Timolol Maleate and Hydrochlorothiazide Tablets »Timolol Maleate and Hydrochlorothiazide Tablets contain not less than 90.0percent and not more than 110.0percent of the labeled amounts of timolol maleate (C17H28N4O7S)and hydrochlorothiazide (C7H8ClN3O4S2). Packaging and storage— Preserve in well-closed,light-resistant containers. USP Reference standards á11ñ USP Timolol Maleate RS.USP Hydrochlorothiazide RS.USP Benzothiadiazine Related Compound A RS. Identification— Transfer a portion of powdered Tablets,equivalent to about 20mg of timolol maleate,to a suitable centrifuge tube containing about 5mLof methanol.Agitate for 20minutes,and centrifuge.Separately dissolve suitable quantities of USP Timolol Maleate RSand USP Hydrochlorothiazide RSin methanol to obtain Standard solutions each having a concentration of 10mg per mL.Separately apply 3µLof the test solution and of each Standard solution to a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Develop the chromatogram using a solvent system consisting of a mixture of chloroform,methanol,and ammonium hydroxide (80:20:1)until the solvent front has moved about three-fourths of the length of the plate.Air-dry,and examine under short-wavelength UVlight:the RFvalues of the principal spots obtained from the Standard solutions correspond to those obtained from the test solution. Dissolution á711ñ 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. Determine the amount of hydrochlorothiazide (C7H8ClN3O4S2)dissolved,employing the following procedure:Filter the ethyl acetate layer obtained previously from the solution under test through filter paper.Determine the amount of C7H8ClN3O4S2dissolved from UVabsorbances at the wavelength of maximum absorbance at about 270nm of the ethyl acetate layer from the solution under test in comparison with a Standard solution in ethyl acetate having a known concentration of USP Hydrochlorothiazide RS. Tolerances— Not less than 80%(Q)of each of the labeled amounts of C13H24N4O3S·C4H4O4and C7H8ClN3O4S2,respectively,is dissolved in 20minutes. Uniformity of dosage units á905ñ: meet the requirements for Content Uniformitywith respect to timolol maleate and to hydrochlorothiazide. Related compounds— 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: 100(C/L)(rU/rS), 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. Assay — 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: 1000C(rU/rS), 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. Auxiliary Information— Staff Liaison:Andrzej Wilk,Ph.D.,Senior Scientific Associate Expert Committee:(PA5)Pharmaceutical Analysis 5 USP28–NF23Page 1935 Pharmacopeial Forum:Volume No.28(6)Page 1750 Phone Number:1-301-816-8305