GUID - 6 en-US PDF

Printed on: Tue Jan 03 2023, 02:46:15 AM(EST) Status: Currently Official on 03-Jan-2023 DocId: GUID-DEC96CEA-BD0D-402A-BC13-8762D0532BC9_6_en-US
Printed by: Mahmud Riyadi Official Date: Official as of 01-Nov-2020 Document Type: DIETARY SUPPLEMENTS @2023 USPC
Do Not Distribute DOI Ref: 0bd9g DOI: https://doi.org/10.31003/USPNF_M88698_06_01
1
method used being stated in the labeling only if Method

Oil- and Water-Soluble Vitamins with 1 is not used.]
Minerals Tablets
Change to read:
DEFINITION
• VITAMIN A, Method 1
Change to read: ▲
Proceed as directed in Vitamin A Assay á571ñ, Assay,
Chromatographic Methods, Procedure 1, except for the
Oil- and Water-Soluble Vitamins with Minerals Tablets contain
Sample stock solution and Sample solution.▲ (USP 1-May-2020)
one or more of the following oil-soluble vitamins: Vitamin A
Sample stock solution: Finely powder NLT 20 Tablets.
▲
as retinyl acetate or retinyl palmitate,▲ (USP 1-May-2020)
Transfer a portion of the powder, equivalent to 5 Tablets,
vitamin D as Ergocalciferol (vitamin D2) or Cholecalciferol
to a container having a polytef-lined screw-cap. Add 10 mL
(vitamin D3), Vitamin E ▲as RRR- or all-rac-alpha-tocopherol, of dimethyl sulfoxide and 15 mL of n-hexane, and shake for
RRR- or all-rac-alpha-tocopheryl acetate, or RRR- or 45 min on a wrist-action shaker in a water bath maintained
all-rac-alpha-tocopheryl acid succinate,▲ (USP 1-May-2020) at 60°. [NOTE—Set up the wrist-action shaker to ensure that
Phytonadione (vitamin K1), and Beta Carotene; and one or the contents of the container are mixed vigorously and
more of the following water-soluble vitamins: Ascorbic Acid thoroughly.] Centrifuge at 3000 rpm for 10 min, and
or its equivalent as Calcium Ascorbate or Sodium Ascorbate, transfer the hexane layer by means of a pipet to a 100-mL
Biotin, Cyanocobalamin, Folic Acid, Niacin or Niacinamide, volumetric flask. Add 15 mL of n-hexane to the dimethyl
pantothenic acid (as Calcium Pantothenate or Racemic sulfoxide layer, shake thoroughly for 5 min, and transfer the
Calcium Pantothenate), Pyridoxine Hydrochloride, hexane layer by means of a pipet to the 100-mL volumetric
Riboflavin, and Thiamine Hydrochloride or Thiamine flask. Repeat this extraction with 3 additional 15-mL
Mononitrate; and one or more minerals derived from portions of n-hexane. Dilute the extracts in the volumetric
al
substances generally recognized as safe, furnishing one or flask with n-hexane to volume.
more of the following elements in ionizable form: boron, Sample solution: Dilute a 10-mL volume of the Sample stock
calcium, chromium, copper, fluorine, iodine, iron, solution with n-hexane to obtain a solution with a
magnesium, manganese, molybdenum, nickel, concentration of 15 µg/mL of vitamin A as retinol
phosphorus, potassium, selenium, tin, vanadium, and zinc. (C20H30O).
Tablets contain NLT 90.0% and NMT 165.0% of the
labeled amount of vitamin A ▲as retinol▲ (USP 1-May-2020)
(C20H30O); vitamin D as cholecalciferol (C27H44O) or
ci ▲
▲ (USP 1-May-2020)
Acceptance criteria: 90.0%–165.0% of the labeled amount
of vitamin A as retinol (C20H30O)
ergocalciferol (C28H44O); vitamin E ▲as
2R-alpha-tocopherol▲ (USP 1-May-2020) (C29H50O2); Change to read:
ffi
phytonadione (C31H46O2); and beta carotene (C40H56); and
NLT 90.0% and NMT 150.0% of the labeled amount of • VITAMIN A, Method 2
ascorbic acid (C6H8O6); ▲▲ (USP 1-May-2020) biotin
▲
(C10H16N2O3S), cyanocobalamin (C63H88CoN14O14P), folic Chromatographic Methods, Procedure 2.▲ (USP 1-May-2020)
acid (C19H19N7O6), niacin (C6H5NO2) or niacinamide Acceptance criteria: 90.0%–165.0% of the labeled amount
of vitamin A as retinol (C20H30O)
O
(C6H6N2O), calcium pantothenate (C18H32CaN2O10),

pyridoxine ▲▲ (USP 1-May-2020) (C8H11NO3▲▲ (USP 1-May-2020)),
Change to read:
riboflavin (C17H20N4O6), and thiamine ▲as thiamine ion
(C12H17N4OS)▲ (USP 1-May-2020) and NLT 90.0% and NMT • VITAMIN A, Method 3
125.0% of the labeled amount of calcium (Ca), copper ▲
(Cu), iron (Fe), manganese (Mn), magnesium (Mg), Chromatographic Methods, Procedure 3.▲ (USP 1-May-2020)
phosphorus (P), potassium (K), and zinc (Zn); and NLT Acceptance criteria: 90.0%–165.0% of the labeled amount
90.0% and NMT 160.0% of the labeled amount of boron of vitamin A as retinol (C20H30O)
(B), chromium (Cr), fluorine (F), iodine (I), molybdenum
(Mo), nickel (Ni), selenium (Se), tin (Sn), and vanadium (V). Change to read:
They may contain other labeled added substances that are
generally recognized as safe, in amounts that are • VITAMIN D (CHOLECALCIFEROL OR ERGOCALCIFEROL),
unobjectionable. Method 1
▲
Proceed as directed in Vitamin D Assay á581ñ, Assay,
IDENTIFICATION Chromatographic Methods, Procedure 1, except for the
Add the following: Sample solution.▲ (USP 1-May-2020)
Sample solution: Prepare as directed for the Sample stock
▲
• A. Characteristic absorption of each mineral as obtained solution in Vitamin A, Method 1. Transfer NLT 20 mL of this
in the tests in Strength▲ (USP 1-May-2020) solution to a suitable container, and if necessary, evaporate
under vacuum at room temperature to obtain a
Add the following: concentration of 2 µg/mL of cholecalciferol or
ergocalciferol.
▲
• B. The retention times of the vitamin peaks of the Sample ▲
▲ (USP 1-May-2020)
solutions correspond to those of the corresponding vitamin Acceptance criteria: 90.0%–165.0% of the labeled amount
peaks of the Standard solutions as obtained in the tests for of vitamin D as cholecalciferol (C27H44O) or ergocalciferol
Strength.▲ (USP 1-May-2020) (C28H44O)
STRENGTH
[NOTE—Where more than one assay method is given for an
individual ingredient, the requirements may be met by
following any one of the specified methods, with the
https://online.uspnf.com/uspnf/document/1_GUID-DEC96CEA-BD0D-402A-BC13-8762D0532BC9_6_en-US 1/24
2
Change to read: rS = peak area of the relevant vitamin E form from the
Standard solution
• VITAMIN D (CHOLECALCIFEROL or ERGOCALCIFEROL), CS = concentration of ▲all-rac-alpha-tocopherol from
Method 2 either USP Alpha Tocopherol RS or USP Alpha
▲
Proceed as directed in Vitamin D Assay á581ñ, Assay, Tocopheryl Acetate RS or RRR-alpha-tocopherol
Chromatographic Methods, Procedure 2.▲ (USP 1-May-2020) from USP Alpha Tocopheryl Acid
Acceptance criteria: 90.0%–165.0% of the labeled amount Succinate RS▲ (USP 1-May-2020) in the Standard
of vitamin D as cholecalciferol (C27H44O) or ergocalciferol solution (mg/mL)
(C28H44O) CU = nominal concentration of the corresponding form
of vitamin E ▲as
Change to read: 2R-alpha-tocopherol▲ (USP 1-May-2020) in the Sample
solution (mg/mL)
• VITAMIN D (CHOLECALCIFEROL or ERGOCALCIFEROL), ▲
F = conversion factor for the content of
Method 3 all-rac-alpha-tocopherol to 2R-alpha-tocopherol,
▲
Proceed as directed in Vitamin D Assay á581ñ, Assay, 1/2 (for products labeled to contain all-rac
Chromatographic Methods, Procedure 3, except for the vitamin E sources) and 1 (for products labeled
Analysis.▲ (USP 1-May-2020) to contain RRR vitamin E sources)▲ (USP 1-May-2020)
Analysis
Samples: Standard solution and Sample solution Acceptance criteria: 90.0%–165.0% of the labeled amount
Measure the peak areas for vitamin D. of alpha-tocopherol (C29H50O2), alpha-tocopheryl acetate
Calculate the percentage of the labeled amount of (C31H52O3), or alpha-tocopheryl acid succinate (C33H54O5)
cholecalciferol (C27H44O) or ergocalciferol (C28H44O) in the as ▲2R-alpha-tocopherol▲ (USP 1-May-2020)
al
portion of Tablets taken:
Change to read:
Result = (rU/rS) × (CS/CU) × 100
• VITAMIN E, Method 2
rU = peak area of cholecalciferol or ergocalciferol ▲
Proceed as directed in Vitamin E Assay á551ñ, Assay,
from the Sample solution Procedure 2, except for the Analysis.▲ (USP 1-May-2020)
rS
CS
= peak area of cholecalciferol or ergocalciferol
from the Standard solution
= concentration of USP Cholecalciferol RS or USP
ci Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
Ergocalciferol RS in the Standard solution alpha-tocopherol (C29H50O2), alpha-tocopheryl acetate
(µg/mL) (C31H52O3), or alpha-tocopheryl acid succinate (C33H54O5)
ffi
CU = nominal concentration of cholecalciferol or as 2R-alpha-tocopherol (C29H50O2) in the portion of
ergocalciferol in the Sample solution (µg/mL) Tablets taken as follows:
Acceptance criteria: 90.0%–165.0% of the labeled amount Result = (rU/rS) × (CS/CU) × F × 100
of vitamin D as cholecalciferol (C27H44O) or ergocalciferol
(C28H44O) rU = peak area of the relevant vitamin E form from the
O
Sample solution
Change to read: rS = peak area of the relevant vitamin E form from the
Standard solution
• VITAMIN E, Method 1 CS = concentration of all-rac-alpha-tocopherol from
▲
Proceed as directed in Vitamin E Assay á551ñ, Assay, either USP Alpha Tocopherol RS or USP Alpha
Procedure 1, except for the Sample solution and Tocopheryl Acetate RS or RRR-alpha-tocopherol
Analysis.▲ (USP 1-May-2020) from USP Alpha Tocopheryl Acid Succinate RS in
Sample solution: Prepare as directed for the Sample stock the Standard solution (mg/mL)
solution in Vitamin A, Method 1. Transfer NLT 20 mL of this CU = nominal concentration of the corresponding form
solution to a suitable container, and evaporate under of vitamin E as 2R-alpha-tocopherol in the Sample
vacuum at room temperature to dryness. Transfer the solution (mg/mL)
residue with the aid of methanol to a suitable volumetric F = conversion factor for the content of
flask, and dilute with methanol to volume to obtain a all-rac-alpha-tocopherol to 2R-alpha-tocopherol,
concentration of 2 mg/mL of alpha-tocopherol, 1/2 (for products labeled to contain all-rac
alpha-tocopheryl acetate, or alpha-tocopheryl acid vitamin E sources) and 1 (for products labeled
succinate. to contain RRR vitamin E sources)
▲
▲ (USP 1-May-2020)
Analysis Acceptance criteria: 90.0%–165.0% of the labeled amount
Samples: Standard solution and Sample solution of alpha-tocopherol (C29H50O2), alpha-tocopheryl acetate
Measure the peak areas. (C31H52O3), or alpha-tocopheryl acid succinate (C33H54O5)
Calculate the percentage of the labeled amount of as ▲2R-alpha-tocopherol▲ (USP 1-May-2020)
alpha-tocopherol (C29H50O2), alpha-tocopheryl acetate
(C31H52O3), or alpha-tocopheryl acid succinate (C33H54O5) Change to read:
▲
as 2R-alpha-tocopherol (C29H50O2)▲ (USP 1-May-2020) in the
portion of Tablets taken: • VITAMIN E, Method 3
▲
Proceed as directed in Vitamin E Assay á551ñ, Assay,
Result = (rU/rS) × (CS/CU) ▲× F▲ (USP 1-May-2020) × 100 Procedure 3, except for the Analysis.
Analysis
rU = peak area of the relevant vitamin E form from the Samples: Standard solution and Sample solution
Sample solution
3
Calculate the percentage of the labeled amount of Analysis

alpha-tocopherol (C29H50O2), alpha-tocopheryl acetate Samples: Standard solution and Sample solution
(C31H52O3), or alpha-tocopheryl acid succinate (C33H54O5) Measure the peak areas. Calculate the percentage of the
as 2R-alpha-tocopherol (C29H50O2) in the portion of labeled amount of phytonadione (C31H46O2) in the portion
Tablets taken as follows: of Tablets taken:
Result = (rU/rS) × (CS/CU) × F × 100 Result = (rU/rS) × (CS/CU) × 100
rU = peak area of the relevant vitamin E form from the rU = peak area of phytonadione from the Sample
Sample solution solution
rS = peak area of the relevant vitamin E form from the rS = peak area of phytonadione from the Standard
Standard solution solution
CS = concentration of all-rac-alpha-tocopherol from CS = concentration of USP Phytonadione RS in the
either USP Alpha Tocopherol RS and USP Alpha Standard solution (µg/mL)
Tocopheryl Acetate RS or RRR-alpha-tocopherol CU = nominal concentration of phytonadione in the
from USP Alpha Tocopheryl Acid Succinate RS in Sample solution (µg/mL)
the Standard solution (mg/mL)
CU = nominal concentration of the corresponding form Acceptance criteria: 90.0%–165.0% of the labeled amount
of vitamin E as 2R-alpha-tocopherol in the Sample of phytonadione (C31H46O2)
solution (mg/mL) • PHYTONADIONE, Method 2
F = conversion factor for the content of [NOTE—Use low-actinic glassware throughout this
all-rac-alpha-tocopherol to 2R-alpha-tocopherol, procedure.]
Solvent: Methanol and isopropyl alcohol (19:1)
al
1/2 (for products labeled to contain all-rac
vitamin E sources) and 1 (for products labeled Mobile phase: Mix 800 mL of methanol, 200 mL of
to contain RRR vitamin E sources)▲ (USP 1-May-2020) methylene chloride, 0.1 mL of glacial acetic acid, 1.36 g of
zinc chloride, and 0.41 g of sodium acetate.
Acceptance criteria: 90.0%–165.0% of the labeled amount Internal standard solution: 5 µg/mL of menaquinone 4
of alpha-tocopherol (C29H50O2), alpha-tocopheryl acetate (vitamin K2) in the Solvent. [NOTE—A concentrated stock
(C31H52O3), or alpha-tocopheryl acid succinate (C33H54O5)
as ▲2R-alpha-tocopherol▲ (USP 1-May-2020)
• PHYTONADIONE, Method 1
ci solution of menaquinone 4 (100 µg/mL) can be stored for
2 months in a refrigerator.]
Standard stock solution: 5 µg/mL of USP
[NOTE—Use low-actinic glassware throughout this Phytonadione RS, prepared by dissolving in methylene
procedure.] chloride with the aid of sonication and diluting with Solvent
ffi
Mobile phase: Methanol and water (19:1) to final volume
Standard stock solution: 200 µg/mL of USP Standard solution: Transfer 1.0 mL of the Standard stock
Phytonadione RS in methanol. Dissolve with the aid of solution and 1.0 mL of the Internal standard solution to a
sonication if necessary. suitable flask, and dilute with Solvent to 5 mL. Pass
System suitability solution: 0.65 mg/mL of USP Alpha through a membrane filter of 0.45-µm or finer pore size.
Tocopheryl Acetate RS and 20 µg/mL of USP Sample solution: Finely powder NLT 20 Tablets. To a
O
Phytonadione RS from the Standard stock solution diluted centrifuge tube fitted with a cap transfer an amount of
with methanol. [NOTE—Dissolve USP Alpha Tocopheryl powder not exceeding 800 mg and equivalent to an
Acetate RS in a portion of methanol, add the Standard stock amount of phytonadione not exceeding 50 µg. Add 4 mL
solution, and then dilute with methanol to volume.] of water. Insert the stopper, and mix using a vortex mixer
Standard solution: 20 µg/mL of USP Phytonadione RS until the sample is dispersed. Place the tube in a water bath
from the Standard stock solution diluted with methanol at 60° for 5 min. Remove from the bath, and again shake
Sample solution: Prepared as directed for the Sample stock or mix using a vortex mixer for 1 min while the preparation
solution in Vitamin A, Method 1. Transfer NLT 20 mL of the is still hot. Add 8 mL of alcohol, and swirl the contents to
solution to a suitable container, and, if necessary, evaporate mix. Place the tube in a water bath at 60° for 5 min. Remove
under vacuum at room temperature to dryness. Transfer from the bath, and again shake or mix using a vortex mixer
the residue to a suitable volumetric flask with the aid of for 2 min while the preparation is still hot. Cool to room
methanol, and dilute with methanol to volume to obtain a temperature. Add a volume of the Internal standard
concentration of 20 µg/mL of phytonadione. solution, equivalent to 1.0 mL per each 5 µg of the expected
Chromatographic system amount of phytonadione in the aliquot taken. Add 20.0 mL
(See Chromatography á621ñ, System Suitability.) of petroleum ether, and cap the tube tightly. Shake or mix
Mode: LC using a vortex mixer for 15 min to thoroughly mix the
Detector: UV 254 nm contents. Centrifuge to separate the 2 layers. Transfer a
Column: 8-mm × 10-cm; 5-µm packing L1 volume of the top layer of petroleum ether, equivalent to
Flow rate: 2 mL/min 5–50 µg of the nominal amount of phytonadione, to an
Injection volume: 100 µL appropriate flask. Place the flask in a water bath at 35°–45°,
System suitability and evaporate the solvent under a stream of nitrogen until
Samples: System suitability solution and Standard solution an oily residue is left. Dissolve the residue in a volume of the
[NOTE—The relative retention times for Solvent to obtain a concentration of 1 µg/mL of
alpha-tocopheryl acetate and phytonadione are phytonadione.
0.68 and 1.0, respectively.] Chromatographic system
Suitability requirements (See Chromatography á621ñ, System Suitability.)
Resolution: NLT 5 between alpha-tocopheryl acetate Mode: LC
and phytonadione, System suitability solution Detector: Fluorometric detector
Relative standard deviation: NMT 3.0%, Standard Excitation: 320 nm
solution Emission: 420 nm
4
Column: 4.6-mm × 25-cm; 5-µm, end-capped packing L1, Vitamin D standard stock solution: 0.05 mg/mL of USP
and a postcolumn reactor constituted with a 4.6-mm × Cholecalciferol RS or USP Ergocalciferol RS in isopropyl
3-cm PEEK column tightly packed with zinc powder. alcohol. Dissolve with the aid of sonication if necessary.
[NOTE—Prepare the postcolumn reactor daily, or as Vitamin E standard stock solution: 3.0 mg/mL of USP
necessary, to meet the System suitability requirements.] Alpha Tocopherol RS, USP Alpha Tocopheryl Acetate RS, or
Flow rate: 1 mL/min USP Alpha Tocopheryl Acid Succinate RS in isopropyl
Injection volume: 25 µL alcohol. Dissolve with the aid of sonication if necessary.
System suitability Phytonadione standard stock solution: 0.04 mg/mL of
Sample: Standard solution USP Phytonadione RS in isopropyl alcohol. Dissolve with the
[NOTE—The relative retention times for the internal aid of sonication if necessary.
standard and phytonadione are 1.0 and 1.4, Sample solution 1: Finely powder NLT 20 Tablets. Transfer
respectively.] an accurately weighed portion of the powder into a 50-mL
Suitability requirements volumetric flask to obtain a solution containing known
Column efficiency: NLT 2500 theoretical plates for the nominal concentrations in the ranges of 0.05–0.1 mg/mL
phytonadione peak of vitamin A and 0.5–0.15 mg/mL of vitamin E. Add 10 mL
Tailing factor: NMT 1.5 for the phytonadione peak of dimethyl sulfoxide and sonicate for 30 min with vigorous
Relative standard deviation: NMT 3.0% intermittent shaking. Cool to room temperature, dilute
Analysis with isopropyl alcohol to volume, and mix well. Pass a
Samples: Standard solution and Sample solution portion of the solution through a nylon filter of 0.45-µm
Calculate the percentage of the labeled amount of pore size, and discard the first milliliter of the filtrate.
phytonadione (C31H46O2) in the portion of Tablets taken: Sample solution 2: Transfer an accurately weighed portion
of the powder from NLT 20 finely powdered Tablets into a
Result = (RU/RS) × (CS/CU) × 100 50-mL volumetric flask to obtain a solution containing
al
known nominal concentrations in the ranges of 1.0–3.5 µg/
RU = peak response ratio of phytonadione to the mL of vitamin D and 1.0–3.5 µg/mL of phytonadione. Add
internal standard from the Sample solution 5 mL of water and sonicate for 10 min with intermittent
RS = peak response ratio of phytonadione to the shaking. Add 10 mL of dimethyl sulfoxide and sonicate for
internal standard from the Standard solution 30 min with vigorous intermittent shaking. Cool to room
CS
CU
= concentration of USP Phytonadione RS in the
Standard solution (µg/mL)
= nominal concentration of phytonadione in the
ci temperature, dilute with isopropyl alcohol to volume, and
mix well. Pass a portion of the solution through a nylon filter
of 0.45-µm pore size, and discard the first milliliter of the
Sample solution (µg/mL) filtrate.
Standard solution: Transfer calculated volumes of Vitamin A
ffi
Acceptance criteria: 90.0%–165.0% of labeled amount of standard stock solution, Vitamin D standard stock solution,
phytonadione (C31H46O2) Vitamin E standard stock solution, and Phytonadione
standard stock solution into a suitable volumetric flask, and
Add the following: dilute with isopropyl alcohol to volume to obtain a solution
with final vitamin concentrations similar to those obtained
▲
• VITAMIN A, VITAMIN D (CHOLECALCIFEROL OR for the corresponding vitamins in Sample solution 1 and
O
ERGOCALCIFEROL), and VITAMIN E, Method 4; Sample solution 2.

