Thiamine Assay

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 ASSAY

1.1 CHEMICAL METHODS, PROCEDURE 1

The following procedure is provided for the determination of thiamine as an ingredient of pharmacopeial preparations containing other active constituents. The procedure involves thiamine reacting with potassium ferricyanide and subsequently being determined by fluorescence detection. Throughout the procedure, protect solutions containing and derived from the test specimen and the Reference Standard from the atmosphere and light, preferably by the use of low-actinic glassware.

Potassium ferricyanide solution: Dissolve 1.0 g of potassium ferricyanide in water to make 100 mL. Prepare fresh on the day of use. Oxidizing reagent: Mix 4.0 mL of Potassium ferricyanide solution with a sufficient amount of 3.5 N sodium hy droxide to make 100 mL. Use this solution within 4 h.

Quinine sulfate stock solution: Dissolve 10 mg of quinine sulfate in 0.1 N sulfuric acid to make 1000 mL. Preserve this solution, protected from light, in a refrigerator.

Quinine sulfate standard solution: Dilute 0.1 N sulfuric acid with Quinine sulfate stock solution (39:1). This solution fluoresces to approximately the same degree as the thiochrome obtained from 1 µg of thiamine hydrochloride and is used to correct the fluorometer at frequent intervals for variation in sensitivity from reading to reading within an assay. Prepare this solution fresh on the day of use.

Standard thiamine hydrochloride stock solution: Transfer about 25 mg of USP Thiamine Hy drochloride RS, accurately weighed, to a 1000- mL volumetric flask. Dissolve the weighed Standard in about 300 mL of dilute alcohol solution (1 in 5) adjusted with 3 N hydrochloric acid to a pH of 4.0, and add the acidified, dilute alcohol to volume. Store in a refrigerator in a light-resistant container. Prepare this stock solution fresh each month.

Standard solution: Dilute a portion of Standard thiamine hydrochloride stock solution quantitatively and stepwise with 0.2 N hydrochloric acid to obtain the Standard solution, each mL of which represents 0.2 µg of USP Thiamine Hy drochloride RS.

Sample solution: Place in a suitable volumetric flask a sufficient amount of the material to be assayed, accurately weighed or measured by volume as directed, such that when diluted with 0.2 N hy drochloric acid to volume, the resulting solution will contain about 100 µg of thiamine hydrochloride (or mononitrate) per mL. If the sample is difficult to dissolve, the solution may be heated on a steam bath, and then cooled and diluted with the acid to volume. Dilute 5 mL of this solution, quantitatively and stepwise, using 0.2 N hydrochloric acid, to an estimated concentration of 0.2 µg of thiamine hydrochloride (or mononitrate) per mL.

Instrumental conditions

(See Fluorescence Spectroscopy 〈853〉.) Mode: Fluorescence

Excitation wavelength: 365 nm Emission wavelength: 435 nm

Analysis: To each of three or more tubes (or other suitable vessels) of about 40-mL capacity, pipet 5 mL of Standard solution. To each of two of these tubes, add rapidly (within 1 to 2 s), with mixing, 3.0 mL of Oxidizing reagent, and within 30 s add 20.0 mL of isobutyl alcohol, then mix vigorously for 90 s by shaking the capped tubes manually, or by bubbling a stream of air through the mixture. Prepare a blank in the remaining tube of the standard by substituting for the Oxidizing reagent with an equal volume of 3.5 N sodium hydroxide and proceed in the same manner. Into each of three or more similar tubes, pipet 5 mL of the Sample solution. Treat these tubes in the same manner as directed for the tubes containing the Standard solution. Into each of the six tubes, pipet 2 mL of dehydrated alcohol, swirl for a few seconds, allow the phases to separate, and decant or draw off about 10 mL of the clear, supernatant isobutyl alcohol solution into the standardized cells; then measure the fluorescence in a suitable fluorometer, having an input filter of narrow transmittance range with a maximum at about 365 nm and an output filter of narrow transmittance range with a maximum at about 435 nm.

