Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 2: 50rpm.

Time: 20minutes.

Procedure— Determine the amount of timolol maleate (C13H24N4O3S·C4H4O4)dissolved,employing the following procedure:Prepare a Standard solution of USP Timolol Maleate RSin 0.1Nhydrochloric acid having a known concentration of about 11µg per mL.Filter a portion of the solution under test,and transfer 10.0mLof the clear filtrate to a suitable separator.Transfer 10.0mLeach of the Standard solution and 0.1Nhydrochloric acid,to provide the blank,to individual separators,and treat each of three separators as follows:Add 20.0mLof ethyl acetate,mix for 1minute,allow the phases to separate,and filter the aqueous layer into a suitable vessel,retaining the ethyl acetate layer from the solution under test for the hydrochlorothiazide determination.Determine the amount of C13H24N4O3S·C4H4O4dissolved from UVabsorbances of the aqueous layer from the solution under test at the wavelength of maximum absorbance at about 293nm in comparison with the aqueous layer from the Standard solution.

Tolerances— Not less than 80%(Q)of each of the labeled amounts of C13H24N4O3S·C4H4O4and C7H8ClN3O4S2,respectively,is dissolved in 20minutes.

pH3.0Bufferand Mobile phase— Proceed as directed underAssay.

Standard solution— Dissolve an accurately weighed quantity of USP Benzothiadiazine Related Compound A RSin methanol to obtain a solution having a known concentration of about 0.5mg per mL.Transfer an accurately measured volume of this solution,and dilute quantitatively,and stepwise if necessary,withMobile phase to obtain a solution having a known concentration of about 0.5µg per mL.

Test solution— Use theAssay preparation prepared as directed in theAssay.

Chromatographic system— Proceed as directed underAssay,except to chromatograph theStandard solution:the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 50µL)of theStandard solution and theTest solution into the chromatograph,record the chromatograms,and measure the peak areas.Calculate the percentage of benzothiadiazine related compound Ain the portion of Tablets taken by the formula: in whichCis the concentration in µg per mL,of USP Benzothiadiazine Related Compound A RSin theStandard solution;Lis the amount,in mg,of hydrochlorothiazide in the portion of Tablets taken,based on the labeled amount;andrUandrSare the peak areas of benzothiadiazine related compound Aobtained from theTest solution andStandard solution,respectively:not more than 1.0%is found.

pH3.0phosphate buffer— Dissolve 13.6g of monobasic potassium phosphate in 100mLof water,adjust with phosphoric acid to a pHof 3.0±0.05,and filter.

Mobile phase— Prepare a suitable filtered and degassed mixture of water,acetonitrile,methanol,andpH3.0phosphate buffer (38:8:2:2),making adjustments if necessary (seeSystem SuitabilityunderChromatography á621ñ).

Standard preparation— Transfer about 50mg of USP Timolol Maleate RS,accurately weighed,to a 500-mLvolumetric flask.Add 50Jmg of USP Hydrochlorothiazide RS,accurately weighed,Jbeing the ratio of the labeled amount,in mg,of hydrochlorothiazide to the labeled amount,in mg,of timolol maleate per Tablet.Add 50mLof 0.05Mmonobasic sodium phosphate,and 125mLof acetonitrile,sonicate for 4minutes,dilute with water to volume,and mix.Pipet 5mLinto a 25-mLvolumetric flask,dilute with acetonitrile solution (1in 10)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 20mg of timolol maleate,to a 1-liter volumetric flask,add about 100mLof 0.05Mmonobasic sodium phosphate,125mLof acetonitrile,and 100mLof water,and mix by mechanical means.Allow to stand for 16hours,dilute with water to volume,mix,and filter.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 295-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph replicate injections of theStandard preparation,and record the peak responses as directed forProcedure:the relative standard deviation is not more than 1.5%;and the resolution,R,between hydrochlorothiazide and timolol maleate is not less than 4.0.

Procedure— Separately inject equal volumes (about 50µL)of theStandard preparation and the Assay preparationinto the chromatograph by means of a suitable microsyringe or sampling valve,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.5for benzothiadiazine related compound A,0.6for hydrochlorothiazide,and 1.0for timolol maleate.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;and rUand rSare the responses of the hydrochlorothiazide peak obtained from theAssay preparationand theStandard preparation,respectively.Calculate the quantity,in mg,of timolol maleate (C17H28N4O7S)by the same formula,changing the terms to refer to timolol maleate.