PHYTONADIONE, Method 3 Chromatographic system
[NOTE—Where vitamin A (retinyl acetate or retinyl (See Chromatography á621ñ, System Suitability.)
palmitate), vitamin D (cholecalciferol or Mode: LC
ergocalciferol), or vitamin E (alpha-tocopherol, Detectors
alpha-tocopheryl acetate, or alpha-tocopheryl acid For Sample solution 1 containing retinyl acetate: UV
succinate) are specified in the following procedure, use 325 nm for 7 min, then switch to 265 nm
the chemical form present in the formulation and the For Sample solution 1 containing retinyl palmitate: UV
relevant Reference Standard. Use low-actinic 265 nm for 18 min, then switch to 325 nm
glassware.] For Sample solution 2: UV 265 nm
Diluent: Methanol and water (90:10, v/v) Column: 4.6-mm × 10-cm; 3-µm packing L1
Solution A: 0.1% (v/v) trifluoroacetic acid in Diluent Temperatures
Solution B: 0.1% (v/v) trifluoroacetic acid in methanol Sample: 4°
Mobile phase: See Table 1. Column: 35°
Flow rate: 1.5 mL/min
Table 1 Injection volume: 20 µL
Time Solution A Solution B System suitability
(min) (%) (%) Sample: Sample solution 1, Sample solution 2, and Standard
0 100 0 solution
Suitability requirements
19 0 100 Relative standard deviation: NMT 2.0% for each
27 0 100 individual peak, Standard solution
Peak purity of vitamin D and vitamin A (as retinyl
28 100 0 palmitate): Monitor the chromatograms of Sample
30 100 0 solution 1 and Sample solution 2 at 450 nm. The area of
any peak at the retention time of retinyl palmitate from
Sample solution 1 and any peak at the retention time of
Vitamin A standard stock solution: A solution containing vitamin D (as cholecalciferol or ergocalciferol) from
the equivalent of 0.2 mg/mL of retinol from USP Retinyl Sample solution 2 should be NMT 5% of the
Acetate RS or USP Retinyl Palmitate RS in isopropyl alcohol. corresponding peak areas detected at 325 nm and
Dissolve with the aid of sonication if necessary.
5
265 nm, respectively. [NOTE—This test is required Potassium hydroxide solution: Dissolve 58.8 g potassium
because of possible interference with carotenoids hydroxide in 50 mL of water.
(cryptoxanthin and lycopene) that may be present in the Iodine solution: 0.01 mg/mL of iodine in cyclohexane.
formulation. Use another procedure in the monograph [NOTE—Prepare this solution fresh daily.]
for analysis of vitamin D and vitamin A (as retinyl Sample solution: Weigh NLT 20 Tablets. Grind the Tablets
palmitate) if the system is not suitable] to a fine powder, and transfer a quantity of the powder,
Analysis equivalent to 2 mg of beta carotene, to a 500-mL
Samples: Sample solution 1, Sample solution 2, and saponification flask. Add 100 mL of alcohol, 6 mL of
Standard solution Potassium hydroxide solution, and a magnetic stirring bar.
Calculate the percentage of the labeled amount of Attach an air condenser to the flask, and heat under reflux
vitamin A activity, as retinol (C20H30O), in the portion of for 45 min with constant stirring. Cool to room
Tablets taken: temperature. Add 170 mL of solvent hexane, and stir for
30 min. Quantitatively transfer the contents of the flask to a
Result = (rU/rS) × (CS/CU) × 100 500-mL separatory funnel with portions of solvent hexane.
Allow the layers to separate for 5–10 min, and transfer the
rU = peak area of the all-trans-retinyl ester from the upper organic layer to a 500-mL volumetric flask. Transfer
Sample solution the lower aqueous layer into the saponification flask. Add
rS = peak area of the all-trans-retinyl ester from the 170 mL of solvent hexane, and stir for an additional 20 min.
Standard solution Quantitatively transfer the contents of the saponification
CS = concentration of retinol in the Standard solution flask to the separatory funnel with the aid of portions of
(mg/mL) solvent hexane. Allow the layers to separate for 10 min.
CU = nominal concentration of vitamin A, as retinol, Drain the lower aqueous layer, and discard. Transfer the
in the Sample solution (mg/mL) organic layer to the volumetric flask containing the
al
previously collected organic layer. Rinse the separatory
Calculate the percentage of the labeled amount of vitamin funnel with small portions of solvent hexane, and transfer
E, as 2R-alpha-tocopherol (C29H50O2), in the portion of the washings to the volumetric flask. Dilute the hexane
Tablets taken: extracts with solvent hexane to volume. Add 3 g of
anhydrous sodium sulfate, shake, and allow to settle.
rU
Result = (rU/rS) × (CS/CU) × F × 100
= peak area of the relevant vitamin E form from the

ci Quantitatively transfer a volume of this solution, equivalent
to 100 µg of beta carotene, to a 50-mL volumetric flask.
Evaporate under a stream of nitrogen to dryness, and
Sample solution immediately add cyclohexane. Add 2 mL of Iodine solution,
rS = peak area of the relevant vitamin E form from the and heat for 15 min in a water bath maintained at 65°. Cool
ffi
Standard solution rapidly, and dilute with cyclohexane to volume.
CS = concentration of all-rac-alpha-tocopherol from Instrumental conditions
either USP Alpha Tocopherol RS or USP Alpha (See Ultraviolet-Visible Spectroscopy á857ñ.)
Tocopheryl Acetate RS or RRR-alpha-tocopherol Mode: Vis
from USP Alpha Tocopheryl Acid Succinate RS in Analytical wavelength: 452 nm
the Standard solution (mg/mL) Blank: Cyclohexane
O
CU = nominal concentration of vitamin E, as Analysis

2R-alpha-tocopherol, in the Sample solution (mg/ Sample: Sample solution
mL) Determine the absorbance against the Blank.
F = conversion factor for the content of Calculate the percentage of the labeled amount of beta
all-rac-alpha-tocopherol to RRR-alpha-tocopherol carotene (C40H56) in the portion of Tablets taken:
equivalent, 1/2 (for products labeled to contain
all-rac vitamin E sources) and 1 (for products Result = (AU/F) × (100/CU)
labeled to contain RRR vitamin E sources)
AU = absorbance of the Sample solution
Calculate the percentage of the labeled amount of vitamin F = absorptivity of beta carotene at 452 nm, 223
D, as cholecalciferol (C27H44O) or ergocalciferol (C28H44O), CU = nominal concentration of beta carotene in the
and phytonadione (C31H46O2) in the portion of Tablets Sample solution (mg/mL)
taken:
Result = (rU/rS) × (CS/CU) × 100 of beta carotene (C40H56)
rU = peak area of the related vitamin from the Sample Change to read:
solution
rS = peak area of the related vitamin from the Standard • ASCORBIC ACID, CALCIUM ASCORBATE, and SODIUM
solution ASCORBATE
CS = concentration of related vitamin in the Standard (See Vitamin C Assay á580ñ.)
solution (µg/mL) [NOTE—For labeling purposes, consider á580ñ, Method I
CU = nominal concentration of related vitamin in the —Titrimetric Method as Method 1; ▲ á580ñ, Method II—
Sample solution (µg/mL) Chromatographic Method, Procedure 1 as Method 2; and
á580ñ, Method II—Chromatographic Method, Procedure
Acceptance criteria: 90.0%–165.0% of the labeled amount 2 as Method 3.▲ (USP 1-May-2020)]
of each individual vitamin▲ (USP 1-May-2020) Acceptance criteria: 90.0%–150.0% of the labeled amount
• BETA CAROTENE of ascorbic acid (C6H8O6), or its salts as calcium ascorbate
[NOTE—Use low-actinic glassware throughout this (C12H14CaO12 · 2H2O) or sodium ascorbate (C6H7NaO6)
procedure.]
6
• BIOTIN, Method 1 Acid-hydrolyzed casein solution: Mix 100 g of vitamin-free

[NOTE—Use low-actinic glassware throughout this casein with 500 mL of 6 N hydrochloric acid, and reflux the
procedure.] mixture for 8–12 h. Remove the hydrochloric acid from the
Mobile phase: Mix 85 mL of acetonitrile, 1 g of sodium mixture by distillation under reduced pressure until a thick
perchlorate, 1 mL of phosphoric acid, and dilute with water paste remains. Redissolve the resulting paste in water,
to 1000 mL. adjust the solution with 1 N sodium hydroxide to a pH of
Standard stock solution: 0.333 mg/mL of USP Biotin RS in 3.5 ± 0.1, and dilute with water to make 1000 mL. Add 20 g
dimethyl sulfoxide of activated charcoal, stir for 1 h, and filter. Repeat the
Standard solution: 5 µg/mL of USP Biotin RS prepared by treatment with activated charcoal. Store under toluene in a
diluting the Standard stock solution in water cool place at a temperature not below 10°. Filter the
Sample solution: Finely powder NLT 20 Tablets. Transfer a solution if a precipitate forms during storage.
portion of the powder, equivalent to 1 mg of biotin, to a Cystine–tryptophan solution: Suspend 4.0 g of L-cystine
200-mL volumetric flask. Add 3 mL of dimethyl sulfoxide, in a solution of 1.0 g of L-tryptophan (or 2.0 g of
and swirl to wet the contents. Place the flask in a water bath D,L-tryptophan) in 700–800 mL of water. Heat to 70°–80°,
at 60°–70° for 5 min. Sonicate for 5 min, dilute with water and add dilute hydrochloric acid (1 in 2) dropwise, with
to volume, and filter. stirring, until the solids are dissolved. Cool, and add water
Chromatographic system to make 1000 mL. Store under toluene in a cool place at a
(See Chromatography á621ñ, System Suitability.) temperature NLT 10°.
Mode: LC Adenine–guanine–uracil solution: Dissolve 200 mg each
Detector: UV 200 nm of adenine sulfate, guanine hydrochloride, and uracil, with
Column: 4.6-mm × 15-cm; 3-µm packing L7 the aid of heat, in 10 mL of 4 N hydrochloric acid. Cool,
Flow rate: 1.2 mL/min and add water to make 200 mL. Store under toluene in a
refrigerator.
al
Injection volume: 100 µL
System suitability Polysorbate 80 solution: 100 mg/mL of polysorbate 80 in
Sample: Standard solution alcohol
Suitability requirements Calcium pantothenate solution: 10 µg/mL of calcium
Relative standard deviation: NMT 3.0% pantothenate in 50% alcohol. Store in a refrigerator.
Analysis Riboflavin–thiamine hydrochloride solution: 20 µg/mL of
Measure the peak areas of biotin. Calculate the percentage
of the labeled amount of biotin (C10H16N2O3S) in the
ci riboflavin and 10 µg/mL of thiamine hydrochloride in
0.02 N acetic acid. Store under toluene, protected from
light, in a refrigerator.
portion of Tablets taken: p-Aminobenzoic acid–niacin–pyridoxine hydrochloride
solution: 10 µg/mL of p-aminobenzoic acid, 50 µg/mL of
ffi
Result = (rU/rS) × (CS/CU) × 100 niacin, and 40 µg/mL of pyridoxine hydrochloride in a
mixture of neutralized alcohol and water (1:3). Store in a
rU = peak area of biotin from the Sample solution refrigerator.
rS = peak area of biotin from the Standard solution Salt solution A: 25 g of monobasic potassium phosphate
CS = concentration of USP Biotin RS in the Standard and 25 g of dibasic potassium phosphate in water to make
solution (µg/mL) 500 mL. Add 5 drops of hydrochloric acid. Store under
O
CU = nominal concentration of biotin in the Sample toluene.

