A: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the Standard preparationobtained as directed in the Assay for enalapril maleate.

B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the Standard preparationobtained as directed in the Assay for hydrochlorothiazide.

Medium: water;900mL.

Apparatus 2: 50rpm.

Time: 30minutes.

Tolerances— Not less than 80%(Q)of the labeled amount of enalapril maleate (C20H28N2O5·C4H4O4)and not less than 60%(Q)of the labeled amount of hydrochlorothiazide (C7H8ClN3O4S2)are dissolved in 30minutes.

Procedure for content uniformity of enalapril maleate—

Procedure for content uniformity of hydrochlorothiazide—

Buffer solution and Mobile phase— Proceed as directed in the Assay for enalapril maleate.

Test solution— Use the Assay preparationprepared as directed in the Assay for enalapril maleate.

Enalaprilat solution— Transfer about 10mg of USP Enalaprilat RS,accurately weighed,to a 25-mLvolumetric flask,dilute with water to volume,and mix.

Enalapril diketopiperazine solution— Carefully place about 20mg of USP Enalapril Maleate RSin a 100-mLbeaker to form a mound on the bottom of the beaker.Place the beaker on a hot plate at about one-half the maximum hot plate temperature setting.Heat for about 5to 10minutes until the solid is melted.Immediately remove the beaker from the hot plate,and allow to cool.[NOTE—Avoid overheating to prevent heat-induced degradation,which would give rise to a brown color.]To the cooled residue in the beaker add 50mLof acetonitrile,and sonicate for a few minutes to dissolve.The solution typically contains,in each mL,between 0.2mg and 0.4mg of enalapril diketopiperazine.

Standard solution— Transfer about 40mg of USP Enalapril Maleate RS,accurately weighed,to a 200-mLvolumetric flask,and dissolve with about 50mLof methanol.Pipet 1mLeach of Enalaprilat solutionand Enalapril diketopiperazine solutioninto the volumetric flask,dilute with Buffer solutionto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L7.The column temperature is maintained at 65,and the flow rate is about 1.5mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.3,0.4,and 1.0for enalaprilat,enalapril diketopiperazine,and enalapril,respectively;the resolution,R,between any of the peaks is not less than 1.3;the column efficiency is not less than 700theoretical plates for enalapril,1500for enalaprilat,and 1500for enalapril diketopiperazine;the tailing factor is not more than 3.5;and the relative standard deviation for replicate injections is not more than 5.0%for enalaprilat and enalapril diketopiperazine and not more than 2.0%for enalapril.

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the peaks.Calculate the quantity,in mg,of enalaprilat (enalapril related compound A)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of enalaprilat in the Standard solution;and rUand rSare the peak responses of enalaprilat obtained from the Test solutionand the Standard solution,respectively.

0.2C(rU/rS),

Buffer solution— Transfer 136mg of monobasic potassium phosphate to a 1000-mLvolumetric flask,add 800mLof water,adjust with phosphoric acid to a pHof 2.0,dilute with water to volume,and mix.

Mobile phase— Prepare a degassed and filtered solution of Buffer solutionand acetonitrile (6:4).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 40mg of USP Enalapril Maleate RS,accurately weighed,to a 200-mLvolumetric flask,add about 50mLof methanol to dissolve,dilute with Buffer solutionto volume,and mix.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 40mg of enalapril maleate,to a 200-mLvolumetric flask,add about 50mLof Buffer solution,and sonicate for 15minutes.Add about 50mLof methanol to the flask,sonicate for 15minutes,dilute with Buffer solutionto volume,mix,and filter.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L7.The column temperature is maintained at 65,and the flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 700theoretical plates;the tailing factor is not more than 3.5;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Assay preparationand the Standard preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of enalapril maleate (C20H28N2O5·C4H4O4)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Enalapril Maleate RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.

Buffer solution— Prepare as directed under Assay for enalapril maleate.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solutionand acetonitrile (9:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 20mg of USP Hydrochlorothiazide RS,accurately weighed,to a 200-mLvolumetric flask,add about 50mLof methanol to dissolve,dilute with Buffer solutionto volume,and mix.

Assay preparation— Prepare as directed for the Assay preparationunder Assay for enalapril maleate,except to weigh a portion of the powdered Tablets equivalent to about 20mg of hydrochlorothiazide.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 310-nm detector and a 4.6-mm ×20-cm column that contains 10-µm packing L7.The column temperature is maintained at 30,and the flow rate is about 2.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the capacity factor,k¢,is not less than 2.0,the column efficiency is not less than 1000theoretical plates,the tailing factor is not more than 2.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Assay preparationand the Standard preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of hydrochlorothiazide (C7H8ClN3O4S2)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydrochlorothiazide RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.