A: Triturate a quantity of Capsule contents,equivalent to about 50mg of rifampin,with 5mLof chloroform,and filter.Apply 3µLeach of the filtrate (test solution)and of a solution of USP Rifampin RSin chloroform containing 10mg per mLto a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in an equilibrated chromatographic chamber containing a solvent system consisting of a mixture of chloroform and methanol (90:10)until the solvent front has moved about one-half of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the red spots on the plate:the RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

B: The chromatogram of the Assay preparationexhibits a major peak for rifampin,the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparation,as obtained in the Assay.

Medium: 0.1Nhydrochloric acid;900mL.

Apparatus 1: 100rpm.

Time: 45minutes.

Procedure— Determine the amount of C43H58N4O12dissolved from absorbances at the wavelength of maximum absorbance at about 475nm on filtered portions of the solution under test,suitably diluted,if necessary,with Dissolution Medium,in comparison with a Standard solution having a known concentration of USP Rifampin RS,calculated on the dried basis,in the same Medium,prepared concomitantly and held in the water bath for the Timespecified.

Tolerances— Not less than 75%(Q)of the labeled amount of C43H58N4O12is dissolved in 45minutes.

Procedure for content uniformity—

Phosphate buffer ,Mobile phase,Solvent mixture,Diluent,Standard preparation,Resolution solution,and Chromatographic system—Proceed as directed in the Assay.

Test preparation— Transfer the contents of 1Capsule to a suitable volumetric flask so that when diluted to volume as directed below,each mLof solution contains about 1.5mg of rifampin.Rinse the Capsule shell with a small quantity of Solvent mixture,and add the washing to the volumetric flask.Add Solvent mixtureuntil the flask is about four-fifths full.Proceed as directed for Assay preparationin the Assay,beginning with “Sonicate for about 5minutes.”

Procedure— Proceed as directed for Procedurein the Assay.Calculate the quantity,in mg,of C43H58N4O12in the Capsule content by the formula: in which Lis the labeled quantity,in mg,of rifampin in the Capsule,Cis the concentration,in mg per mL,of USP Rifampin RS,calculated on the dried basis,in the Standard preparation,Dis the concentration,in mg per mL,of rifampin in the Test preparation,based on the labeled quantity per Capsule and the extent of dilution,and rUand rSare the rifampin peak responses obtained from the Test preparationand the Standard preparation,respectively.

Phosphate buffer— Dissolve 136.1g of monobasic potassium phosphate in about 500mLof water,add 6.3mLof phosphoric acid,dilute with water to 1000mL,and mix (pH3.1±0.1).

Mobile phase— Prepare a suitable mixture of water,acetonitrile,Phosphate buffer,1.0Mcitric acid,and 0.5Msodium perchlorate (510:350:100:20:20),filter through a suitable filter of 0.7µm or finer porosity,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Solvent mixture— Prepare a mixture of acetonitrile and methanol (1:1).

Diluent— Prepare a suitable mixture of water,acetonitrile,1.0Mdibasic sodium phosphate,1.0Mmonobasic potassium phosphate,and 1.0Mcitric acid (640:250:77:23:10).

Standard preparation— Dissolve an accurately weighed quantity of USP Rifampin RSin Solvent mixtureto obtain a solution having a known concentration of about 1.5mg per mL,sonicating for about 30seconds,if necessary,to ensure dissolution.Transfer 10.0mLof this solution to a 50-mLvolumetric flask,dilute with acetonitrile to volume,and mix.[NOTE—Use this working solution within 5hours.]Transfer 5.0mLof the working solution to a 50-mLvolumetric flask,dilute with Diluentto volume,and mix.Each mLof this solution contains about 0.03mg of USP Rifampin RS.[NOTE—Inject this Standard preparation into the chromatograph within 30to 60seconds after preparation.]

Assay preparation— Remove,as completely as possible,the contents of not less than 20Capsules,and weigh accurately.Mix the Capsule contents,and transfer an accurately weighed portion of the powder,equivalent to about 300mg of rifampin,to a 200-mLvolumetric flask,and add about 180mLof Solvent mixture.Sonicate for about 5minutes,allow to equilibrate to room temperature,dilute with Solvent mixtureto volume,and mix.Transfer 10.0mLof the resulting solution to a 50-mLvolumetric flask,dilute with acetonitrile to volume,and mix.[NOTE—Use this solution within 5hours.]Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute with Diluentto volume,and mix.[NOTE—Inject this Assay preparation into the chromatograph within 30to 60seconds after preparation.]

Resolution solution— Dissolve USP Rifampin Quinone RSin Solvent mixtureto obtain a solution containing about 0.1mg per mL.Transfer 1.5mLof this solution and 5.0mLof the working solution used to prepare the Standard preparationto a 50-mLvolumetric flask,dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×10-cm column that contains 5-µm packing L7.The flow rate is about 1.5mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for rifampin quinone and 1.0for rifampin,and the resolution,R,between the rifampin quinone and rifampin peaks is not less than 4.0.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the area responses for the major peaks.Calculate the quantity,in mg,of C43H58N4O12in 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,in the Standard preparation,and rUand rSare the rifampin peak area responses obtained from the Assay preparationand the Standard preparation,respectively.