solution (µg/mL) Salt solution B: 10 g of magnesium sulfate, 0.5 g of sodium
chloride, 0.5 g of ferrous sulfate, and 0.5 g of manganese
Acceptance criteria: 90.0%–150.0% of the labeled amount sulfate in water to make 500 mL. Add 5 drops of
of biotin (C10H16N2O3S) hydrochloric acid, and mix. Store under toluene.
Basal medium stock solution: Dissolve the anhydrous
Change to read: dextrose and anhydrous sodium acetate in the solutions
previously mixed according to ▲Table 2,▲ (USP 1-May-2020)and
• BIOTIN, Method 2 adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute
[NOTE—Use low-actinic glassware throughout this with water to 250 mL.
procedure.]
Dehydrated mixtures yielding formulations similar to the ▲
Table 2▲ (USP 1-May-2020)
media described herein may be used provided that, when Acid-hydrolyzed casein solution 25 mL
constituted as directed, they have growth-promoting
properties equal to or superior to those obtained with the Cystine–tryptophan solution 25 mL
media prepared as described herein. Polysorbate 80 solution 0.25 mL
Standard stock solution: 50 µg/mL of USP Biotin RS in 50%
alcohol. Store in a refrigerator. Dextrose, anhydrous 10 g
Standard solution: 0.1 ng/mL of USP Biotin RS in water, Sodium acetate, anhydrous 5g
prepared by dilution of the Standard stock solution with
water on the day of the assay Adenine–guanine–uracil solution 5 mL
Sample solution: Finely powder NLT 30 Tablets. Transfer a Calcium pantothenate solution 5 mL
portion of the powder, equivalent to 100 µg of biotin, to a
200-mL volumetric flask. Add 3 mL of 50% alcohol, and Riboflavin–thiamine hydrochloride solution 5 mL
swirl to wet the contents. Heat the flask in a water bath at p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solu-
60°–70° for 5 min. Sonicate for 5 min, dilute with 50% tion 5 mL
alcohol to volume, and filter. Dilute a volume of the filtrate Salt solution A 5 mL
quantitatively, and stepwise if necessary, with water to
obtain a solution with a concentration of 0.1 ng/mL. Salt solution B 5 mL
7
Stock culture of Lactobacillus plantarum: Dissolve 2.0 g of Calculation: Prepare a standard concentration-response
yeast extract in 100 mL of water. Add 500 mg of anhydrous curve as follows. For each level of the Standard, calculate
dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g the response from the sum of the duplicate values of the
of agar, and heat the mixture on a steam bath, with stirring, transmittance (ΣS) as the difference, y = 2.00 − ΣS. Plot this
until the agar dissolves. Add 10-mL portions of the hot response on the ordinate of cross-section paper against the
solution to test tubes, close or cover the tubes, sterilize in logarithm of the milliliter of the Standard solution per tube
an autoclave at 121° for 15 min, and allow the tubes to cool on the abscissa, using for the ordinate either an arithmetic
in an upright position. Prepare stab cultures in 3 or more of or a logarithmic scale, whichever gives the better
the tubes, using a pure culture of Lactobacillus plantarum, 1 approximation to a straight line. Draw the straight line or
incubating for 16–24 h at a temperature between 30° and smooth curve that best fits the plotted points.
37° held constant to within ±0.5°. Store in a refrigerator. Calculate the response, y = 2.00 − ΣU, adding together the
Prepare a fresh stab of the stock culture every week, and do two transmittances (ΣU) for each level of the Sample
not use for Inoculum if the culture is more than 1 week old. solution. Read from the standard curve the logarithm of
Culture medium: To each of a series of test tubes containing the volume of the Standard solution corresponding to each
5.0 mL of Basal medium stock solution add 5.0 mL of water of those values of y that falls within the range of lowest
containing 0.5 ng of biotin. Plug the tubes with cotton, and highest points plotted for the Standard. Subtract from
sterilize in an autoclave at 121° for 15 min, and cool. each logarithm so obtained the logarithm of the volume,
Inoculum: [NOTE—A frozen suspension of Lactobacillus in mL, of the Sample solution to obtain the difference, X,
plantarum may be used as the stock culture, provided it for each dosage level. Average the values of X for each of
yields an inoculum comparable to a fresh culture.] Transfer 3 or more dosage levels to obtain X, which equals the
cells from the Stock culture of Lactobacillus plantarum to a log-relative potency, M′, of the Sample solution.
sterile tube containing 10 mL of Culture medium. Incubate Determine the quantity, in µg, of biotin (C10H16N2O3S) in
al
this culture for 16–24 h at a temperature between 30° and the portion of Tablets taken:
37° held constant to within ±0.5°. The cell suspension is the
Inoculum. antilog M = antilog (M′ + log R)
Analysis
Samples: Standard solution and Sample solution R = amount of biotin assumed to be present in the
To similar separate test tubes add, in duplicate, 1.0 and/or portion of the Tablets taken (µg)
1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. To
each tube and to 4 similar but empty tubes add 5.0 mL of
Basal medium stock solution and sufficient water to make
ci Calculate the percentage of the labeled amount of biotin
(C10H16N2O3S) in the portion of the Tablets taken:
10 mL.
To similar test tubes add, in duplicate, volumes of the Result = [(antilog M)/N] × 100
ffi
Sample solution corresponding to 3 or more of the levels
specified for the Standard solution, including the levels of N = nominal amount of biotin in the portion of the
2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL of the Basal Tablets taken (µg)
medium stock solution and sufficient water to make 10 mL.
Place one complete set of Standard and sample tubes Replication: Repeat the entire determination at least once,
together in one tube rack and the duplicate set in a second using separately prepared Sample solutions. If the difference
O
rack or section of a rack, preferably in random order. between the two log-potencies M is NMT 0.08, their mean,
Cover the tubes of both series to prevent contamination, M, is the assayed log-potency of the test material (see
and sterilize in an autoclave at 121° for 5 min. Cool. Add Design and Analysis of Biological Assays á111ñ, The Confidence
1 drop of Inoculum to each tube, except 2 of the 4 tubes Interval and Limits of Potency). If the two determinations
containing no Standard solution (the uninoculated differ by more than 0.08, conduct one or more additional
blanks). Incubate the tubes at a temperature between 30° determinations. From the mean of two or more values of M
and 37° held constant to within ±0.5° until, following 16– that do not differ by more than 0.15, compute the mean
24 h of incubation, there has been no substantial increase potency of the preparation under assay.
in turbidity in the tubes containing the highest level of Acceptance criteria: 90.0%–150.0% of the labeled amount
Standard during a 2-h period. of biotin (C10H16N2O3S)
Determine the transmittance of the tubes in the following • BIOTIN, Method 3
manner. Mix the contents of each tube, and transfer to a [NOTE—Use low-actinic glassware throughout this
spectrophotometer cell. Place the cell in a procedure.]
spectrophotometer that has been set at a specific Solution A: Transfer 800 mL of water and 100 mL of
wavelength of 540–660 nm, and read the transmittance triethylamine to a 1000-mL volumetric flask. Add 80 mL of
when a steady state is reached. This steady state is 85% phosphoric acid, and dilute with water to volume.
observed a few seconds after agitation when the Mobile phase: Transfer 80 mL of acetonitrile and 10 mL of
galvanometer reading remains constant for 30 s or more. Solution A to a 1000-mL volumetric flask. Dilute with water
Allow approximately the same time interval for the to volume.
reading on each tube. Standard solution: 0.6 µg/mL of USP Biotin RS in water.
With the transmittance set at 1.00 for the uninoculated [NOTE—A portion of the Standard solution will be used to
blank, read the transmittance of the inoculated blank. determine the percent recovery of biotin from the
With the transmittance set at 1.00 for the inoculated Solid-phase extraction procedure.]
blank, read the transmittance for each of the remaining Sample solution: Finely powder NLT 20 Tablets. Transfer an
tubes. If there is evidence of contamination with a foreign amount of powdered Tablets to a volumetric flask to
microorganism, disregard the result of the assay. obtain a nominal concentration of 0.6 µg/mL of biotin. Add
water up to 80% of the flask capacity, and sonicate for 30–
40 min, with occasional mixing, to dissolve. Dilute with
water to volume, and filter. Adjust the pH of the solution
1 ATCC No. 8014 is suitable. This strain was formerly known as Lactobacillus
arabinosus 17-5.
8
with either dilute acetic acid or 0.1 N sodium hydroxide to a Sample solution: Finely powder NLT 30 Tablets. Transfer a
pH of 6.0–7.0. portion of the powder, equivalent to 100 µg of
Solid-phase extraction: [NOTE—Condition the extraction cyanocobalamin, to a 250-mL flask. Quantitatively add
column specified in this procedure in the following manner. 100.0 mL of water, and carefully extract for 2 min. Filter
Wash the column with a 2-mL portion of methanol. 10 mL of the extract, and use the filtrate.
Equilibrate with a 2-mL portion of water.] Separately pipet Chromatographic system
5.0 mL each of the Sample solution and Standard solution (See Chromatography á621ñ, System Suitability.)
into freshly conditioned solid-phase extraction columns Mode: LC
consisting of a mixed-mode packing with a sorbent-mass Detector: 550 nm
of 60 mg. [NOTE—The mixed-mode packing consists of Column: 4.6-mm × 15-cm; 5-µm packing L1
anion-exchange and reversed-phase sorbents. The Flow rate: 0.5 mL/min
reverse-phase component is a polymer of copolymer Injection volume: 200 µL
N-vinylpyrrolidone and divinylbenzene. The anion System suitability
exchange moiety is a trialkylamino group.2 ] Sample: Standard solution
Wash the column with 10 mL of 30% (v/v) methanol in Suitability requirements
water. Apply an appropriate volume (4.9 mL) of 30% Relative standard deviation: NMT 3.0%
(v/v) methanol in 0.1 N hydrochloric acid to the column. Analysis
Collect the eluate in a 5-mL volumetric flask, containing Samples: Standard solution and Sample solution
100 µL of 40% (w/v) sodium acetate in water, and dilute Measure the peak areas of cyanocobalamin.
with 30% (v/v) methanol in 0.1 N hydrochloric acid to Calculate the percentage of the labeled amount of
volume. cyanocobalamin (C63H88CoN14O14P) in the portion of
Chromatographic system Tablets taken:
(See Chromatography á621ñ, System Suitability.)
al
Mode: LC Result = (rU/rS) × (CS/CU) × 100
Detector: UV 200 nm
Column: 4.6-mm × 25-cm; packing L1 rU = peak area from the Sample solution
Flow rate: 2 mL/min rS = peak area from the Standard solution
Injection volume: 100 µL CS = concentration of USP Cyanocobalamin
System suitability
Samples: Standard solution and portion of the Standard
solution that has undergone Solid-phase extraction
ci CU
(Crystalline) RS in the Standard solution (µg/mL)
= nominal concentration of cyanocobalamin in the
Sample solution (µg/mL)
Suitability requirements
Tailing factor: NMT 1.5, Standard solution Acceptance criteria: 90.0%–150.0% of the labeled amount
ffi
Relative standard deviation: NMT 2.0%, Standard of cyanocobalamin (C63H88CoN14O14P)
solution and Standard solution that has undergone
Solid-phase extraction Change to read:
Recovery: 95%–100%, Standard solution that has
undergone Solid-phase extraction • CYANOCOBALAMIN, Method 2
Analysis [NOTE—Use low-actinic glassware throughout this
O
Samples: Standard solution and the Sample solution that procedure.]

have both undergone Solid-phase extraction Standard stock solution: 1.0 µg/mL of USP
Measure the peak areas of biotin. Calculate the percentage Cyanocobalamin (Crystalline) RS in 25% alcohol. Store in a
of the labeled amount of biotin (C10H16N2O3S) in the refrigerator.
portion of Tablets taken: Standard solution: Dilute a suitable volume of the Standard
stock solution with water to a measured volume such that
Result = (rU/rS) × (CS/CU) × 100 after the incubation period as described in the Analysis, the
difference in transmittance between the inoculated blank
rU = peak area of biotin from the Sample solution and the 5.0-mL level of the Standard solution is NLT that
rS = peak area of biotin from the Standard solution which corresponds to a difference of 1.25 mg in dried cell
CS = concentration of USP Biotin RS in the Standard weight. This concentration usually falls between 0.01 and
solution (µg/mL) 0.04 ng/mL of the Standard solution. Prepare this solution
CU = nominal concentration of biotin in the Sample fresh for each assay.
solution (µg/mL) Sample solution: Finely powder NLT 20 Tablets. Transfer a
portion of the powdered Tablets, equivalent to 1.0 µg of
Acceptance criteria: 90.0%–150.0% of the labeled amount cyanocobalamin, to an appropriate vessel containing, for
of biotin (C10H16N2O3S) each gram of powdered Tablets taken, 25 mL of an aqueous
• CYANOCOBALAMIN, Method 1 extracting solution prepared just before use to contain, in
[NOTE—Use low-actinic glassware throughout this each 100 mL, 1.29 g of dibasic sodium phosphate, 1.1 g of
procedure.] anhydrous citric acid, and 1.0 g of sodium metabisulfite.
Mobile phase: Methanol and water (7:13) Autoclave the mixture at 121° for 10 min. Allow any
Standard stock solution: 10 µg/mL of USP undissolved particles of the extract to settle, and filter or
Cyanocobalamin (Crystalline) RS in water. [NOTE—Store in a centrifuge if necessary. Dilute an aliquot of the clear
dark place, and discard after 1 week.] solution with water to obtain a final solution containing
Standard solution: 1 µg/mL of USP Cyanocobalamin vitamin B12 activity equivalent to the nominal activity of the
(Crystalline) RS from Standard stock solution diluted with Standard solution.
water Acid-hydrolyzed casein solution: Prepare as directed in
Biotin, Method 2.
Asparagine solution: Dissolve 2.0 g of L-asparagine in water
2 A suitable cartridge is the Waters Oasis MAX Vac RC, particle size 30 µm,
to make 200 mL. Store under toluene in a refrigerator.
part #186000371.
9
Adenine–guanine–uracil solution: Prepare as directed in pass with the aid of reduced pressure through a layer of the
Biotin, Method 2. filter aid. Repeat if necessary until a clear, straw-colored
Xanthine solution: Suspend 0.20 g of xanthine in 30– filtrate is obtained. Store under toluene in a refrigerator.
40 mL of water, heat to 70°, add 6.0 mL of 6 N ammonium Culture medium: [NOTE—A dehydrated mixture containing
hydroxide, and stir until the solid is dissolved. Cool, and the same ingredients may be used provided that, when
dilute with water to make 200 mL. Store under toluene in a constituted as directed in the labeling, it yields a medium
refrigerator. equivalent to that obtained from the formula given
Salt solution A: Dissolve 10 g of monobasic potassium herein.] Dissolve 0.75 g of yeast extract, 0.75 g of dried
phosphate and 10 g of dibasic potassium phosphate in peptone, 1.0 g of anhydrous dextrose, and 0.20 g of
water to make 200 mL, and add 2 drops of hydrochloric monobasic potassium phosphate in 60–70 mL of water.
acid. Store this solution under toluene. Add 10 mL of Tomato juice preparation and 1 mL of
Salt solution B: Dissolve 4.0 g of magnesium sulfate, 0.20 g Polysorbate 80 solution. Adjust with 1 N sodium hydroxide
of sodium chloride, 0.20 g of ferrous sulfate, and 0.20 g of to a pH of 6.8, and add water to make 100 mL. Place 10-mL
manganese sulfate in water to make 200 mL. Add 2 drops portions of the solution in test tubes, and plug with cotton.
of hydrochloric acid. Store this solution under toluene. Sterilize the tubes and contents in an autoclave at 121° for
Polysorbate 80 solution: Dissolve 20 g of polysorbate 80 in 15 min. Cool as rapidly as possible to avoid color formation
alcohol to make 200 mL. Store in a refrigerator. resulting from overheating the medium.
Vitamin solution A: Dissolve 10 mg of riboflavin, 10 mg of Suspension medium: Dilute a measured volume of the
thiamine hydrochloride, 100 µg of biotin, and 20 mg of Basal medium stock solution with an equal volume of water.
niacin in 0.02 N acetic acid to make 400 mL. Store under Place 10-mL portions of the diluted medium in test tubes.
toluene, protected from light, in a refrigerator. Sterilize, and cool as directed for Culture medium.
Vitamin solution B: Dissolve 20 mg of p-aminobenzoic Stock culture of Lactobacillus leichmannii: To 100 mL of
Culture medium add 1.0–1.5 g of agar, and heat the mixture
al
acid, 10 mg of calcium pantothenate, 40 mg of pyridoxine
hydrochloride, 40 mg of pyridoxal hydrochloride, 8 mg of on a steam bath, with stirring, until the agar dissolves. Place
pyridoxamine dihydrochloride, and 2 mg of folic acid in a 10-mL portions of the hot solution in test tubes, cover the
mixture of water and neutralized alcohol (3:1) to make tubes, sterilize at 121° for 15 min in an autoclave, and allow
400 mL. Store, protected from light, in a refrigerator. the tubes to cool in an upright position. Inoculate 3 or more
Basal medium stock solution: Prepare the medium of the tubes by stab transfer of a pure culture of Lactobacillus
according to the following formula and directions. A
dehydrated mixture containing the same ingredients may
be used provided that, when constituted as directed in the
ci leichmannii.3 [NOTE—Before first using a fresh culture in this
assay, make NLT 10 successive transfers of the culture in a
2-week period.] Incubate for 16–24 h at a temperature
labeling, it yields a medium comparable to that obtained between 30° and 40° held constant to within ±0.5°. Store
from the formula given herein. in a refrigerator.
ffi
Add the ingredients in the order listed in ▲Table Prepare fresh stab cultures at least 3 times each week, and
3,▲ (USP 1-May-2020)carefully dissolving cystine and do not use them for preparing the Inoculum if more than
tryptophan in the 1N hydrochloric acid before adding the 4 days old. The activity of the microorganism can be
next 8 solutions to the resulting solution. Add 100 mL of increased by daily or twice-daily transfer of the stab
water, and dissolve the dextrose, sodium acetate, and culture, to the point where definite turbidity in the liquid
ascorbic acid. Filter, if necessary. Add the Polysorbate 80 Inoculum can be observed 2–4 h after inoculation. A
O
solution, adjust with 1 N sodium hydroxide to a pH of 5.5– slow-growing culture seldom gives a suitable response
6.0, and dilute with Purified Water to make 250 mL. curve and may lead to erratic results.
Inoculum: [NOTE—A frozen suspension of Lactobacillus
▲
Table 3▲ (USP 1-May-2020) leichmannii may be used as the stock culture, provided it
L-Cystine 0.1 g
yields an Inoculum comparable to a fresh culture.] Transfer
cells from the Stock culture of Lactobacillus leichmannii to 2
L-Tryptophan 0.05 g sterile tubes containing 10 mL each of the Culture medium.
1 N hydrochloric acid 10 mL Incubate these cultures for 16–24 h at a temperature
between 30° and 40° held constant to within ±0.5°. Under
Adenine–guanine–uracil solution 5 mL aseptic conditions centrifuge the cultures, and decant the
Xanthine solution 5 mL supernatant. Suspend the cells from the culture in 5 mL of
sterile Suspension medium, and combine. Using sterile
Vitamin solution A 10 mL Suspension medium, adjust the volume so that a 1-in-20
Vitamin solution B 10 mL dilution in saline TS produces 70% transmittance when
read on a suitable spectrophotometer that has been set at a
Salt solution A 5 mL wavelength of 530 nm, equipped with a 10-mm cell, and
Salt solution B 5 mL read against saline TS set at 100% transmittance. Prepare a
1-in-400 dilution of the adjusted suspension using sterile
Asparagine solution 5 mL Basal medium stock solution. [NOTE—This dilution may be
Acid-hydrolyzed casein solution 25 mL altered, when necessary, to obtain the desired test
response.] The cell suspension is the Inoculum.
Dextrose, anhydrous 10 g
Calibration of spectrophotometer: Check the wavelength
Sodium acetate, anhydrous 5g of the spectrophotometer periodically using a standard
wavelength cell or other suitable device. Before reading any
Ascorbic acid 1g
tests, calibrate the spectrophotometer for 0% and 100%
Polysorbate 80 solution 5 mL transmittance, using water, and with the wavelength set at
530 nm.
Tomato juice preparation: Centrifuge commercially 3 Pure cultures of Lactobacillus leichmannii (listed as Lactobacillus delbruekii)
canned tomato juice so that most of the pulp is removed.
may be obtained as #7830 from ATCC, 10801 University Blvd., Manassas,
Suspend 5 g/L of analytical filter aid in the supernatant, and VA 20110-2209 (www.atcc.org).
10
Analysis of the volume, in mL, of the Sample solution to obtain

