1.0 and/toluene in a refrigerator. or 1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard

Polysorbate 80 solution: 100 mg/mL of polysorbate 80 solution. To each tube and to four similar empty tubesin alcohol add 5.0 mL of Basal medium stock solution and sufficient

Riboflavin–thiamine hydrochloride–biotin solution: 20 water to make 10 mL.µg/mL of riboflavin, 10 µg/mL of thiamine To similar separate test tubes add, in duplicate, volumeshydrochloride, and 0.04 µg/mL of biotin in 0.02 N acetic of the Sample solution corresponding to three or moreacid. Store under toluene, protected from light, in a of the levels specified for the Standard solution,refrigerator. including the levels of 2.0, 3.0, and 4.0 mL. To each

p-Aminobenzoic acid–niacin–pyridoxine hydrochloride tube add 5.0 mL of the Basal medium stock solution andsolution: 10 µg/mL of p-aminobenzoic acid, 50 µg/mL sufficient water to make 10 mL. Place one complete setof niacin, and 40 µg/mL of pyridoxine hydrochloride in a of Standard and sample tubes together in one tube rackmixture of neutralized alcohol and water (1:3). Store in a and the duplicate set in a second rack or section of arefrigerator. rack, preferably in random order.

Salt solution A: Dissolve 25 g of monobasic potassium Cover the tubes of both series to prevent contamination,phosphate and 25 g of dibasic potassium phosphate in and sterilize in an autoclave at 121° for 5 min. Cool,water to make 500 mL. Add 5 drops of hydrochloric and add 1 drop of Inoculum to each tube, except twoacid. Store under toluene. of the four tubes containing no Standard solution (the

Salt solution B: Dissolve 10 g of magnesium sulfate, 0.5 uninoculated blanks). Incubate the tubes at ag of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g temperature between 30° and 37°, held constant toof manganese sulfate in water to make 500 mL. Add 5

4 ATCC No. 8014 is suitable. This strain was formerly known as Lactobacillusdrops of hydrochloric acid. Store under toluene.arabinosus 17-5.




within ±0.5° until, following 16–24 h of incubation, • CALCIUM PANTOTHENATE, Method 3there has been no substantial increase in turbidity in Buffer solution: Dissolve 10.0 g of monobasic potassiumthe tubes containing the highest level of Standard phosphate in 2000 mL of water, and adjust withduring a 2-h period. phosphoric acid to a pH of 3.5.

Determine the transmittance of the tubes in the Mobile phase: Methanol and Buffer solution (1:9)following manner. Mix the contents of each tube, and Standard stock solution: 0.25 mg/mL of USP Calciumtransfer to an optical container if necessary. Read the Pantothenate RS in water. Prepare fresh every 4 weeks.transmittance between 540 and 660 nm when a steady Store in a refrigerator.state is reached. This steady state is observed a few Standard solution: 40 µg/mL of USP Calciumseconds after agitation when the galvanometer reading Pantothenate RS from Standard stock solution dilutedremains constant for 30 s or more. Allow approximately with waterthe same time interval for the reading on each tube. Sample solution: Finely powder NLT 20 Tablets. Transfer

With the transmittance set at 1.00 for the uninoculated a portion of the powder, equivalent to 10 mg of calciumblank, read the transmittance of the inoculated blank. pantothenate, to a 250-mL volumetric flask. Add 10 mLWith the transmittance set at 1.00 for the inoculated of methanol, and swirl the flask to disperse. Dilute withblank, read the transmittance for each of the remaining water to volume, mix, and filter.tubes. If there is evidence of contamination with a Chromatographic systemforeign microorganism, disregard the result of the (See Chromatography ⟨621⟩, System Suitability.)assay. Mode: LC

Calculation: Prepare a standard concentration-response Detector: UV 205 nmcurve as follows. For each level of the Standard, Column: 3.9-mm × 30-cm; 5-µm packing L1calculate the response from the sum of the duplicate Column temperature: 50°values of the transmittance (ΣS) as the difference, y = Flow rate: 2 mL/min2.00 − ΣS. Plot this response on the ordinate of cross- Injection size: 25 µLsection paper against the logarithm of the mL of System suitabilityStandard solution per tube on the abscissa, using for the Sample: Standard solutionordinate either an arithmetic or a logarithmic scale, Suitability requirementswhichever gives the better approximation to a straight Relative standard deviation: NMT 3.0%line. Draw the straight line or smooth curve that best Analysisfits the plotted points. Samples: Standard solution and Sample solutionCalculate the response, y = 2.00 − ΣU, adding together Measure the peak areas for calcium pantothenate.the two transmittances for each level of the Sample Calculate the percentage of the labeled amount ofsolution (ΣU). Read from the standard curve the calcium pantothenate (C18H32CaN2O10) in the portionlogarithm of the volume of the Standard solution of Tablets taken:corresponding to each of those values of y that fall

Result = (rU/rS) × (CS/CU) × 100within the range of the lowest and highest pointsplotted for the Standard. Subtract from each logarithm

rU = peak area from the Sample solutionso obtained the logarithm of the volume, in mL, of therS = peak area from the Standard solutionSample solution to obtain the difference, X, for eachCS = concentration of USP Calcium Pantothenatedosage level. Average the values of X for each of three

RS in the Standard solution (mg/mL)or more dosage levels to obtain X, which equals theCU = nominal concentration of calciumlog-relative potency, M′, of the Sample solution.

pantothenate in the Sample solution (mg/mL)Determine the quantity, in mg, of calciumAcceptance criteria: 90.0%–150.0% of the labeledpantothenate (C18H32CaN2O10) in the portion of Tabletsamount of calcium pantothenate (C18H32CaN2O10)taken:

• NIACIN or NIACINAMIDE, PYRIDOXINE HYDROCHLORIDE,RIBOFLAVIN, and THIAMINE, Method 1antilog M = antilog (M′ + log R)[NOTE—Use low-actinic glassware throughout this

R = number of mg of calcium pantothenate that procedure.]was assumed to be present in the portion of Diluent: Acetonitrile, glacial acetic acid, and waterthe Tablets taken (5:1:94)

Calculate the percentage of the labeled amount of Mobile phase: A mixture of methanol, glacial aceticcalcium pantothenate in the portion of the Tablets acid, and water (27:1:73) containing 140 mg of sodiumtaken: 1-hexanesulfonate per 100 mL

Standard solution: [NOTE—Use USP Niacin RS in placeResult = [(antilog M)/N] × 100 of USP Niacinamide RS for formulations containing

niacin.] Transfer 80 mg of USP Niacinamide RS, 20 mgN = nominal amount of calcium pantothenate in of USP Pyridoxine Hydrochloride RS, 20 mg of USP

the portion of the Tablets taken (mg) Riboflavin RS, and 20 mg of USP Thiamine HydrochlorideReplication: Repeat the entire determination at least RS, to a 200-mL volumetric flask, and add 180 mL ofonce, using separately prepared Sample solutions. If the Diluent. Immerse the flask in a hot water bathdifference between the two log-potencies M is NMT maintained at 65°–70° for 10 min with regular shaking0.08, their mean, M, is the assayed log-potency of the or using a vortex mixer, until all the solid materials aretest material (see Design and Analysis of Biological Assays dissolved. Chill rapidly in a cold water bath for 10 min⟨111⟩, The Confidence Interval and Limits of Potency). If to room temperature, and dilute with Diluent to volume.the two determinations differ by more than 0.08, Sample solution: Finely powder NLT 30 Tablets.Transferconduct one or more additional determinations. From a portion of the powder, equivalent to 10 mg ofthe mean of two or more values of M that do not differ niacinamide and 2.5 mg each of pyridoxineby more than 0.15, compute the mean potency of the hydrochloride, riboflavin, and thiamine hydrochloride, topreparation under assay. a 50-mL centrifuge tube. Add 25.0 mL of Diluent, and

Acceptance criteria: 90.0%–150.0% of the labeled mix using a vortex mixer for 30 s to completely suspendamount of calcium pantothenate (C18H32CaN2O10) the powder. Immerse the centrifuge tube in a hot water

bath maintained at 65°–70°, heat for 5 min, and mix ona vortex mixer for 30 s. Return the tube to the hot waterbath, heat for another 5 min, and mix on a vortex mixer




for 30 s. Filter a portion of the solution, cool to room Mr2 = molecular weight of thiamine hydrochloride,temperature, and use the clear filtrate. [NOTE—Use the 337.27filtrate within 3 h of filtration.] Acceptance criteria: 90.0%–150.0% of the labeled

Chromatographic system amount of niacinamide (C6H6N2O) or niacin (C6H5NO2),(See Chromatography ⟨621⟩, System Suitability.) pyridoxine hydrochloride (C8H11NO3 · HCl), riboflavinMode: LC (C17H20N4O6), and thiamine as thiamine hydrochlorideDetector: UV 280 nm (C12H17ClN4OS · HCl) or thiamine mononitrateColumn: 3.9-mm × 30-cm; packing L1 (C12H17N5O4S)Flow rate: 1 mL/min • NIACIN, Method 2Injection size: 10 µL [NOTE—Use low-actinic glassware throughout this

System suitability procedure.]Sample: Standard solution Solution A: Transfer 1 mL of glacial acetic acid and 2.5[NOTE—The relative retention times for niacinamide, g of edetate disodium to a 100-mL volumetric flask.pyridoxine, riboflavin, and thiamine are about 0.3, 0.5, Dissolve in and dilute with water to volume.0.8, and 1.0, respectively.] Extraction solvent: Solution A and methanol (3:1)

Suitability requirements Mobile phase: 0.1 M sodium acetate solution (13.6Relative standard deviation: NMT 3.0% mg/mL of sodium acetate in water). Adjust with acetic

Analysis acid to a pH of 5.4. [NOTE—A small amount of methanolSamples: Standard solution and Sample solution (up to 1%) may be added to the Mobile phase toMeasure the peak areas for niacin or niacinamide, improve resolution.]pyridoxine, riboflavin, and thiamine. Calculate the Standard stock solution: 1 mg/mL of USP Niacin RS inpercentage of the labeled amount of niacinamide Extraction solvent(C6H6N2O) in the portion of Tablets taken: Standard solution: Transfer 5.0 mL of Standard stock

solution to a 25-mL volumetric flask, and dilute withResult = (rU/rS) × (CS/CU) × 100 Extraction solvent to volume.

