A: Infrared Absorption 197M.

B: Prepare a solution in a mixture of equal volumes of toluene and alcohol containing 1 mg per mL. Apply 10 µL of this solution and 10 µL of a Standard solution of USP Trichlormethiazide RS in the same medium having a known concentration of 1 mg per mL to a suitable thin-layer chromatographic plate (see Thin-Layer Chromatography under Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram, using ethyl acetate as the solvent, until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the solvent to evaporate. Locate the spots using short-wavelength UV light: the RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

Standard solution— Prepare a solution in a mixture of equal volumes of toluene and alcohol containing 250 µg of USP Benzothiadiazine Related Compound A RS in each mL.

Test solution— Transfer 100.0 mg of Trichlormethiazide, accurately weighed, to a 10-mL volumetric flask, dissolve in 1 mL of acetone, dilute with a mixture of equal volumes of toluene and alcohol to volume, and mix.

Procedure— Apply 5 µL each of the Standard solution and of the Test solution to a suitable thin-layer chromatographic plate (see Thin-Layer Chromatography, under Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram in ethyl acetate until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and locate the spots on the plate by spraying first with a 1 in 20 solution of sodium nitrite in dilute hydrochloric acid (1 in 12), and then with a 1 in 1000 solution of N-(1-naphthyl)ethylenediamine dihydrochloride in alcohol: after 3 minutes, any spot from the Test solution, occurring at the RF value corresponding to that produced by the Standard solution, is not greater in size or intensity than the spot produced by the Standard solution, corresponding to not more than 2.5% of diazotizable substances.

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of 0.05 M monobasic potassium phosphate and methanol (7:3). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Transfer about 120 mg of methylparaben to a 50-mL volumetric flask, add methanol to volume, and mix.

Standard preparation— Transfer about 25 mg of USP Trichlormethiazide RS, accurately weighed, to a 50-mL volumetric flask, add 4.0 mL of Internal standard solution, dilute with methanol to volume, and mix to obtain a solution having a known concentration of about 0.5 mg of USP Trichlormethiazide RS per mL.

Assay preparation— Transfer about 50 mg of Trichlormethiazide, accurately weighed, to a 100-mL volumetric flask, add 8.0 mL of Internal standard solution, dilute with methanol 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 2.3 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 2.0, and the relative standard deviation for the response ratios calculated for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (5 to 25 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 0.56 for trichlormethiazide and 1.0 for methylparaben. Calculate the quantity, in mg, of C8H8Cl3N3O4S2 in the portion of Trichlormethiazide taken by the formula: in which C is the concentration, in mg per mL, of USP Trichlormethiazide RS in the Standard preparation, and RU and RS are the peak response ratios obtained from the Assay preparation and the Standard preparation, respectively.