A: Place a quantity of finely powdered Tablets, equivalent to about 100 mg of metoprolol tartrate, in a 50-mL volumetric flask. Add about 30 mL of 0.1 N sodium hydroxide, shake for 20 minutes, dilute with 0.1 N sodium hydroxide to volume, and mix. Filter a portion of this mixture, discarding the first 10 mL of the filtrate. Place 25 mL of the filtrate into a separator, and extract with three 15-mL portions of chloroform, filtering the chloroform extracts through chloroform-prerinsed anhydrous sodium sulfate, and combining the extracts in a suitable container. [NOTE—Retain the aqueous layer remaining after extraction for Identification test B.] Evaporate the chloroform to dryness, and place in a freezer to congeal the residue: the IR absorption spectrum of a potassium bromide dispersion of the residue so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Metoprolol Tartrate RS.

B: Pass the aqueous layer from Identification test A through 0.1 N sodium hydroxide-prerinsed cotton. Dilute a portion of the filtrate quantitatively and stepwise with 0.1 N sodium hydroxide to obtain a solution having a concentration of about 0.01 mg of hydrochlorothiazide per mL: the UV absorption spectrum of this solution exhibits maxima and minima at the same wavelengths as a Standard solution prepared as follows. Dissolve 25 mg of USP Hydrochlorothiazide RS in 50 mL of 0.1 N sodium hydroxide in a separator, and extract with three 15-mL portions of chloroform. Discard the chloroform extracts, and pass the aqueous solution through 0.1 N sodium hydroxide-prerinsed cotton. Pipet 2 mL of the filtrate into a 100-mL volumetric flask, dilute with 0.1 N sodium hydroxide to volume, and mix.

Medium: simulated gastric fluid TS (without enzyme); 900 mL.

Apparatus 1: 100 rpm.

Time: 30 minutes.

Determination of dissolved metoprolol tartrate— Remove about 125 mL of the solution under test, allow to cool to room temperature, and filter, discarding the first 25 mL of the filtrate. [NOTE—Retain about 30 mL of the remaining filtrate of the solution under test for the Determination of dissolved hydrochlorothiazide.] If necessary, quantitatively dilute a portion of the filtrate with fresh Dissolution Medium to obtain a solution having a concentration of about 0.05 mg of metoprolol tartrate per mL. Transfer to separate separators 50.0 mL of the filtrate, 50.0 mL of a Standard solution in Dissolution Medium having a known concentration of about 0.05 mg of USP Metoprolol Tartrate RS per mL, and 50.0 mL of Dissolution Medium to provide the blank. Add 10 mL of 2.5 N sodium hydroxide to each separator, and extract each with three 15-mL portions of chloroform, filtering the chloroform extracts through pledgets of chloroform-prerinsed glass wool into individual 50-mL volumetric flasks. Dilute the contents of each flask with chloroform to volume, and mix. Determine the absorbances of the solutions from the filtrate and the Standard solution in 2-cm cells at the wavelength of maximum absorbance at about 276 nm, with a suitable spectrophotometer, using the solution from the blank to set the instrument. Calculate the quantity, in mg, of (C15H25NO3)2·C4H6O6 dissolved by the formula: in which C is the concentration, in mg per mL, of USP Metoprolol Tartrate RS in the Standard solution; f is the dilution factor for the solution from the filtrate; and AU and AS are the absorbances of the solution from the filtrate and of the Standard solution, respectively.

Determination of dissolved hydrochlorothiazide— Filter a portion of the filtrate retained from the Determination of dissolved metoprolol tartrate through a filter of 0.8 µm or finer porosity, discarding the first 5 mL of the filtrate. If necessary, quantitatively dilute a portion of the filtrate with fresh Dissolution Medium to obtain a solution having a concentration of about 0.03 mg of hydrochlorothiazide per mL. Prepare a Standard solution in Dissolution Medium having a known concentration of about 0.03 mg of USP Hydrochlorothiazide RS per mL. Determine the absorbances of these solutions in 2-cm cells at the wavelength of maximum absorbance at about 316 nm, using Dissolution Medium as the blank. Calculate the quantity, in mg, of C7H8ClN3O4S2 dissolved by the formula: in which C is the concentration, in mg per mL, of USP Hydrochlorothiazide RS in the Standard solution; f is the dilution factor for the solution from the filtrate; and AU and AS are the absorbances of the solution from the filtrate and of the Standard solution, respectively.