Calculate the quantity, in µg, of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) in each 5 mL of the Sample solution:

Result = (A − b)/(S − d)

A = average fluorometer readings of the portions of the Sample solution treated with the Oxidizing reagent

b = reading for the blank of the Sample solution

S = average fluorometer readings of the portions of the Standard solution treated with the Oxidizing reagent

d = reading for the blank of the Standard solution

Calculate the quantity, in mg, of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) in the assay material on the basis of the aliquots taken. Where indicated, the quantity, in mg, of thiamine mononitrate (C₁₂H₁₇ClN₄OS) may be calculated by multiplying the quantity of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) found by 0.9706.

The following liquid chromatographic procedures are provided for the determination of thiamine as an active pharmaceutical ingredient, a dietary supplement ingredient, or a component in dietary supplements or pharmaceutical dosage forms.

Throughout these procedures, protect solutions containing and derived from the test specimen and the Reference Standards from the atmosphere and light, preferably by the use of low-actinic glassware.

1.2 CHROMATOGRAPHIC METHODS, PROCEDURE 1

This procedure can be used to determine thiamine in:

Oil- and Water-Soluble Vitamins Capsules   Oil- and Water-Soluble Vitamins Tablets

Oil- and Water-Soluble Vitamins with Minerals Capsules

Oil- and Water-Soluble Vitamins with Minerals Tablets   Water-Soluble Vitamins Capsules

Water-Soluble Vitamins Tablets

Water-Soluble Vitamins with Minerals Capsules   Water-Soluble Vitamins with Minerals Tablets

This is the procedure that involves the extraction of thiamine from the formulation by the Diluent, heat, and mechanical shaking. Unless specified in the individual monographs, the Standard solution, Sample solutions, and reagent solutions are prepared as follows.

Diluent: Acetonitrile, glacial acetic acid, and water (5:1:94)

Mobile phase: A mixture of methanol, glacial acetic acid, and water (27:1:73) containing 140 mg of sodium 1-hexanesulfonate per 100 mL Standard solution: Transfer 20 mg of USP Thiamine Hy drochloride RS to a 200-mL 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 of the solid materials are dissolved. Chill rapidly in a cold water bath for 10 min to room temperature, and dilute with Diluent to volume.

Sample solution for capsules: Weigh NLT 20 capsules in a tared weighing bottle. Open the capsules, without loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the shells by washing with several portions of ether. Discard the washings, and dry the capsule shells with the aid of a current of dry air until the odor of ether is no longer perceptible. Weigh the empty capsule shells in the tared weighing bottle, and calculate the average net weight per capsule. Transfer a portion of the capsule contents, equivalent to 2.5 mg of thiamine hydrochloride, to a 50-mL centrifuge tube. Add 25.0 mL of Diluent, and mix using a vortex mixer for 30 s to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at 65°–70°, heat for 5 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 5 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear filtrate. [NOTE—Use the filtrate within 3 h of filtration.]

Sample solution for tablets: Finely powder NLT 30 tablets. Transfer a portion of the powder, equivalent to 2.5 mg of thiamine hydrochloride, to a 50-mL centrifuge tube. Add 25.0 mL of Diluent, and mix using a vortex mixer for 30 s to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at 65°–70°, heat for 5 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 5 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear filtrate. [NOTE—Use the filtrate within 3 h of filtration.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection volume: 10 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and appropriate Sample solution

For products containing thiamine hydrochloride, calculate the percentage of the labeled amount of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) in the portion of sample taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of thiamine from the appropriate Sample solution

r_S = peak response of thiamine from the Standard solution

C_S = concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL)

C_U = nominal concentration of thiamine hydrochloride in the appropriate Sample solution (mg/mL)

For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C₁₂H₁₇ClN₄OS) in the portion of sample taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of thiamine from the appropriate Sample solution

r_S = peak response of thiamine from the Standard solution

C_S = concentration of USP Thiamine Hy drochloride RS in the Standard solution (mg/mL)

C_U = nominal concentration of thiamine mononitrate in the appropriate Sample solution (mg/mL)

