A: Thin-Layer Chromatographic Identification Test 201—

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

Medium: pH 6.8 phosphate buffer; 1000 mL.

Apparatus 2: 50 rpm.

Time: 30 minutes.

Procedure— Determine the amounts of valsartan (C24H29N5O3) and hydrochlorothiazide (C7H8ClN3O4S2) dissolved by employing UV absorption at the wavelengths of maximum absorbance at about 250 nm for valsartan and at about 272 nm for hydrochlorothiazide on portions of the solution under test passed through a 1-µm glass fiber filter, diluted with Medium if necessary, using a 0.2-cm quartz cell. Calculate the amount of valsartan (C24H29N5O3) dissolved, in percentage, by the formula: Calculate the amount of hydrochlorothiazide (C7H8ClN3O4S2) dissolved, in percentage, by the formula: in which AT1 is the absorbance of the solution under test at 272 nm; AT2 is the absorbance of the solution under test at 250 nm; A1%V272nm is the absorptivity (1%, 0.2 cm, 272 nm) of valsartan in Medium; A1% V250nm is the absorptivity (1%, 0.2 cm, 250 nm) of valsartan in Medium; A1%H272 nm is the absorptivity (1%, 0.2 cm, 272 nm) of hydrochlorothiazide in Medium; A1%H250nm is the absorptivity (1%, 0.2 cm, 250 nm) of hydrochlorothiazide in Medium.

Tolerances— Not less than 80% (Q) of the labeled amounts of C24H29N5O3 and C7H8ClN3O4S2 is dissolved in 30 minutes.

procedure for content uniformity—

Diluent, Solution A, Solution B, and Mobile phase— Prepare as directed in the Assay.

Standard stock solution— Dissolve accurately weighed quantities of USP Benzothiadiazine Related Compound A RS, USP Hydrochlorothiazide RS, USP Valsartan RS, and USP Valsartan Related Compound B RS in Diluent to obtain a solution having known concentrations of about 0.03 mg per mL, 0.06 mg per mL, 0.08 mg per mL, and 0.2 mg per mL, respectively.

Resolution solution— Dilute 5.0 mL of Standard stock solution with Diluent to 100.0 mL, and mix.

Standard solution— Dilute 10.0 mL of the Resolution solution with Diluent to 100.0 mL, and mix.

Test solution— Use the Assay preparation as specified.

Chromatographic system— Prepare as directed in the Assay. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between valsartan related compound B and valsartan, and between benzothiadiazine related compound A and hydrochlorothiazide is not less than 1.4. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation, determined from the valsartan and hydrochlorothiazide peaks, for replicate injections is not more than 10.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks, disregarding the peak, if any, with a retention time of about 22 minutes. Calculate the quantity, in mg, of each impurity in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Benzothiadiazine Related Compound A RS, or the relevant USP Reference Standard (when determining the quantity of other impurities) in the Standard solution; and rU and rS are the corresponding peak responses obtained from the Test solution and the Standard solution, respectively: not more than 1.0% of benzothiadiazine related compound A is found; not more than 0.2% of any other impurity, excluding valsartan related compound A, is found; and not more than 1.3% of total impurities, excluding valsartan related compound A, is found. [note—Valsartan related compound A is the enantiomer of valsartan and coelutes with valsartan in this test.]

Diluent— Prepare a mixture of acetonitrile and water (1:1).

Solution A— Prepare a filtered and degassed mixture of water, acetonitrile, and trifluoroacetic acid (90:10:0.1).

Solution B— Prepare a filtered and degassed mixture of acetonitrile, water, and trifluoroacetic acid (90:10:0.1).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer about 12.5 mg of USP Hydrochlorothiazide RS, accurately weighed, to a 200-mL volumetric flask. Add about 12.5J mg of USP Valsartan RS, accurately weighed, J being the ratio of the labeled amount, in mg, of valsartan to the labeled amount, in mg, of hydrochlorothiazide per Tablet. Add about 100 mL of Diluent, sonicate for 15 minutes, dilute with Diluent to volume, and mix. Transfer 25.0 mL of this solution to a 50-mL volumetric flask, dilute with Diluent to volume, and mix. Quantitatively dilute a volume of this solution with Diluent to obtain a solution having a known concentration of about 0.2 mg of USP Valsartan RS per mL.

Assay preparation— Transfer a number of Tablets, equivalent to about 62.5 mg of hydrochlorothiazide, to a 250-mL volumetric flask. Add 5 mL of water, and allow to stand for 5 minutes. Then add about 100 mL of Diluent, sonicate for 15 minutes, and shake for 30 minutes. Dilute with Diluent to volume, mix, and centrifuge a portion of this solution at 3000 rpm. Dilute 25.0 mL of the clear supernatant with Diluent to 200.0 mL, and mix (Solution 1). [note—Retain a portion of Solution 1 to use as the Test solution in the test for Related compounds.] Dilute an accurately measured volume of Solution 1 with Diluent to obtain a solution containing about 0.2 mg of valsartan per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 265-nm detector and a 3.0-mm × 12.5-cm column that contains 5-µm packing L1. The flow rate is about 0.4 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Separately calculate the quantities, in mg, of valsartan (C24H29N5O3) and hydrochlorothiazide (C7H8ClN3O4S2) in the portion of Tablets taken by the formula: in which L is the labeled quantity, in mg, of the relevant analyte in each Tablet; CS is the concentration, in mg per mL, of the appropriate USP Reference Standard in the Standard preparation; CU is the concentration, in mg per mL, of the corresponding analyte in the Assay preparation, based on the labeled quantity per Tablet and the extent of dilution; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.