Tolerances— Not less than 80% (Q) of the labeled amount of metoprolol tartrate (C15H25NO3)2·C4H6O6 and not less than 80% (Q) of the labeled amount of hydrochlorothiazide (C7H8ClN3O4S2) are dissolved in 30 minutes.

Procedure for content uniformity of metoprolol tartrate— Transfer 1 Tablet to a suitable volumetric flask, add 0.1 N hydrochloric acid to about 60% of the nominal volume, sonicate for 15 minutes, and shake by mechanical means for 30 minutes. Dilute with 0.1 N hydrochloric acid to volume to obtain a final solution having a concentration of approximately 1000 µg per mL. Mix, and filter, discarding the first 20 mL of the filtrate. Pipet 10 mL of the filtrate, 10 mL of a Standard solution of USP Metoprolol Tartrate RS in the same medium having a known concentration of about 1000 µg per mL, and 10 mL of 0.1 N hydrochloric acid to provide a blank into individual separators. To each separator add 2.0 mL of 2.5 N sodium hydroxide, and extract with three 25-mL portions of chloroform, passing the chloroform extracts through chloroform-prerinsed glass wool into individual 100-mL volumetric flasks. Dilute with chloroform to volume, and mix. Determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 276 nm, with a suitable spectrophotometer, using the blank to set the instrument. Calculate the quantity, in mg, of (C15H25NO3)2·C4H6O6 in the Tablet by the formula: in which T is the labeled quantity, in mg, of metoprolol tartrate in the Tablet; C is the concentration, in µg per mL, of USP Metoprolol Tartrate RS in the Standard solution; and AU and AS are the absorbances of the solution from the Tablet and the Standard solution, respectively.

Procedure for content uniformity of hydrochlorothiazide— Transfer 1 Tablet to a 100-mL volumetric flask containing 15 mL of water, and shake by mechanical means for 15 minutes. Add 60 mL of methanol, sonicate for 5 minutes, and shake by mechanical means for 30 minutes. Dilute with methanol to volume, and mix. Centrifuge 40 mL of this suspension. Dilute an accurately measured portion of the supernatant quantitatively with methanol to obtain a solution having a concentration of about 0.05 mg of hydrochlorothiazide per mL. Filter a portion of this solution through a 0.5-µm porosity filter, discarding the first few mL of the filtrate. Use the filtrate as the test solution. Transfer about 25 mg of USP Hydrochlorothiazide RS, accurately weighed, to a 100-mL volumetric flask containing 15 mL of water, dilute with methanol to volume, and mix. Transfer 10.0 mL of this solution to a 50-mL volumetric flask, dilute with methanol to volume, and mix (Standard solution). Concomitantly determine the absorbances of the test solution and the Standard solution at the wavelength of maximum absorbance at about 316 nm, with a suitable spectrophotometer, using methanol as the blank. Calculate the quantity, in mg, of hydrochlorothiazide (C7H8ClN3O4S2) in the Tablet taken by the formula: in which L is the labeled quantity, in mg, of hydrochlorothiazide in the Tablet; C is the concentration, in mg per mL, of USP Hydrochlorothiazide RS in the Standard solution; D is the concentration, in mg per mL, of hydrochlorothiazide in the test solution based on the labeled quantity in the Tablet and the extent of dilution; and AU and AS are the absorbances of the Test solution and the Standard solution, respectively.

Standard solution— Weigh accurately 5 mg of USP Benzothiadiazine Related Compound A RS, and dissolve in 2 mL of methanol contained in a 50-mL volumetric flask. Dilute with water to volume, and mix. Pipet 5 mL of the resulting solution into a 100-mL volumetric flask, add 20 mL of methanol, dilute with water to volume, and mix. Each mL of Standard solution contains 5 µg of the Reference Standard.

Test solution— Transfer a portion of the powdered Tablets prepared for the Assay, accurately weighed and equivalent to 50 mg of hydrochlorothiazide, to a 100-mL volumetric flask containing a mixture of 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 immediately after preparation.

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 a 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 10), and allow to stand for 5 minutes. Add 2 mL of ammonium sulfamate solution (1 in 50), allow to stand for 5 minutes with frequent swirling, 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 obtained from the Standard solution and the Test solution at 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.