M_r1 = molecular weight of thiamine mononitrate, 327.36

M_r2 = molecular weight of thiamine hydrochloride, 337.27

1.3 CHROMATOGRAPHIC METHODS, PROCEDURE 2

This procedure can be used to determine thiamine in:

Oil- and Water-Soluble Vitamins Capsules   Oil- and Water-Soluble Vitamins Tablets

Oil- and Water-Soluble Vitamins with Minerals Capsules

Oil- and Water-Soluble Vitamins with Minerals Tablets   Water-Soluble Vitamins Capsules

Water-Soluble Vitamins Tablets

Water-Soluble Vitamins with Minerals Capsules   Water-Soluble Vitamins with Minerals Tablets

This is the procedure that involves the extraction of thiamine from the formulation by dilute hydrochloric acid, heat, and mechanical shaking.

Unless specified in the individual monographs, the Standard solution, Sample solutions, and reagent solutions are prepared as follows.

Solution A: 1.88 g/L of sodium 1-hexanesulfonate in 0.1% phosphoric acid Mobile phase: Solution A and acetonitrile (46:9)

Standard stock solution: 0.1 mg/mL of USP Thiamine Hydrochloride RS in 0.2 N hydrochloric acid

Standard solution: 0.02 mg/mL of USP Thiamine Hydrochloride RS from Standard stock solution diluted with 0.2 N hydrochloric acid Sample solution for capsules: Weigh NLT 20 capsules in a tared weighing bottle. Open the capsules, without the loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing, if necessary, with several portions of ether. Discard the washings, and dry the capsule shells with the aid of a current of dry air until the odor of ether is no longer perceptible. Weigh the empty capsule shells in the tared weighing bottle, and calculate the average net weight per capsule. Mix a portion of the capsule contents with a volume of 0.2 N hydrochloric acid to obtain a concentration of 0.02 mg/mL of thiamine. Shake the solution for 15 min with a wrist-action shaker, and heat to boiling for 30 min. Cool to room temperature, and filter. Use the clear filtrate.

Sample solution for tablets: Weigh and finely powder NLT 20 tablets. Mix a portion of the powder with a volume of 0.2 N hydrochloric acid to obtain a concentration of 0.02 mg/mL of thiamine. Shake for 15 min with a wrist-action shaker, and heat to boiling for 30 min. Cool to room temperature, and filter. Use the clear filtrate.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 2 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and appropriate Sample solution

For products containing thiamine hydrochloride, calculate the percentage of the labeled amount of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) in the portion of sample taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of thiamine from the appropriate Sample solution

r_S = peak response of thiamine from the Standard solution

C_S = concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL)

C_U = nominal concentration of thiamine hydrochloride in the appropriate Sample solution (mg/mL)

For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C₁₂H₁₇ClN₄OS) in the portion of sample taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of thiamine from the appropriate Sample solution

r_S = peak response of thiamine from the Standard solution

C_S = concentration of USP Thiamine Hydrochloride RS in the Standard solution (mg/mL)

C_U = nominal concentration of thiamine mononitrate in the appropriate Sample solution (mg/mL)

M_r1 = molecular weight of thiamine mononitrate, 327.36

M_r2 = molecular weight of thiamine hydrochloride, 337.27

1.4 CHROMATOGRAPHIC METHODS, PROCEDURE 3

This procedure can be used to determine thiamine in:

Oil- and Water-Soluble Vitamins Capsules

Oil- and Water-Soluble Vitamins Oral Solution   Oil- and Water-Soluble Vitamins Tablets

Oil- and Water-Soluble Vitamins with Minerals Capsules

Oil- and Water-Soluble Vitamins with Minerals Oral Solution   Oil- and Water-Soluble Vitamins with Minerals Tablets

Water-Soluble Vitamins Capsules   Water-Soluble Vitamins Tablets

Water-Soluble Vitamins with Minerals Capsules

Water-Soluble Vitamins with Minerals Oral Solution   Water-Soluble Vitamins with Minerals Tablets

This is the procedure that involves the extraction of thiamine from the formulation by mixtures of organic solvents, heat, and mechanical shaking.

