Assay
Mobile phase
Prepare a filtered and degassed mixture of 0.04
M aqueous monobasic potassium phosphate and methanol (55:45). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Internal standard solution
Prepare a solution of methylparaben in Mobile phase having a concentration of about 100 µg per mL.
Standard preparation
Prepare a solution of
USP Thiamine Hydrochloride RS in
Mobile phase having an accurately known concentration of about 500 µg per mL. Pipet 10 mL of this solution and 10 mL of
Internal standard solution into a 100-mL volumetric flask, dilute with
Mobile phase to volume, and mix to obtain a
Standard preparation having a known concentration of about 50 µg per mL.
Assay preparation
Quantitatively dilute an accurately measured volume of Oral Solution with Mobile phase to obtain a solution containing about 500 µg of thiamine hydrochloride per mL. Pipet 10 mL of the resulting solution and 10 mL of Internal standard solution into a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 0.35 for thiamine and 1.0 for methylparaben; the resolution,
R, between the thiamine and methylparaben peaks is not less than 6.0; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject equal volumes (about 25 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of thiamine hydrochloride (C
12H
17ClN
4OS·HCl) in each mL of the Oral Solution taken by the formula:
C(L/D)(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Thiamine Hydrochloride RS in the
Standard preparation; L is the labeled quantity, in mg per mL, of thiamine hydrochloride in the Oral Solution;
D is the concentration, in mg per mL, of thiamine hydrochloride in the
Assay preparation on the basis of the labeled quantity and the extent of dilution; and
RU and
RS are the ratios of the peak responses of thiamine to methylparaben obtained from the
Assay preparation and the
Standard preparation, respectively.