Sample solution: [NOTE—This preparation is suitable forrU = peak area of niacinamide from the Sample the determination of niacin or niacinamide, pyridoxine,

solution and riboflavin, when present in the formulation.] FinelyrS = peak area of niacinamide from the Standard powder NLT 20 Tablets. Transfer a portion of the

solution powder, equivalent to 2 mg of riboflavin, to a 200-mLCS = concentration of USP Niacinamide RS in the volumetric flask. If riboflavin is not present in the

Standard solution (mg/mL) formulation, transfer a portion of the powder, equivalentCU = nominal concentration of niacinamide in the to 2 mg of pyridoxine. If pyridoxine is not present in the

Sample solution (mg/mL) formulation, transfer a portion of the powder, equivalentFor formulations containing niacin: to 20 mg of niacin or niacinamide. Add 100.0 mL of

Extraction solvent, and mix for 20 min, using a wrist-Result = (rU/rS) × (CS/CU) × 100 action shaker. Immerse the flask in a water bath

maintained at 70°–75°, and heat for 20 min. Mix on arU = peak area of niacin from the Sample solution vortex mixer for 30 s, cool to room temperature, andrS = peak area of niacin from the Standard solution filter. Use the clear filtrate.CS = concentration of USP Niacin RS in the Chromatographic systemStandard solution (mg/mL) (See Chromatography ⟨621⟩, System Suitability.)CU = nominal concentration of niacin in the Sample Mode: LCsolution (mg/mL) Detector: UV 254 nmSeparately calculate the percentage of the labeled Column: 4.6-mm × 25-cm; packing L1amount of pyridoxine hydrochloride (C8H11NO3 · HCl), Flow rate: 1 mL/minriboflavin (C17H20N4O6), and thiamine hydrochloride Injection size: 20 µL(C12H17ClN4OS · HCl) in the portion of Tablets taken: System suitabilitySample: Standard solutionResult = (rU/rS) × (CS/CU) × 100 Suitability requirements

Relative standard deviation: NMT 3.0%rU = peak area of the corresponding vitamin from[NOTE—If necessary, flush the column with methanolthe Sample solutionbetween injections.]rS = peak area of the corresponding vitamin from

Analysisthe Standard solutionSamples: Standard solution and Sample solutionCS = concentration of the relevant USP ReferenceMeasure the peak areas for niacin. Calculate theStandard in the Standard solution (mg/mL)percentage of the labeled amount of niacin (C6H5NO2)CU = nominal concentration of the correspondingin the portion of Tablets taken:vitamin in the Sample solution (mg/mL)

For products containing thiamine mononitrate, calculate Result = (rU/rS) × (CS/CU) × 100the percentage of the labeled amount of thiaminemononitrate (C12H17N5O4S) in the portion of Tablets rU = peak area from the Sample solutiontaken: rS = peak area from the Standard solution

CS = concentration of USP Niacin RS in theResult = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100 Standard solution (mg/mL)CU = nominal concentration of niacin in the SamplerU = peak area of thiamine from the Sample

solution (mg/mL)solutionAcceptance criteria: 90.0%–150.0% of the labeledrS = peak area of thiamine from the Standardamount of niacin (C6H5NO2)solution

• NIACINAMIDE, Method 2CS = concentration of USP Thiamine Hydrochloride[NOTE—Use low-actinic glassware throughout thisRS in the Standard solution (mg/mL)procedure.]CU = nominal concentration of thiamine

Solution A, Extraction solvent, Mobile phase, Standardmononitrate in the Sample solution (mg/mL)stock solution, Standard solution, Sample solution,Mr1 = molecular weight of thiamine mononitrate,Chromatographic system, and System suitability:327.36




Using USP Niacinamide RS in place of USP Niacin RS, Standard solution: Dilute 5.0 mL of Standard stockproceed as directed for Niacin, Method 2. solution with Extraction solvent to 25.0 mL.

Analysis Chromatographic systemSamples: Standard solution and Sample solution (See Chromatography ⟨621⟩, System Suitability.)Measure the peak areas for niacinamide. Calculate the Mode: LCpercentage of the labeled amount of niacinamide Detector: UV 254 nm(C6H6N2O) in the portion of Tablets taken: Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/minResult = (rU/rS) × (CS/CU) × 100 Injection size: 20 µL

System suitabilityrU = peak area from the Sample solution Sample: Standard solutionrS = peak area from the Standard solution Suitability requirementsCS = concentration of USP Niacinamide RS in the Relative standard deviation: NMT 3.0%

Standard solution (mg/mL) AnalysisCU = nominal concentration of niacinamide in the Samples: Standard solution and Sample solution

Sample solution (mg/mL) Measure the peak areas for riboflavin. Calculate theAcceptance criteria: 90.0%–150.0% of the labeled percentage of the labeled amount of riboflavinamount of niacinamide (C6H6N2O) (C17H20N4O6) in the portion of Tablets taken:

• PYRIDOXINE HYDROCHLORIDE, Method 2[NOTE—Use low-actinic glassware throughout this Result = (rU/rS) × (CS/CU) × 100procedure.]

Extraction solvent, Mobile phase, and Sample rU = peak area from the Sample solutionsolution: Prepare as directed for Niacin, Method 2. rS = peak area from the Standard solution

Standard stock solution: 0.1 mg/mL of USP Pyridoxine CS = concentration of USP Riboflavin RS in theHydrochloride RS in Extraction solvent Standard solution (mg/mL)

Standard solution: Transfer 5.0 mL of Standard stock CU = nominal concentration of riboflavin in thesolution to a 25-mL volumetric flask, and dilute with Sample solution (mg/mL)Extraction solvent to volume. Acceptance criteria: 90.0%–150.0% of the labeled

Chromatographic system amount of riboflavin (C17H20N4O6)(See Chromatography ⟨621⟩, System Suitability.) • THIAMINE, Method 2Mode: LC [NOTE—Use low-actinic glassware throughout thisDetector: UV 254 nm procedure.]Column: 4.6-mm × 25-cm; packing L1 Solution A: 1.88 g/L of sodium 1-hexanesulfonate inFlow rate: 1 mL/min 0.1% phosphoric acidInjection size: 20 µL Mobile phase: Solution A and acetonitrile (46:9)

System suitability Standard stock solution: 0.1 mg/mL of USP ThiamineSample: Standard solution Hydrochloride RS in 0.2 N hydrochloric acidSuitability requirements Standard solution: 0.02 mg/mL of USP Thiamine

Relative standard deviation: NMT 3.0% Hydrochloride RS from Standard stock solution dilutedAnalysis with 0.2 N hydrochloric acidSamples: Standard solution and Sample solution Sample solution: Weigh and finely powder NLT 20Measure the peak areas for pyridoxine. Calculate the Tablets. Mix a portion of the Tablets’ powder with apercentage of the labeled amount of pyridoxine volume of 0.2 N hydrochloric acid to obtain ahydrochloride (C8H11NO3 · HCl) in the portion of concentration of 0.02 mg/mL of thiamine. Shake for 15Tablets taken: min with a wrist-action shaker, and heat to boiling for

30 min. Cool to room temperature, and filter. Use theResult = (rU/rS) × (CS/CU) × 100 clear filtrate.

Chromatographic systemrU = peak area from the Sample solution (See Chromatography ⟨621⟩, System Suitability.)rS = peak area from the Standard solution Mode: LCCS = concentration of USP Pyridoxine Hydrochloride Detector: UV 254 nm

RS in the Standard solution (mg/mL) Column: 4.6-mm × 25-cm; packing L1CU = nominal concentration of pyridoxine Flow rate: 2 mL/min

hydrochloride in the Sample solution Injection size: 20 µL(mg/mL) System suitability

Acceptance criteria: 90.0%–150.0% of the labeled Sample: Standard solutionamount of pyridoxine hydrochloride (C8H11NO3 · HCl) Suitability requirements

• RIBOFLAVIN, Method 2 Relative standard deviation: NMT 3.0%[NOTE—Use low-actinic glassware throughout this Analysisprocedure.] Samples: Standard solution and Sample solution

Extraction solvent and Sample solution: Prepare as Measure the areas for the major peaks. For productsdirected for Niacin, Method 2. containing thiamine hydrochloride, calculate the

Solution A: 6.8 mg/mL of sodium acetate in water percentage of the labeled amount of thiamineMobile phase: Prepare a mixture of Solution A and hydrochloride (C12H17ClN4OS · HCl) in the portion ofmethanol (13:7). Add 2 mL of triethylamine per L of the Tablets taken:mixture, and adjust with glacial acetic acid to a pH of5.2. Result = (rU/rS) × (CS/CU) × 100

