A: Transfer the contents of a number of Capsules, equivalent to about 100 mg of hydrochlorothiazide, to a 20-mesh sieve. Break up any large lumps with the aid of a spatula, and collect the powder that passes through the sieve. [NOTE—Retain the material on the screen for Identification test B.] Transfer the powder that passed through the sieve to a screw-capped, 35-mL centrifuge tube, add 5 mL of solvent hexane, and shake for 5 minutes. Centrifuge, and discard the solvent. To the residue in the centrifuge tube add 10 mL of 1 N sodium hydroxide, shake, and filter, collecting the filtrate in a separator. Wash the filter with 5 mL of water, and collect the washing in the separator. Add 50 mL of ether to the separator, shake for 2 minutes, and allow the phases to separate. Drain the aqueous layer into a beaker, adjust with 6 N hydrochloric acid to a pH of about 2, induce crystallization by scratching the inner surface of the beaker with a glass rod, and allow to stand until crystallization is complete. Collect the crystals on a filter, and dry at 105 for 30 minutes. Grind the crystals to a fine powder: the IR absorption spectrum of a mineral oil dispersion of the powder so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Hydrochlorothiazide RS.

B: Wash the material retained on the screen from Identification test A with a small amount of water, discarding the washings. Transfer the particles remaining on the screen to a glass mortar, add about 5 mL of water, and triturate the mixture with a glass pestle. Transfer the suspension, with the aid of about 10 mL of water, to a 35-mL screw-capped centrifuge tube, add about 1 mL of 1 N sodium hydroxide, and mix. Add about 15 mL of ether, and shake by mechanical means for 5 minutes. Centrifuge the mixture, and transfer as much of the ether layer as possible to a second centrifuge tube. Add 0.1 mL of hydrochloric acid to the ether extract, and shake. Centrifuge, and discard the ether. Add about 15 mL of ether to the residue, and shake by mechanical means for 5 minutes. Centrifuge, and discard the ether layer. Dry the residue in vacuum at 45 for 30 minutes. Grind the crystals to a fine powder: the IR absorption spectrum of a mineral oil dispersion of the powder so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Propranolol Hydrochloride RS.

C: The retention times of the major peaks for propanolol hydrochloride and hydrochlorothiazide in the chromatogram of the Assay preparation correspond to those exhibited in the chromatogram of the Standard preparation, as obtained in the Assay.

pH 1.5 buffer solution, pH 6.8 buffer solution, Media, and Apparatus— Proceed as directed in the test for Drug release under Propranolol Hydrochloride Extended-Release Capsules.

Analytical method— Determine the amounts of hydrochlorothiazide (C7H8ClN3O4S2) and propranolol hydrochloride (C16H21NO2·HCl) dissolved, using the following method.

Standard stock solution A— Prepare a solution of USP Propranolol Hydrochloride RS in dilute hydrochloric acid (1 in 100) having a known concentration of about 0.4 mg per mL.

Standard stock solution B— Dissolve an accurately weighed quantity of USP Hydrochlorothiazide RS in 0.25 N sodium hydroxide to obtain a solution having a concentration of about 25 mg per mL. Quantitatively dilute this solution with water to obtain a solution having a known concentration of about 0.5 mg per mL.

Standard solution— Prepare, by combining aliquots of Standard stock solutions A and B, and diluting with dilute hydrochloric acid (1 in 100), solutions bracketing the expected concentration of the samples at the various time points.

Times: 30 minutes; 1.5, 4, 8, 14, and 24 hours.

Procedure— Use an automatic analyzer consisting of a liquid sampler, a proportioning pump, two UV spectrophotometers, and a manifold consisting of the components illustrated in the diagram under Automated Methods of Analysis 16. Start the sampler and conduct determinations at a rate of 30 per hour, using a ratio of about 1:1 for the sample to wash time. Calculate the amounts of C7H8ClN3O4S2 and C16H21NO2·HCl dissolved by comparison with the Standard solution.

Tolerances (Hydrochlorothiazide)— Use the Acceptance Table under Dissolution 711. Not less than 80% (Q) of the labeled amount of C7H8ClN3O4S2 is dissolved in 30 minutes.

Tolerances (Propranolol Hydrochloride)— The percentages of the labeled amount of C16H21NO2·HCl dissolved at the times specified conform to Acceptance Table 1 under Drug Release 724.

pH 1.5 Buffer solution, pH 6.8 Buffer solution, Media, and Apparatus— Proceed as directed in the test for Dissolution under Propranolol Hydrochloride Extended-Release Capsules.

Analytical method— Determine the amounts of hydrochlorothiazide (C7H8ClN3O4S2) and propranolol hydrochloride (C16H21NO2·HCl) dissolved, using the following method.

Standard stock solution A— Prepare a solution of USP Propranolol Hydrochloride RS in dilute hydrochloric acid (1 in 100) having a known concentration of about 0.4 mg per mL.

Standard stock solution B— Dissolve an accurately weighed quantity of USP Hydrochlorothiazide RS in 0.25 N sodium hydroxide to obtain a solution having a concentration of about 25 mg per mL. Quantitatively dilute this solution with water to obtain a solution having a known concentration of about 0.5 mg per mL.

Standard solution— Prepare, by combining aliquots of Standard stock solutions A and B, and diluting with dilute hydrochloric acid (1 in 100), solutions bracketing the expected concentration of the samples at the various time points.