Samples: Standard solution and Sample solution the difference, X, for each dosage level. Average the
Because of the high sensitivity of the test organism to values of X for each of 3 or more dosage levels to obtain
minute amounts of vitamin B12 activity and to traces of X, which equals the log-relative potency, M′, of the
many cleansing agents, cleanse meticulously by suitable Sample solution.
means, followed preferably by heating at 250° for 2 h Determine the quantity, in µg, of cyanocobalamin
using hard-glass 20-mm × 150-mm test tubes and other (C63H88CoN14O14P) in the portion of Tablets taken:
necessary glassware.
To separate test tubes add, in duplicate, 1.0, 1.5, 2.0, 3.0, antilog M = antilog (M′ + log R)
4.0, and 5.0 mL of the Standard solution. To each of these
tubes and to 4 similar but empty tubes add 5.0 mL of the R = amount of cyanocobalamin assumed to be
Basal medium stock solution and sufficient water to make present in the portion of Tablets taken (µg)
10 mL.
To similar separate test tubes add, in duplicate, 1.0, 1.5, Calculate the percentage of the labeled amount of
2.0, 3.0, and 4.0 mL of the Sample solution. To each tube cyanocobalamin (C63H88CoN14O14P) in the portion of the
add 5.0 mL of the Basal medium stock solution and Tablets taken:
sufficient water to make 10 mL. Place one complete set of
Standard and sample tubes together in one tube rack and Result = [(antilog M)/N] × 100
the duplicate set in a second rack or section of a rack,
N = nominal amount of cyanocobalamin in the
preferably in random order.
portion of the Tablets taken (µg)
Cover the tubes to prevent bacterial contamination, and
sterilize in an autoclave at 121° for 5 min, arranging to Replication: Repeat the entire determination at least once,
al
reach this temperature in NMT 10 min by preheating the using separately prepared Sample solutions. If the
autoclave if necessary. Cool as rapidly as possible to avoid difference between the two log-potencies M is NMT 0.08,
color formation resulting from overheating the medium. their mean, M, is the assayed log-potency of the test
Take precautions to maintain uniformity of sterilizing and
material (see ▲▲ (USP 1-May-2020) Design and Analysis of
cooling conditions throughout the assay, because packing
Biological Assays á111ñ, The Confidence Interval and Limits
the tubes too closely in the autoclave or overloading it
may cause variation in the heating rate.
Aseptically add 0.5 mL of Inoculum to each tube so
prepared, except 2 of the 4 containing no Standard
ci of Potency). If the two determinations differ by more than
0.08, conduct one or more additional determinations.
From the mean of two or more values of M that do not
differ by more than 0.15, compute the mean potency of
solution (the uninoculated blanks). Incubate the tubes at a
the preparation under assay.
temperature between 30° and 40°, held constant to
ffi
within ±0.5°, for 16–24 h.
of cyanocobalamin (C63H88CoN14O14P)
Terminate growth by heating to a temperature NLT 80° for
5 min. Cool to room temperature. After agitating its
contents, read the transmittance at 530 nm when a steady Change to read:
state is reached. This steady state is observed a few • FOLIC ACID, Method 1
seconds after agitation when the reading remains
Proceed as directed in Folic Acid Assay á411ñ, Assay,
O
▲
constant for 30 s or more. Allow approximately the same
Procedure 1.▲ (USP 1-May-2020)
time interval for the reading on each tube.
With the transmittance set at 100% for the uninoculated
of folic acid (C19H19N7O6)
blank, read the transmittance of the inoculated blank. If
the difference is greater than 5% or if there is evidence of
contamination with a foreign microorganism, disregard Change to read:
the results of the assay. • FOLIC ACID, Method 2
With the transmittance set at 100% for the uninoculated ▲
Proceed as directed in Folic Acid Assay á411ñ, Assay,
blank, read the transmittance of each of the remaining
Procedure 2.▲ (USP 1-May-2020)
tubes. Disregard the results of the assay if the slope of the
standard curve indicates a problem with sensitivity.
of folic acid (C19H19N7O6)
Calculation: Prepare a standard concentration-response
curve by the following procedure. Test for and replace any • CALCIUM PANTOTHENATE, Method 1
aberrant individual transmittances. For each level of the Mobile phase: Phosphoric acid and water (1:1000)
Standard, calculate the response from the sum of the Internal standard solution: 80 mg of p-hydroxybenzoic
duplicate values of the transmittances (ΣS) as the acid in 3 mL of alcohol. Add 50 mL of water and 7.1 g of
dibasic sodium phosphate, and dilute with water to
difference, y = 2.00 − ΣS. Plot this response on the ordinate
1000 mL. Adjust with phosphoric acid to a pH of 6.7.
of cross-section paper against the logarithm of the Standard solution: 0.6 mg/mL of USP Calcium
milliliter of the Standard solution per tube on the abscissa, Pantothenate RS in the Internal standard solution
using for the ordinate either an arithmetic or a logarithmic Sample solution: Finely powder NLT 30 Tablets. Transfer a
scale, whichever gives the better approximation to a portion of the powder, equivalent to 15 mg of calcium
straight line. Draw the straight line or smooth curve that pantothenate, to a centrifuge tube. Add 25.0 mL of the
best fits the plotted points. Internal standard solution, and shake vigorously for 10 min.
Calculate the response, y = 2.00 − ΣU, adding together the Centrifuge, filter, and use the clear filtrate.
two transmittances (ΣU) for each level of the Sample Chromatographic system
solution. Read from the standard curve the logarithm of (See Chromatography á621ñ, System Suitability.)
the volume of the Standard solution corresponding to Mode: LC
each of those values of y that falls within the range of the Detector: UV 210 nm
lowest and highest points plotted for the Standard. Column: 3.9-mm × 15-cm; packing L1
Subtract from each logarithm so obtained the logarithm Flow rate: 1.5 mL/min
11
Injection volume: 10 µL D,L-tryptophan) in 700–800 mL of water, heat to 70°–80°,

System suitability and add dilute hydrochloric acid (1 in 2) dropwise, with
Sample: Standard solution stirring, until the solids are dissolved. Cool, and dilute with
[NOTE—The relative retention times for calcium water to make 1000 mL. Store under toluene in a cool place
pantothenate and p-hydroxybenzoic acid are about at a temperature not below 10°.
0.5 and 1.0, respectively.] Adenine–guanine–uracil solution: Dissolve 200 mg each
Suitability requirements of adenine sulfate, guanine hydrochloride, and uracil, with
Relative standard deviation: NMT 3.0% the aid of heat, in 10 mL of 4 N hydrochloric acid. Cool,
Analysis and dilute with water to make 200 mL. Store under toluene
Samples: Standard solution and Sample solution in a refrigerator.
Measure the peak areas of calcium pantothenate and the Polysorbate 80 solution: 100 mg/mL of polysorbate 80 in
internal standard. alcohol
Calculate the percentage of the labeled amount of calcium Riboflavin–thiamine hydrochloride–biotin solution: 20
pantothenate (C18H32CaN2O10) in the portion of Tablets µg/mL of riboflavin, 10 µg/mL of thiamine hydrochloride,
taken: and 0.04 µg/mL of biotin in 0.02 N acetic acid. Store under
toluene, protected from light, in a refrigerator.
Result = (RU/RS) × (CS/CU) × 100 p-Aminobenzoic acid–niacin–pyridoxine hydrochloride
solution: 10 µg/mL of p-aminobenzoic acid, 50 µg/mL of
RU = peak area ratio of calcium pantothenate to niacin, and 40 µg/mL of pyridoxine hydrochloride in a
p-hydroxybenzoic acid from the Sample solution mixture of neutralized alcohol and water (1:3). Store in a
RS = peak area ratio of calcium pantothenate to refrigerator.
p-hydroxybenzoic acid from the Standard Salt solution A: Dissolve 25 g of monobasic potassium
solution
al
phosphate and 25 g of dibasic potassium phosphate in
CS = concentration of USP Calcium Pantothenate RS in water to make 500 mL. Add 5 drops of hydrochloric acid.
the Standard solution (mg/mL) Store under toluene.
CU = nominal concentration of calcium pantothenate Salt solution B: Dissolve 10 g of magnesium sulfate, 0.5 g
in the Sample solution (mg/mL) of sodium chloride, 0.5 g of ferrous sulfate, and 0.5 g of
manganese sulfate in water to make 500 mL. Add 5 drops
of calcium pantothenate (C18H32CaN2O10)
ci of hydrochloric acid. Store under toluene.
Basal medium stock solution: Dissolve the anhydrous
dextrose and anhydrous sodium acetate in the solutions
Change to read: previously mixed according to ▲Table 4,▲ (USP 1-May-2020)and
adjust with 1 N sodium hydroxide to a pH of 6.8. Dilute
ffi
• CALCIUM PANTOTHENATE, Method 2 with water to 250 mL.
Standard stock solution: Dissolve 50 mg of USP Calcium
Pantothenate RS ▲▲ (USP 1-May-2020) in 500 mL of water in a ▲
Table 4▲ (USP 1-May-2020)
1000-mL volumetric flask. Add 10 mL of 0.2 N acetic acid
Acid-hydrolyzed casein solution 25 mL
and 100 mL of sodium acetate solution (1 in 60), and dilute
with water to volume to obtain a concentration of 50 Cystine–tryptophan solution 25 mL
O
µg/mL of USP Calcium Pantothenate RS. Store under