Standard stock solution: Transfer 20 mg of USPRiboflavin RS to a 200-mL volumetric flask, and add 180 rU = peak area of thiamine from the SamplemL of Extraction solvent. Immerse the flask for 5 min in a solutionwater bath maintained at 65°–75°. Mix well, and repeat rS = peak area of thiamine from the Standardif necessary until dissolved. Chill rapidly in a cold water solutionbath to room temperature, and dilute with Extraction CS = concentration of USP Thiamine Hydrochloridesolvent to volume. RS in the Standard solution (mg/mL)




CU = nominal concentration of thiamine or niacinamide (C6H6N2O) in the portion of Tabletshydrochloride in the Sample solution taken:(mg/mL)

Result = (rU/rS) × (CS/CU) × 100For products containing thiamine mononitrate, calculatethe percentage of the labeled amount of thiamine

rU = peak area of niacin or niacinamide from themononitrate (C12H17N5O4S) in the portion of TabletsSample solutiontaken:

rS = peak area of niacin or niacinamide from theStandard solutionResult = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100

CS = concentration of USP Niacin RS or USPrU = peak area of thiamine from the Sample Niacinamide RS in the Standard solution

solution (mg/mL)rS = peak area of thiamine from the Standard CU = nominal concentration of niacin or

solution niacinamide in the Sample solution (mg/mL)CS = concentration of USP Thiamine Hydrochloride Separately calculate the percentage of the labeled

RS in the Standard solution (mg/mL) amount of pyridoxine hydrochloride (C8H11NO3 · HCl),CU = nominal concentration of thiamine riboflavin (C17H20N4O6), and thiamine hydrochloride

mononitrate in the Sample solution (mg/mL) (C12H17ClN4OS · HCl) in the portion of Tablets taken:Mr1 = molecular weight of thiamine mononitrate,

Result = (rU/rS) × (CS/CU) × 100327.36Mr2 = molecular weight of thiamine hydrochloride,

rU = peak area of the corresponding vitamin from337.27the Sample solutionAcceptance criteria: 90.0%–150.0% of the labeled

rS = peak area of the corresponding vitamin fromamount of thiamine as thiamine hydrochloridethe Standard solution(C12H17ClN4OS · HCl) or thiamine mononitrate

CS = concentration of the relevant USP Reference(C12H17N5O4S)Standard in the Standard solution (mg/mL)• NIACIN or NIACINAMIDE, PYRIDOXINE HYDROCHLORIDE,

CU = nominal concentration of the correspondingRIBOFLAVIN, and THIAMINE, Method 3vitamin in the Sample solution (mg/mL)[NOTE—Use low-actinic glassware throughout this

For products containing thiamine mononitrate, calculateprocedure.]the percentage of the labeled amount of thiamineReagent: 25 mg/mL of edetate disodium in watermononitrate (C12H17N5O4S) in the portion of TabletsMobile phase: Transfer 0.4 mL of triethylamine, 15.0 mLtaken:of glacial acetic acid, and 350 mL of methanol to a

2000-mL volumetric flask. Dilute with 0.008 M sodiumResult = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 1001-hexanesulfonate to volume.

Standard stock solution: 1.5 mg/mL of USP Niacin RS rU = peak area of thiamine from the Sampleor USP Niacinamide RS, 0.24 mg/mL of USP Pyridoxine solutionHydrochloride RS, 0.08 mg/mL of USP Riboflavin RS, and rS = peak area of thiamine from the Standard0.24 mg/mL of USP Thiamine Hydrochloride RS in the solutionReagent, with heating if necessary CS = concentration of USP Thiamine HydrochlorideStandard solution: Transfer 5.0 mL of Standard stock RS in the Standard solution (mg/mL)solution to a stoppered 125-mL flask. Add 10.0 mL of a CU = nominal concentration of thiaminemixture of methanol and glacial acetic acid (9:1) and mononitrate in the Sample solution (mg/mL)30.0 mL of a mixture of methanol and ethylene glycol Mr1 = molecular weight of thiamine mononitrate,(1:1). Insert the stopper, shake for 15 min in a water 327.36bath maintained at 60°, and cool. Filter, discarding the Mr2 = molecular weight of thiamine hydrochloride,first few mL of the filtrate. 337.27Sample solution: Weigh and finely powder NLT 20 Acceptance criteria: 90.0%–150.0% of the labeledTablets. Transfer a portion of the powder, equivalent to amount of niacinamide (C6H6N2O) or niacin (C6H5NO2),7.5 mg of niacin or niacinamide, 1.2 mg of pyridoxine pyridoxine hydrochloride (C8H11NO3 · HCl), riboflavinhydrochloride, 0.4 mg of riboflavin, and 1.2 mg of (C17H20N4O6), and thiamine as thiamine hydrochloridethiamine hydrochloride to a stoppered 125-mL flask. Add (C12H17ClN4OS · HCl) or thiamine mononitrate10.0 mL of a mixture of methanol and glacial acetic acid (C12H17N5O4S)(9:1) and 30.0 mL of a mixture of methanol and [NOTE—Commercially available atomic absorptionethylene glycol (1:1). Insert the stopper, shake for 15 standard solutions for the minerals, where applicable,min in a water bath maintained at 60°, and cool. Filter, may be used where preparation of a Standard stockdiscarding the first few mL of the filtrate. solution is described in the following assays. UseChromatographic system deionized water where water is specified. Where atomic(See Chromatography ⟨621⟩, System Suitability.) absorption spectrophotometry is specified in the assay,Mode: LC the Standard solutions and the Sample solution may beDetector: UV 270 nm diluted quantitatively with the solvent specified, ifColumn: 4.6-mm × 25-cm; packing L7 necessary, to yield solutions of suitable concentrationsColumn temperature: 50° adaptable to the linear or working range of theFlow rate: 2.0 mL/min instrument.]Injection size: 5 µL • CALCIUM, Method 1System suitability Lanthanum chloride solution: 267 mg/mL ofSample: Standard solution lanthanum chloride heptahydrate in 0.125 NSuitability requirements hydrochloric acidRelative standard deviation: NMT 2.0% Calcium standard solution: 400 µg/mL of calcium.Analysis Dissolve 1.001 g of calcium carbonate, previously driedSamples: Standard solution and Sample solution at 300° for 3 h and cooled in a desiccator for 2 h, in 25Measure the areas of the peaks. Calculate the mL of 1 N hydrochloric acid. Boil to expel carbonpercentage of the labeled amount of niacin (C6H5NO2) dioxide, and dilute with water to 1000 mL.




Standard stock solution: 100 µg/mL of calcium from Mode: Atomic absorption spectrophotometryCalcium standard solution diluted with 0.125 N Analytical wavelength: Chromium emission line athydrochloric acid 357.9 nm

Standard solutions: Into separate 100-mL volumetric Lamp: Chromium hollow-cathodeflasks pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Flame: Air–acetyleneStandard stock solution. To each flask add 1.0 mL of Blank: 0.125 N hydrochloric acidLanthanum chloride solution, and dilute with water to Analysisvolume to obtain concentrations of 1.0, 1.5, 2.0, 2.5, Samples: Standard solutions and Sample solutionand 3.0 µg/mL of calcium. Determine the absorbances of the solutions against the

Sample solution: Finely powder NLT 20 Tablets. Transfer Blank. Plot the absorbances of the Standard solutionsa portion of the powder, equivalent to 5 Tablets, to a versus the concentration, in µg/mL, of chromium, andporcelain crucible. Heat the crucible in a muffle furnace draw the straight line best fitting the four plottedmaintained at 550° for 6–12 h, and cool. Add 60 mL of points. From the graph so obtained, determine thehydrochloric acid, and boil gently on a hot plate or concentration, C, in µg/mL, of chromium in thesteam bath for 30 min, intermittently rinsing the inner Sample solution.surface of the crucible with 6 N hydrochloric acid. Cool, Calculate the percentage of the labeled amount ofand quantitatively transfer the contents of the crucible to chromium (Cr) in the portion of Tablets taken:a 100-mL volumetric flask. Rinse the crucible with small

Result = (C/CU) × 100portions of 6 N hydrochloric acid, and add the rinsingsto the flask. Dilute with water to volume, and filter,

C = measured concentration of chromium in thediscarding the first 5 mL of the filtrate. Dilute thisSample solution (µg/mL)solution quantitatively, with 0.125 N hydrochloric acid,

CU = nominal concentration of chromium in theto obtain a concentration of 2 µg/mL of calcium, addingSample solution (µg/mL)1 mL of Lanthanum chloride solution per 100 mL of the

Acceptance criteria: 90.0%–160.0% of the labeledfinal volume.amount of chromium (Cr)Instrumental conditions

• COPPER, Method 1(See Spectrophotometry and Light-Scattering ⟨851⟩.)Copper standard solution: Dissolve 1.00 g of copperMode: Atomic absorption spectrophotometryfoil in a minimum volume of a 50% solution of nitricAnalytical wavelength: Calcium emission line at 422.7acid, and dilute with a 1% solution of nitric acid to 1000nmmL. This solution contains 1000 µg/mL of copper.Lamp: Calcium hollow-cathode

Standard stock solution: 100 µg/mL of copper fromFlame: Nitrous oxide–acetyleneCopper standard solution diluted with 0.125 NBlank: 0.125 N hydrochloric acid containing 1 mL ofhydrochloric acidLanthanum chloride solution per 100 mL

Standard solutions: To separate 200-mL volumetricAnalysisflasks transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of theSamples: Standard solutions and Sample solutionStandard stock solution. Dilute with water to volume toDetermine the absorbances of the solutions, against theobtain concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0Blank. Plot the absorbances of the Standard solutionsµg/mL of copper.versus the concentration, in µg/mL, of calcium, and

Sample solution: Proceed as directed for Calcium,draw the straight line best fitting the five plottedMethod 1, except to prepare the Sample solution topoints. From the graph so obtained, determine thecontain 2 µg/mL of copper and to omit the use of theconcentration, C, in µg/mL, of calcium in the SampleLanthanum chloride solution.solution.

