A: Infrared Absorption 197K—Dry specimens at 105 for 2 hours.

B: A solution (1 in 50) responds to the tests for Chloride 191.

Solution A , Solution B, and Mobile phase—Prepare as directed in the Assay.

Test solution— Dissolve quantitatively an accurately weighed quantity of Thiamine Hydrochloride in Mobile phase to obtain a solution having a concentration of about 1.0 mg per mL.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.0-mm × 15-cm column that contains packing L1. The flow rate is about 0.75 mL per minute.

Procedure— Inject about 10 µL of the Test solution into the chromatograph, and allow the Test solution to elute for not less than three times the retention time of the main peak. Record the chromatogram and measure the areas of the peak responses: the total of the responses of all secondary peaks is not greater than 1.0% of the total of the responses of all of the peaks.

Solution A— Prepare a 0.005 M solution of sodium 1-octanesulfonate in dilute glacial acetic acid (1 in 100).

Solution B— Prepare a mixture of methanol and acetonitrile (3:2).

Mobile phase— Prepare a mixture of Solution A and Solution B (60:40), filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Transfer 2.0 mL of methylbenzoate to a 100-mL volumetric flask, dilute with methanol to volume, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Thiamine Hydrochloride RS in Mobile phase to obtain a solution having a known concentration of about 1 mg per mL. Transfer 20.0 mL of this solution to a 50-mL volumetric flask, add 5.0 mL of Internal standard solution, dilute with Mobile phase to volume, and mix to obtain a Standard preparation having a known concentration of about 400 µg per mL.

Assay preparation— Transfer an accurately weighed quantity of about 200 mg of Thiamine Hydrochloride to a 100-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix. Transfer 10.0 mL of this solution to a 50-mL volumetric flask, add 5.0 mL of Internal standard solution, 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 4-mm × 30-cm column that contains packing L1. The flow rate is about 1 mL per minute. [note—The flow rate may be adjusted as needed to obtain a retention time of about 12 minutes for thiamine hydrochloride.] Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between the thiamine and methylbenzoate peaks is not less than 4.0; the tailing factor for the thiamine peak is not more than 2.0; the column efficiency determined from the thiamine peak is not less than 1500 theoretical plates; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas of the major peaks. Calculate the quantity, in mg, of C12H17ClN4OS·HCl in the Thiamine Hydrochloride taken by the formula: in which C is the concentration, in µg per mL, of USP Thiamine Hydrochloride RS in the Standard preparation; and RU and RS are the ratios of the peak areas of thiamine to methylbenzoate obtained from the Assay preparation and the Standard preparation, respectively.