Unless specified in the individual monographs, the Standard solutions, Sample solutions, and reagent solutions are prepared as follows.

Diluent: 25 mg/mL of edetate disodium in water

Mobile phase: Transfer 0.4 mL of triethylamine, 15.0 mL of glacial acetic acid, and 350 mL of methanol to a 2000-mL volumetric flask. Dilute with 0.008 M sodium 1-hexanesulfonate to volume.

Standard stock solution: 0.24 mg/mL of USP Thiamine Hy drochloride RS in Diluent, with heating if necessary

Standard solution for capsules/tablets: Transfer 5.0 mL of Standard stock solution to a stoppered 125-mL flask. Add 10.0 mL of a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene gl y col (1:1). Insert the stopper, shake for 15 min in a water bath maintained at 60°, and cool. Filter, and discard the first few mL of the filtrate.

Standard solution for oral solution: 24 µg/mL of USP Thiamine Hy drochloride RS, diluted from the Standard stock solution with Diluent Sample solution for capsules: Weigh NLT 20 capsules in a tared weighing bottle. Open the capsules, without the loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing, if necessary, with several portions of ether. Discard the washings, and dry the capsule shells with the aid of a current of dry air until the odor of ether is no longer perceptible. Weigh the empty capsule shells in the tared weighing bottle, and calculate the average net weight per capsule. Transfer a portion of the capsule contents, equivalent to 1.2 mg of thiamine hydrochloride, to a stoppered 125-mL flask. Add 10.0 mL of a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene gly col (1:1). Insert the stopper, shake for 15 min in a water bath maintained at 60°, and cool. Filter, and discard the first few mL of the filtrate.

Sample solution for oral solution: Equivalent to 24 µg/mL of thiamine hydrochloride or thiamine mononitrate from oral solution in Diluent Sample solution for tablets: Weigh and finely powder NLT 20 tablets. Transfer a portion of the powder, equivalent to 1.2 mg of thiamine, to a stoppered 125-mL flask. Add 10.0 mL of a mixture of methanol and glacial acetic acid (9:1) and 30.0 mL of a mixture of methanol and ethylene gly col (1:1). Insert the stopper, shake for 15 min in a water bath maintained at 60°, and cool. Filter, and discard the first few mL of the filtrate.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 270 nm

Column: 4.6-mm × 25-cm; packing L7

Column temperature: 50°

Flow rate: 2 mL/min Injection volume: 5 µL

System suitability

Sample: Standard solution Suitability requirements

Relative standard deviation: NMT 2.0%

Analysis

Samples: Appropriate Standard solution and appropriate Sample solution

For products containing thiamine hydrochloride, calculate the percentage of the labeled amount of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) in the portion of sample taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of thiamine from the appropriate Sample solution

r_S = peak response of thiamine from the appropriate Standard solution

C_S = concentration of USP Thiamine Hydrochloride RS in the appropriate Standard solution (mg/mL)

C_U = nominal concentration of thiamine in the appropriate Sample solution (mg/mL)

For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C₁₂H₁₇ClN₄OS) in the portion of sample taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of thiamine from the appropriate Sample solution

r_S = peak response of thiamine from the appropriate Standard solution

C_S = concentration of USP Thiamine Hydrochloride RS in the appropriate Standard solution (mg/mL)

C_U = nominal concentration of thiamine mononitrate in the appropriate Sample solution (mg/mL)

M_r1 = molecular weight of thiamine mononitrate, 327.36

M_r2 = molecular weight of thiamine hydrochloride, 337.27

1.5 CHROMATOGRAPHIC METHODS, PROCEDURE 4

This procedure can be used to determine thiamine in:

 An active pharmaceutical ingredient   A dietary ingredient

This is the procedure that involves the dissolution of the sample directly into the Mobile phase. The procedure is applicable for the determination of thiamine hydrochloride or thiamine mononitrate as active pharmaceutical or dietary supplement ingredients.

