A: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay for enalapril maleate.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay for hydrochlorothiazide.

Medium: water; 900 mL.

Apparatus 2: 50 rpm.

Time: 30 minutes.

(TC/D)(A320 – A360)U /(A320 – A360)S

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 30 minutes.

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 preparation prepared as directed in the Assay for enalapril maleate.

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

Enalapril diketopiperazine solution— Carefully place about 20 mg of USP Enalapril Maleate RS in a 100-mL beaker 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 5 to 10 minutes 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 50 mL of acetonitrile, and sonicate for a few minutes to dissolve. The solution typically contains, in each mL, between 0.2 mg and 0.4 mg of enalapril diketopiperazine.

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

Chromatographic system (see Chromatography 621)— 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 1.5 mL per 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 1.0 for 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 700 theoretical plates for enalapril, 1500 for enalaprilat, and 1500 for 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 50 µL) of the Standard solution and the Test solution into 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 C is the concentration, in µg per mL, of enalaprilat in the Standard solution; and rU and rS are the peak responses of enalaprilat obtained from the Test solution and the Standard solution, respectively.

0.2C(rU / rS)

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

Mobile phase— Prepare a degassed and filtered solution of Buffer solution and acetonitrile (6:4). Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

Chromatographic system (see Chromatography 621)— 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 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 700 theoretical 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 50 µL) of the Assay preparation and the Standard preparation into 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 C is the concentration, in µg per mL, of USP Enalapril Maleate RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.

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

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (9:1). Make adjustments if necessary (see System Suitability under Chromatography 621).

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

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

Chromatographic system (see Chromatography 621)— 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 2.5 mL per 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 1000 theoretical 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 50 µL) of the Assay preparation and the Standard preparation into 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 C is the concentration, in µg per mL, of USP Hydrochlorothiazide RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.