531 THIAMINE ASSAY
Special Solutions and Solvents
POTASSIUM FERRICYANIDE SOLUTION
Dissolve 1.0 g of potassium ferricyanide in water to make 100 mL. Prepare fresh on the day of use.
Mix 4.0 mL of Potassium Ferricyanide Solution
with sufficient 3.5 N sodium hydroxide to make 100 mL. Use this solution within 4 hours.
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 Hydrochloride 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 light-resistant container, in a refrigerator. Prepare this stock solution fresh each month.
Dilute a portion of Standard Thiamine Hydrochloride Stock Solution
quantitatively and stepwise with 0.2 N hydrochloric acid to obtain the Standard Preparation,
each mL of which represents 0.2 µg of USP Thiamine Hydrochloride RS
Place in a suitable volumetric flask sufficient amount of the material to be assayed, accurately weighed or measured by volume as directed, such that when diluted to volume with 0.2 N hydrochloric acid, the resulting solution will contain about 100 µg of thiamine hydrochloride (or mononitrate) per mL. If the sample is difficultly soluble, 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.
Into each of three or more tubes (or other suitable vessels), of about 40-mL capacity, pipet 5 mL of Standard Preparation.
To each of two of these tubes add rapidly (within 1 to 2 seconds), with mixing, 3.0 mL of Oxidizing Reagent
, and within 30 seconds add 20.0 mL of isobutyl alcohol, then mix vigorously for 90 seconds 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
an equal volume of 3.5 N sodium hydroxide and proceeding in the same manner.
Into each of three or more similar tubes pipet 5 mL of the Assay Preparation. Treat these tubes in the same manner as directed for the tubes containing the Standard Preparation.
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 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.
The number of µg of C12
OS·HCl in each 5 mL of the Assay Preparation
is given by the formula:
) / (S d
in which A
are the average fluorometer readings of the portions of the Assay Preparation
and the Standard Preparation
treated with Oxidizing Reagent
, respectively, and b
are the readings for the blanks of the Assay Preparation
and the Standard Preparation,
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 (C12H17N5O4S) may be calculated by multiplying the quantity of C12H17ClN4OS·HCl found by 0.9706.
: Lawrence Evans, III, Ph.D., Scientist
Expert Committee : (DSN05) Dietary Supplements - Non-Botanicals
USP29NF24 Page 2593
Phone Number : 1-301-816-8389