A:Thin-Layer Chromatographic Identification Test á201ñ—

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

Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 1: 100rpm.

Time: 45minutes.

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

Isoniazid standard solution— Accurately weigh about 66mg of USP Isoniazid RSinto a 100-mLvolumetric flask.Dissolve in and dilute with 0.1Nhydrochloric acid to volume,and mix.

Standard stock solution— Accurately weigh about 66mg of USP Rifampin RSinto a 200-mLvolumetric flask,dissolve in 10mLof 0.1Nhydrochloric acid,and mix.Add 50.0mLof Isoniazid standard solution,dilute with 0.1Nhydrochloric 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-mLaliquot of the Standard stock solutionand 10.0mLof Phosphate buffer solutionto a 50-mLvolumetric flask.Dilute with water to volume,and mix.[NOTE—Analyze the solution immediately,if possible,and if not,within 3hours after final dilution.]

Test solution— At the end of the test run,withdraw a 25-mLaliquot,and filter,discarding the first 10mLof the filtrate.Allow to cool for about 10minutes,and transfer 5.0mLof the filtrate and 10.0mLof the Phosphate buffer solutionto a 50-mLvolumetric flask.Dilute with water to volume,and mix.[NOTE—Analyze the solution immediately,if possible,and if not,within 3hours 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 Suitabilityunder Chromatography á621ñ).

Chromatographic system(see Chromatography á621ñ)— 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 1.5mLper minute.

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 isoniazid peaks.

Tolerances— Not less than 75%(Q)of the labeled amount of C43H58N4O12and not less than 80%(Q)of the labeled amount of C6H7N3Oare dissolved in 45minutes.

Change to read:

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

Solution A— Prepare a filtered and degassed mixture of Buffer solutionand 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 Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve accurately weighed quantities of USP Rifampin RSand USP Isoniazid RSin a mixture of Buffer solutionand methanol (96:4)to obtain a solution having known concentrations of about 0.16mg per mLand 0.08mg per mL,respectively.[NOTE—Use this solution within 10minutes.]USP28

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

Chromatographic system (see Chromatography á621ñ)— 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 1.5mLper 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 2.6and 1.0for rifampin and isoniazid,respectively;the column efficiency is not less than 50,000and not less than 6,000theoretical 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 20µL)of the Standard preparationand 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 Cis 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 rUand rSare the peak responses obtained from the corresponding analytes obtained from the Assay preparationand the Standard preparation,respectively.