Polysorbate 80 solution 0.25 mL
toluene in a refrigerator.
Standard solution: On the day of the assay, dilute a volume Dextrose, anhydrous 10 g
of the Standard stock solution with water to obtain a Sodium acetate, anhydrous 5g
concentration of 0.01–0.04 µg/mL of calcium
pantothenate, the exact concentration being such that the Adenine–guanine–uracil solution 5 mL
responses obtained as directed in the Analysis, using Riboflavin–thiamine hydrochloride–biotin solution 5 mL
2.0 and 4.0 mL of the Standard solution, are within the
linear portion of the log-concentration-response curve. p-Aminobenzoic acid–niacin–pyridoxine hydrochloride solu-
Sample solution: Finely powder NLT 30 Tablets. Transfer a tion 5 mL
portion of the powder, equivalent to 50 mg of calcium Salt solution A 5 mL
pantothenate, to a 1000-mL volumetric flask containing
Salt solution B 5 mL
500 mL of water. Add 10 mL of 0.2 N acetic acid and
100 mL of sodium acetate solution (1 in 60), dilute with
water to volume, and filter. Dilute a volume of this solution Stock culture of Lactobacillus plantarum: Dissolve 2.0 g of
to obtain a solution having approximately the same yeast extract in 100 mL of water. Add 500 mg of anhydrous
concentration as that of the Standard solution. dextrose, 500 mg of anhydrous sodium acetate, and 1.5 g
Acid-hydrolyzed casein solution: Mix 100 g of vitamin-free of agar, and heat the mixture on a steam bath, with stirring,
casein with 500 mL of 6 N hydrochloric acid, and reflux the until the agar dissolves. Add 10-mL portions of the hot
mixture for 8–12 h. Remove the hydrochloric acid from the solution to the test tubes, close or cover the tubes, sterilize
mixture by distillation under reduced pressure until a thick in an autoclave at 121° for 15 min, and allow the tubes to
paste remains. Redissolve the resulting paste in water, cool in an upright position. Prepare stab cultures in 3 or
adjust the solution with 1 N sodium hydroxide to a pH of more of the tubes using a pure culture of Lactobacillus
3.5 ± 0.1, and dilute with water to make 1000 mL. Add 20 g plantarum, 1 incubating for 16–24 h at a temperature
of activated charcoal, stir for 1 h, and filter. Repeat the between 30° and 37° held constant to within ±0.5°. Store
treatment with activated charcoal. Store under toluene in a in a refrigerator. Prepare a fresh stab of the stock culture
cool place at a temperature not below 10°. Filter the every week, and do not use for the Inoculum if the culture
solution if a precipitate forms during storage. is more than 1 week old.
Cystine–tryptophan solution: Suspend 4.0 g of L-cystine Culture medium: To each of a series of test tubes containing
in a solution of 1.0 g of L-tryptophan (or 2.0 g of 5.0 mL of Basal medium stock solution add 5.0 mL of water
12
containing 0.2 µg of calcium pantothenate. Plug the tubes values of X for each of 3 or more dosage levels to obtain
with cotton, sterilize in an autoclave at 121° for 15 min, X, which equals the log-relative potency, M′, of the
and cool. Sample solution. Determine the quantity, in mg, of
Inoculum: [NOTE—A frozen suspension of Lactobacillus calcium pantothenate (C18H32CaN2O10) in the portion of
plantarum may be used as the stock culture, provided it Tablets taken:
yields an Inoculum comparable to a fresh culture.]
Transfer cells from the Stock culture of Lactobacillus antilog M = antilog (M′ + log R)
plantarum to a sterile tube containing 10 mL of the Culture
medium. Incubate this culture for 16–24 h at a R = amount of calcium pantothenate assumed to be
temperature between 30° and 37° held constant to within present in the portion of the Tablets taken (mg)
±0.5°. The cell suspension is the Inoculum.
Analysis Calculate the percentage of calcium pantothenate
Samples: Standard solution and Sample solution (C18H32CaN2O10) in the portion of the Tablets taken:
To similar separate test tubes add, in duplicate, 1.0 and/or
1.5, 2.0, 3.0, 4.0, and 5.0 mL of the Standard solution. To Result = [(antilog M)/N] × 100
each tube and to 4 similar but empty tubes, add 5.0 mL
of the Basal medium stock solution and sufficient water to N = nominal amount of calcium pantothenate in the
make 10 mL. portion of the Tablets taken (mg)
To similar separate test tubes add, in duplicate, volumes
of the Sample solution corresponding to 3 or more of the Replication: Repeat the entire determination at least once,
levels specified for the Standard solution, including the using separately prepared Sample solutions. If the
levels of 2.0, 3.0, and 4.0 mL. To each tube add 5.0 mL difference between the two log-potencies M is NMT 0.08,
of the Basal medium stock solution and sufficient water to their mean, M, is the assayed log-potency of the test
al
make 10 mL. Place one complete set of Standard and material (see Design and Analysis of Biological Assays á111ñ,
sample tubes together in one tube rack and the duplicate The Confidence Interval and Limits of Potency). If the two
set in a second rack or section of a rack, preferably in determinations differ by more than 0.08, conduct one or
random order. more additional determinations. From the mean of two or
Cover the tubes of both series to prevent contamination,
ci more values of M that do not differ by more than 0.15,
and sterilize in an autoclave at 121° for 5 min. Cool, and compute the mean potency of the preparation under
add 1 drop of Inoculum to each tube, except 2 of the 4 assay.
tubes containing no Standard solution (the uninoculated Acceptance criteria: 90.0%–150.0% of the labeled amount
blanks). Incubate the tubes at a temperature between 30° of calcium pantothenate (C18H32CaN2O10)
and 37°, held constant to within ±0.5° until, following 16– • CALCIUM PANTOTHENATE, Method 3
ffi
24 h of incubation, until there has been no substantial Buffer solution: Dissolve 10.0 g of monobasic potassium
increase in turbidity in the tubes containing the highest phosphate in 2000 mL of water, and adjust with phosphoric
level of Standard during a 2-h period. acid to a pH of 3.5.
Determine the transmittance of the tubes in the following Mobile phase: Methanol and Buffer solution (1:9)
manner. Mix the contents of each tube, and transfer to an Standard stock solution: 0.25 mg/mL of USP Calcium
optical container if necessary. Read the transmittance Pantothenate RS in water. Prepare fresh every 4 weeks.
O
between 540 and 660 nm when a steady state is reached. Store in a refrigerator.
This steady state is observed a few seconds after agitation Standard solution: 40 µg/mL of USP Calcium
when the galvanometer reading remains constant for 30 s Pantothenate RS from the Standard stock solution diluted
or more. Allow approximately the same time interval for with water
the reading on each tube. Sample solution: Finely powder NLT 20 Tablets. Transfer a
With the transmittance set at 1.00 for the uninoculated portion of the powder, equivalent to 10 mg of calcium
blank, read the transmittance of the inoculated blank. pantothenate, to a 250-mL volumetric flask. Add 10 mL of
With the transmittance set at 1.00 for the inoculated methanol, and swirl the flask to disperse. Dilute with water
blank, read the transmittance for each of the remaining to volume, mix, and filter.
tubes. If there is evidence of contamination with a foreign Chromatographic system
microorganism, disregard the result of the assay. (See Chromatography á621ñ, System Suitability.)
Calculation: Prepare a standard concentration-response Mode: LC
curve as follows. For each level of the Standard calculate Detector: UV 205 nm
the response from the sum of the duplicate values of the Column: 3.9-mm × 30-cm; 5-µm packing L1
transmittance (ΣS) as the difference, y = 2.00 − ΣS. Plot this Column temperature: 50°
response on the ordinate of cross-section paper against Flow rate: 2 mL/min
the logarithm of the milliliter of the Standard solution per Injection volume: 25 µL
tube on the abscissa, using for the ordinate either an System suitability
arithmetic or a logarithmic scale, whichever gives the Sample: Standard solution
better approximation to a straight line. Draw the straight Suitability requirements
line or smooth curve that best fits the plotted points. Relative standard deviation: NMT 3.0%
Calculate the response, y = 2.00 − ΣU, adding together the Analysis
two transmittances (ΣU) for each level of the Sample
Measure the peak areas of calcium pantothenate.
solution. Read from the standard curve the logarithm of Calculate the percentage of the labeled amount of calcium
the volume of the Standard solution corresponding to pantothenate (C18H32CaN2O10) in the portion of Tablets
each of those values of y that falls within the range of the
taken:
lowest and highest points plotted for the Standard.
Subtract from each logarithm so obtained the logarithm Result = (rU/rS) × (CS/CU) × 100
of the volume, in mL, of the Sample solution to obtain
the difference, X, for each dosage level. Average the
13
rU = peak area of calcium pantothenate from the rU = peak area of the relevant vitamin from the
Sample solution corresponding Sample solution
rS = peak area of calcium pantothenate from the rS = peak area of the relevant vitamin from the
Standard solution corresponding Standard solution
CS = concentration of USP Calcium Pantothenate RS in CS = concentration of the relevant vitamin Reference
the Standard solution (mg/mL) Standard in the corresponding Standard solution
CU = nominal concentration of calcium pantothenate (µg/mL)
in the Sample solution (mg/mL) CU = nominal concentration of the relevant vitamin in
the corresponding Sample solution (µg/
Acceptance criteria: 90.0%–150.0% of the labeled amount mL)▲ (USP 1-May-2020)
of calcium pantothenate (C18H32CaN2O10)
Change to read: of ▲each individual vitamin▲ (USP 1-May-2020)
• NIACIN OR NIACINAMIDE, PYRIDOXINE HYDROCHLORIDE, Change to read:
RIBOFLAVIN, and THIAMINE, Method 1
[NOTE—Use low-actinic glassware throughout this • NIACIN, Method 2
procedure.] ▲
Proceed as directed in Niacin or Niacinamide Assay á441ñ,
Diluent: Acetonitrile, glacial acetic acid, and water Assay, Chromatographic Methods, Procedure
(5:1:94) 2.▲ (USP 1-May-2020)
Mobile phase: A mixture of methanol, glacial acetic acid, Acceptance criteria: 90.0%–150.0% of the labeled amount
and water (27:1:73) containing 140 mg of sodium of niacin (C6H5NO2)
1-hexanesulfonate per 100 mL
al
Standard solution: [NOTE—Use USP Niacin RS in place of Change to read:
USP Niacinamide RS for formulations containing niacin.]
Transfer 80 mg of USP Niacinamide RS, 20 mg of USP • NIACINAMIDE, Method 2
Pyridoxine Hydrochloride RS, 20 mg of USP Riboflavin RS, ▲
Proceed as directed in Niacin or Niacinamide Assay á441ñ,
and 20 mg of USP Thiamine Hydrochloride RS, to a 200-mL Assay, Chromatographic Methods, Procedure
volumetric flask, and add 180 mL of Diluent. Immerse the
flask in a hot water bath maintained at 65°–70° for 10 min
with regular shaking or using a vortex mixer, until all the
ci 2.▲ (USP 1-May-2020)
of niacinamide (C6H6N2O)
solid materials are dissolved. Chill rapidly in a cold water • PYRIDOXINE HYDROCHLORIDE, Method 2
bath for 10 min to room temperature, and dilute with [NOTE—Use low-actinic glassware throughout this
ffi
Diluent to volume. procedure.]
Sample solution: Finely powder NLT 30 Tablets. Transfer a Extraction solvent, Mobile phase, and Sample solution:
portion of the powder, equivalent to 10 mg of niacinamide Prepare as directed for Niacin, Method 2.
and 2.5 mg each of pyridoxine hydrochloride, riboflavin, Standard stock solution: 0.1 mg/mL of USP Pyridoxine
and thiamine hydrochloride, to a 50-mL centrifuge tube. Hydrochloride RS in the Extraction solvent
Add 25.0 mL of Diluent, and mix using a vortex mixer for Standard solution: Transfer 5.0 mL of Standard stock
O
30 s to completely suspend the powder. Immerse the solution to a 25-mL volumetric flask, and dilute with the
centrifuge tube in a hot water bath maintained at 65°–70°, Extraction solvent to volume.
heat for 5 min, and mix on a vortex mixer for 30 s. Return Chromatographic system
the tube to the hot water bath, heat for another 5 min, and (See Chromatography á621ñ, System Suitability.)
mix on a vortex mixer for 30 s. Filter a portion of the Mode: LC
solution, cool to room temperature, and use the clear Detector: UV 254 nm
filtrate. [NOTE—Use the filtrate within 3 h of filtration.] Column: 4.6-mm × 25-cm; packing L1
Chromatographic system Flow rate: 1 mL/min
(See Chromatography á621ñ, System Suitability.) Injection volume: 20 µL
Mode: LC System suitability
Detector: UV 280 nm Sample: Standard solution
Column: 3.9-mm × 30-cm; packing L1 Suitability requirements
Flow rate: 1 mL/min Relative standard deviation: NMT 3.0%
Injection volume: 10 µL Analysis
System suitability Samples: Standard solution and Sample solution
Sample: Standard solution▲▲ (USP 1-May-2020) Measure the peak areas of pyridoxine. Calculate the
Suitability requirements percentage of the labeled amount of pyridoxine
Relative standard deviation: NMT 3.0% hydrochloride (C8H11NO3 · HCl) in the portion of Tablets
Analysis taken:
▲
Calculate the percentage of the labeled amount of Result = (rU/rS) × (CS/CU) × 100
vitamin B1 as thiamine ion (C12H17N4OS+), vitamin B2 as
riboflavin (C17H20N4O6), vitamin B3 as niacin (C6H5NO2) or rU = peak area of pyridoxine from the Sample solution
niacinamide (C6H6N2O), vitamin B6 as pyridoxine rS = peak area of pyridoxine from the Standard solution
(C8H11NO3) ▲▲ (ERR 1-May-2020) in the portion of Tablets CS = concentration of USP Pyridoxine
taken: Hydrochloride RS in the Standard solution
(mg/mL)
Result = (rU/rS) × (CS/CU) × 100 CU = nominal concentration of pyridoxine
hydrochloride in the Sample solution (mg/mL)
14
Acceptance criteria: 90.0%–150.0% of the labeled amount or niacinamide (C6H6N2O), vitamin B6 as pyridoxine
of pyridoxine hydrochloride (C8H11NO3 · HCl) (C8H11NO3) ▲▲ (ERR 1-May-2020) in the portion of Tablets
taken:
Change to read:
• RIBOFLAVIN, Method 2
▲
Proceed as directed in Riboflavin Assay á481ñ, Assay, rU = peak area of the relevant vitamin from the
Chromatographic Methods, Procedure 2.▲ (USP 1-May-2020) corresponding Sample solution
Acceptance criteria: 90.0%–150.0% of the labeled amount rS = peak area of the relevant vitamin from the
of riboflavin (C17H20N4O6) corresponding Standard solution
CS = concentration of the relevant vitamin Reference
Change to read: Standard in the corresponding Standard solution
(µg/mL)
• THIAMINE, Method 2 CU = nominal concentration of the relevant vitamin in
▲
Proceed as directed in Thiamine Assay á531ñ, Assay, the corresponding Sample solution (µg/
Chromatographic Methods, Procedure 2.▲ (USP 1-May-2020) mL)▲ (USP 1-May-2020)
of thiamine ▲as thiamine ion (C12H17N4OS+)▲ (USP 1-May-2020) Acceptance criteria: 90.0%–150.0% of the labeled amount
of ▲each individual vitamin▲ (USP 1-May-2020)
Change to read:
Add the following:
• NIACIN OR NIACINAMIDE, PYRIDOXINE HYDROCHLORIDE,
RIBOFLAVIN, and THIAMINE, Method 3 ▲
• FOLIC ACID Method 3; ASCORBIC ACID, NIACIN OR
al
[NOTE—Use low-actinic glassware throughout this NIACINAMIDE, PYRIDOXINE HYDROCHLORIDE, CALCIUM
procedure.] PANTOTHENATE, RIBOFLAVIN, and THIAMINE, Method 4
Reagent: 25 mg/mL of edetate disodium in water [NOTE—Use low-actinic glassware.]
Mobile phase: Transfer 0.4 mL of triethylamine, 15.0 mL of Solution A: 0.56 g of edetate disodium and 2.04 g of
glacial acetic acid, and 350 mL of methanol to a 2000-mL potassium phosphate monobasic per 1000 mL of water.
volumetric flask. Dilute with 0.008 M sodium
1-hexanesulfonate to volume.
Standard stock solution: 1.5 mg/mL of USP Niacin RS or
ci Adjust with phosphoric acid to a pH of 3.0.
Solution B: Acetonitrile, acetic acid, glacial, and water
(5:1:94)
USP Niacinamide RS, 0.24 mg/mL of USP Pyridoxine Solution C: Aqueous solution containing 2 mL of
Hydrochloride RS, 0.08 mg/mL of USP Riboflavin RS, and ammonium hydroxide, 1 g of sodium perchlorate per
ffi
0.24 mg/mL of USP Thiamine Hydrochloride RS in the 100 mL
Reagent, with heating if necessary Solution D: Aqueous solution containing 2 mL of
Standard solution: Transfer 5.0 mL of the Standard stock ammonium hydroxide, 1 g of sodium perchlorate and 2 g
solution to a stoppered 125-mL flask. Add 10.0 mL of a of sodium ascorbate per 100 mL
mixture of methanol and glacial acetic acid (9:1) and Mobile phase A: 0.02% trifluoroacetic acid in water
30.0 mL of a mixture of methanol and ethylene glycol (1:1). Mobile phase B: Acetonitrile
O
Insert the stopper, shake for 15 min in a water bath Mobile phase: See Table 5.
maintained at 60°, and cool. Filter, discarding the first few
milliliters of the filtrate. Table 5
Sample solution: Weigh and finely powder NLT 20 Tablets. Time Mobile Phase A Mobile Phase B
Transfer a portion of the powder, equivalent to 7.5 mg of (min) (%) (%)
niacin or niacinamide, 1.2 mg of pyridoxine hydrochloride,
0 100 0
0.4 mg of riboflavin, and 1.2 mg of thiamine hydrochloride
to a stoppered 125-mL flask. Add 10.0 mL of a mixture of 1.0 100 0
methanol and glacial acetic acid (9:1), and 30.0 mL of a 6.0 95 5
mixture of methanol and ethylene glycol (1:1). Insert the
stopper, shake for 15 min in a water bath maintained at 60°, 15.0 83 17
and cool. Filter, discarding the first few milliliters of the 17.0 80 20
filtrate.
Chromatographic system 19.0 80 20
(See Chromatography á621ñ, System Suitability.) 19.1 100 0
Mode: LC
Detector: UV 270 nm 25.0 100 0
Column: 4.6-mm × 25-cm; packing L7
Column temperature: 50° Ascorbic acid standard stock solution: 1.0 mg/mL of
Flow rate: 2.0 mL/min ascorbic acid from USP Ascorbic Acid RS in Solution A
Injection volume: 5 µL Thiamine standard stock solution: 0.2 mg/mL of
System suitability thiamine from USP Thiamine Hydrochloride RS in Solution
Sample: Standard solution B. The solution can be placed on a steam bath for about 5–
Suitability requirements 10 min with frequent swirling to assist in dissolution.
Relative standard deviation: NMT 2.0% Riboflavin standard stock solution: 0.2 mg/mL of USP
Analysis Riboflavin RS in Solution B. Heat the solution to 65°–70° for
Samples: Standard solution and Sample solution 10 min to assist in dissolution.
▲
Calculate the percentage of the labeled amount of Niacin or niacinamide standard stock solution:
vitamin B1 as thiamine ion (C12H17N4OS+), vitamin B2 as 1.0 mg/mL of USP Niacin RS or USP Niacinamide RS in
riboflavin (C17H20N4O6), vitamin B3 as niacin (C6H5NO2) Solution A
15
Pyridoxine standard stock solution: 0.2 mg/mL of corresponding vitamins, dilute with Solution C to volume,
pyridoxine from USP Pyridoxine Hydrochloride RS in and mix well.
Solution C Chromatographic system
Folic acid standard stock solution: 0.2 mg/mL of folic (See Chromatography á621ñ, System Suitability.)
acid from USP Folic Acid RS in Solution C Mode: LC
Calcium pantothenate standard stock solution: Detectors
1.0 mg/mL of calcium pantothenate from USP Calcium For calcium pantothenate: UV 210 nm
Pantothenate RS in Mobile phase A For thiamine, ascorbic acid, niacin or niacinamide, and
System suitability solution: Transfer 0.5 mL of Ascorbic Acid riboflavin: UV 254 nm
standard stock solution, 2.5 mL of Thiamine standard stock For pyridoxine hydrochloride and folic acid: UV
solution, and 0.5 mL of Niacin or niacinamide standard stock 280 nm
solution into 10-mL volumetric flask and dilute to volume Column: 4.6-mm × 15-cm; 5-µm packing L1
with Mobile phase A. Column temperature: 25°
Sample solution 1: Weigh and finely powder NLT 20 Flow rate: 1.0 mL/min
Tablets. Transfer a calculated amount of accurately Injection volume: 20 µL
weighed powder into a 100-mL volumetric flask to obtain a System suitability
solution containing known nominal concentrations of Samples: System suitability solution, Standard solution 1,
thiamine, riboflavin, and calcium pantothenate in the Standard solution 2 and Standard solution 3
ranges of 5–30 µg/mL for thiamine, 10–60 µg/mL for Suitability requirements
riboflavin, and 25–150 µg/mL for calcium pantothenate. Resolution: NLT 3.5 between the thiamine and ascorbic
Add Solution B to about half the volume and mix on a vortex acid peaks and NLT 4.0 between the ascorbic acid and
mixer for 5 min. Heat the flask at 65°–70° for 5 min, swirling niacin or niacinamide peaks, System suitability solution
frequently and sonicate additionally for 5 min. Cool to
al
Relative standard deviation: NMT 2.0% for each
room temperature, dilute with Mobile phase A to volume, individual peak, Standard solution 1, Standard solution
and mix well. Pass a portion of the solution through a 2, and Standard solution 3
suitable filter of 0.45-µm pore size and discard the first Analysis
milliliter of the filtrate. Samples: Sample solutions 1–3 and Standard solutions 1–3
Sample solution 2: Transfer a portion of finely powdered Calculate the percentage of the labeled amount of vitamin
Tablets into a 100-mL volumetric flask to obtain a solution
containing known nominal concentrations of ascorbic acid
and niacin or niacinamide in the ranges of 50–300 µg/mL
ci B1 as thiamine ion (C12H17N4OS+), vitamin B2 as riboflavin
(C17H20N4O6), vitamin B3 as niacin (C6H5NO2) or
niacinamide (C6H6N2O), vitamin B6 as pyridoxine
for ascorbic acid and 12–65 µg/mL for niacin or (C8H11NO3), calcium pantothenate (C18H32CaN2O10),
niacinamide, add Solution A to about half the volume and ▲
ascorbic acid (C6H8O6),▲ (ERR 1-May-2020) and folic acid
ffi
mix on a vortex mixer for 5 min, sonicate for 10 min with
occasional shaking. Cool to room temperature, dilute with (C19H19N7O6), in the portion of Tablets taken:
Solution A to volume, mix well. Pass a portion of the solution
through a suitable filter of 0.45-µm pore size and discard
the first milliliter of the filtrate. rU = peak area of the relevant vitamin from the
Sample solution 3: Transfer a portion of finely powdered corresponding Sample solution
O
Tablets into a 100-mL volumetric flask to obtain a solution rS = peak area of the relevant vitamin from the
containing known nominal concentrations of pyridoxine corresponding Standard solution
hydrochloride and folic acid in the ranges of 5–30 µg/mL CS = concentration of the relevant vitamin Reference
for pyridoxine hydrochloride and 2–12 µg/mL for folic Standard in the corresponding Standard solution
acid, add Solution D to about half the volume, heat on a (µg/mL)
water bath at 40° for 5 min and sonicate for 10 min with CU = nominal concentration of the relevant vitamin in
occasional shaking. Cool to room temperature, dilute with the corresponding Sample solution (µg/mL)
Solution D to volume, and mix well. Pass a portion of the
solution through a suitable filter of 0.45-µm pore size and Acceptance criteria: 90.0%–150.0% of the labeled amount
discard the first milliliter of the filtrate. of each individual vitamin▲ (USP 1-May-2020)
Standard solution 1: Accurately transfer calculated volumes [NOTE—In the following assays commercially available
of Thiamine standard stock solution, Riboflavin standard stock atomic absorption standard solutions for the minerals,
solution, and Calcium pantothenate standard stock solution where applicable, may be used where preparation of a
into a suitable volumetric flask to obtain a solution with final Standard stock solution is described. Use deionized water
vitamin concentrations similar to the nominal where water is specified. Where atomic absorption
concentrations in the Sample solution 1 for the spectrophotometry is specified in the assay, the Standard
corresponding vitamins, dilute with Mobile phase A to solutions and the Sample solution may be diluted
volume, mix well. quantitatively with the solvent specified, if necessary, to
Standard solution 2: Accurately transfer calculated volumes yield solutions of suitable concentrations adaptable to
of Ascorbic acid standard stock solution and Niacin or the linear or working range of the instrument.]
niacinamide standard stock solution, into a suitable • CALCIUM, Method 1
volumetric flask to obtain a solution with final vitamin Lanthanum chloride solution: 267 mg/mL of lanthanum
concentrations similar to the nominal concentrations in the chloride heptahydrate in 0.125 N hydrochloric acid
Sample solution 2 for the corresponding vitamins, dilute Calcium standard solution: 400 µg/mL of calcium. Dissolve
with Mobile phase A to volume, mix well. 1.001 g of calcium carbonate, previously dried at 300° for
Standard solution 3: Accurately transfer calculated volumes 3 h and cooled in a desiccator for 2 h, in 25 mL of 1 N
of Pyridoxine standard stock solution and Folic acid standard hydrochloric acid. Boil to expel carbon dioxide, and dilute
stock solution into a suitable volumetric flask to obtain a with water to 1000 mL.
solution with final vitamin concentrations similar to the
nominal concentrations in Sample solution 3 for the
16
Standard stock solution: 100 µg/mL of calcium from the Sample solution: Prepare as directed for Calcium, Method
Calcium standard solution diluted with 0.125 N 1, except obtain a concentration of 1 µg/mL of chromium
hydrochloric acid and omit the use of the Lanthanum chloride solution.
Standard solutions: Into separate 100-mL volumetric flasks Instrumental conditions
pipet 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Standard stock (See Atomic Absorption Spectroscopy á852ñ.)
solution. To each flask add 1.0 mL of the Lanthanum chloride Mode: Atomic absorption spectrophotometry
solution, and dilute with water to volume to obtain Analytical wavelength: Chromium emission line at
concentrations of 1.0, 1.5, 2.0, 2.5, and 3.0 µg/mL of 357.9 nm
calcium. Lamp: Chromium hollow-cathode
Sample solution: Finely powder NLT 20 Tablets. Transfer a Flame: Air–acetylene
portion of the powder, equivalent to 5 Tablets, to a Blank: 0.125 N hydrochloric acid
porcelain crucible. Heat the crucible in a muffle furnace Analysis
maintained at 550° for 6–12 h, and cool. Add 60 mL of Samples: Standard solutions and Sample solution
hydrochloric acid, and boil gently on a hot plate or steam Determine the absorbances of the solutions against the
bath for 30 min, intermittently rinsing the inner surface of Blank. Plot the absorbances of the Standard solutions
the crucible with 6 N hydrochloric acid. Cool, and versus the concentration, in µg/mL, of chromium, and
quantitatively transfer the contents of the crucible to a draw the straight line best fitting the 4 plotted points.
100-mL volumetric flask. Rinse the crucible with small From the graph, determine the concentration (C), in
portions of 6 N hydrochloric acid, and add the rinsings to µg/mL, of chromium in the Sample solution.
the flask. Dilute with water to volume, and filter, discarding Calculate the percentage of the labeled amount of
the first 5 mL of the filtrate. Dilute this solution chromium (Cr) in the portion of Tablets taken:
quantitatively, with 0.125 N hydrochloric acid, to obtain a
concentration of 2 µg/mL of calcium, adding 1 mL of the Result = (C/CU) × 100
al
Lanthanum chloride solution per 100 mL of the final volume.
Instrumental conditions C = measured concentration of chromium in the
(See Atomic Absorption Spectroscopy á852ñ.) Sample solution (µg/mL)
Mode: Atomic absorption spectroscopy CU = nominal concentration of chromium in the
Analytical wavelength: Calcium emission line at Sample solution (µg/mL)
422.7 nm
Lamp: Calcium hollow-cathode
Flame: Nitrous oxide–acetylene
ci Acceptance criteria: 90.0%–160.0% of the labeled amount
of chromium (Cr)
Blank: 0.125 N hydrochloric acid containing 1 mL of • COPPER, Method 1
Lanthanum chloride solution per 100 mL Copper standard solution: Dissolve 1.00 g of copper foil
ffi
Analysis in a minimum volume of a 50% solution of nitric acid, and
Samples: Standard solutions and Sample solution dilute with a 1% solution of nitric acid to 1000 mL. This
Determine the absorbances of the solutions against the solution contains 1000 µg/mL of copper.
Blank. Plot the absorbances of the Standard solutions Standard stock solution: 100 µg/mL of copper from the
versus the concentration, in µg/mL, of calcium, and draw Copper standard solution diluted with 0.125 N
the straight line best fitting the 5 plotted points. From the hydrochloric acid
O
graph, determine the concentration (C), in µg/mL, of Standard solutions: To separate 200-mL volumetric flasks
calcium in the Sample solution. transfer 1.0, 2.0, 4.0, 6.0, and 8.0 mL of the Standard stock
Calculate the percentage of the labeled amount of calcium solution. Dilute with water to volume to obtain
(Ca) in the portion of Tablets taken: concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0 µg/mL of
copper.
Result = (C/CU) × 100 Sample solution: Prepare as directed for Calcium, Method
1, except obtain a concentration of 2 µg/mL of copper and
C = measured concentration of calcium in the Sample omit the use of the Lanthanum chloride solution.
solution (µg/mL) Instrumental conditions
CU = nominal concentration of calcium in the Sample (See Atomic Absorption Spectroscopy á852ñ.)
solution (µg/mL) Mode: Atomic absorption spectrophotometry
Analytical wavelength: Copper emission line at 324.7 nm
Acceptance criteria: 90.0%–125.0% of the labeled amount Lamp: Copper hollow-cathode
of calcium (Ca) Flame: Air–acetylene
• CHROMIUM, Method 1 Blank: 0.125 N hydrochloric acid
Chromium standard solution: 1000 µg/mL of chromium Analysis
from potassium dichromate, previously dried at 120° for 4 h Samples: Standard solutions and Sample solution
in water. Store in a polyethylene bottle. Determine the absorbances of the solutions against the
Standard stock solution: 10 µg/mL of chromium from the Blank. Plot the absorbances of the Standard solutions
Chromium standard solution diluted with 6 N hydrochloric versus the concentration, in µg/mL, of copper, and draw
acid and water (1 in 20) the straight line best fitting the 5 plotted points. From the
Standard solutions: Transfer 10.0 and 20.0 mL of the graph, determine the concentration (C), in µg/mL, of
Standard stock solution to separate 100-mL volumetric copper in the Sample solution.
flasks, and transfer 15.0 and 20.0 mL of the Standard stock Calculate the percentage of the labeled amount of copper
solution to separate 50-mL volumetric flasks. Dilute the (Cu) in the portion of Tablets taken:
contents of each of the 4 flasks with 0.125 N hydrochloric
acid to volume to obtain concentrations of 1.0, 2.0, 3.0, Result = (C/CU) × 100
and 4.0 µg/mL of chromium.
C = concentration of copper in the Sample solution
from the graph (µg/mL)
17
CU = nominal concentration of copper in the Sample pH 10.0 buffer: Add 214 mL of 0.1 N sodium hydroxide to
solution (µg/mL) 1000 mL of 0.05 M sodium bicarbonate.
Mobile phase: Alcohol, 0.1 N sulfuric acid, and water
Acceptance criteria: 90.0%–125.0% of the labeled amount (20:5:175)
of copper (Cu) Standard stock solution: 220 µg/mL of USP Sodium
• FLUORIDE, Method 1 Fluoride RS in water. This solution contains 100 µg/mL of
[NOTE—Store all solutions in plastic containers.] fluoride.
3 M sodium acetate solution: Dissolve 408 g of sodium Standard solution: [NOTE—Condition the solid-phase
acetate in 600 mL of water contained in a 1000-mL extraction column specified for use in the Standard solution
volumetric flask. Allow the solution to equilibrate to room and the Sample solution in the following manner. Using a
temperature, and dilute with water to volume. Adjust with a vacuum at a pressure not exceeding 5 mm of mercury,
few drops of acetic acid to a pH of 7.0. wash the column with 1 column volume of methanol
Sodium citrate solution: Dissolve 222 g of sodium citrate followed by 1 column volume of pH 10.0 buffer. Do not
in 250 mL of water in a 1000-mL volumetric flask. Add allow the column top to dry. If the top of the column
28 mL of perchloric acid, and dilute with water to volume. becomes dry, recondition the column.] Transfer 10.0 mL of
Fluoride standard stock solution: 500 µg/mL of fluoride the Standard stock solution to a 100-mL volumetric flask.
from a quantity of sodium fluoride, previously dried at 100° Add 75 mL of water, and adjust with 0.1 N sodium
for 4 h and cooled in a desiccator in water hydroxide to a pH of 10.4 ± 0.1. Dilute with water to
Intermediate stock solution A: 100 µg/mL of fluoride from volume. Filter, discarding the first 15 mL of the filtrate.
the Fluoride standard stock solution diluted with water Transfer 25.0 mL of the filtrate to a 50-mL volumetric flask,
Intermediate stock solution B: 10 µg/mL of fluoride from add 15.0 mL of water, and adjust with 0.1 N sodium
the Fluoride standard stock solution diluted with water hydroxide to a pH of 10.0. Dilute with pH 10.0 buffer to
Standard solutions: To 5 separate 100-mL volumetric flasks volume. Elute a portion of this solution through a 3-mL
al
transfer 3.0, 5.0, and 10.0 mL of Intermediate stock solution solid-phase extraction column containing L1 packing that
B and 5.0 and 10.0 mL of Intermediate stock solution A. To is connected through an adapter to a second solid-phase
each flask add 10.0 mL of 1 N hydrochloric acid, 25 mL of extraction column containing sulfonylpropyl strong
3 M sodium acetate solution, and 25.0 mL of Sodium citrate cation-exchange packing. Discard the first 3 mL of the
solution. Dilute the contents of each flask with water to eluate, and collect the rest of the eluate in a suitable flask
volume to obtain concentrations of 0.3, 0.5, 1.0, 5.0, and
10.0 µg/mL of fluoride.
Sample solution: Transfer a quantity of the finely powdered
ci for injection into the chromatograph.
Sample solution: Finely powder NLT 20 Tablets. Transfer a
portion of powdered Tablets, equivalent to 1 mg of
Tablets, equivalent to 200 µg of fluoride, to a 100-mL fluorine, in 15 mL of water, and shake vigorously. Rinse the
volumetric flask. Add 10.0 mL of 1 N hydrochloric acid, sides of the flask with 15 mL of water, and allow to stand
ffi
25.0 mL of 3 M sodium acetate solution, and 25.0 mL of for 10 min. Dilute with water to 85 mL, adjust with 1 N
Sodium citrate solution, and dilute with water to volume. sodium hydroxide to a pH of 10.4 ± 0.1, and dilute with
Analysis water to 100 mL. Prepare as directed for the Standard
Samples: Standard solutions and Sample solution solution, beginning with “Filter, discarding the first 15 mL
To separate plastic beakers, each containing a of the filtrate”.
plastic-coated stirring bar, transfer 50.0 mL each of the Chromatographic system
O
Standard solutions and the Sample solution. Measure the (See Chromatography á621ñ, System Suitability.)
potentials (see pH á791ñ), in mV, of the Standard solutions Mode: LC
and the Sample solution, with a pH meter capable of a Detector: Conductivity
minimum reproducibility of ±0.2 mV and equipped with a Columns
fluoride-specific ion-indicating electrode and a calomel Guard: 4.6-mm × 3-cm; packing L17
reference electrode. [NOTE—When taking measurements, Analytical: 7.8-mm × 30-cm; packing L17
immerse the electrodes in the solution, stir on a magnetic Flow rate: 0.5 mL/min
stirrer having an insulated top until equilibrium is attained Injection volume: 100 µL
(1–2 min), and record the potential. Rinse and dry the System suitability
electrodes between measurements, taking care to avoid Sample: Standard solution
damaging the crystal of the specific-ion electrode.] Suitability requirements
Plot the logarithms of fluoride concentrations, in µg/mL, Relative standard deviation: NMT 2.0%
of the Standard solutions versus potential, in mV. From the Analysis
standard response curve and the measured potential of Samples: Standard solution and Sample solution
the Sample solution, determine the concentration (C), in Measure the peak areas of fluoride. Calculate the
µg/mL, of fluoride in the Sample solution. percentage of the labeled amount of fluorine (F) in the
Calculate the percentage of the labeled amount of fluorine portion of Tablets taken:
(F) in the portion of Tablets taken:
Result = (C/CU) × 100
rU = peak area from the Sample solution
C = measured concentration of fluoride in the Sample rS = peak area from the Standard solution
solution (µg/mL) CS = concentration of fluoride in the Standard solution
CU = nominal concentration of fluorine in the Sample (µg/mL)
solution (µg/mL) CU = nominal concentration of fluorine in the Sample
solution (µg/mL)
of fluorine (F) Acceptance criteria: 90.0%–160.0% of the labeled amount
• FLUORIDE, Method 2 of fluorine (F)
[NOTE—Use plastic containers and deionized water
throughout this procedure.]
18
• IODIDE Determine the absorbances of the solutions against the

