A: Thin-Layer Chromatographic Identification Test á201ñ—

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

Medium: simulated gastric fluid TS,without pepsin;900mL.

Apparatus 1: 100rpm.

Time: 30minutes.

Standard stock solution— Prepare a solution in Mediumhaving known concentrations of about 0.22mg of USP Isoniazid RSand 1.3mg of USP Pyrazinamide RSper mL.Use this solution on the day prepared.

Intermediate standard solution— Transfer about 27mg of USP Rifampin RS,accurately weighed,to a 200-mLvolumetric flask,add 50.0mLof the Standard stock solution,and swirl to dissolve.Dilute with Mediumto volume,and mix.Place this flask into the dissolution bath immediately prior to starting the tablet dissolution.Withdraw the flask from the dissolution bath at the same time that the solutions under test are withdrawn.

Standard solution— Transfer 10.0mLof the Intermediate standard solutionto a 50-mLvolumetric flask,dilute with Mediumto volume,and mix.

Procedure— Transfer 10.0mLof the filtered solution under test to a separate 50-mLvolumetric flask,dilute with Mediumto volume,and mix.Concomitantly determine the UVabsorbances at 475nm of the solution obtained and the Standard solution,using the Mediumas the blank.Calculate the quantity,in mg,of rifampin (C43H58N4O12)dissolved by the formula: in which Cis the concentration,in mg per mL,of USP Rifampin RSin the Standard solution;and AUand ASare the absorbances of the solution under test and the Standard solution,respectively.

Tolerances— Not less than 80%(Q)of the labeled amount of rifampin (C43H58N4O12)is dissolved in 30minutes.

Mobile phase— Prepare a filtered and degassed mixture of water,1Mmonobasic potassium phosphate,and acetonitrile (860:100:40).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Prepare a solution of isonicotinic acid in Mediumcontaining about 0.125mg per mL.Transfer 10mLof this solution and 4mLof the Standard stock solutionto a 100-mLvolumetric flask containing 15mLof 1Mdibasic potassium phosphate and 30mLof Mobile phase.Dilute with Mobile phaseto volume,and mix.

Standard solution— Transfer 15.0mLof the Intermediate standard solutionto a 100-mLvolumetric flask containing 15mLof 1Mdibasic potassium phosphate and 30mLof Mobile phase.Dilute with Mobile phaseto volume,and mix.This solution may be used for 20hours.

Test solution— Withdraw 60mLof the solution under test,and filter,discarding the first 20mLof the filtrate.Centrifuge the filtrate for 5minutes.Transfer 15.0mLof this solution to a 100-mLvolumetric flask containing 15mLof 1Mdibasic potassium phosphate and 30mLof Mobile phase.Dilute with Mobile phaseto volume,and mix.This solution may be used for 20hours.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×30-cm column that contains packing L44.The flow rate is about 1mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.7for isonicotinic acid,1.0for pyrazinamide,and 1.8for isoniazid;and the resolution,R,between isonicotinic acid and pyrazinamide is not less than 2.5and between pyrazinamide and isoniazid not less than 4.0.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviations determined from the pyrazinamide and isoniazid responses for replicate injections are not more than 1.5%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of isoniazid (C6H7N3O)dissolved by the formula: in which Cis the concentration,in mg per mL,of USP Isoniazid RSin the Standard solution;and rUand rSare the isoniazid peak areas obtained from the Test solutionand the Standard solution,respectively.Calculate the quantity,in mg,of pyrazinamide (C5H5N3O)dissolved by the same formula,except to read “USP Pyrazinamide RS”where “USP Isoniazid RS”is specified,and “pyrazinamide”where “isoniazid”is specified.

Tolerances— Not less than 80%(Q)of the labeled amount of isoniazid (C6H7N3O)and not less than 75%of the labeled amount of pyrazinamide (C5H5N3O)are dissolved in 30minutes.

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 RS,USP Isoniazid RS,and USP Pyrazinamide RSin a mixture of Buffer solutionand methanol (96:4)to obtain a solution having known concentrations of about 0.16mg per mL,0.08mg per mL,and 0.43mg per mL,respectively.[NOTE—Use this solution within 10minutes.]USP28

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed quantity 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 1.8,0.7,and 1.0for rifampin,isoniazid,and pyrazinamide,respectively;the resolution,R,between isoniazid and pyrazinamide is not less than 4;the column efficiency is not less than 50,000,not less than 6,000,and not less than 10,000theoretical plates for rifampin,isoniazid,and pyrazinamide,respectively;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 20µL)of the Standard preparationand the Assay preparation into the chromatograph,record the chromatograms,and measure the peak responses.Calculate the quantities,in mg,of rifampin (C43H58N4O12),isoniazid (C6H7N3O),and pyrazinamide (C5H5N3O)in the portion of Tablets 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 RSor of USP Pyrazinamide 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.