A: Infrared Absorption 197M.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, both relative to the internal standard, as obtained in the Assay.

Mobile phase— Prepare a filtered and degassed mixture of 0.01 M sodium 1-pentanesulfonate, methanol, and glacial acetic acid (80:20:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Transfer about 50 mg of hydrochlorothiazide to a 25-mL volumetric flask, add methanol to volume, and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Diazoxide RS in methanol, and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 1 mg per mL. Transfer 5.0 mL of this solution to a 100-mL volumetric flask, add 2.0 mL of Internal standard solution, dilute with a mixture of water and methanol (4:1) to volume, and mix to obtain a solution having a known concentration of about 50 µg of USP Diazoxide RS per mL.

Assay preparation— Transfer about 50 mg of Diazoxide, accurately weighed, to a 50-mL volumetric flask, add methanol to volume, and mix. Transfer 5.0 mL of this solution to a 100-mL volumetric flask, add 2.0 mL of Internal standard solution, dilute with a mixture of water and methanol (4:1) 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 L11. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between the analyte and internal standard peaks is not less than 5; and the relative standard deviation for replicate injections is not more than 1.5%.

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 responses for the major peaks. The relative retention times are about 0.4 for hydrochlorothiazide and 1.0 for diazoxide. Calculate the quantity, in mg, of C8H7ClN2O2S in the portion of Diazoxide taken by the formula: in which C is the concentration, in µg per mL, of USP Diazoxide RS in the Standard preparation; and RU and RS are the peak response ratios obtained from the Assay preparation and the Standard preparation, respectively.