A: Place a quantity of finely powdered Tablets,equivalent to about 100mg of metoprolol tartrate,in a 50-mLvolumetric flask.Add about 30mLof 0.1Nsodium hydroxide,shake for 20minutes,dilute with 0.1Nsodium hydroxide to volume,and mix.Filter a portion of this mixture,discarding the first 10mLof the filtrate.Place 25mLof the filtrate into a separator,and extract with three 15-mLportions 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 forIdentification testB.]Evaporate the chloroform to dryness,and place in a freezer to congeal the residue:the IRabsorption 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 fromIdentification testAthrough 0.1Nsodium hydroxide-prerinsed cotton.Dilute a portion of the filtrate quantitatively and stepwise with 0.1Nsodium hydroxide to obtain a solution having a concentration of about 0.01mg of hydrochlorothiazide per mL:the UVabsorption spectrum of this solution exhibits maxima and minima at the same wavelengths as a Standard solution prepared as follows.Dissolve 25mg of USP Hydrochlorothiazide RSin 50mLof 0.1Nsodium hydroxide in a separator,and extract with three 15-mLportions of chloroform.Discard the chloroform extracts,and pass the aqueous solution through 0.1Nsodium hydroxide-prerinsed cotton.Pipet 2mLof the filtrate into a 100-mLvolumetric flask,dilute with 0.1Nsodium hydroxide to volume,and mix.

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

Apparatus 1: 100rpm.

Time: 30minutes.

Determination of dissolved metoprolol tartrate— Remove about 125mLof the solution under test,allow to cool to room temperature,and filter,discarding the first 25mLof the filtrate.[NOTE—Retain about 30mLof the remaining filtrate of the solution under test for theDetermination of dissolved hydrochlorothiazide.]If necessary,quantitatively dilute a portion of the filtrate with freshDissolution Medium to obtain a solution having a concentration of about 0.05mg of metoprolol tartrate per mL.Transfer to separate separators 50.0mLof the filtrate,50.0mLof a Standard solution inDissolution Medium having a known concentration of about 0.05mg of USP Metoprolol Tartrate RSper mL,and 50.0mLofDissolution Medium to provide the blank.Add 10mLof 2.5Nsodium hydroxide to each separator,and extract each with three 15-mLportions of chloroform,filtering the chloroform extracts through pledgets of chloroform-prerinsed glass wool into individual 50-mLvolumetric 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 276nm,with a suitable spectrophotometer,using the solution from the blank to set the instrument.Calculate the quantity,in mg,of (C15H25NO3)2·C4H6O6dissolved by the formula: in whichCis the concentration,in mg per mL,of USP Metoprolol Tartrate RSin theStandard solution;f is the dilution factor for the solution from the filtrate;andAUandASare the absorbances of the solution from the filtrate and of theStandard solution,respectively.

Determination of dissolved hydrochlorothiazide— Filter a portion of the filtrate retained from theDetermination of dissolved metoprolol tartratethrough a filter of 0.8µm or finer porosity,discarding the first 5mLof the filtrate.If necessary,quantitatively dilute a portion of the filtrate with freshDissolution Medium to obtain a solution having a concentration of about 0.03mg of hydrochlorothiazide per mL.Prepare aStandard solution inDissolution Medium having a known concentration of about 0.03mg of USP Hydrochlorothiazide RSper mL.Determine the absorbances of these solutions in 2-cm cells at the wavelength of maximum absorbance at about 316nm,usingDissolution Medium as the blank.Calculate the quantity,in mg,of C7H8ClN3O4S2dissolved by the formula: in whichCis the concentration,in mg per mL,of USP Hydrochlorothiazide RSin theStandard solution;f is the dilution factor for the solution from the filtrate;andAUandASare the absorbances of the solution from the filtrate and of theStandard solution,respectively.

Tolerances— Not less than 80%(Q)of the labeled amount of metoprolol tartrate (C15H25NO3)2·C4H6O6and not less than 80%(Q)of the labeled amount of hydrochlorothiazide (C7H8ClN3O4S2)are dissolved in 30minutes.

Procedure for content uniformity of metoprolol tartrate— Transfer 1Tablet to a suitable volumetric flask,add 0.1Nhydrochloric acid to about 60%of the nominal volume,sonicate for 15minutes,and shake by mechanical means for 30minutes.Dilute with 0.1Nhydrochloric acid to volume to obtain a final solution having a concentration of approximately 1000µg per mL.Mix,and filter,discarding the first 20mLof the filtrate.Pipet 10mLof the filtrate,10mLof a Standard solution of USP Metoprolol Tartrate RSin the same medium having a known concentration of about 1000µg per mL,and 10mLof 0.1Nhydrochloric acid to provide a blank into individual separators.To each separator add 2.0mLof 2.5Nsodium hydroxide,and extract with three 25-mLportions of chloroform,passing the chloroform extracts through chloroform-prerinsed glass wool into individual 100-mLvolumetric 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 276nm,with a suitable spectrophotometer,using the blank to set the instrument.Calculate the quantity,in mg,of (C15H25NO3)2·C4H6O6in the Tablet by the formula: in whichTis the labeled quantity,in mg,of metoprolol tartrate in the Tablet;Cis the concentration,in µg per mL,of USP Metoprolol Tartrate RSin theStandard solution;andAUandASare the absorbances of the solution from the Tablet and theStandard solution,respectively.