Bromine water: To 20 mL of bromine in a glass-stoppered Blank. Plot the absorbances of the Standard solutions
bottle add 100 mL of water. Insert the stopper into the versus the concentration, in µg/mL, of iron, and draw the
bottle, and shake. Allow to stand for 30 min, and use the straight line best fitting the 5 plotted points. From the
supernatant. graph, determine the concentration (C), in µg/mL, of
Analysis: Transfer an amount of finely powdered Tablets, iron in the Sample solution.
equivalent to 3 mg of iodide, to a nickel crucible. Add 5 g Calculate the percentage of the labeled amount of iron (Fe)
of sodium carbonate, 5 mL of 50% (w/v) sodium hydroxide in the portion of Tablets taken:
solution, and 10 mL of alcohol, taking care that the entire
specimen is moistened. Heat the crucible on a steam bath Result = (C/CU) × 100
to evaporate the alcohol, then dry the crucible at 100° for
30 min to prevent spattering upon subsequent heating. C = measured concentration of iron in the Sample
Transfer the crucible with its contents to a furnace heated solution (µg/mL)
to 500°, and heat the crucible for 15 min. [NOTE—Heating CU = nominal concentration of iron in the Sample
at 500° is necessary to carbonize any organic matter solution (µg/mL)
present; a higher temperature may be used, if necessary, to
ensure complete carbonization of all organic matter.] Acceptance criteria: 90.0%–125.0% of the labeled amount
Cool the crucible, add 25 mL of water, cover the crucible of iron (Fe)
with a watchglass, and boil gently for 10 min. Filter the • MAGNESIUM, Method 1
solution, and wash the crucible with boiling water, Lanthanum chloride solution: Prepare as directed in
collecting the filtrate and washings in a beaker. Add Calcium, Method 1.
phosphoric acid until the solution is neutral to methyl Magnesium standard solution: Transfer 1.0 g of
orange, then add 1 mL excess of phosphoric acid. Add magnesium ribbon to a 1000-mL volumetric flask, dissolve
al
excess of Bromine water, and boil the solution gently until in 50 mL of 6 N hydrochloric acid, dilute with water to
colorless and then for 5 min longer. Add a few crystals of volume, and mix to obtain a solution with a known
salicylic acid, and cool the solution to 20°. Add 1 mL of concentration of 1000 µg/mL of magnesium.
phosphoric acid and 0.5 g of potassium iodide, and titrate Standard stock solution: 20 µg/mL of magnesium from the
the liberated iodine with 0.005 N sodium thiosulfate VS, Magnesium standard solution diluted with 0.125 N
adding starch TS when the liberated iodine color has
nearly disappeared.
Calculate the percentage of the labeled amount of iodine
ci hydrochloric acid
Standard solutions: To separate 100-mL volumetric flasks
transfer 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the Standard stock
(I) in the portion of Tablets taken: solution. To each flask add 1.0 mL of the Lanthanum chloride
solution, and dilute with 0.125 N hydrochloric acid to
ffi
Result = V × NA × F × Ime × (Aw/W) × (100/L) volume to obtain concentrations of 0.2, 0.3, 0.4, 0.5, and
0.6 µg/mL of magnesium.
V = volume of sodium thiosulfate consumed (mL) Sample solution: Prepare as directed for Calcium, Method
NA = actual normality of the sodium thiosulfate 1, except obtain a concentration of 0.4 µg/mL of
solution used magnesium.
F = correction factor to convert mg to µg, 1000 Instrumental conditions
O
µg/mL (See Atomic Absorption Spectroscopy á852ñ.)

