á531ñTHIAMINE ASSAY

USP Reference Standards á11ñ— USP Thiamine Hydrochloride RS.
The following procedure is provided for the determination of thiamine as an ingredient of Pharmacopeial preparations containing other active constituents.

Special Solutions and Solvents—
POTASSIUM FERRICYANIDE SOLUTION— Dissolve 1.0g of potassium ferricyanide in water to make 100mL.Prepare fresh on the day of use.
OXIDIZING REAGENT— Mix 4.0mLof Potassium Ferricyanide Solutionwith sufficient 3.5Nsodium hydroxide to make 100mL.Use this solution within 4hours.
QUININE SULFATE STOCK SOLUTION— Dissolve 10mg of quinine sulfate in 0.1Nsulfuric acid to make 1000mL.Preserve this solution,protected from light,in a refrigerator.
QUININE SULFATE STANDARD SOLUTION— Dilute 0.1Nsulfuric acid with Quinine Sulfate Stock Solution(39:1).This solution fluoresces to approximately the same degree as the thiochrome obtained from 1µg 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 25mg of USP Thiamine Hydrochloride RS,accurately weighed,to a 1000-mLvolumetric flask.Dissolve the weighed Standard in about 300mLof dilute alcohol solution (1in 5)adjusted with 3Nhydrochloric acid to a pHof 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.

Standard Preparation— Dilute a portion of Standard Thiamine Hydrochloride Stock Solutionquantitatively and stepwise with 0.2Nhydrochloric acid to obtain the Standard Preparation,each mLof which represents 0.2µg of USP Thiamine Hydrochloride RS.

Assay Preparation— 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 0.2Nhydrochloric acid,the resulting solution will contain about 100µg 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 5mLof this solution,quantitatively and stepwise,using 0.2Nhydrochloric acid,to an estimated concentration of 0.2µg of thiamine hydrochloride (or mononitrate)per mL.

Procedure— Into each of three or more tubes (or other suitable vessels),of about 40-mLcapacity,pipet 5mLof Standard Preparation.To each of two of these tubes add rapidly (within 1to 2seconds),with mixing,3.0mLof Oxidizing Reagent,and within 30seconds add 20.0mLof isobutyl alcohol,then mix vigorously for 90seconds 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 Reagentan equal volume of 3.5Nsodium hydroxide and proceeding in the same manner.
Into each of three or more similar tubes pipet 5mLof 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 2mLof dehydrated alcohol,swirl for a few seconds,allow the phases to separate,and decant or draw off about 10mLof 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 365nm and an output filter of narrow transmittance range with a maximum at about 435nm.

Calculation— The number of µg of C12H17ClN4OS·HCl in each 5mLof the Assay Preparationis given by the formula:
(A-b)/(S-d),
in which Aand Sare the average fluorometer readings of the portions of the Assay Preparationand the Standard Preparationtreated with Oxidizing Reagent,respectively,and band dare the readings for the blanks of the Assay Preparationand the Standard Preparation,respectively.
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.