A: Transfer a quantity of powdered Tablets, equivalent to about 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 so obtained 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 chlorothiazide, 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 Chlorothiazide 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 chlorothiazide).

Medium: mixture of pH 8.0 phosphate buffer (see Buffer Solutions in the section Reagents, Indicators, and Solutions) and n-propyl alcohol (3:2); 900 mL.

Apparatus 2: 75 rpm.

Time: 60 minutes.

Determination of dissolved reserpine—

STANDARD PREPARATION —Dissolve about 34 mg of USP Reserpine RS, accurately weighed, in a 50-mL volumetric flask containing 5 mL of chloroform, dilute with n-propyl alcohol to volume (Solution 1), and mix. Pipet 1 mL of Solution 1 into a 50-mL volumetric flask, dilute with n-propyl alcohol to volume (Solution 2), and mix. Pipet 1 mL of Solution 2 into a 100-mL volumetric flask, dilute with Dissolution Medium to volume, and mix to obtain the Standard preparation.

PROCEDURE —Filter a portion of the solution under test through paper, and transfer 5.0 mL of the clear filtrate to a 25-mL volumetric flask. Pipet 5 mL of the Standard preparation into a separate 25-mL volumetric flask. Treat each flask as follows: Add 5 drops of hydrochloric acid and 5 mL of a mixture of water, alcohol, and sulfuric acid (29:20:1), and mix. Add by pipet 5 mL of sodium nitrite solution (3 in 1000), dilute with alcohol to volume, mix, and allow to stand for 30 minutes. Concomitantly determine the fluorescences of the solution under test and the Standard preparation 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 amount of reserpine (C33H40N2O9) dissolved.

Determination of dissolved chlorothiazide—

STANDARD PREPARATION —Transfer about 27 mg of USP Chlorothiazide RS, accurately weighed, to a 50-mL volumetric flask containing 5 mL of methanol, swirl to dissolve, dilute with Dissolution Medium to volume, and mix. Pipet 2 mL of this solution into a 100-mL volumetric flask, dilute with Dissolution Medium to volume, and mix.

PROCEDURE —Determine the amount of chlorothiazide (C7H6ClN3O4S2) dissolved from UV absorbances at the wavelength of maximum absorbance at about 292 nm of filtered portions of the solution under test, suitably diluted with Dissolution Medium, in comparison with the Standard preparation.

Tolerances— Not less than 75% (Q) of the labeled amount of C33H40N2O9 is dissolved in 60 minutes, and not less than 75% (Q) of the labeled amount of C7H6ClN3O4S2 is dissolved in 60 minutes.

Procedure for content uniformity for reserpine—

Standard preparation— Prepare as directed for Standard preparation in the Assay for reserpine.

Test preparation— Weigh 1 Tablet, grind to a fine powder, and transfer to a stoppered, 50-mL centrifuge tube. Add 25.0 mL of chloroform and methanol solution (1:1), shake by mechanical means for 15 minutes, and centrifuge. Pipet 4 mL of the clear supernatant into a 100-mL volumetric flask, dilute with the chloroform and methanol solution to volume, and mix.

Procedure— Proceed as directed for Procedure in the Assay for reserpine. Calculate the quantity, in mg, of C33H40N2O9, in the Tablet taken by the formula: in which Wt is the weight, in mg, of the Tablet, WU is the weight, in mg, of the portion of Tablet taken, T is the labeled quantity, in mg, of reserpine in the Tablet, C is the concentration, in µg per mL, of USP Reserpine RS in the Standard preparation, D is the concentration, in µg per mL, of reserpine in the Test preparation, based upon the labeled quantity per Tablet and the extent of dilution, and IU and IS are the fluorescence intensities of the solutions from the Test preparation and the Standard preparation, respectively.

Procedure for content uniformity for chlorothiazide—

Standard preparation— Prepare as directed for Standard preparation under Assay for chlorothiazide.

Test preparation— Transfer 1 Tablet to a 500-mL volumetric flask, add 300 mL of 0.1 N sodium hydroxide, and sonicate, swirling the flask intermittently, until the Tablet is dissolved. Dilute with 0.1 N sodium hydroxide to volume, mix, and filter, discarding the first 15 mL of the filtrate. Dilute a portion of the clear filtrate quantitatively with 0.1 N sodium hydroxide to obtain a solution having a concentration of about 10 µg of chlorothiazide per mL.

Procedure— Proceed as directed for Procedure in the Assay for chlorothiazide. Calculate the quantity, in mg, of C7H6ClN3O4S2, in the Tablet taken by the formula: in which T is the labeled quantity, in mg, of chlorothiazide in the Tablet, C is the concentration, in µg per mL, of USP Chlorothiazide RS in the Standard preparation, D is the concentration, in µg per mL, of chlorothiazide in the Test preparation, based upon the labeled quantity per Tablet and the extent of dilution, and AU and AS are the absorbances of the solutions from the Test preparation and the Standard preparation, respectively.

(Official January 1, 2007)

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 less than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 1 mg of reserpine, to a stoppered, 50-mL centrifuge tube, add 25.0 mL 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 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 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 Chlorothiazide 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 per mL. Use a freshly prepared solution.

Assay preparation— Weigh and finely powder not less than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 250 mg of chlorothiazide, to a 500-mL volumetric flask, add about 300 mL of 0.1 N sodium hydroxide, and shake by mechanical means for 15 minutes. Dilute with 0.1 N sodium hydroxide to volume, and mix. Filter through paper, discarding the first 15 mL of the filtrate. Pipet 2 mL of the clear filtrate into 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 292 nm, with a suitable spectrophotometer, using 0.1 N sodium hydroxide as the blank. Calculate the quantity, in mg, of C7H6ClN3O4S2, in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Chlorothiazide RS in the Standard preparation, and AU and AS are the absorbances of the Assay preparation and the Standard preparation, respectively.