Instrumental conditionsCalculate the percentage of the labeled amount of(See Spectrophotometry and Light-Scattering ⟨851⟩.)calcium (Ca) in the portion of Tablets taken:Mode: Atomic absorption spectrophotometryAnalytical wavelength: Copper emission line at 324.7Result = (C/CU) × 100nm

C = measured concentration of calcium in the Lamp: Copper hollow-cathodeSample solution (µg/mL) Flame: Air–acetylene

CU = nominal concentration of calcium in the Blank: 0.125 N hydrochloric acidSample solution (µg/mL) Analysis

Acceptance criteria: 90.0%–125.0% of the labeled Samples: Standard solutions and Sample solutionamount of calcium (Ca) Determine the absorbances of the solutions against the

• CHROMIUM, Method 1 Blank. Plot the absorbances of the Standard solutionsChromium standard solution: 1000 µg/mL of versus the concentration, in µg/mL, of copper, andchromium from potassium dichromate, previously dried draw the straight line best fitting the five plottedat 120° for 4 h in water. Store in a polyethylene bottle. points. From the graph so obtained, determine the

Standard stock solution: 10 µg/mL of chromium from concentration, C, in µg/mL, of copper in the SampleChromium standard solution diluted with 6 N hydrochloric solution.acid and water (1 in 20) Calculate the percentage of the labeled amount of

Standard solutions: Transfer 10.0 and 20.0 mL of the copper (Cu) in the portion of Tablets taken:Standard stock solution to separate 100-mL volumetric

Result = (C/CU) × 100flasks, and transfer 15.0 and 20.0 mL of the Standardstock solution to separate 50-mL volumetric flasks. Dilute

C = measured concentration of copper in thethe contents of each of the four flasks with 0.125 NSample solution (µg/mL)hydrochloric acid to volume to obtain concentrations of

CU = nominal concentration of copper in the1.0, 2.0, 3.0, and 4.0 µg/mL of chromium.Sample solution (µg/mL)Sample solution: Proceed as directed for Calcium,

Acceptance criteria: 90.0%–125.0% of the labeledMethod 1, except to prepare the Sample solution toamount of copper (Cu)contain 1 µg/mL of chromium and to omit the use of

• FLUORIDE, Method 1the Lanthanum chloride solution.[NOTE—Store all solutions in plastic containers.]Instrumental conditions

3 M sodium acetate solution: Dissolve 408 g of sodium(See Spectrophotometry and Light-Scattering ⟨851⟩.)acetate in 600 mL of water contained in a 1000-mL




volumetric flask. Allow the solution to equilibrate to mercury, wash the column with one column volume ofroom temperature, and dilute with water to volume. methanol followed by one column volume of pH 10.0Adjust with a few drops of acetic acid to a pH of 7.0. buffer. Do not allow the column top to dry. If the top of

Sodium citrate solution: Dissolve 222 g of sodium the column becomes dry, recondition the column.]citrate in 250 mL of water in a 1000-mL volumetric flask. Transfer 10.0 mL of Standard stock solution to a 100-mLAdd 28 mL of perchloric acid, and dilute with water to volumetric flask. Add 75 mL of water, and adjust withvolume. 0.1 N sodium hydroxide to a pH of 10.4 ±0.1. Dilute

Fluoride standard stock solution: 500 µg/mL of fluoride with water to volume. Filter, discarding the first 15 mLfrom a quantity of sodium fluoride, previously dried at of the filtrate. Transfer 25.0 mL of the filtrate to a 50-mL100° for 4 h and cooled in a desiccator, in water volumetric flask, add 15.0 mL of water, and adjust with

Intermediate stock solution A: 100 µg/mL of fluoride 0.1 N sodium hydroxide to a pH of 10.0. Dilute with pHfrom Fluoride standard stock solution diluted with water 10.0 buffer to volume. Elute a portion of this solution

Intermediate stock solution B: 10 µg/mL of fluoride through a 3-mL solid-phase extraction columnfrom Fluoride standard stock solution diluted with water containing L1 packing that is connected through an

Standard solutions: To five separate 100-mL volumetric adaptor to a second solid-phase extraction columnflasks transfer 3.0, 5.0, and 10.0 mL of Intermediate stock containing sulfonylpropyl strong cation-exchangesolution B and 5.0 and 10.0 mL of Intermediate stock packing. Discard the first 3 mL of the eluate, and collectsolution A. To each flask add 10.0 mL of 1 N hydrochloric the rest of the eluate in a suitable flask for injection intoacid, 25 mL of 3 M sodium acetate solution, and 25.0 mL the chromatograph.of Sodium citrate solution. Dilute the contents of each Sample solution: Finely powder NLT 20 Tablets. Transferflask with water to volume to obtain concentrations of a portion of powdered Tablets, equivalent to 1 mg of0.3, 0.5, 1.0, 5.0, and 10.0 µg/mL of fluoride. fluorine, in 15 mL of water, and shake vigorously. Rinse

Sample solution: Transfer a quantity of the finely the sides of the flask with 15 mL of water, and allow topowdered Tablets, equivalent to 200 µg of fluoride, to a stand for 10 min. Dilute with water to 85 mL, adjust100-mL volumetric flask. Add 10.0 mL of 1 N with 1 N sodium hydroxide to a pH of 10.4 ±0.1, andhydrochloric acid, 25.0 mL of 3 M sodium acetate dilute with water to 100 mL. Proceed as directed for thesolution, and 25.0 mL of Sodium citrate solution, and Standard solution, beginning with “Filter, discarding thedilute with water to volume. first 15 mL of the filtrate.”

Analysis Chromatographic systemSamples: Standard solutions and Sample solution (See Chromatography ⟨621⟩, System Suitability.)To separate plastic beakers, each containing a plastic- Mode: LCcoated stirring bar, transfer 50.0 mL each of the Detector: ConductivityStandard solutions and the Sample solution. Measure Guard column: 4.6-mm × 3-cm; packing L17the potentials (see pH ⟨791⟩), in mV, of the Standard Analytical column: 7.8-mm × 30-cm; packing L17solutions and the Sample solution, with a pH meter Flow rate: 0.5 mL/mincapable of a minimum reproducibility of ±0.2 mV and Injection size: 100 µLequipped with a fluoride-specific ion-indicating System suitabilityelectrode and a calomel reference electrode. [NOTE— Sample: Standard solutionWhen taking measurements, immerse the electrodes in Suitability requirementsthe solution, stir on a magnetic stirrer having an Relative standard deviation: NMT 2.0%insulated top until equilibrium is attained (1–2 min), Analysisand record the potential. Rinse and dry the electrodes Samples: Standard solution and Sample solutionbetween measurements, taking care to avoid Measure the peak areas for fluoride. Calculate thedamaging the crystal of the specific-ion electrode.] percentage of the labeled amount of fluorine (F) in the

Plot the logarithms of fluoride concentrations, in µg/ portion of Tablets taken:mL, of the Standard solutions versus potential, in mV.

Result = (rU/rS) × (CS/CU) × 100From the standard response curve so obtained and themeasured potential of the Sample solution, determine

rU = peak area from the Sample solutionthe concentration, C, in µg/mL, of fluoride in therS = peak area from the Standard solutionSample solution.CS = concentration of fluoride in the StandardCalculate the percentage of the labeled amount of

solution (µg/mL)fluorine (F) in the portion of Tablets taken:CU = nominal concentration of fluorine in the

Sample solution (µg/mL)Result = (C/CU) × 100Acceptance criteria: 90.0%–160.0% of the labeled

C = measured concentration of fluoride in the amount of fluorine (F)Sample solution (µg/mL) • IODIDE, Method 1

CU = nominal concentration of fluorine in the Bromine water: To 20 mL of bromine in a glass-Sample solution (µg/mL) stoppered bottle add 100 mL of water. Insert the

Acceptance criteria: 90.0%–160.0% of the labeled stopper into the bottle, and shake. Allow to stand for 30amount of fluorine (F) min, and use the supernatant.

• FLUORIDE, Method 2 Analysis[NOTE—Use plastic containers and deionized water Sample: Tabletsthroughout this procedure.] Transfer an amount of finely powdered Tablets,

pH 10.0 buffer: Add 214 mL of 0.1 N sodium hydroxide equivalent to 3 mg of iodide, to a nickel crucible. Addto 1000 mL of 0.05 M sodium bicarbonate. 5 g of sodium carbonate, 5 mL of 50% (w/v) sodium

Mobile phase: Alcohol, 0.1 N sulfuric acid, and water hydroxide solution, and 10 mL of alcohol, taking care(20:5:175) that the entire specimen is moistened. Heat the crucible

Standard stock solution: 220 µg/mL of USP Sodium on a steam bath to evaporate the alcohol, then dry theFluoride RS in water. This solution contains 100 µg/mL crucible at 100° for 30 min to prevent spattering uponof fluoride. subsequent heating. Transfer the crucible with its

Standard solution: [NOTE—Condition the solid-phase contents to a furnace heated to 500°, and heat theextraction column specified for use in the Standard crucible for 15 min. [NOTE—Heating at 500° issolution and the Sample solution in the following manner. necessary to carbonize any organic matter present; aUsing a vacuum at a pressure not exceeding 5 mm of




higher temperature may be used, if necessary, to ensure Acceptance criteria: 90.0%–125.0% of the labeledcomplete carbonization of all organic matter.] amount of iron (Fe)