Unless specified in the individual monographs, the Standard solution, Sample solution, and reagent solutions are prepared as follows.

Solution A: 0.005 M sodium 1-octanesulfonate in dilute glacial acetic acid (1 in 100)

Solution B: Methanol and acetonitrile (3:2)

Mobile phase: Solution A and Solution B (60:40)

Internal standard solution: 2% (v/v) of methylbenzoate in methanol

Standard solution: Prepare a 1-mg/mL solution of USP Thiamine Hy drochloride RS in Mobile phase. Transfer 20.0 mL of this solution and 5.0 mL of Internal standard solution to a 50-mL volumetric flask, and dilute with Mobile phase to volume. The Standard solution contains 400 µg/mL of thiamine hydrochloride.

Sample solution: Prepare a 2-mg/mL solution of thiamine hydrochloride or thiamine mononitrate in Mobile phase. Transfer 10.0 mL of this solution and 5.0 mL of Internal standard solution to a 50-mL volumetric flask, and dilute with Mobile phase to volume.

Flow rate: 1 mL/min. [NOTE—The flow rate may be adjusted to obtain a retention time of about 12 min for thiamine.] Injection volume: 10 µL

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 4.0 between the thiamine and methylbenzoate peaks

Tailing factor: NMT 2.0 for the thiamine peak

Column efficiency: NLT 1500 theoretical plates for thiamine

Relative standard deviation: NMT 2.0% for the ratios of thiamine peak response to the internal standard peak response Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) in the portion of sample taken:

Result = (R_U/R_S) × (C_S/C_U) × 100

R_U = peak response ratio of thiamine to methylbenzoate from the Sample solution

R_S = peak response ratio of thiamine to methylbenzoate from the Standard solution

C_S = concentration of USP Thiamine Hy drochloride RS in the Standard solution (mg/mL)

C_U = concentration of thiamine hydrochloride in the Sample solution (mg/mL)

If the active pharmaceutical or dietary supplement ingredient is thiamine mononitrate, calculate the percentage of thiamine mononitrate (C₁₂H₁₇ClN₄OS) in the portion of sample taken:

Result = (R_U/R_S) × (C_S/C_U) × (M_r1/M_r2) × 100

R_U = peak response ratio of thiamine to methylbenzoate from the Sample solution

R_S = peak response ratio of thiamine to methylbenzoate from the Standard solution

C_S = concentration of USP Thiamine Hy drochloride RS in the Standard solution (mg/mL)

C_U = concentration of thiamine mononitrate in the Sample solution (mg/mL)

M_r1 = molecular weight of thiamine mononitrate, 327.36

M_r2 = molecular weight of thiamine hydrochloride, 337.27

1.6 CHROMATOGRAPHIC METHODS, PROCEDURE 5

This procedure can be used to determine thiamine in: Thiamine Hy drochloride Injection

Thiamine Hydrochloride Oral Solution Thiamine Mononitrate Oral Solution

This is the procedure that involves the dissolution of the sample directly into the Mobile phase. The procedure is applicable for the determination of thiamine hydrochloride or thiamine mononitrate as an active ingredient in the formulations such as those used in Thiamine Hydrochloride Injection, Thiamine Hydrochloride Oral Solution, and Thiamine Mononitrate Oral Solution.

Unless specified in the individual monographs, the Standard solution, Internal standard solution, and Sample solution are prepared as follows.

Mobile phase: Methanol and 0.04 M aqueous monobasic potassium phosphate (45:55) Internal standard solution: 100 µg/mL of methylparaben in Mobile phase

Standard stock solution: 500 µg/mL of USP Thiamine Hy drochloride RS in Mobile phase

Standard solution: Dilute a mixture of equal volumes of the Standard stock solution and Internal standard solution with Mobile phase to obtain a concentration of USP Thiamine Hy drochloride RS of about 50 µg/mL.