Times: 30 minutes; 1.5, 4, 8, 14, and 24 hours.

Procedure— Use an automatic analyzer consisting of a liquid sampler, a proportioning pump, two UV spectrophotometers, and a manifold consisting of the components illustrated in the diagram under Automated Methods of Analysis 16. Start the sampler and conduct determinations at a rate of 30 per hour, using a ratio of about 1:1 for the sample to wash time. Calculate the amounts of C7H8ClN3O4S2 and C16H21NO2·HCl dissolved by comparison with the Standard solution.

Tolerances (Hydrochlorothiazide)— Use Acceptance Table 1. Not less than 80% (Q) of the labeled amount of C7H8ClN3O4S2 is dissolved in 30 minutes.

Tolerances (Propranolol Hydrochloride)— The percentages of the labeled amount of C16H21NO2·HCl dissolved at the times specified conform to Acceptance Table 2.

(Official April 1, 2006)

Procedure for content uniformity—

Tetrabutylammonium hydroxide solution, Buffer, Mobile phase, Standard preparation, and Chromatographic system— Prepare as directed in the Assay.

Standard solution— Transfer about 25 mg of USP Benzothiadiazine Related Compound A RS, accurately weighed, to a 200-mL volumetric flask, add 30 mL of methanol, and swirl to dissolve. Dilute with Buffer to volume, and mix. Dilute an accurately measured volume of this solution quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.5 µg per mL.

Test solution— Use the Assay preparation prepared as directed in the Assay.

Procedure— Proceed as directed for Procedure in the Assay, except to inject equal volumes (about 50 µL) of the Standard solution and the Test solution. Calculate the percentage of benzothiadiazine related compound A in the Capsules 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; N is the number of Capsules taken to prepare the Test solution; L is the labeled amount, in mg, of hydrochlorothiazide in each Capsule taken; and rU 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.

(Official January 1, 2007)

Tetrabutylammonium hydroxide solution— Use a suitable aqueous or methanolic solution having a known concentration of tetrabutylammonium hydroxide.

Buffer— Dissolve 31.25 g of monobasic potassium phosphate in 500 mL of water in a 1000-mL volumetric flask. Add 18.75 mL of phosphoric acid, mix, and add a volume of Tetrabutylammonium hydroxide solution equivalent to about 13 g of tetrabutylammonium hydroxide. Dilute with water to volume, and mix. Dilute 100 mL of this solution with water to obtain 1000 mL of solution, adjusting, if necessary, with phosphoric acid or 10 N potassium hydroxide to a pH of 2.4 ± 0.1.

Mobile phase— Prepare a suitable mixture of Buffer and methanol (850:150). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard hydrochlorothiazide stock solution— Transfer about 25 mg of USP Hydrochlorothiazide RS, accurately weighed, to a 100-mL volumetric flask, add 15 mL of methanol, and sonicate for 5 minutes, adding ice to the bath, if necessary, to maintain the temperature at not more than 20. Dilute with Buffer to volume, and mix. Use this solution within 3 days.

Standard propranolol hydrochloride stock solution— Dissolve an accurately weighed quantity of USP Propranolol Hydrochloride RS in Mobile phase to obtain a solution having a known concentration of about 0.25J mg per mL, J being the ratio of the labeled quantity, in mg, of propranolol hydrochloride to the labeled quantity, in mg, of hydrochlorothiazide per Capsule.

Standard preparation— Transfer 5.0 mL of Standard hydrochlorothiazide stock solution and 5.0 mL of Standard propranolol hydrochloride stock solution to a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix. This solution contains about 50 µg of hydrochlorothiazide and 50J µg of propranolol hydrochloride per mL. Use this solution within 3 days.

Assay preparation— Carefully open an accurately counted number of Capsules, equivalent to about 500 mg of hydrochlorothiazide, and transfer the contents and the Capsule shells to a 500-mL volumetric flask. Add 5.0 mL of water to the flask, and allow to stand for 5 minutes. Dilute with methanol to volume, mix, and sonicate for 10 minutes, adding ice to the bath, if necessary, to maintain the temperature at not more than 20. Remove the flask from the bath, and shake it occasionally for 1 hour. Centrifuge a portion of the contents of the flask, if necessary, to obtain a clear solution. Transfer 5.0 mL of the clear solution to a 100-mL volumetric flask, add 10.0 mL of methanol, dilute with Buffer to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4-mm × 15-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency determined from the propranolol peak is not less than 2500 theoretical plates when calculated by the formula: the tailing factor for the hydrochlorothiazide and propranolol peaks is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. The retention time for propranolol is between 12 and 25 minutes; and the relative retention times are about 0.25 for benzothiadiazine related compound A, 0.4 for hydrochlorothiazide, and 1.0 for propranolol. Calculate the quantities, in mg, of hydrochlorothiazide (C7H8ClN3O4S2) and propranolol hydrochloride (C16H21NO2·HCl) in each Capsule taken by the same formula: in which C is the concentration, in µg per mL, ofUSP Propranolol Hydrochloride RS or USP Hydrochlorothiazide RS in the Standard preparation; N is the number of Capsules taken to prepare the Assay preparation; and rU and rS are the peak responses of the corresponding analyte obtained from the Assay preparation and the Standard preparation, respectively.