Cool the crucible, add 25 mL of water, cover the • MAGNESIUM, Method 1crucible with a watchglass, and boil gently for 10 min. Lanthanum chloride solution: Prepare as directed inFilter the solution, and wash the crucible with boiling Calcium, Method 1.water, collecting the filtrate and washings in a beaker. Magnesium standard solution: Transfer 1.0 g ofAdd phosphoric acid until the solution is neutral to magnesium ribbon to a 1000-mL volumetric flask,methyl orange, then add 1 mL excess of phosphoric dissolve in 50 mL of 6 N hydrochloric acid, dilute withacid. Add excess of Bromine water, and boil the solution water to volume, and mix to obtain a solution with agently until colorless and then for 5 min longer. Add a known concentration of 1000 µg/mL of magnesium.few crystals of salicylic acid, and cool the solution to Standard stock solution: 20 µg/mL of magnesium from20°. Add 1 mL of phosphoric acid and 0.5 g of Magnesium standard solution diluted with 0.125 Npotassium iodide, and titrate the liberated iodine with hydrochloric acid0.005 N sodium thiosulfate VS, adding starch TS when Standard solutions: To separate 100-mL volumetricthe liberated iodine color has nearly disappeared. flasks transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of Standard

Calculate the percentage of the labeled amount of iodine stock solution. To each flask add 1.0 mL of Lanthanum(I) in the portion of Tablets taken: chloride solution, and dilute with 0.125 N hydrochloric

acid to volume to obtain concentrations of 0.2, 0.3, 0.4,Result = V × NA × F × Ime × (Aw/W) × (100/L) 0.5, and 0.6 µg/mL of magnesium.

Sample solution: Proceed as directed for Calcium,V = volume of sodium thiosulfate consumed (mL) Method 1, except to prepare the Sample solution toNA = actual normality of the sodium thiosulfate contain a concentration of 0.4 µg/mL of magnesium.

solution used Instrumental conditionsF = correction factor to convert mg to µg (1000 (See Spectrophotometry and Light-Scattering ⟨851⟩.)

µg/mL) Mode: Atomic absorption spectrophotometryIme = milliequivalent of I (21.16 mg/meq) Analytical wavelength: Magnesium emission line atAw = average weight of the Tablets 285.2 nmW = weight of the portion of Tablets taken Lamp: Magnesium hollow-cathodeL = labeled amount of iodine (µg/Tablet) Flame: Air–acetylene

Acceptance criteria: 90.0%–160.0% of the labeled Blank: 0.125 N hydrochloric acid containing 1 mL ofamount of iodine (I) Lanthanum chloride solution per 100 mL

• IODIDE, Method 2: Proceed as directed in Automated AnalysisMethods of Analysis ⟨16⟩, Assay for Iodide. Samples: Standard solutions and Sample solution

Acceptance criteria: 90.0%–160.0% of the labeled Determine the absorbances of the solutions against theamount of iodine (I) Blank. Plot the absorbances of the Standard solutions

• IRON, Method 1 versus the concentration, in µg/mL, of magnesium,Iron standard stock solution: Transfer 100 mg of iron and draw the straight line best fitting the five plottedpowder to a 1000-mL volumetric flask. Dissolve in 25 mL points. From the graph so obtained, determine theof 6 N hydrochloric acid, dilute with water to volume, concentration, C, in µg/mL, of magnesium in theand mix. Sample solution.

Standard solutions: To separate 100-mL volumetric Calculate the percentage of the labeled amount offlasks transfer 2.0, 4.0, 5.0, 6.0, and 8.0 mL of Iron magnesium (Mg) in the portion of Tablets taken:standard stock solution. Dilute the contents of each flaskwith water to volume to obtain concentrations of 2.0, Result = (C/CU) × 1004.0, 5.0, 6.0, and 8.0 µg/mL of iron.

Sample solution: Proceed as directed for Calcium, C = measured concentration of magnesium in theMethod 1, except to prepare the Sample solution to Sample solution (µg/mL)contain a concentration of 5 µg/mL of iron and to omit CU = nominal concentration of magnesium in thethe use of the Lanthanum chloride solution. Sample solution (µg/mL)

Instrumental conditions Acceptance criteria: 90.0%–125.0% of the label claim (See Spectrophotometry and Light-Scattering ⟨851⟩.) • MANGANESE, Method 1Mode: Atomic absorption spectrophotometry Manganese standard stock solution: Transfer 1.00 g ofAnalytical wavelength: Iron emission line at 248.3 nm manganese to a 1000-mL volumetric flask. Dissolve in 20Lamp: Iron hollow-cathode mL of nitric acid, dilute with 6 N hydrochloric acid toFlame: Air–acetylene volume, and mix to obtain a solution with aBlank: 0.125 N hydrochloric acid concentration of 1000 µg/mL of manganese.

Analysis Standard stock solution: 50 µg/mL of manganese fromSamples: Standard solutions and Sample solution Manganese standard stock solution diluted with 0.125 NDetermine the absorbances of the solutions against the hydrochloric acidBlank. Plot the absorbances of the Standard solutions Standard solutions: To separate 100-mL volumetricversus the concentration, in µg/mL, of iron, and draw flasks transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of Standardthe straight line best fitting the five plotted points. stock solution. Dilute the contents of each flask withFrom the graph so obtained, determine the 0.125 N hydrochloric acid to volume to obtain solutionsconcentration, C, in µg/mL, of iron in the Sample with known concentrations of 0.5, 0.75, 1.0, 1.5, andsolution. 2.0 µg/mL of manganese.

Calculate the percentage of the labeled amount of iron Sample solution: Proceed as directed for Calcium,(Fe) in the portion of Tablets taken: Method 1, except to prepare the Sample solution to

contain a concentration of 1 µg/mL of manganese andResult = (C/CU) × 100 to omit the use of the Lanthanum chloride solution.

Instrumental conditionsC = measured concentration of iron in the Sample (See Spectrophotometry and Light-Scattering ⟨851⟩.)

solution (µg/mL) Mode: Atomic absorption spectrophotometryCU = nominal concentration of iron in the Sample Analytical wavelength: Manganese emission line at

solution (µg/mL) 279.5 nm




Lamp: Manganese hollow-cathode Calculate the percentage of the labeled amount ofFlame: Air–acetylene molybdenum (Mo) in the portion of Tablets taken:Blank: 0.125 N hydrochloric acid

Result = (C/CU) × 100AnalysisSamples: Standard solutions and Sample solution

C = measured concentration of molybdenum inDetermine the absorbances of the solutions against thethe Sample solution (µg/mL)Blank. Plot the absorbances of the Standard solutions

CU = nominal concentration of molybdenum in theversus the concentration, in µg/mL, of manganese,Sample solution (µg/mL)and draw the straight line best fitting the five plotted

Acceptance criteria: 90.0%–160.0% of the labeledpoints. From the graph so obtained, determine theamount of molybdenum (Mo)concentration, C, in µg/mL, of manganese in the

• MOLYBDENUM, Method 2Sample solution.Sodium fluoride solution: Add 200 mL of water to 10 gCalculate the percentage of the labeled amount ofof sodium fluoride, stir until the solution is saturated,manganese (Mn) in the portion of Tablets taken:and filter. Store in a polyethylene bottle.

Ferrous sulfate solution: 4.98 mg/mL of ferrous sulfateResult = (C/CU) × 100in water

C = measured concentration of manganese in the Potassium thiocyanate solution: 200 mg/mL ofSample solution (µg/mL) potassium thiocyanate in water

CU = nominal concentration of manganese in the 20% stannous chloride solution: Transfer 40 mg ofSample solution (µg/mL) stannous chloride to a beaker, add 20 mL of 6.5 N

Acceptance criteria: 90.0%–125.0% of the labeled hydrochloric acid solution, and heat the solution untilamount of manganese (Mn) the stannous chloride is dissolved. Cool and dilute with

• MOLYBDENUM, Method 1 water to 100 mL.Diluent: 20 mg/mL of ammonium chloride in water Diluted stannous chloride solution: 20% stannousMolybdenum standard solution: Transfer 1.0 g of chloride solution diluted with water (1 in 25). Prepare thismolybdenum wire to a 1000-mL volumetric flask, and solution fresh at the time of use.dissolve in 50 mL of nitric acid, warming if necessary. Standard solution: 20 µg/mL of molybdenum in waterDilute with water to volume, and mix to obtain a Sample: A portion of finely powdered Tablets, equivalentsolution with a concentration of 1000 µg/mL of to 40 µg of molybdenummolybdenum. Instrumental conditions

Standard stock solution: 100 µg/mL of molybdenum (See Spectrophotometry and Light-Scattering ⟨851⟩.)from Molybdenum standard solution diluted with water Mode: Vis

Standard solutions: To separate 100-mL volumetric Analytical wavelength: 465 nmflasks transfer 2.0, 10.0, and 25.0 mL of the Standard Cell: 1 cmstock solution, and add 5.0 mL of perchloric acid to each Blank: Amyl alcoholflask. Gently boil the solution in each flask for 15 min, Analysiscool to room temperature, and dilute each with Diluent Samples: Standard solution and Sampleto volume to obtain concentrations of 5.0, 10.0, and Transfer the Sample and 2.0 mL of the Standard solution25.0 µg/mL of molybdenum. to separate 200-mL beakers. Add 20 mL of nitric acid