Ime = milliequivalent weight of iodine, 21.16 mg/meq Mode: Atomic absorption spectrophotometry
Aw = average weight of the Tablets Analytical wavelength: Magnesium emission line at
W = weight of the portion of the Tablets taken 285.2 nm
L = labeled amount of iodine (µg/Tablet) Lamp: Magnesium hollow-cathode
Flame: Air–acetylene
Acceptance criteria: 90.0%–160.0% of the labeled amount Blank: 0.125 N hydrochloric acid containing 1 mL of
of iodine (I) Lanthanum chloride solution per 100 mL
• IRON, Method 1 Analysis
Iron standard stock solution: Transfer 100 mg of iron Samples: Standard solutions and Sample solution
powder to a 1000-mL volumetric flask. Dissolve in 25 mL of Determine the absorbances of the solutions against the
6 N hydrochloric acid, and dilute with water to volume. Blank. Plot the absorbances of the Standard solutions
Standard solutions: To separate 100-mL volumetric flasks, versus the concentration, in µg/mL, of magnesium, and
transfer 2.0, 4.0, 5.0, 6.0, and 8.0 mL of the Iron standard draw the straight line best fitting the 5 plotted points.
stock solution. Dilute the contents of each flask with water From the graph, determine the concentration (C), in
to volume to obtain concentrations of 2.0, 4.0, 5.0, 6.0, µg/mL, of magnesium in the Sample solution.
and 8.0 µg/mL of iron. Calculate the percentage of the labeled amount of
Sample solution: Prepare as directed for Calcium, Method magnesium (Mg) in the portion of Tablets taken:
1, except obtain a concentration of 5 µg/mL of iron and
omit the use of the Lanthanum chloride solution. Result = (C/CU) × 100
Instrumental conditions
(See Atomic Absorption Spectroscopy á852ñ.) C = measured concentration of magnesium in the
Mode: Atomic absorption spectrophotometry Sample solution (µg/mL)
Analytical wavelength: Iron emission line at 248.3 nm CU = nominal concentration of magnesium in the
Lamp: Iron hollow-cathode Sample solution (µg/mL)
Flame: Air–acetylene
Blank: 0.125 N hydrochloric acid Acceptance criteria: 90.0%–125.0% of the labeled amount
Analysis of magnesium (Mg)
Samples: Standard solutions and Sample solution • MANGANESE, Method 1
Manganese standard stock solution: Transfer 1.00 g of
manganese to a 1000-mL volumetric flask. Dissolve in
19
20 mL of nitric acid, dilute with 6 N hydrochloric acid to heating and swirling until the fumes appear again. Cool to
volume, and mix to obtain a solution with a concentration room temperature. Quantitatively transfer the contents of
of 1000 µg/mL of manganese. the flask to a 100-mL volumetric flask with the aid of the
Standard stock solution: 50 µg/mL of manganese from the Diluent, and dilute with Diluent to volume.
Manganese standard stock solution diluted with 0.125 N Instrumental conditions
hydrochloric acid (See Atomic Absorption Spectroscopy á852ñ.)
Standard solutions: To separate 100-mL volumetric flasks Mode: Atomic absorption spectrophotometry
transfer 1.0, 1.5, 2.0, 3.0, and 4.0 mL of the Standard stock Analytical wavelength: Molybdenum emission line at
solution. Dilute the contents of each flask with 0.125 N 313.3 nm
hydrochloric acid to volume to obtain solutions with known Lamp: Molybdenum hollow-cathode
concentrations of 0.5, 0.75, 1.0, 1.5, and 2.0 µg/mL of Flame: Nitrous oxide–acetylene
manganese. Blank: Diluent and perchloric acid (20:1)
Sample solution: Prepare as directed for Calcium, Method Analysis
1, except obtain a concentration of 1 µg/mL of manganese Samples: Standard solutions and Sample solution
and omit the use of the Lanthanum chloride solution. Determine the absorbances of the solutions against the
Instrumental conditions Blank. Plot the absorbances of the Standard solutions
(See Atomic Absorption Spectroscopy á852ñ.) versus the concentration, in µg/mL, of molybdenum, and
Mode: Atomic absorption spectrophotometry draw the straight line best fitting the 3 plotted points.
Analytical wavelength: Manganese emission line at From the graph, determine the concentration (C), in
279.5 nm µg/mL, of molybdenum in the Sample solution.
Lamp: Manganese hollow-cathode Calculate the percentage of the labeled amount of
Flame: Air–acetylene molybdenum (Mo) in the portion of Tablets taken:
al
Blank: 0.125 N hydrochloric acid
Analysis Result = (C/CU) × 100
Samples: Standard solutions and Sample solution
Determine the absorbances of the solutions against the C = measured concentration of molybdenum in the
Blank. Plot the absorbances of the Standard solutions Sample solution (µg/mL)
versus the concentration, in µg/mL, of manganese, and CU = nominal concentration of molybdenum in the
From the graph, determine the concentration (C), in

µg/mL, of manganese in the Sample solution.
ci
draw the straight line best fitting the 5 plotted points. Sample solution (µg/mL)

Calculate the percentage of the labeled amount of of molybdenum (Mo)
manganese (Mn) in the portion of Tablets taken:
ffi
Change to read:
Result = (C/CU) × 100
• MOLYBDENUM, Method 2
C = measured concentration of manganese in the Sodium fluoride solution: Add 200 mL of water to 10 g of
Sample solution (µg/mL) sodium fluoride, stir until the solution is saturated, and
CU = nominal concentration of manganese in the filter. Store in a polyethylene bottle.
O
Sample solution (µg/mL) Ferrous sulfate solution: 4.98 mg/mL of ferrous sulfate in
water
Acceptance criteria: 90.0%–125.0% of the labeled amount Potassium thiocyanate solution: 200 mg/mL of potassium
of manganese (Mn) thiocyanate in water
• MOLYBDENUM, Method 1 20% stannous chloride solution: Transfer 40 mg of
Diluent: 20 mg/mL of ammonium chloride in water stannous chloride to a beaker, add 20 mL of 6.5 N
Molybdenum standard solution: Transfer 1.0 g of hydrochloric acid solution, and heat the solution until the
molybdenum wire to a 1000-mL volumetric flask, and stannous chloride is dissolved. Cool, and dilute with water
dissolve in 50 mL of nitric acid, warming if necessary. Dilute to 100 mL.
with water to volume, and mix to obtain a solution with a Diluted stannous chloride solution: 20% stannous chloride
concentration of 1000 µg/mL of molybdenum. solution diluted with water (1 in 25). Prepare this solution
Standard stock solution: 100 µg/mL of molybdenum from fresh at the time of use.
the Molybdenum standard solution diluted with water Standard solution: 20 µg/mL of molybdenum in water
Standard solutions: To separate 100-mL volumetric flasks Sample: A portion of finely powdered Tablets equivalent to
transfer 2.0, 10.0, and 25.0 mL of the Standard stock 40 µg of molybdenum
solution, and add 5.0 mL of perchloric acid to each flask. Instrumental conditions
Gently boil the solution in each flask for 15 min, cool to (See ▲Ultraviolet-Visible Spectroscopy á857ñ.)▲ (ERR 1-May-2020)
room temperature, and dilute each with Diluent to volume Mode: Vis
to obtain concentrations of 5.0, 10.0, and 25.0 µg/mL of Analytical wavelength: 465 nm
molybdenum. Cell: 1 cm
Sample solution: Transfer a portion of the powder, Blank: Amyl alcohol
equivalent to 1000 µg of molybdenum, to a suitable flask, Analysis
and add 12 mL of nitric acid. [NOTE—The volume of nitric Samples: Standard solution and Sample
acid may be varied to ensure that the powder is uniformly Transfer the Sample and 2.0 mL of the Standard solution to
dispersed.] Carefully swirl the flask to disperse the test separate 200-mL beakers. Add 20 mL of nitric acid to each
specimen. Sonicate for 10 min or until the test specimen is beaker. Cover each beaker with a watchglass, and boil
completely dissolved. Gently boil the solution for 15 min, slowly on a hot plate for 45 min. Cool to room
and cool to room temperature. Carefully add 8 mL of temperature. Add 6 mL of perchloric acid, cover the
perchloric acid, heat until perchloric acid fumes appear, beakers with a watchglass, and continue the heating until
and swirl the flask to dissipate the fumes. Repeat the digestion is complete, as indicated when the liquid
becomes colorless or pale yellow. Evaporate the solutions
20
in the beakers to dryness. Rinse the sides of the beakers 10.0 mL of this solution to a 100-mL volumetric flask, and
and the watchglasses with water, and add more water to dilute with water to volume.
complete 50 mL in each beaker. Gently boil the water Instrumental conditions
solution for a few min. Cool to room temperature. Add 2 (See Ultraviolet-Visible Spectroscopy á857ñ.)
drops of methyl orange TS, and neutralize with Mode: Vis
ammonium hydroxide. Add 8.2 mL of hydrochloric acid. Analytical wavelength: 650 nm
Quantitatively transfer the contents of the beakers to Cell: 1 cm
separate 100-mL volumetric flasks, rinse the beakers with Analysis
water, transfer the rinsings to the corresponding Samples: Standard solution and Sample solution
volumetric flasks, and dilute with water to volume. To 3 separate 25-mL volumetric flasks transfer 5.0 mL each
Transfer 50.0 mL of each solution to separatory funnels. of the Standard solution, the Sample solution, and water to
To each separatory funnel add 1.0 mL of Sodium fluoride provide the blank. To each of the 3 flasks add 1.0 mL
solution, 0.5 mL of Ferrous sulfate solution, 4.0 mL of each of Ammonium molybdate solution, Hydroquinone
Potassium thiocyanate solution, 1.5 mL of 20% stannous solution, and Sodium bisulfite solution, and swirl to mix.
chloride solution, and 15.0 mL of amyl alcohol, and shake Dilute the contents of each flask with water to volume,
the separatory funnel for 1 min. Allow the layers to and allow the flasks to stand for 30 min. Determine the
separate, and discard the aqueous layers. Add 25 mL of absorbances of the solutions against the blank.
Diluted stannous chloride solution to each separatory Calculate the percentage of the labeled amount of
funnel, and shake gently for 15 s. Allow the layers to phosphorus (P) in the portion of Tablets taken:
separate, and discard the aqueous layers. Transfer the
organic layer from each separatory funnel to a centrifuge Result = (AU/AS) × (CS/CU) × 100
tube, and centrifuge at 2000 rpm for 10 min. Determine
the absorbances of the organic phases obtained from the AU = absorbance of the Sample solution
al
Standard solution and the Sample, and correct with the AS = absorbance of the Standard solution
Blank. CS = concentration of phosphorus in the Standard
Calculate the percentage of the labeled amount of solution (µg/mL)
molybdenum (Mo) in the portion of Tablets taken: CU = nominal concentration of phosphorus in the
AU
Result = (AU/AS) × [(V × CS)/MU] × 100
= absorbance of the Sample

ci Acceptance criteria: 90.0%–125.0% of the labeled amount
of phosphorus (P)
AS = absorbance of the Standard solution • POTASSIUM
V = volume of the Standard solution analyzed, 2.0 mL Potassium standard solution: 100 µg/mL of potassium
ffi
CS = concentration of molybdenum in the Standard from potassium chloride, previously dried at 105° for 2 h,
solution (µg/mL) in water
MU = nominal amount of molybdenum in the Sample Standard stock solution: 10 µg/mL of potassium from the
(µg) Potassium standard solution diluted with 0.125 N
hydrochloric acid
Acceptance criteria: 90.0%–160.0% of the labeled amount Standard solutions: Transfer 5.0, 10.0, 15.0, 20.0, and
O
of molybdenum (Mo) 25.0 mL of the Standard stock solution to separate 100-mL

• PHOSPHORUS, Method 1 volumetric flasks. Dilute the contents of each flask with
Sulfuric acid solution: Cautiously add sulfuric acid to water 0.125 N hydrochloric acid to volume to obtain solutions
(37.5: 100), and mix. containing 0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of potassium.
Ammonium molybdate solution: 50 mg/mL of Sample solution: Prepare as directed for Calcium, Method
ammonium molybdate in the Sulfuric acid solution and 1, except obtain a concentration of 1 µg/mL of potassium
water (2:3). [NOTE—Dissolve in water first, and then dilute and omit the use of the Lanthanum chloride solution.
with the Sulfuric acid solution to volume.] Instrumental conditions
Hydroquinone solution: 5 mg/mL of hydroquinone in (See Atomic Absorption Spectroscopy á852ñ.)
water. Add 1 drop of sulfuric acid per 100 mL of solution. Mode: Atomic absorption spectrophotometry
Sodium bisulfite solution: 200 mg/mL of sodium bisulfite Analytical wavelength: Potassium emission line at
in water 766.5 nm
Phosphorus standard stock solution: Weigh 4.395 g of Lamp: Potassium hollow-cathode
monobasic potassium phosphate, previously dried at 105° Flame: Air–acetylene
for 2 h and stored in a desiccator, and transfer to a 1000-mL Blank: Water
volumetric flask. Dissolve in water, add 6 mL of sulfuric acid Analysis
as a preservative, dilute with water to volume, and mix to Samples: Standard solutions and Sample solution
obtain a solution with a concentration of 1000 µg/mL of Determine the absorbances of the solutions against the
phosphorus. Blank. Plot the absorbances of the Standard solutions
Standard solution: 20 µg/mL of phosphorus from versus the concentration, in µg/mL, of potassium, and
Phosphorus standard stock solution diluted with water draw the straight line best fitting the 5 plotted points.
Sample solution: [NOTE—Finely powder and weigh a From the graph, determine the concentration (C), in
counted number of Tablets.] Transfer a portion of the µg/mL, of potassium in the Sample solution.
powder, equivalent to 100 mg of phosphorus, to 25 mL of Calculate the percentage of the labeled amount of
nitric acid, and digest on a hot plate for 30 min. Add 15 mL potassium (K) in the portion of Tablets taken:
of hydrochloric acid, and continue the digestion to the
cessation of brown fumes. Cool, and transfer the contents Result = (C/CU) × 100
of the flask to a 500-mL volumetric flask with the aid of small
portions of water. Dilute with water to volume. Transfer C = measured concentration of potassium in the
21
CU = nominal concentration of potassium in the 50% ammonium hydroxide solution: Ammonium

Sample solution (µg/mL) hydroxide diluted with water (1 in 2)
Reagent A: 9 mg/mL of edetate disodium and 25 mg/mL of
Acceptance criteria: 90.0%–125.0% of the labeled amount hydroxylamine hydrochloride in water. [NOTE—Dissolve
of potassium (K) edetate disodium in a portion of water first, then add
• SELENIUM, Method 1 hydroxylamine hydrochloride, and dilute with water to
Diluent: Prepare as directed in Molybdenum, Method 1. volume.]
Selenium standard solution: [CAUTION—Selenium is toxic; Reagent B: Transfer 200 mg of 2,3-diaminonaphthalene
handle it with care.] Dissolve 1 g of metallic selenium in a to a 250-mL separatory funnel, and add 200 mL of 0.1 N
minimum volume of nitric acid. Evaporate to dryness. Add hydrochloric acid. Wash the solution with 3 40-mL portions
2 mL of water, and evaporate to dryness. Repeat the of cyclohexane, and discard the cyclohexane layer. Filter
addition of water and the evaporation to dryness 3 times. the solution into a brown bottle, and cover the solution
Dissolve the residue in 3 N hydrochloric acid, transfer to a with a 1-cm layer of cyclohexane. This solution is stable for
1000-mL volumetric flask, and dilute with 3 N hydrochloric 1 week if stored in a refrigerator.
acid to volume to obtain a concentration of 1000 µg/mL of Standard stock solution: [CAUTION—Selenium is toxic;
selenium. handle it with care.] Dissolve 1 g of metallic selenium in a
Standard stock solution: 100 µg/mL of selenium from the minimum volume of nitric acid. Evaporate to dryness. Ádd
Selenium standard solution diluted with water 2 mL of water, and evaporate to dryness. Repeat the
Standard solutions: To separate 100-mL volumetric flasks addition of water and evaporation to dryness 3 times.
transfer 5.0, 10.0, and 25.0 mL of the Standard stock Dissolve the residue in 3 N hydrochloric acid, transfer to a
solution, and add 5.0 mL of perchloric acid to each flask. 1000-mL volumetric flask, and dilute with 3 N hydrochloric
Gently boil the solutions for 15 min, cool to room acid to volume to obtain a solution with a concentration of
temperature, and dilute each with Diluent to volume to 1000 µg/mL of selenium. Dilute a volume of the solution
al
obtain solutions with concentrations of 5.0, 10.0, and 25.0 with 0.125 N hydrochloric acid to obtain a concentration
µg/mL of selenium. of 2.0 µg/mL of selenium.
Sample solution: Transfer a portion of the powder, Standard solution: Transfer 10 mL of the Standard stock
equivalent to 1000 µg of selenium, to a suitable flask, and solution to a glass-stoppered flask. Add 1 mL of perchloric
add 12 mL of nitric acid. [NOTE—The volume of nitric acid acid and 1 mL of Hydrochloric acid solution, and dilute with
may be varied to ensure that the powder is uniformly
dispersed.] Carefully swirl the flask to disperse the test
specimen. Sonicate for 10 min or until the test specimen is
ci water to 20 mL.
Sample solution: Transfer a portion of finely powdered
Tablets, equivalent to 20 µg of selenium, to a suitable flask.
completely dissolved. Gently boil the solution for 15 min, Add 10 mL of nitric acid, and warm gently on a hot plate.
and cool to room temperature. Carefully add 8 mL of Continue heating until the initial nitric acid reaction has
ffi
perchloric acid to the flask, heat the flask until perchloric subsided, then add 3 mL of perchloric acid.
acid fumes appear, and swirl the flask to dissipate the [CAUTION—Exercise care at this stage because the
fumes. Repeat the heating and swirling until the fumes perchloric acid reaction becomes vigorous.]
appear again. Cool to room temperature. Transfer the Continue heating on the hot plate until the appearance of
contents of the flask to a 50-mL volumetric flask with the white fumes of perchloric acid or until the digest begins
aid of the Diluent, and dilute with Diluent to volume. to darken. Add 0.5 mL of nitric acid and resume heating,
O
Instrumental conditions adding additional amounts of nitric acid if further

