A: Transfer a quantity of powdered Tablets, equivalent to 1 mg of reserpine, to a stoppered, 50-mL centrifuge tube. Add 20 mL of citric acid solution (1 in 50), and shake until the powder is suspended. Extract the mixture with two 20-mL portions of chloroform, centrifuge, and withdraw the chloroform, filtering each extract through a pledget of cotton into a 50-mL volumetric flask. Dilute with chloroform to volume, and mix: the UV absorption spectrum of the solution exhibits maxima and minima at the same wavelengths as that of a similar solution of USP Reserpine RS, concomitantly measured (presence of reserpine).

B: Transfer a quantity of powdered Tablets, equivalent to about 50 mg of hydrochlorothiazide, to a test tube containing 10 mL of acetone, agitate for 5 minutes, and centrifuge. Use the clear supernatant as the Test solution. Separately apply 10 µL each of the Test solution and a Standard solution of USP Hydrochlorothiazide RS in acetone containing 5 mg per mL to a thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture and previously washed with methanol. Develop the chromatogram in a solvent system consisting of a mixture of ethyl acetate and isopropyl alcohol (17:3) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, air-dry, and examine under short-wavelength UV light: the RF value of the principal spot in the chromatogram of the Test solution corresponds to that obtained from the Standard solution (presence of hydrochlorothiazide).

Medium: mixture of 0.1 N hydrochloric acid and n-propyl alcohol (3:2); 900 mL.

Apparatus 2: 50 rpm.

Times: 45 minutes, 60 minutes.

Determination of dissolved reserpine—

Determination of dissolved hydrochlorothiazide—

Procedure for content uniformity for reserpine—

Procedure for content uniformity for hydrochlorothiazide—

Standard solution— Accurately weigh 25 mg of USP Benzothiadiazine Related Compound A RS, dissolve in 5 mL of methanol contained in a 100-mL volumetric flask, dilute with water to volume, and mix. Pipet 4 mL of this solution into a 100-mL volumetric flask, dilute with water to volume, and mix.

Test solution— Transfer a portion of finely powdered Tablets, accurately weighed and equivalent to about 100 mg of hydrochlorothiazide, to a 100-mL volumetric flask, and add 20 mL of methanol and 20 mL of water. Shake continuously for 5 to 10 minutes, dilute with water to volume, mix, and filter. Use the filtrate as the Test solution.

Procedure— Pipet 5 mL each of the Standard solution and the Test solution into separate, 50-mL volumetric flasks. Pipet 5 mL of water into a third 50-mL volumetric flask to provide the blank. To each flask add 1 mL of freshly prepared sodium nitrite solution (1 in 100) and 5 mL of dilute hydrochloric acid (1 in 12), and allow to stand for 5 minutes. Add 2 mL of ammonium sulfamate solution (1 in 50), shake vigorously, allow to stand for 5 minutes, then add 2 mL of freshly prepared disodium chromotropate solution (1 in 100) and 10 mL of sodium acetate TS. Dilute with water to volume, and mix. Concomitantly determine the absorbances of the solutions from the Standard solution and the Test solution in 1-cm cells at the wavelength of maximum absorbance at about 500 nm, with a suitable spectrophotometer, against the blank. The absorbance of the solution from the Test solution does not exceed that of the solution from the Standard solution, corresponding to not more than 1.0% of diazotizable substances.

Standard preparation— Dissolve about 25 mg of USP Reserpine RS, accurately weighed, in 1 mL of chloroform contained in a 50-mL volumetric flask, dilute with methanol to volume, and mix. Dilute a portion of this solution quantitatively and stepwise with chloroform and methanol solution (1:1) to obtain a solution having a known concentration of about 0.2 µg of reserpine per mL.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer to a stoppered, 50-mL centrifuge tube an accurately weighed portion of the powder, equivalent to about 1 mg of reserpine, add 25.0 mL of a mixture of chloroform and methanol solution (1:1), shake by mechanical means for 15 minutes, and centrifuge. Dilute a portion of the clear supernatant quantitatively and stepwise with a mixture of chloroform and methanol solution (1:1) to obtain a solution having a concentration of about 0.2 µg of reserpine per mL.

Procedure— Separately transfer 5.0 mL of the Assay preparation, 5.0 mL of the Standard preparation, and 5.0 mL of a mixture of chloroform and methanol solution (1:1) to provide the reagent blank, respectively, to three 25-mL volumetric flasks. To each flask add 0.5 mL of hydrochloric acid, 1.0 mL of a 3 in 1000 solution of sodium nitrite in dilute methanol (1 in 2), mix, and allow to stand for 30 minutes. Add 1 mL of ammonium sulfamate solution (1 in 20) to each flask, add chloroform and methanol solution (1:1) to volume, mix, and allow to stand for 10 minutes. Concomitantly determine the fluorescence intensities of the solutions in a suitable spectrophotometer arranged to deliver activation radiation at 405 nm and to measure the resultant fluorescence at the emission wavelength of about 500 nm. Calculate the quantity, in mg, of C33H40N2O9 in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Reserpine RS in the Standard preparation, and IU and IS are the fluorescence intensities of the solutions from the Assay preparation and the Standard preparation, respectively.

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrochlorothiazide RS in 0.1 N sodium hydroxide, and dilute quantitatively and stepwise with 0.1 N sodium hydroxide to obtain a solution having a known concentration of about 10 µg of hydrochlorothiazide per mL. Use a freshly prepared solution.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Weigh accurately a portion of the powder, equivalent to about 50 mg of hydrochlorothiazide, and transfer to a 500-mL volumetric flask. Add 200 mL of 0.1 N sodium hydroxide, shake by mechanical means for 15 minutes, dilute with the same solvent to volume, and mix. Filter a portion of the solution through paper, discarding the first 15 mL of the filtrate, and transfer 10.0 mL of the clear filtrate to a 100-mL volumetric flask. Dilute with 0.1 N sodium hydroxide to volume, and mix.

Procedure— Concomitantly determine the absorbances of the Standard preparation and the Assay preparation in 1-cm cells at the wavelength of maximum absorbance, at about 274 nm, with a suitable spectrophotometer, using 0.1 N sodium hydroxide as the blank. Calculate the quantity, in mg, of C7H8ClN3O4S2 in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Hydrochlorothiazide RS in the Standard preparation, and AU and AS are the absorbances of the solutions from the Assay preparation and the Standard preparation, respectively.