Sample solution: Transfer a portion of the powder, to each beaker. Cover each beaker with a watchglass,equivalent to 1000 µg of molybdenum, to a suitable and boil slowly on a hot plate for 45 min. Cool toflask, and add 12 mL of nitric acid. [NOTE—The volume room temperature. Add 6 mL of perchloric acid, coverof nitric acid may be varied to ensure that the powder is the beakers with a watchglass, and continue theuniformly dispersed.] Carefully swirl the flask to disperse heating until digestion is complete, as indicated whenthe test specimen. Sonicate for 10 min, or until the test the liquid becomes colorless or pale yellow. Evaporatespecimen is completely dissolved. Gently boil the the solutions in the beakers to dryness. Rinse the sidessolution for 15 min, and cool to room temperature. of the beakers and the watchglasses with water, andCarefully add 8 mL of perchloric acid, heat until add more water to complete 50 mL in each beaker.perchloric acid fumes appear, and swirl the flask to Gently boil the water solution for a few min. Cool todissipate the fumes. Repeat the heating and swirling room temperature. Add 2 drops of methyl orange TS,until the fumes appear again. Cool to room temperature. and neutralize with ammonium hydroxide. Add 8.2Quantitatively transfer the contents of the flask to a 100- mL of hydrochloric acid. Quantitatively transfer themL volumetric flask with the aid of the Diluent, and contents of the beakers to separate 100-mL volumetricdilute with Diluent to volume. flasks, rinse the beakers with water, transfer the

Instrumental conditions rinsings to the corresonding volumetric flasks, and(See Spectrophotometry and Light-Scattering ⟨851⟩.) dilute with water to volume. Transfer 50.0 mL of eachMode: Atomic absorption spectrophotometry solution to separatory funnels. To each separatoryAnalytical wavelength: Molybdenum emission line at funnel add 1.0 mL of Sodium fluoride solution, 0.5 mL313.3 nm of Ferrous sulfate solution, 4.0 mL of Potassium

Lamp: Molybdenum hollow-cathode thiocyanate solution, 1.5 mL of 20% Stannous chlorideFlame: Nitrous oxide–acetylene solution, and 15.0 mL of amyl alcohol, and shake theBlank: Diluent and perchloric acid (20:1) separatory funnel for 1 min. Allow the layers to

Analysis separate, and discard the aqueous layers. Add 25 mLSamples: Standard solutions and Sample solution of Diluted stannous chloride solution to each separatoryDetermine the absorbances of the solutions against the funnel, and shake gently for 15 s. Allow the layers toBlank. Plot the absorbances of the Standard solutions separate, and discard the aqueous layers. Transfer theversus the concentration, in µg/mL, of molybdenum, organic layer from each separatory funnel to aand draw the straight line best fitting the three plotted centrifuge tube, and centrifuge at 2000 rpm for 10points. From the graph so obtained, determine the min. Determine the absorbances of the organic phasesconcentration, C, in µg/mL, of molybdenum in the obtained from the Standard solution and the Sample,Sample solution. and correct with the Blank.




Calculate the percentage of the labeled amount of • POTASSIUMmolybdenum (Mo) in the portion of Tablets taken: Potassium standard solution: 100 µg/mL of potassium

from potassium chloride, previously dried at 105° for 2Result = (AU/AS) × [(V × CS)/MU] × 100 h, in water

Standard stock solution: 10 µg/mL of potassium fromAU = absorbance of the Sample Potassium standard solution diluted with 0.125 NAS = absorbance of the Standard solution hydrochloric acidV = volume of the Standard solution analyzed, 2.0 Standard solutions: Transfer 5.0, 10.0, 15.0, 20.0, and

mL 25.0 mL of the Standard stock solution to separate 100-CS = concentration of molybdenum in the Standard mL volumetric flasks. Dilute the contents of each flask

solution (µg/mL) with 0.125 N hydrochloric acid to volume to obtainMU = nominal amount of molybdenum in the solutions containing 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of

Sample (µg) potassium.Acceptance criteria: 90.0%–160.0% of the labeled Sample solution: Proceed as directed for Calcium,amount of molybdenum (Mo) Method 1, except to prepare the Sample solution to

• PHOSPHORUS, Method 1 contain a concentration of 1 µg/mL of potassium and toSulfuric acid solution: Cautiously add sulfuric acid to omit the use of the Lanthanum chloride solution.water (37.5:100), and mix. Instrumental conditions

Ammonium molybdate solution: 50 mg/mL of (See Spectrophotometry and Light-Scattering ⟨851⟩.)ammonium molybdate in Sulfuric acid solution and water Mode: Atomic absorption spectrophotometry(2:3). [NOTE—Dissolve in water first, and then dilute with Analytical wavelength: Potassium emission line atSulfuric acid solution to volume.] 766.5 nm

Hydroquinone solution: 5 mg/mL of hydroquinone in Lamp: Potassium hollow-cathodewater. Add one drop of sulfuric acid per 100 mL of Flame: Air–acetylenesolution. Blank: Water

Sodium bisulfite solution: 200 mg/mL of sodium Analysisbisulfite in water Samples: Standard solutions and Sample solution

Phosphorus standard stock solution: Weigh 4.395 g of Determine the absorbances of the solutions against themonobasic potassium phosphate, previously dried at Blank. Plot the absorbances of the Standard solutions105° for 2 h and stored in a desiccator, and transfer to a versus the concentration, in µg/mL, of potassium, and1000-mL volumetric flask. Dissolve in water, add 6 mL of draw the straight line best fitting the five plottedsulfuric acid as a preservative, dilute with water to points. From the graph so obtained, determine thevolume, and mix to obtain a solution with a concentration, C, in µg/mL, of potassium in theconcentration of 1000 µg/mL of phosphorus. Sample solution.

Standard solution: 20 µg/mL of phosphorus from Calculate the percentage of the labeled amount ofPhosphorus standard stock solution diluted with water potassium (K) in the portion of Tablets taken:

Sample solution: [NOTE—Finely powder and weigh acounted number of Tablets.] Transfer a portion of the Result = (C/CU) × 100powder, equivalent to 100 mg of phosphorus, to 25 mLof nitric acid, and digest on a hot plate for 30 min. Add C = measured concentration of potassium in the15 mL of hydrochloric acid, and continue the digestion Sample solution (µg/mL)to the cessation of brown fumes. Cool, and transfer the CU = nominal concentration of potassium in thecontents of the flask to a 500-mL volumetric flask with Sample solution (µg/mL)the aid of small portions of water. Dilute with water to Acceptance criteria: 90.0%–125.0% of the labeledvolume. Transfer 10.0 mL of this solution to a 100-mL amount of potassium (K)volumetric flask, and dilute with water to volume. • SELENIUM, Method 1

Instrumental conditions Diluent: Prepare as directed in Molybdenum, Method 1.(See Spectrophotometry and Light-Scattering ⟨851⟩.) Selenium standard solution: [CAUTION—Selenium isMode: Vis toxic; handle it with care.] Dissolve 1 g of metallicAnalytical wavelength: 650 nm selenium in a minimum volume of nitric acid. EvaporateCell: 1 cm to dryness, add 2 mL of water, and evaporate to

Analysis dryness. Repeat the addition of water and theSamples: Standard solution and Sample solution evaporation to dryness three times. Dissolve the residueTo three separate 25-mL volumetric flasks transfer 5.0 in 3 N hydrochloric acid, transfer to a 1000-mLmL each of the Standard solution, the Sample solution, volumetric flask, and dilute with 3 N hydrochloric acid toand water to provide the blank. To each of the three volume to obtain a concentration of 1000 µg/mL offlasks add 1.0 mL each of Ammonium molybdate selenium.solution, Hydroquinone solution, and Sodium bisulfite Standard stock solution: 100 µg/mL of selenium fromsolution, and swirl to mix. Dilute the contents of each Selenium standard solution diluted with waterflask with water to volume, and allow the flasks to Standard solutions: To separate 100-mL volumetricstand for 30 min. Determine the absorbances of the flasks transfer 5.0, 10.0, and 25.0 mL of the Standardsolutions against the blank. stock solution, and add 5.0 mL of perchloric acid to each

Calculate the percentage of the labeled amount of flask. Gently boil the solutions for 15 min, cool to roomphosphorus (P) in the portion of Tablets taken: temperature, and dilute each with Diluent to volume to

obtain solutions with concentrations of 5.0, 10.0, andResult = (AU/AS) × (CS/CU) × 100 25.0 µg/mL of selenium.

Sample solution: Transfer a portion of the powder,AU = absorbance of the Sample solution equivalent to 1000 µg of selenium, to a suitable flask,AS = absorbance of the Standard solution and add 12 mL of nitric acid. [NOTE—The volume ofCS = concentration of phosphorus in the Standard nitric acid may be varied to ensure that the powder is

solution (µg/mL) uniformly dispersed.] Carefully swirl the flask to disperseCU = nominal concentration of phosphorus in the the test specimen. Sonicate for 10 min or until the test

Sample solution (µg/mL) specimen is completely dissolved. Gently boil theAcceptance criteria: 90.0%–125.0% of the labeled solution for 15 min, and cool to room temperature.amount of phosphorus (P) Carefully add 8 mL of perchloric acid to the flask, heat




the flask until perchloric acid fumes appear, and swirl the begins to darken. Add 0.5 mL of nitric acid and resumeflask to dissipate the fumes. Repeat the heating and heating, adding additional amounts of nitric acid ifswirling until the fumes appear again. Cool to room further darkening occurs. Digest for 10 min after thetemperature. Transfer the contents of the flask to a 50- first appearance of perchloric acid fumes or until themL volumetric flask with the aid of the Diluent, and digest becomes colorless. Cool the flask, add 2.5 mL ofdilute with Diluent to volume. Hydrochloric acid solution, and return the flask to the hot

Instrumental conditions plate to expel residual nitric acid. Heat the mixture for(See Spectrophotometry and Light-Scattering ⟨851⟩.) 3 min after it begins to boil. Cool the flask to roomMode: Atomic absorption spectrophotometry temperature, and dilute with water to 20 mL.Analytical wavelength: Selenium emission line at 196.0 Instrumental conditionsnm (See Spectrophotometry and Light-Scattering ⟨851⟩.)