Sample stock solution: Equivalent to 500 µg/mL of thiamine hydrochloride or thiamine mononitrate in Mobile phase from an accurately measured volume of oral solution or injection

Sample solution: Dilute a mixture of equal volumes of the Internal standard solution and Sample stock solution with Mobile phase to obtain a concentration of thiamine hydrochloride or thiamine mononitrate of about 50 µg/mL.

Flow rate: 1 mL/min Injection volume: 25 µL

System suitability

Sample: Standard solution

[NOTE—The relative retention times for thiamine and methylparaben are about 0.35 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 6.0 between thiamine and methylparaben Column efficiency: NLT 1500 theoretical plates for thiamine

Relative standard deviation: NMT 2.0% for the ratios of thiamine peak response to the methylparaben peak response

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) in the portion of sample taken:

Result = (R_U/R_S) × (C_S/C_U) × 100

R_U = peak response ratio of thiamine to methylparaben from the Sample solution

R_S = peak response ratio of thiamine to methylparaben from the Standard solution

C_S = concentration of USP Thiamine Hy drochloride RS in the Standard solution (mg/mL)

C_U = nominal concentration of thiamine hydrochloride in the Sample solution (mg/mL)

If the products contain thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C H N O S) in the portion of sample taken:

Result = (R_U/R_S) × (C_S/C_U) × (M_r1/M_r2) × 100

R_U = peak response ratio of thiamine to methylparaben from the Sample solution

R_S = peak response ratio of thiamine to methylparaben from the Standard solution

C_S = concentration of USP Thiamine Hy drochloride RS in the Standard solution (mg/mL)

C_U = concentration of thiamine mononitrate in the Sample solution (mg/mL)

M_r1 = molecular weight of thiamine mononitrate, 327.36

M_r2 = molecular weight of thiamine hydrochloride, 337.27

1.7 CHROMATOGRAPHIC METHODS, PROCEDURE 6

This procedure can be used to determine thiamine in the following: Thiamine Hy drochloride Tablets

This is the procedure that involves the dissolution of the sample directly into water. The procedure is applicable to the performance (dissolution) test for Thiamine Hy drochloride Tablets.

Unless specified in the individual monographs, the Standard solution and Sample solution are prepared as follows.

Medium: Water, 900 mL

Apparatus 2: 50 rpm

Time: 45 min

Mobile phase: A mixture of methanol, glacial acetic acid, and water (27:1:73) containing 140 mg of sodium 1-hexanesulfonate per 100 mL Standard solution: A known concentration of USP Thiamine Hy drochloride RS in Medium (water)

Sample solution: Filtered portion of the solution under test, suitably diluted with Medium (water) if necessary

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1 mL/min

Injection volume: 10 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of thiamine hydrochloride (C₁₂H₁₇ClN₄OS · HCl) in the portion of sample taken:

Result = (r_U/r_S) × (C_S × D × V/L) × 100

r_U = peak response of thiamine from the Sample solution

r_S = peak response of thiamine from the Standard solution

C_S = concentration of USP Thiamine Hy drochloride RS in the Standard solution (mg/mL)

D = dilution factor for the Sample solution

V = volume of Medium, 900 mL

L = labeled amount of thiamine hydrochloride (mg/Tablet)

1.8 CHROMATOGRAPHIC METHODS, PROCEDURE 7

This procedure can be used to determine thiamine in: Oil- and Water-Soluble Vitamins Capsules

Oil- and Water-Soluble Vitamins Oral Solution Oil- and Water-Soluble Vitamins Tablets

Oil- and Water-Soluble Vitamins with Minerals Capsules

Oil- and Water-Soluble Vitamins with Minerals Oral Solution Oil- and Water-Soluble Vitamins with Minerals Tablets Water-Soluble Vitamins Capsules

Water-Soluble Vitamins Tablets

Water-Soluble Vitamins with Minerals Capsules Water-Soluble Vitamins with Minerals Oral Solution Water-Soluble Vitamins with Minerals Tablets

This is a newly added procedure as part of the USP monograph modernization efforts. The procedure uses hydrophilic interaction liquid chromatography (HILIC), and the sample preparation involves the extraction of thiamine from the formulation by the Diluent, heat, and mechanical shaking.

