A: Shake a quantity of powdered Tablets,equivalent to about 50mg of chlorthalidone,with 5mLof methanol for 15minutes,and filter.Apply 10µLof this test solution,10µLof a Standard solution of USP Chlorthalidone RSin methanol containing 10mg per mL,and 10µLof a second Standard solution of USP Atenolol RSin methanol containing 10Jmg per mL,Jbeing the ratio of the labeled amount,in mg,of atenolol to the labeled amount,in mg,of chlorthalidone per Tablet to a thin-layer chromatographic plate (seeChromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in a solvent system consisting of a mixture of n-butyl alcohol and 1Nammonium hydroxide (5:1)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,and air-dry.Locate the spots on the plate by viewing under short-wavelength UVlight:the principal spots obtained from the test solution correspond in RFvalue,size,and intensity to those obtained from the respective Standard solutions.

B: The retention times of the major peaks in the chromatogram of theAssay preparation correspond to those in the chromatogram of theStandard preparation,as obtained in theAssay.

Medium: 0.01Nhydrochloric acid;900mL.

Apparatus 2: 50rpm.

Time: 45minutes.

Mobile phaseand Chromatographic system— Prepare as directed in theAssay.

Diluent— Prepare a mixture of 1000mLof acetonitrile and 32mLof 3.6Nsulfuric acid.

Standard solvent— Prepare a mixture of water andDiluent(750:225).

Standard solution— Dissolve accurately weighed quantities of USP Atenolol RSand USP Chlorthalidone RSinStandard solvent to obtain a solution having known concentrations of about 0.00085Lmg of USP Atenolol RSand 0.00085L¢mg of USP Chlorthalidone RSper mL,Land L¢being the labeled amounts,in mg,of atenolol and chlorthalidone,respectively,per Tablet.

Test solution— Mix 10.0mLof the filtered solution under test and 3.0mLofDiluent.

Procedure— Separately inject equal volumes (about 10µL)of theStandard solution and theTest solution into the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantities,in mg,of atenolol (C14H22N2O3)and chlorthalidone (C14H11ClN2O4S)dissolved by the same formula: in which Cis the concentration,in mg per mL,of the appropriate Reference Standard in theStandard solution;and rUand rSare the responses of the corresponding analyte obtained from theTest solution and theStandard solution,respectively.

Tolerances— Not less than 80%(Q)of the labeled amount of atenolol (C14H22N2O3)is dissolved in 45minutes,and not less than 70%(Q)of the labeled amount of chlorthalidone (C14H11ClN2O4S)is dissolved in 45minutes.

Procedure for content uniformity— Proceed as directed in theAssay,except to prepare theAssay preparation as follows.Transfer 1Tablet to a volumetric flask of such capacity that when filled to volume,a concentration of about 0.25mg of chlorthalidone per mLis obtained.Add a mixture of water and acetonitrile (1:1)to about half the capacity of the flask,and shake by mechanical means for not less than 15minutes to disintegrate the Tablet.Dilute with water to volume,and mix.Pass a portion of this solution through a filter having a 0.5-µm or finer porosity,and use the filtrate as theAssay preparation.Calculate the quantities,in mg,of atenolol (C14H22N2O3)and chlorthalidone (C14H11ClN2O4S)in the Tablet taken by the formula: in which Vis the volume,in mL,of the volumetric flask used to prepare theAssay preparation;and the other terms are as defined in theAssay.

Mobile phase— Prepare a mixture of 740mLof water,250mLof acetonitrile,8mLof 3.6Nsulfuric acid,and 930mg of sodium octyl sulfate.Make adjustments if necessary (seeSystem SuitabilityunderChromatography á621ñ).

Standard preparation— Dissolve accurately weighed quantities of USP Atenolol RSand USP Chlorthalidone RSin a mixture of water and acetonitrile (3:1)to obtain a solution having known concentrations of about 0.25mg of USP Chlorthalidone RSand 0.25Jmg of USP Atenolol RSper mL,Jbeing the ratio of the labeled amount,in mg,of atenolol to the labeled amount,in mg,of chlorthalidone per Tablet.

Assay preparation— Transfer 10Tablets to a volumetric flask of such capacity that when filled to volume,a concentration of about 0.5mg of chlorthalidone per mLis obtained.Add a mixture of water and acetonitrile (1:1)to about half the capacity of the flask,and shake by mechanical means for not less than 15minutes to disintegrate the Tablets.Dilute with a mixture of water and acetonitrile (1:1)to volume,and mix.Pass a portion of this stock solution through a filter having a 0.5-µm or finer porosity.Transfer 25.0mLof the clear filtrate to a 50-mLvolumetric flask,dilute with water to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 275-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1.7mLper minute.Chromatograph theStandard preparation,and record the peak responses as directed forProcedure:the relative retention times are about 0.8for atenolol and 1.0for chlorthalidone;the resolution,R,between the atenolol and chlorthalidone peaks is not less than 3.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of theAssay preparation and theStandard preparation into the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantities,in mg,of atenolol (C14H22N2O3)and chlorthalidone (C14H11ClN2O4S)in each Tablet taken by the formula: in which Cis the concentration,in mg per mL,of the appropriate USP Reference Standard in theStandard preparation;Vis the volume,in mL,of the volumetric flask used to prepare the stock solution for theAssay preparation;and rUand rSare the responses for the corresponding analyte obtained from theAssay preparation and theStandard preparation,respectively.