Lamp: Selenium hollow-cathode Mode: UVFlame: Air–acetylene Analytical wavelength: 380 nmBlank: Diluent and perchloric acid (20:1) Cell: 1 cm

Analysis Blank: 1 mL of perchloric acid and 1 mL of HydrochloricSamples: Standard solutions and Sample solution acid solution diluted with water to 20 mLDetermine the absorbances of the solutions against the AnalysisBlank. Plot the absorbances of the Standard solutions Samples: Standard solution and Sample solutionversus the concentration, in µg/mL, of selenium, and Treat the Sample solution, the Standard solution, and thedraw the straight line best fitting the three plotted Blank as follows. Add 5 mL of Reagent A to each flask,points. From the graph so obtained, determine the and swirl gently to mix. Adjust the solution in eachconcentration, C, in µg/mL, of selenium in the Sample flask with 50% Ammonium hydroxide solution to a pHsolution. of 1.1 ±0.1. Add 5 mL of Reagent B to each flask, and

Calculate the percentage of the labeled amount of swirl gently to mix. Place the flasks in a water bathselenium (Se) in the portion of Tablets taken: maintained at 50°, and equilibrate for 30 min, taking

care that the flasks are covered to protect them fromResult = (C/CU) × 100 light. Cool to room temperature, and transfer the

contents of each flask to separate separatory funnels.C = measured concentration of selenium in the Transfer 10.0 mL of cyclohexane to each separatory

Sample solution (µg/mL) funnel, and extract vigorously for 1 min. Discard theCU = nominal concentration of selenium in the aqueous layer. Transfer the cyclohexane layer to a

Sample solution (µg/mL) centrifuge tube, and centrifuge at 1000 rpm for 1 minAcceptance criteria: 90.0%–160.0% of the labeled to remove any remaining water. Determine theamount of selenium (Se) absorbances of the solutions obtained from the

• SELENIUM, Method 2 Samples against the solution obtained from the Blank.Hydrochloric acid solution: Hydrochloric acid diluted Calculate the percentage of the labeled amount ofwith water (1 in 10) selenium (Se) in the portion of Tablets taken:

50% ammonium hydroxide solution: Ammoniumhydroxide diluted with water (1 in 2) Result = (AU/AS) × [(V × CS)/MU] × 100

Reagent A: 9 mg/mL of edetate disodium and 25mg/mL of hydroxylamine hydrochloride in water. AU = absorbances of the cyclohexane layer from the[NOTE—Dissolve edetate disodium in a portion of water Sample solutionfirst, then add hydroxylamine hydrochloride, and dilute AS = absorbances of the cyclohexane layer from thewith water to volume.] Standard solution

Reagent B: Transfer 200 mg of 2,3-diaminonaphthalene V = volume of the Standard stock solution used toto a 250-mL separatory funnel, and add 200 mL of 0.1 prepare the Standard solution, 10 mLN hydrochloric acid. Wash the solution with three 40-mL CS = concentration of selenium in the Standardportions of cyclohexane, and discard the cyclohexane stock solution (µg/mL)layer. Filter the solution into a brown bottle, and cover MU = nominal amount of selenium in the Samplethe solution with a 1-cm layer of cyclohexane. This solution (µg)solution is stable for 1 week if stored in a refrigerator. Acceptance criteria: 90.0%–160.0% of the labeled

Standard stock solution: [CAUTION—Selenium is toxic; amount of selenium (Se)handle it with care.] Dissolve 1 g of metallic selenium in • ZINC, Method 1a minimum volume of nitric acid. Evaporate to dryness, Zinc standard solution: 1000 µg/mL of zinc from zincadd 2 mL of water, and evaporate to dryness. Repeat the oxide in 5 M hydrochloric acid (3.89 mg/mL) andaddition of water and evaporation to dryness three diluted with water to final volume. [NOTE—Dissolve intimes. Dissolve the residue in 3 N hydrochloric acid, 5 M hydrochloric acid by warming, if necessary, cool,transfer to a 1000-mL volumetric flask, and dilute with and then dilute to final volume.]3 N hydrochloric acid to volume to obtain a solution Standard stock solution: 50 µg/mL of zinc from Zincwith a concentration of 1000 µg/mL of selenium. Dilute standard stock solution diluted with 0.125 N hydrochlorica volume of the solution with 0.125 N hydrochloric acid acidto obtain a concentration of 2.0 µg/mL of selenium. Standard solutions: Transfer 1.0, 2.0, 3.0, 4.0, and 5.0

Standard solution: Transfer 10 mL of the Standard stock mL of Standard stock solution to separate 100-mLsolution to a glass-stoppered flask. Add 1 mL of volumetric flasks. Dilute the contents of each flask withperchloric acid and 1 mL of Hydrochloric acid solution, 0.125 N hydrochloric acid to volume to obtainand dilute with water to 20 mL. concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of

Sample solution: Transfer a portion of finely powdered zinc.Tablets, equivalent to 20 µg of selenium, to a suitable Sample solution: Proceed as directed for the Sampleflask. Add 10 mL of nitric acid, and warm gently on a solution in Calcium, Method 1, except to prepare thehot plate. Continue heating until the initial nitric acid Sample solution to contain a concentration of 2 µg/mL ofreaction has subsided, then add 3 mL of perchloric acid. zinc and to omit the use of the Lanthanum chloride[CAUTION—Exercise care at this stage, because the solution.

perchloric acid reaction becomes vigorous.] Instrumental conditionsContinue heating on the hot plate until the appearance (See Spectrophotometry and Light-Scattering ⟨851⟩.)of white fumes of perchloric acid or until the digest




Mode: Atomic absorption spectrophotometry Diluent to prepare a six-point calibration curve to bracketAnalytical wavelength: Zinc emission line at 213.8 nm the concentration range of each mineral of interest.Lamp: Zinc hollow-cathode Sample solution 1 (for Tablets containing minerals foundFlame: Air–acetylene in Standard stock solution 1 and Standard stock solutionBlank: 0.125 N hydrochloric acid 2): Weigh and finely powder NLT 20 Tablets. Transfer a

Analysis portion, equal to 3.5 times the average Tablet weight, toSamples: Standard solutions and Sample solution a 250-mL volumetric flask. Slowly add 25 mL of StockDetermine the absorbances of the solutions against the aqua regia solution in 5-mL increments followed byBlank. Plot the absorbances of the Standard solutions mixing. [NOTE—If the sample contains a carbonate,versus the concentration, in µg/mL, of zinc, and draw bubbling will occur. Wait until bubbling ends tothe straight line best fitting the five plotted points. proceed.] Bring the solution to a boil on a hot plate.From the graph so obtained, determine the Continue to heat gently until fumes cease (about 1 h).concentration, C, in µg/mL, of zinc in the Sample [NOTE—If the sample contains selenium, digest for NMTsolution. 15 min.] Remove from heat, cool, and dilute with water

Calculate the percentage of the labeled amount of zinc to volume. Filter about 30 mL into a centrifuge tube(Zn) in the portion of Tablets taken: using a 5-µm pore size nylon syringe filter. If necessary,

make any further dilutions using the Diluent.Result = (C/CU) × 100 Sample solution 2 (for Tablets containing minerals found

only in Standard stock solution 2): Weigh and finelyC = measured concentration of zinc in the Sample powder NLT 20 Tablets. Transfer a portion, equal to 3.5

solution (µg/mL) times the average Tablet weight, to a 250-mL volumetricCU = nominal concentration of zinc in the Sample flask. Slowly add 25 mL of Stock aqua regia solution in 5-

solution (µg/mL) mL increments followed by mixing. [NOTE—If the sampleAcceptance criteria: 90.0%–125.0% of the labeled contains a carbonate, bubbling will occur. Wait untilamount of zinc (Zn) bubbling ends to proceed.] Bring the solution to a boil

• BORON, NICKEL, TIN, and VANADIUM, Method 1; CALCIUM, on a hot plate. Continue to heat gently until fumesCHROMIUM, COPPER, IRON, MAGNESIUM, MANGANESE, cease (about 1 h). [NOTE—If the sample containsPHOSPHORUS, and ZINC, Method 2; MOLYBDENUM and selenium, digest for NMT 15 min.] Remove from heat,SELENIUM, Method 3 cool, and dilute with water to volume. Filter about 30

Stock aqua regia solution: Prepare a mixture of mL into a centrifuge tube using a 5-µm pore size nylonhydrochloric acid and nitric acid (3:1) by adding the syringe filter. If necessary, make any further dilutionsnitric acid to the hydrochloric acid. [NOTE—Periodically using the Diluent.vent the solution in an appropriate fume hood.] Sample solution 3 (for Tablets containing minerals found

Diluent: Prepare a mixture of Stock aqua regia solution only in Standard stock solution 1): Weigh and finelyand water (1:9) by adding one volume of Stock aqua powder NLT 20 Tablets. Transfer a portion, equal to theregia solution to two volumes of water. Dilute with average Tablet weight, to a 250-mL volumetric flask.additional water to volume, and mix well. Slowly add 25 mL of Stock aqua regia solution in 5-mL