Unless specified in the individual monographs, the Standard solution, Sample solutions, and reagent solutions are prepared as follows.

Diluent: Methanol, glacial acetic acid, and water (50:1:49)

Solution A: 50 mM ammonium formate. Adjust with ammonium hy droxide to a pH of 9.0. Solution B: Acetonitrile

Mobile phase: Gradient elution. See Table 1.

Table 1

Standard solution: Transfer 20 mg of USP Thiamine Hy drochloride RS to a 200-mL volumetric flask, and add 160 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 of the solid materials are dissolved. Chill rapidly in a cold water bath for 10 min to room temperature, and dilute with Diluent to volume.

Sample solution for capsules: Weigh NLT 20 capsules in a tared weighing bottle. Open the capsules, without the loss of shell material, and transfer the contents to a 100-mL beaker. Remove any contents adhering to the empty shells by washing, if necessary, with several portions of ether. Discard the washings, and dry the capsule shells with the aid of a current of dry air until the odor of ether is no longer perceptible. Weigh the empty capsule shells in the tared weighing bottle, and calculate the average net weight per capsule. Transfer a portion of the capsule contents, equivalent to 2.5 mg of thiamine hydrochloride, to a 50-mL centrifuge tube. Add 25.0 mL of Diluent, and mix using a vortex mixer for 30 s to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at 68°, heat for 10 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 10 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear filtrate.

Sample solution for oral solution: Equivalent to 0.1 mg/mL of thiamine hydrochloride in Diluent, from an accurately measured volume of oral solution. In an appropriate volumetric flask, dissolve the sample with Diluent to about 80% of the total volume, immerse the flask in a water bath maintained at 68° for 10 min, chill rapidly in a cold water bath to room temperature, and dilute with Diluent. Mix well, and filter a portion of the solution; finally, use the clear filtrate.

Sample solution for tablets: Finely powder NLT 30 tablets. Transfer a portion of the powder, equivalent to 2.5 mg of thiamine hydrochloride, to a 50-mL centrifuge tube. Add 25.0 mL of Diluent, and mix using a vortex mixer for 30 s to completely suspend the powder. Immerse the centrifuge tube in a hot water bath maintained at 65°–70°, heat for 10 min, and mix on a vortex mixer for 30 s. Return the tube to the hot water bath, heat for another 10 min, and mix on a vortex mixer for 30 s. Filter a portion of the solution, cool to room temperature, and use the clear filtrate. [NOTE—Use the filtrate within 3 h of filtration.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 267 nm

Column: 4.6-mm × 15-cm; 3.5-µm packing L68

Column temperature: 40°

Flow rate: 1.2 mL/min

Injection volume: 10 µL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and appropriate Sample solution

For products containing thiamine hydrochloride, calculate the percentage of the labeled amount of thiamine hydrochloride (C H ClN OS · HCl) in the portion of sample taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of thiamine from the appropriate Sample solution

r_S = peak response of thiamine from the Standard solution

C_S = concentration of USP Thiamine Hy drochloride RS in the Standard solution (mg/mL)

C_U = nominal concentration of thiamine in the appropriate Sample solution (mg/mL)

For products containing thiamine mononitrate, calculate the percentage of the labeled amount of thiamine mononitrate (C H N O S) in the portion of sample taken:

Result = (r_U/r_S) × (C_S/C_U) × (M_r1/M_r2) × 100

r_U = peak response of thiamine from the appropriate Sample solution

r_S = peak response of thiamine from the Standard solution

C_S = concentration of USP Thiamine Hy drochloride RS in the Standard solution (mg/mL)

C_U = nominal concentration of thiamine mononitrate in the appropriate Sample solution (mg/mL)

M_r1 = molecular weight of thiamine mononitrate, 327.36

M_r2 = molecular weight of thiamine hydrochloride, 337.27

2 ADDITIONAL REQUIREMENTS

USP REFERENCE STANDARDS 〈11〉

USP Thiamine Hydrochloride RS