A: Infrared Absorption á197Mñ.

B: Prepare a solution in a mixture of equal volumes of toluene and alcohol containing 1mg per mL.Apply 10µLof this solution and 10µLof a Standard solution of USP Trichlormethiazide RSin the same medium having a known concentration of 1mg per mLto a suitable thin-layer chromatographic plate (see Thin-Layer Chromatographyunder Chromatography á621ñ)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 UVlight:the RFvalue 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 250µg of USP Benzothiadiazine Related Compound A RSin each mL.

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

Procedure— Apply 5µLeach of the Standard solutionand of the Test solutionto a suitable thin-layer chromatographic plate (see Thin-Layer Chromatography,under Chromatography á621ñ)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 1in 20solution of sodium nitrite in dilute hydrochloric acid (1in 12),and then with a 1in 1000solution of N-(1-naphthyl)ethylenediamine dihydrochloride in alcohol:after 3minutes,any spot from the Test solution,occurring at the RFvalue 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.

Mobile phase— Prepare a filtered and degassed mixture of 0.05Mmonobasic potassium phosphate and methanol (7:3).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

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

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

Assay preparation— Transfer about 50mg of Trichlormethiazide,accurately weighed,to a 100-mLvolumetric flask,add 8.0mLof Internal standard solution,dilute with methanol to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—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 2.3mLper 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 (5to 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.56for trichlormethiazide and 1.0for methylparaben.Calculate the quantity,in mg,of C8H8Cl3N3O4S2in the portion of Trichlormethiazide taken by the formula: in which Cis the concentration,in mg per mL,of USP Trichlormethiazide RSin the Standard preparation,and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.