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

B: The retention time of the ethambutol peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay for ethambutol hydrochloride.

Medium: 10mMpH6.8sodium phosphate buffer,prepared by dissolving 7g of anhydrous dibasic sodium phosphate in 5Lof water,and adjusting with phosphoric acid to a pHof 6.8;900mL.

Apparatus 2: 100rpm.

Time: 45minutes.

Procedure— Determine the amounts of rifampin (C43H58N4O12),isoniazid (C6H7N3O),pyrazinamide (C5H5N3O),and ethambutol hydrochloride (C10H24N2O2·2HCl)dissolved using filtered portions of the solution under test and by employing the procedures set forth in the Assay for rifampin,isoniazid,and pyrazinamideand the Assay for ethambutol hydrochloride.

Tolerances— Not less than 75%(Q)of the labeled amounts of C43H58N4O12,C6H7N3O,C5H5N3O,and C10H24N2O2·2HCl is dissolved in 45minutes.

Change to read:

Buffer solution— Dissolve 1.4g of anhydrous 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 a 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 for rifampin,isoniazid,and pyrazinamide are about 1.8,0.7,and 1.0,respectively;the resolution,R,between isoniazid and pyrazinamide is not less than 4;the column efficiencies,determined from the rifampin,isoniazid,and pyrazinamide peaks are not less than 50,000theoretical plates,6000theoretical plates,and 10,000theoretical plates,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 preparationinto 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 the appropriate USP Reference Standard in the Standard preparation;and rUand rSare the peak responses of the corresponding analyte obtained from the Standard preparation and the Assay preparation,respectively.

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

Buffer solution— Mix 1.0mLof triethylamine and 1Lof water,and adjust with phosphoric acid to a pHof 7.0.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and Buffer solution(50:50).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Ethambutol Hydrochloride RSin Diluentto obtain a solution having a known concentration of about 0.3mg per mL.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed quantity of the powder,equivalent to about 30mg of ethambutol hydrochloride,to a 100-mLvolumetric flask,and add about 90mLof Diluent.Sonicate for about 10minutes,allow to equilibrate to room temperature,dilute with Diluentto volume,and mix.Pass a portion of this solution through a filter,discarding the first 10mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 200-nm detector and a 4.6-mm ×15-cm column that contains a 5-µm base-deactivated packing L10.The flow rate is about 1.0mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 3;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the quantity,in mg,of ethambutol hydrochloride (C10H24N2O2·2HCl)in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Ethambutol Hydrochloride RSin the Standard preparation;and rUand rSare the ethambutol peak responses obtained from the Assay preparationand the Standard preparation,respectively.