System suitability solution: Prepare a mixture of increments, followed by mixing. [NOTE—If the sample 1000 mg/L of yttrium in 5% nitric acid solution, 1000 contains a carbonate, bubbling will occur. Wait untilmg/L of scandium in 5% nitric acid solution, and Diluent bubbling ends to proceed.] Bring the solution to a boil(1:1:198), and mix. on a hot plate. Continue to heat gently (about 1 h) until

Standard stock solution 1 (Ca, Cu, Fe, Mg, Mn, P, and fumes cease. Remove from heat, cool, and dilute withZn): [NOTE—It is only necessary to include the minerals water to volume. Filter about 30 mL into a centrifugeof interest in the solution.] Using commercially available tube using a 5-µm pore size nylon syringe filter. Ifelement standard (single- or multi-element) solutions in necessary, make any further dilutions using the Diluent.5% nitric acid solution, pipet the appropriate amount of Instrumental conditionselement standard solution into a volumetric flask, and (See Plasma Spectrochemistry ⟨730⟩.)dilute with 5% nitric acid solution to obtain a solution Mode: Inductively coupled plasma spectrometry, usinghaving final concentrations of about 1000 mg/L of a spectrometer set to measure the emission of eachcalcium, 100 mg/L of copper, 250 mg/L of iron, 500 mineral of interest at about the correspondingmg/L of magnesium, 100 mg/L of manganese, 800 wavelength. [NOTE—The operating conditions may bemg/L of phosphorus, and 250 mg/L of zinc. developed and optimized based on the manufacturer’s

Standard stock solution 2 (B, Cr, Mo, Ni, Se, Sn, and V): recommendation. The wavelengths selected should be [NOTE—It is only necessary to include the minerals of demonstrated experimentally to provide sufficient

interest in the solution.] Using commercially available specificity, sensitivity, linearity, accuracy, and precision.]element standard (single- or multi-element) solutions in System suitability20% hydrochloric acid solution, pipet the appropriate [NOTE—Analyze the System suitability solution, andamount of element standard solution into a volumetric obtain the response as directed in the Analysis. ]flask, and dilute with 20% hydrochloric acid solution to Suitability requirementsobtain a solution having final concentrations of about Relative standard deviation: NMT 2.0%200 mg/L of boron, and and 100 mg/L each of Analysischromium, molybdenum, nickel, selenium, tin, and Samples: Standard solutions and Sample solutionvanadium.

Standard solutions: Prepare a mixture of Standard stocksolution 1 and Standard stock solution 2, as required, in




Determine the emission of each mineral of interest in the • USP REFERENCE STANDARDS ⟨11⟩Standard solutions and Sample solution with an USP Alpha Tocopherol RSinductively coupled plasma system using the Diluent as USP Alpha Tocopheryl Acetate RSthe blank. Plot the emission of the Standard solutions USP Alpha Tocopheryl Acid Succinate RSversus the concentration, in mg/L, of the minerals of USP Biotin RSinterest, and draw the straight line best fitting the USP Calcium Pantothenate RSplotted points. From the graph so obtained, determine USP Cholecalciferol RSthe concentration, C, in mg/L, for each mineral of USP Cyanocobalamin RSinterest in the Sample solution. Calculate the percentage USP Ergocalciferol RSof the labeled amount for each mineral taken: USP Folic Acid RS

USP Niacin RSResult = C × (V/W) × F × (CW/L) × 100 USP Niacinamide RS

USP Phytonadione RSC = measured concentration of the relevant USP Pyridoxine Hydrochloride RS

element in the Sample solution (mg/L) USP Riboflavin RSV = volume of the Sample solution (L) USP Sodium Fluoride RSW = sample weight (mg) USP Thiamine Hydrochloride RSF = dilution factor of the Sample solution USP Vitamin A RSCW = average Tablet weight (mg)L = labeled amount per Tablet (mg)

Acceptance criteria: 90.0%–125.0% of the labeled.amount of calcium (Ca), copper (Cu), iron (Fe),

magnesium (Mg), manganese (Mn), phosphorus (P), and Water-Soluble Vitamins Capsuleszinc (Zn); and 90.0%–160.0% of the labeled amounts ofboron (B), chromium (Cr), molybdenum (Mo), nickel DEFINITION(Ni), selenium (Se), tin (Sn), and vanadium (V) Water-Soluble Vitamins Capsules contain two or more of the

following water-soluble vitamins: Ascorbic Acid or itsPERFORMANCE TESTSequivalent as Calcium Ascorbate or Sodium Ascorbate, Bi-• DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTSotin, Cyanocobalamin, Folic Acid, Dexpanthenol or⟨2040⟩: Meet the requirements for DissolutionPanthenol, Pantothenic Acid (as Calcium Pantothenate or• WEIGHT VARIATION OF DIETARY SUPPLEMENTS ⟨2091⟩: MeetRacemic Calcium Pantothenate), Niacin or Niacinamide,the requirementsPyridoxine Hydrochloride, Riboflavin, and Thiamine Hy-drochloride or Thiamine Mononitrate. Capsules containCONTAMINANTSNLT 90.0% and NMT 150.0% of the labeled amounts of• MICROBIAL ENUMERATION TESTS ⟨2021⟩: The total aerobicascorbic acid (C6H8O6), biotin (C10H16N2O3S), cyanocobal-microbial count does not exceed 3000 cfu/g, and theamin (C63H88CoN14O14P), folic acid (C19H19N7O6), dexpan-combined molds and yeasts count does not exceed 300thenol (C9H19NO4) or panthenol (C9H19NO4), calcium pan-cfu/g.tothenate (C18H32CaN2O10), niacin (C6H5NO2) or• MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIEDniacinamide (C6H6N2O), pyridoxine hydrochlorideMICROORGANISMS ⟨2022⟩: Meet the requirements of the(C8H11NO3 · HCl), riboflavin (C17H20N4O6), and thiaminetests for absence of Salmonella species, Escherichia coli,(C12H17ClN4OS) as thiamine hydrochloride or thiamineand Staphylococcus aureusmononitrate.

ADDITIONAL REQUIREMENTS They do not contain any form of Beta Carotene or Vitamin• PACKAGING AND STORAGE: Preserve in tight, light-resistant A, D, E, or K. They do not contain any minerals for which

containers. nutritional value is claimed. They may contain other la-• LABELING:5 The label states that the product is Oil- and beled added substances in quantities that are

Water-Soluble Vitamins with Minerals Tablets. The label unobjectionable.also states the quantity of each vitamin and mineral per

STRENGTHdosage unit and where necessary the chemical form in[NOTE—In the following assays, where more than one assaywhich a vitamin is present and also states the salt form ofmethod is given for an individual ingredient, the require-the mineral used as the source of each element. Wherements may be met by following any one of the specifiedthe product contains vitamin E, the label indicatesmethods, the method used being stated in the labeling onlywhether it is the D- or DL- form. Where more than oneif Method 1 is not used.]assay method is given for a particular vitamin, the• ASCORBIC ACID, Method 1labeling states with which assay method the product

Sample solution: Weigh NLT 20 Capsules in a taredcomplies only if Method 1 is not used.weighing bottle. Open the Capsules, without the loss of

5 USP Units of activity for vitamins, where such exist or formerly existed, are shell material, and transfer the contents to a 100-mLequivalent to the corresponding international units, where such formerlybeaker. Remove any contents adhering to the emptyexisted. 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 shells by washing, if necessary, with several portions oflabels of vitamin products continues. Where articles are labeled in terms of ether. Discard the washings, and dry the Capsule shellsUnits in addition to the required labeling, the relationship of the USP Units or with the aid of a current of dry air until the odor ofIU 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) ether is no longer perceptible. Weigh the empty Capsuleor 0.55 µg of all-trans-retinyl palmitate (vitamin A palmitate), and 1 µg of shells in the tared weighing bottle, and calculate the av-retinol (3.3 USP Vitamin A Units) = 1 retinol equivalent (RE); 1 IU of beta erage net weight per Capsule. Transfer a portion of thecarotene = 0.6 µg of all-trans-beta carotene; 1 USP Vitamin D Unit = 0.025

Capsule contents, equivalent to a nominal amount ofµg of ergocalciferol or cholecalciferol; and 1 mg of dl-alpha tocopherol = 1.1former USP Vitamin E Units, 1 mg of dl-alpha tocopheryl acetate = 1 former 100 mg of ascorbic acid, to a 200-mL volumetric flask,USP Vitamin E Unit, 1 mg of dl-alpha tocopheryl acid succinate = 0.89 former and add 75 mL of metaphosphoric–acetic acids TS. InsertUSP Vitamin E Unit, 1 mg of d-alpha tocopherol = 1.49 former USP Vitamin E a stopper into the flask, and shake by mechanical meansUnits, and 1 mg of d-alpha tocopheryl acetate = 1.36 former USP Vitamin EUnits, 1 mg of d-alpha tocopheryl acid succinate = 1.21 former USP Vitamin E for 30 min. Dilute with water to volume. Transfer a por-Units. In terms of d-alpha tocopherol equivalents, 1 mg of d-alpha tocopheryl tion of the solution to a centrifuge tube, and centrifugeacetate = 0.91, 1 mg of d-alpha tocopheryl acid succinate = 0.81, 1 mg of dl- until a clear supernatant is obtained. Pipet 4.0 mL of thisalpha tocopherol = 0.74, 1 mg of dl-alpha tocopheryl acetate = 0.67, and 1

solution into a 50-mL conical flask, and add 5 mL ofmg of dl-alpha tocopheryl acid succinate = 0.60.metaphosphoric–acetic acids TS.



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Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 20:53:51 … · 2011-12-02 · states the equivalent content of dexpanthenol. Where 3,7-Dimethyl-9-(2,6,6-trimethyl-1- cyclohexen-1-yl)