A: Thin-Layer Chromatographic Identification Test 201—

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

Medium: 0.1 N hydrochloric acid; 900 mL.

Apparatus 1: 100 rpm.

Time: 45 minutes.

Phosphate buffer solution— Dissolve 15.3 g of dibasic potassium phosphate and 80.0 g of monobasic potassium phosphate into a 1-L volumetric flask, mix, dilute with water to volume, and mix.

Isoniazid standard solution— Accurately weigh about 66 mg of USP Isoniazid RS into a 100-mL volumetric flask. Dissolve in and dilute with 0.1 N hydrochloric acid to volume, and mix.

Standard stock solution— Accurately weigh about 66 mg of USP Rifampin RS into a 200-mL volumetric flask, dissolve in 10 mL of 0.1 N hydrochloric acid, and mix. Add 50.0 mL of Isoniazid standard solution, dilute with 0.1 N hydrochloric acid to volume, and mix. [NOTE—Prepare this solution immediately before the test is performed, and place in the dissolution bath at the start of the test.]

Standard solution— At the end of the test run, transfer a 5.0-mL aliquot of the Standard stock solution and 10.0 mL of Phosphate buffer solution to a 50-mL volumetric flask. Dilute with water to volume, and mix. [NOTE—Analyze the solution immediately, if possible, and if not, within 3 hours after final dilution.]

Test solution— At the end of the test run, withdraw a 25-mL aliquot, and filter, discarding the first 10 mL of the filtrate. Allow to cool for about 10 minutes, and transfer 5.0 mL of the filtrate and 10.0 mL of the Phosphate buffer solution to a 50-mL volumetric flask. Dilute with water to volume, and mix. [NOTE—Analyze the solution immediately, if possible, and if not, within 3 hours after final dilution.]

Mobile phase— Prepare a filtered and degassed mixture of water, Phosphate buffer solution, and methanol (850:100:50). Make adjustments if necessary (see System Suitability under Chromatography 621).

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.0-mm × 30-cm column that contains 10-µm packing L1. The flow rate is about 1.5 mL per minute.

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 isoniazid peaks.

Tolerances— Not less than 75% (Q) of the labeled amount of C43H58N4O12 and not less than 80% (Q) of the labeled amount of C6H7N3O are dissolved in 45 minutes.

(Official January 1, 2007)

Buffer solution— Dissolve 1.4 g of dibasic sodium phosphate in 1 L of water, and adjust with phosphoric acid to a pH of 6.8.

Solution A— Prepare a filtered and degassed mixture of Buffer solution and acetonitrile (96:4).

Solution B— Prepare a filtered and degassed mixture of acetonitrile and Buffer solution (55:45).

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— Dissolve accurately weighed quantities of USP Rifampin RS and USP Isoniazid RS in a mixture of Buffer solution and methanol (96:4) to obtain a solution having known concentrations of about 0.16 mg per mL and 0.08 mg per mL, respectively. [NOTE—Use this solution within 10 minutes.]

Assay preparation— Weigh the contents of not fewer than 10 Capsules, mix, and transfer an accurately weighed portion of the powder, equivalent to about 8 mg of isoniazid, to a 100-mL volumetric flask, and add about 90 mL of Buffer solution. Sonicate for about 10 minutes, allow to equilibrate to room temperature, dilute with Buffer solution to volume, and mix. [NOTE—Use this solution within 2 hours.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 238-nm detector and a 4.6-mm × 25-cm column that contains 5-µm base-deactivated packing L1. The flow rate is about 1.5 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 2.6 and 1.0 for rifampin and isoniazid, respectively; the column efficiency is not less than 50,000 and not less than 6,000 theoretical plates for rifampin and isoniazid, respectively; the tailing factors are 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 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the quantity, in mg, of rifampin (C43H58N4O12) and isoniazid (C6H7N3O) in the portion of Capsules taken by the formula: in which C is the concentration, in mg per mL, of USP Rifampin RS, calculated on the dried basis, or of USP Isoniazid RS, as appropriate, in the Standard preparation; and rU and rS are the peak responses obtained from the corresponding analytes obtained from the Assay preparation and the Standard preparation, respectively.