A: Infrared Absorption 197M: previously dried at 105 for 1 hour.

B: Ultraviolet Absorption 197U—

Mobile phase, Resolution solution, and Chromatographic system— Proceed as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Benzothiadiazine Related Compound A RS in Mobile phase to obtain a solution having a known concentration of about 1.5 µg per mL.

Test solution— Proceed as directed for Assay preparation in the Assay.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The relative retention times are about 0.9 for benzothiadiazine related compound A and 1.0 for chlorothiazide. Calculate the quantity, in mg, of benzothiadiazine related compound A in the portion of Chlorothiazide taken by the formula: in which C is the concentration, in µg per mL, of USP Benzothiadiazine Related Compound A RS in the Standard solution; and r U and rS are the peak responses of benzothiadiazine related compound A obtained from the Test solution and the Standard solution, respectively: not more than 1.0% is present.

Solvent— Use dimethyl sulfoxide.

(Official January 1, 2007)

Mobile phase— Prepare a suitable degassed mixture of 0.1 M monobasic sodium phosphate and acetonitrile (9:1), adjust with phosphoric acid to a pH of 3.0 ± 0.1, and filter. Make adjustments if necessary (see System Suitability under Chromatography 621).

Resolution solution— Dissolve quantities of USP Reference Standards in Mobile phase to obtain solutions having known concentrations of about 0.15 mg per mL of USP Chlorothiazide RS and about 1.5 µg per mL of USP Benzothiadiazine Related Compound A RS.

Standard preparation— [NOTE—A volume of acetonitrile not exceeding 10% of the total volume of solution may be used to dissolve the reference standard.] Dissolve an accurately weighed quantity of USP Chlorothiazide RS in Mobile phase to obtain a solution having a known concentration of about 0.15 mg per mL.

Assay preparation— Transfer about 30 mg of Chlorothiazide, accurately weighed, to a 200-mL volumetric flask, dissolve in a small volume of acetonitrile, not exceeding 10% of the total volume of the 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 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.2 mL per minute. Chromatograph replicate injections of the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation is not more than 1.5%. Chromatograph the Resolution solution: the resolution, R, between benzothiadiazine related compound A and chlorothiazide is not less than 3.5.

Procedure— [NOTE—The Standard preparation and the Assay preparation should be injected immediately upon preparation.] Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major chlorothiazide peaks. The relative retention times are about 0.9 for benzothiadiazine related compound A and 1.0 for chlorothiazide. Calculate the quantity, in mg, of C7H6ClN3O4S2 in the portion of Chlorothiazide taken by the formula: in which C is the concentration, in mg per mL, of USP Chlorothiazide RS in the Standard preparation; and rU and rS are the peak responses of chlorothiazide obtained from the Assay preparation and the Standard preparation, respectively.