Procedure for content uniformity of hydrochlorothiazide— Transfer 1Tablet to a 100-mLvolumetric flask containing 15mLof water,and shake by mechanical means for 15minutes.Add 60mLof methanol,sonicate for 5minutes,and shake by mechanical means for 30minutes.Dilute with methanol to volume,and mix.Centrifuge 40mLof this suspension.Dilute an accurately measured portion of the supernatant quantitatively with methanol to obtain a solution having a concentration of about 0.05mg of hydrochlorothiazide per mL.Filter a portion of this solution through a 0.5-µm porosity filter,discarding the first few mLof the filtrate.Use the filtrate as the test solution.Transfer about 25mg of USP Hydrochlorothiazide RS,accurately weighed,to a 100-mLvolumetric flask containing 15mLof water,dilute with methanol to volume,and mix.Transfer 10.0mLof this solution to a 50-mLvolumetric 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 316nm,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 whichLis the labeled quantity,in mg,of hydrochlorothiazide in the Tablet;Cis the concentration,in mg per mL,of USP Hydrochlorothiazide RSin theStandard solution;Dis 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;andAUandASare the absorbances of theTest solution and theStandard solution,respectively.

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

Test solution— Transfer a portion of the powdered Tablets prepared for theAssay,accurately weighed and equivalent to 50mg of hydrochlorothiazide,to a 100-mLvolumetric flask containing a mixture of 20mLof methanol and 20mLof water.Shake continuously for 5to 10minutes,dilute with water to volume,mix,and filter.Use the filtrate as theTest solutionimmediately after preparation.

Procedure— Pipet 5mLeach of theStandard solutionand theTest solutioninto separate,50-mLvolumetric flasks.Pipet 5mLof water into a third 50-mLvolumetric flask to provide a blank.To each flask add 1mLof freshly prepared sodium nitrite solution (1in 100)and 5mLof dilute hydrochloric acid (1in 10),and allow to stand for 5minutes.Add 2mLof ammonium sulfamate solution (1in 50),allow to stand for 5minutes with frequent swirling,then add 2mLof freshly prepared disodium chromotropate solution (1in 100)and 10mLof sodium acetate TS.Dilute with water to volume,and mix.Concomitantly determine the absorbances of the solutions obtained from theStandard solutionand theTest solutionat 500nm,with a suitable spectrophotometer,against the blank.The absorbance of the solution from theTest solutiondoes not exceed that of the solution from theStandard solution,corresponding to not more than 1.0%of diazotizable substances.

Mobile phaseand Chromatographic system— Proceed as directed in theAssayunderMetoprolol Tartrate Injection.

Internal standard solution— Dissolve USP Oxprenolol Hydrochloride RSin freshly preparedMobile phaseto obtain a solution containing about 360µg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Metoprolol Tartrate RSin 0.1Nhydrochloric acid to obtain a stock solution having a known concentration of about 1000µg per mL.Transfer 10.0mLof this stock solution to a suitable separator,add 2.0mLof 2.5Nsodium hydroxide,and extract with three 25-mLportions 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 20.0mLofInternal standard solutionto the flask,sonicate for 3minutes,and gently swirl to dissolve the residue in the flask.

Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 100mg of metoprolol tartrate,to a 100-mLvolumetric flask,add 60mLof 0.1Nhydrochloric acid,heat on a steam bath for 3minutes,and sonicate for 5minutes.Shake for 30minutes.Allow the solution to cool to room temperature,dilute with 0.1Nhydrochloric acid to volume,and mix.Filter a portion of this solution,discarding the first 20mLof the filtrate.Transfer 10.0mLof the filtrate to a separator,add 2.0mLof 2.5Nsodium hydroxide,and extract with three 25-mLportions 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 20.0mLofInternal standard solutionto the flask,sonicate for 3minutes,and gently swirl to dissolve the residue in the flask.Filter a portion of this solution through a filter of 0.5µm or finer porosity,discarding the first few mLof the filtrate.Use the filtrate as theAssay preparation.

Procedure— Proceed as directed forProcedure in theAssay underMetoprolol Tartrate Injection.Calculate the quantity,in mg,of metoprolol tartrate [(C15H25NO3)2·C4H6O6]in the portion of Tablets taken by the formula: in whichCis the concentration,in µg per mL,of USP Metoprolol Tartrate RSin the stock solution used to prepare theStandard preparation;andRUandRSare the peak response ratios of metoprolol tartrate to oxprenolol hydrochloride obtained from theAssay preparationand theStandard preparation,respectively.

Mobile phase— [NOTE—Pass the methanol and water through 0.5-µm porosity filters before use.]Dissolve 1.38g of monobasic sodium phosphate in 780mLof water,add 220mLof methanol,and mix.Degas before use.Make adjustments if necessary (seeSystem SuitabilityunderChromatography á621ñ).

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

System suitability solution— Dissolve a quantity of USP Benzothiadiazine Related Compound A RSinInternal standard solutionto obtain a solution containing about 1mg per mL.Mix 1mLof this solution and 4mLof methanol.

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

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 25mg of hydrochlorothiazide,to a 50-mLvolumetric flask.Add 10.0mLofInternal standard solutionand 20mLof methanol,and sonicate for 5minutes.Shake by mechanical means for 30minutes,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 mLof the filtrate.Use the filtrate as theAssay preparation.

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

Procedure— Separately inject equal volumes (about 4µL)of theStandard preparationand theAssay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.6for sulfanilamide and 1.0for hydrochlorothiazide.Calculate the quantity,in mg,of hydrochlorothiazide (C7H8ClN3O4S2)in the portion of Tablets taken by the formula: in whichCis the concentration,in mg per mL,of USP Hydrochlorothiazide RSin theStandard preparation;andRUandRSare the peak response ratios of hydrochlorothiazide to sulfanilamide obtained from theAssay preparationand theStandard preparation,respectively.