(Official January 1, 2007)

Mobile phase and Chromatographic system— Proceed as directed in the Assay under Metoprolol Tartrate Injection.

Internal standard solution— Dissolve USP Oxprenolol Hydrochloride RS in freshly prepared Mobile phase to obtain a solution containing about 360 µg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Metoprolol Tartrate RS in 0.1 N hydrochloric acid to obtain a stock solution having a known concentration of about 1000 µg per mL. Transfer 10.0 mL of this stock solution to a suitable separator, add 2.0 mL of 2.5 N sodium hydroxide, and extract with three 25-mL portions of chloroform. Pass the chloroform extracts through chloroform-prerinsed glass wool into a round-bottom flask, and evaporate on a rotary evaporator under vacuum to dryness. Add 20.0 mL of Internal standard solution to the flask, sonicate for 3 minutes, and gently swirl to dissolve the residue in the flask.

Assay preparation— Weigh and finely powder not less than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 mg of metoprolol tartrate, to a 100-mL volumetric flask, add 60 mL of 0.1 N hydrochloric acid, heat on a steam bath for 3 minutes, and sonicate for 5 minutes. Shake for 30 minutes. Allow the solution to cool to room temperature, dilute with 0.1 N hydrochloric acid to volume, and mix. Filter a portion of this solution, discarding the first 20 mL of the filtrate. Transfer 10.0 mL of the filtrate to a separator, add 2.0 mL of 2.5 N sodium hydroxide, and extract with three 25-mL portions of chloroform. Pass the chloroform extracts through chloroform-prerinsed glass wool into a round-bottom flask, and evaporate on a rotary evaporator under vacuum to dryness. Add 20.0 mL of Internal standard solution to the flask, sonicate for 3 minutes, and gently swirl to dissolve the residue in the flask. Filter a portion of this solution through a filter of 0.5 µm or finer porosity, discarding the first few mL of the filtrate. Use the filtrate as the Assay preparation.

Procedure— Proceed as directed for Procedure in the Assay under Metoprolol Tartrate Injection. Calculate the quantity, in mg, of metoprolol tartrate [(C15H25NO3)2·C4H6O6] in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Metoprolol Tartrate RS in the stock solution used to prepare the Standard preparation; and RU and RS are the peak response ratios of metoprolol tartrate to oxprenolol hydrochloride obtained from the Assay preparation and the Standard preparation, respectively.

Mobile phase— [NOTE—Pass the methanol and water through 0.5-µm porosity filters before use.] Dissolve 1.38 g of monobasic sodium phosphate in 780 mL of water, add 220 mL of methanol, and mix. Degas before use. Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Dissolve a quantity of sulfanilamide in methanol to obtain a solution containing about 0.4 mg per mL.

System suitability solution— Dissolve a quantity of USP Benzothiadiazine Related Compound A RS in Internal standard solution to obtain a solution containing about 1 mg per mL. Mix 1 mL of this solution and 4 mL of methanol.

Standard preparation— Transfer about 50 mg of USP Hydrochlorothiazide RS, accurately weighed, to a 100-mL volumetric flask, add 20.0 mL of Internal standard solution, dilute with methanol to volume, and mix.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 25 mg of hydrochlorothiazide, to a 50-mL volumetric flask. Add 10.0 mL of Internal standard solution and 20 mL of methanol, and sonicate for 5 minutes. Shake by mechanical means for 30 minutes, dilute with methanol to volume, and mix. Centrifuge a portion of this solution, and pass a portion of the supernatant through a 0.5-µm porosity filter, discarding the first few mL of the filtrate. Use the filtrate as the Assay preparation.

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 0.6 mL per minute. Chromatograph replicate injections of the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for sulfanilamide and 1.0 for benzothiadiazine related compound A; and the resolution, R, between the peaks is not less than 2.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 4 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 0.6 for sulfanilamide and 1.0 for hydrochlorothiazide. Calculate the quantity, in mg, of hydrochlorothiazide (C7H8ClN3O4S2) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrochlorothiazide RS in the Standard preparation; and RU and RS are the peak response ratios of hydrochlorothiazide to sulfanilamide obtained from the Assay preparation and the Standard preparation, respectively.