(See Atomic Absorption Spectroscopy á852ñ.) darkening occurs. Digest for 10 min after the first
Mode: Atomic absorption spectrophotometry appearance of perchloric acid fumes or until the digest
Analytical wavelength: Selenium emission line at becomes colorless. Cool the flask, add 2.5 mL of
196.0 nm Hydrochloric acid solution, and return the flask to the hot
Lamp: Selenium hollow-cathode plate to expel residual nitric acid. Heat the mixture for
Flame: Air–acetylene 3 min after it begins to boil. Cool the flask to room
Blank: Diluent and perchloric acid (20:1) temperature, and dilute with water to 20 mL.
Analysis Instrumental conditions
Samples: Standard solutions and Sample solution (See Ultraviolet-Visible Spectroscopy á857ñ.)
Determine the absorbances of the solutions against the Mode: UV
Blank. Plot the absorbances of the Standard solutions Analytical wavelength: 380 nm
versus the concentration, in µg/mL, of selenium, and draw Cell: 1 cm
the straight line best fitting the 3 plotted points. From the Blank: 1 mL of perchloric acid and 1 mL of Hydrochloric acid
graph, determine the concentration (C), in µg/mL, of solution diluted with water to 20 mL
selenium in the Sample solution. Analysis
Calculate the percentage of the labeled amount of selenium Samples: Standard solution and Sample solution
(Se) in the portion of Tablets taken: Treat the Sample solution, the Standard solution, and the
Blank as follows. Add 5 mL of Reagent A to each flask, and
Result = (C/CU) × 100 swirl gently to mix. Adjust the solution in each flask with
50% ammonium hydroxide solution to a pH of 1.1 ± 0.1.
C = concentration of selenium in the Sample solution Add 5 mL of Reagent B to each flask, and swirl gently to
from the graph (µg/mL) mix. Place the flasks in a water bath maintained at 50°,
CU = nominal concentration of selenium in the Sample and equilibrate for 30 min, taking care that the flasks are
solution (µg/mL) covered to protect them from light. Cool to room
temperature, and transfer the contents of each flask to
Acceptance criteria: 90.0%–160.0% of the labeled amount separate separatory funnels. Transfer 10.0 mL of
of selenium (Se) cyclohexane to each separatory funnel, and extract
• SELENIUM, Method 2 vigorously for 1 min. Discard the aqueous layer. Transfer
Hydrochloric acid solution: Hydrochloric acid diluted with the cyclohexane layer to a centrifuge tube, and centrifuge
water (1 in 10)
22
at 1000 rpm for 1 min to remove any remaining water. PHOSPHORUS, and ZINC, Method 2; MOLYBDENUM and
Determine the absorbances of the solutions obtained SELENIUM, Method 3
from the Samples against the solution obtained from the Stock aqua regia solution: Prepare a mixture of
Blank. hydrochloric acid and nitric acid (3:1) by adding the nitric
Calculate the percentage of the labeled amount of selenium acid to the hydrochloric acid. [NOTE—Periodically vent the
(Se) in the portion of Tablets taken: solution in an appropriate fume hood.]
Diluent: Prepare a mixture of the Stock aqua regia solution
Result = (AU/AS) × [(V × CS)/MU] × 100 and water (1:9) by adding 1 volume of Stock aqua regia
solution to 2 volumes of water. Dilute with additional water
AU = absorbances of the cyclohexane layer from the to volume, and mix well.
Sample solution System suitability solution: Prepare a mixture of 1000 mg/
AS = absorbances of the cyclohexane layer from the L of yttrium in 5% nitric acid solution, 1000 mg/L of
Standard solution scandium in 5% nitric acid solution, and Diluent (1:1:198),
V = volume of the Standard stock solution used to and mix.
prepare the Standard solution, 10 mL Standard stock solution 1 (Ca, Cu, Fe, Mg, Mn, P, and Zn)
CS = concentration of selenium in the Standard stock : [NOTE—It is only necessary to include the minerals of
solution (µg/mL) interest in the solution.] Using commercially available
MU = nominal amount of selenium in the Sample element standard (single- or multi-element) solutions in 5%
solution (µg) nitric acid solution, pipet the appropriate amount of
element standard solution into a volumetric flask, and dilute
Acceptance criteria: 90.0%–160.0% of the labeled amount with 5% nitric acid solution to obtain a solution having final
of selenium (Se) concentrations of about 1000 mg/L of calcium, 100 mg/L
• ZINC, Method 1 of copper, 250 mg/L of iron, 500 mg/L of magnesium,
al
Zinc standard stock solution: 1000 µg/mL of zinc from zinc 100 mg/L of manganese, 800 mg/L of phosphorus, and
oxide in 5 M hydrochloric acid (3.89 mg/mL) and diluted 250 mg/L of zinc.
with water to final volume. [NOTE—Dissolve in 5 M Standard stock solution 2 (B, Cr, Mo, Ni, Se, Sn, and V):
hydrochloric acid by warming, if necessary, cool, and then [NOTE—It is only necessary to include the minerals of interest
dilute to final volume.] in the solution.] Using commercially available element
Standard stock solution: 50 µg/mL of zinc from the Zinc
standard stock solution diluted with 0.125 N
hydrochloric acid
ci standard (single- or multi-element) solutions in 20%
hydrochloric acid solution, pipet the appropriate amount
of element standard solution into a volumetric flask, and
Standard solutions: Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL dilute with 20% hydrochloric acid solution to obtain a
of Standard stock solution to separate 100-mL volumetric solution having final concentrations of about 200 mg/L of
ffi
flasks. Dilute the contents of each flask with 0.125 N boron, and 100 mg/L each of chromium, molybdenum,
hydrochloric acid to volume to obtain concentrations of nickel, selenium, tin, and vanadium.
0.5, 1.0, 1.5, 2.0, and 2.5 µg/mL of zinc. Standard solutions: Prepare a mixture of Standard stock
Sample solution: Prepare as directed for the Sample solution solution 1 and Standard stock solution 2, as required, in the
in Calcium, Method 1, except obtain a concentration of 2 Diluent to prepare a 6-point calibration curve to bracket the
µg/mL of zinc and omit the use of the Lanthanum chloride concentration range of each mineral of interest.
O
solution. Sample solution 1 (for Tablets containing minerals in

Instrumental conditions Standard stock solution 1 and Standard stock solution 2):
(See Atomic Absorption Spectroscopy á852ñ.) Weigh and finely powder NLT 20 Tablets. Transfer a
Mode: Atomic absorption spectrophotometry portion, equal to 3.5 times the average Tablet weight, to a
Analytical wavelength: Zinc emission line at 213.8 nm 250-mL volumetric flask. Slowly add 25 mL of the Stock
Lamp: Zinc hollow-cathode aqua regia solution in 5-mL increments, followed by mixing.
Flame: Air–acetylene [NOTE—If the sample contains a carbonate, bubbling will
Blank: 0.125 N hydrochloric acid occur. Wait until bubbling ends to proceed.] Bring the
Analysis solution to a boil on a hot plate. Continue to heat gently
Samples: Standard solutions and Sample solution until fumes cease (about 1 h). [NOTE—If the sample contains
Determine the absorbances of the solutions against the selenium, digest for NMT 15 min.] Remove from heat, cool,
Blank. Plot the absorbances of the Standard solutions and dilute with water to volume. Pass about 30 mL into a
versus the concentration, in µg/mL, of zinc, and draw the centrifuge tube using a 5-µm pore size nylon syringe filter.
straight line best fitting the 5 plotted points. From the If necessary, make any further dilutions using the Diluent.
graph, determine the concentration (C), in µg/mL, of Sample solution 2 (for Tablets containing minerals only in
zinc in the Sample solution. Standard stock solution 2): Weigh and finely powder NLT
Calculate the percentage of the labeled amount of zinc (Zn) 20 Tablets. Transfer a portion, equal to 3.5 times the
in the portion of Tablets taken: average Tablet weight, to a 250-mL volumetric flask. Slowly
add 25 mL of the Stock aqua regia solution in 5-mL
Result = (C/CU) × 100 increments followed by mixing. [NOTE—If the sample
contains a carbonate, bubbling will occur. Wait until
C = measured concentration of zinc in the Sample bubbling ends to proceed.] Bring the solution to a boil on a
solution (µg/mL) hot plate. Continue to heat gently until fumes cease (about
CU = nominal concentration of zinc in the Sample 1 h). [NOTE—If the sample contains selenium, digest for
solution (µg/mL) NMT 15 min.] Remove from heat, cool, and dilute with
water to volume. Pass about 30 mL into a centrifuge tube
Acceptance criteria: 90.0%–125.0% of the labeled amount using a nylon syringe filter of 5-µm pore size. If necessary,
of zinc (Zn) make any further dilutions using the Diluent.
• BORON, NICKEL, TIN, and VANADIUM, Method 1; CALCIUM, Sample solution 3 (for Tablets containing minerals only in
CHROMIUM, COPPER, IRON, MAGNESIUM, MANGANESE, Standard stock solution 1): Weigh and finely powder NLT
23
20 Tablets. Transfer a portion, equal to the average Tablet ADDITIONAL REQUIREMENTS

weight, to a 250-mL volumetric flask. Slowly add 25 mL • PACKAGING AND STORAGE: Preserve in tight, light-resistant
of the Stock aqua regia solution in 5-mL increments, containers.
followed by mixing. [NOTE—If the sample contains a
carbonate, bubbling will occur. Wait until bubbling ends to Change to read:
proceed.] Bring the solution to a boil on a hot plate.
Continue to heat gently (about 1 h) until fumes cease. • LABELING:4 The label states that the product is Oil- and
Remove from heat, cool, and dilute with water to volume. Water-Soluble Vitamins with Minerals Tablets. The label
Pass about 30 mL into a centrifuge tube using a nylon also states the quantity of each vitamin and mineral ▲in mg/
syringe filter of 5-µm pore size. If necessary, make any Tablet or µg/Tablet▲ (USP 1-May-2020) and where necessary the
further dilutions using the Diluent. chemical form in which a vitamin is present and also states
Instrumental conditions the salt form of the mineral used as the source of each
(See Plasma Spectrochemistry á730ñ.) element. Where the product contains vitamin E, the label
Mode: Inductively coupled plasma spectrometry, using a indicates whether it is the ▲RRR▲ (USP 1-May-2020) or
spectrometer set to measure the emission of each mineral ▲
all-rac▲ (USP 1-May-2020) form. Where more than one assay
of interest at about the corresponding wavelength. method is given for a particular vitamin, the labeling states
[NOTE—The operating conditions may be developed and with which assay method the product complies only if
optimized based on the manufacturer’s recommendation. Method 1 is not used.
The wavelengths selected should be demonstrated
experimentally to provide sufficient specificity, sensitivity, Change to read:
linearity, accuracy, and precision.]
System suitability • USP REFERENCE STANDARDS á11ñ
Sample: System suitability solution USP Alpha Tocopherol RS
al
[NOTE—Analyze the System suitability solution, and obtain 2H-1-Benzopyran-6-ol, 3,4-dihydro-2,5,7,8,-tetramethyl-
the response as directed in the Analysis.] 2-(4,8,12-trimethyltridecyl)-.
Suitability requirements C29H50O2 430.70
Relative standard deviation: NMT 2.0% USP Alpha Tocopheryl Acetate RS
Analysis USP Alpha Tocopheryl Acid Succinate RS
Samples: Standard solutions and Sample solution
Determine the emission of each mineral of interest in the
ci
Standard solutions and Sample solution with an inductively
▲
USP Ascorbic Acid RS▲ (USP 1-May-2020)
USP Biotin RS
1H-Thieno[3,4-d]imidazole-4-pentanoic acid, hexahydro-
coupled plasma system using the Diluent as the blank. Plot 2-oxo-, 3aS-[(3aα,4β,6aα)]-.
the emission of the Standard solutions versus the C10H16N2O3S 244.31
ffi
concentration, in mg/L, of the minerals of interest, and USP Calcium Pantothenate RS
draw the straight line best fitting the plotted points. From β-Alanine, N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-,
the graph, determine the concentration (C), in mg/L, for calcium salt (2:1), (R)-.
each mineral of interest in the Sample solution. C18H32CaN2O10 476.53
Calculate the percentage of the labeled amount for each USP Cholecalciferol RS
mineral taken: 9,10-Secocholesta-5,7,10(19)-trien-3-ol, (3β,5Z,7E)-.
O
C27H44O 384.64
Result = C × (V/W) × F × (CW/L) × 100 USP Cyanocobalamin (Crystalline) RS
Vitamin B12.
C = measured concentration of the relevant element
in the Sample solution (mg/L) C63H88CoN14O14P 1355.37
V = volume of the Sample solution (L) USP Ergocalciferol RS
W = sample weight (mg) 9,10-Secoergosta-5,7,10(19),22-tetraen-3-ol, (3β,5Z,7E,
F = dilution factor of the Sample solution 22E)-.
CW = average Tablet weight (mg) C28H44O 396.65
L = labeled amount (mg/Tablet) 4 ▲
▲ (USP 1-May-2020)Where articles are labeled in terms of Units in addition
Acceptance criteria: 90.0%–125.0% of the labeled amount to the required labeling, the relationship of the USP Units or IU to mass is
as follows. One USP Vitamin A Unit = 0.3 µg of all-trans-retinol (vitamin A
of calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), alcohol) or 0.344 µg of all-trans-retinyl acetate (vitamin A acetate) or 0.55
manganese (Mn), phosphorus (P), and zinc (Zn); and µg of all-trans-retinyl palmitate (vitamin A palmitate), and 1 µg of retinol
90.0%–160.0% of the labeled amount of boron (B), (3.3 USP Vitamin A Units) = 1 retinol equivalent (RE); 1 IU of beta carotene
chromium (Cr), molybdenum (Mo), nickel (Ni), selenium = 0.6 µg of all-trans-beta carotene; 1 USP Vitamin D Unit = 0.025 µg of
(Se), tin (Sn), and vanadium (V) ergocalciferol or cholecalciferol; and 1 mg of ▲all-rac▲ (USP 1-May-2020)-alpha-
tocopherol = 1.1 former USP Vitamin E Units; 1 mg of ▲all-
PERFORMANCE TESTS rac▲ (USP 1-May-2020)-alpha-tocopheryl acetate = 1 former USP Vitamin E unit;
• DISINTEGRATION AND DISSOLUTION á2040ñ, Dissolution: 1 mg of ▲all-rac▲ (USP 1-May-2020)-alpha-tocopheryl acid succinate = 0.89
Meet the requirements former USP Vitamin E Units; 1 mg of ▲RRR▲ (USP 1-May-2020)-alpha-tocopherol
= 1.49 former USP Vitamin E Units; 1 mg of ▲RRR▲ (USP 1-May-2020)-alpha-
• WEIGHT VARIATION á2091ñ: Meet the requirements tocopheryl acetate = 1.36 former USP Vitamin E Units; and 1 mg of
CONTAMINANTS
▲
RRR▲ (USP 1-May-2020)-alpha-tocopheryl acid succinate = 1.21 former USP
• MICROBIAL ENUMERATION TESTS á2021ñ: The total aerobic Vitamin E Units. In terms of ▲RRR▲ (USP 1-May-2020)-alpha-tocopherol
equivalents, 1 mg of ▲RRR▲ (USP 1-May-2020)-alpha-tocopheryl acetate = 0.91;
microbial count does not exceed 3 × 103 cfu/g, and the 1 mg of ▲RRR▲ (USP 1-May-2020)-alpha-tocopheryl acid succinate = 0.81;
combined molds and yeasts count does not exceed 3 × 1 mg of ▲all-rac▲ (USP 1-May-2020)-alpha-tocopherol = 0.74; 1 mg of ▲all-
102 cfu/g. rac▲ (USP 1-May-2020)-alpha-tocopheryl acetate = 0.67; and 1 mg of ▲all-
• ABSENCE OF SPECIFIED MICROORGANISMS á2022ñ, Test rac▲ (USP 1-May-2020)-alpha-tocopheryl acid succinate = 0.60. ▲Note that 1 mg
Procedures, Test for Absence of Salmonella Species and Test of Institute of Medicine (IOM) alpha-tocopherol equivalent = 1 mg of 2R-
for Absence of Escherichia coli: Meet the requirements alpha-tocopherol = 1 mg of RRR-alpha-tocopherol = 2 mg of all-rac-alpha-
tocopherol.▲ (USP 1-May-2020)
24
USP Folic Acid RS USP Riboflavin RS

USP Niacin RS Riboflavine.
3-Pyridinecarboxylic acid. C17H20N4O6 376.36
C6H5NO2 123.11 USP Sodium Fluoride RS
USP Niacinamide RS Sodium fluoride.
3-Pyridinecarboxamide. NaF 41.99
C6H6N2O 122.12 USP Thiamine Hydrochloride RS
USP Phytonadione RS Thiazolium, 3-[(4-amino-2-methyl-5-pyrimidinyl)
USP Pyridoxine Hydrochloride RS methyl]-5-(2-hydroxyethyl)-4-methyl-, chloride,
3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-, monohydrochloride.
hydrochloride. C12H17ClN4OS · HCl 337.27
C8H11NO3 · HCl 205.64 ▲
▲ (USP 1-May-2020)
▲
USP Retinyl Acetate RS
3,7-Dimethyl-9-(2,6,6-trimethyl-1- cyclohexen-1-yl)
2,4,6,8-nonatetraen-1-ol acetate.
USP Retinyl Palmitate RS
[(2E,4E,6E,8E)-3,7-Dimethyl-9-(2,6,6-trimethyl-1-
cyclohex-1-en-1-yl)nona-2,4,6,8-tetraen-1-yl]
hexadecanoate.▲ (USP 1-May-2020)
al
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O

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Printed on: Tue Jan 03 2023, 02:46:15 AM(EST) Status: Currently Official on 03-Jan-2023 DocId: GUID-DEC96CEA-BD0D-402A-BC13-8762D0532BC9_6_en-US

method used being stated in the labeling only if Method

(C6H6N2O), calcium pantothenate (C18H32CaN2O10),

Calculate the percentage of the labeled amount of Analysis

Result = (rU/rS) × (CS/CU) × F × 100 Result = (rU/rS) × (CS/CU) × 100

ERGOCALCIFEROL), and VITAMIN E, Method 4; Sample solution 2.

= peak area of the relevant vitamin E form from the

CU = nominal concentration of vitamin E, as Analysis

• BIOTIN, Method 1 Acid-hydrolyzed casein solution: Mix 100 g of vitamin-free

CU = nominal concentration of biotin in the Sample toluene.

Samples: Standard solution and the Sample solution that procedure.]

Analysis of the volume, in mL, of the Sample solution to obtain

Injection volume: 10 µL D,L-tryptophan) in 700–800 mL of water, heat to 70°–80°,

µg/mL of USP Calcium Pantothenate RS. Store under

• IODIDE Determine the absorbances of the solutions against the

µg/mL (See Atomic Absorption Spectroscopy á852ñ.)

From the graph, determine the concentration (C), in

Acceptance criteria: 90.0%–160.0% of the labeled amount

= absorbance of the Sample

of molybdenum (Mo) 25.0 mL of the Standard stock solution to separate 100-mL

CU = nominal concentration of potassium in the 50% ammonium hydroxide solution: Ammonium

Instrumental conditions adding additional amounts of nitric acid if further

solution. Sample solution 1 (for Tablets containing minerals in

20 Tablets. Transfer a portion, equal to the average Tablet ADDITIONAL REQUIREMENTS

USP Folic Acid RS USP Riboflavin RS

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