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

Stock test preparation— Transfer about 200mg of Rifampin to a 100-mLvolumetric flask,dissolve in and dilute with acetonitrile to volume,and mix.Sonicate for about 30seconds,if necessary,to ensure dissolution.[NOTE—Use this solution within 2hours.]

Test preparation— Transfer 5.0mLof Stock test to a 50-mLvolumetric flask,dilute with Solvent to volume,and mix.[NOTE—Prepare this solution immediately prior to injection into the chromatograph.]

Dilute test preparation— Transfer 10.0mLof Stock test preparationto a 100-mLvolumetric flask,dilute with acetonitrile to volume,and mix.Transfer 5.0mLof the resulting solution to a 50-mLvolumetric flask,dilute with acetonitrile to volume,and mix.Transfer 5.0mLof this solution to another 50-mLvolumetric flask,dilute with Solvent mixtureto volume,and mix.[NOTE—Prepare this final dilution immediately prior to injection into the chromatograph.]

Procedure— Separately inject equal volumes (about 50µL)of the Test preparationand the Dilute test preparationinto the chromatograph,record the chromatograms,and measure the responses for all of the peaks.Calculate the percentage of each related substance by the formula: in which rTiis the area of the peak of the individual related substance in the chromatogram obtained from the Test preparation,rDis the area of the rifampin peak in the chromatogram obtained from the Dilute test preparation,and SrTiis the sum of the areas of all of the peaks of the related substances obtained in the chromatogram of the Test preparation:not more than 1.5%of rifampin quinone is found,not more than 1.0%of any other individual related substance is found,and a total of not more than 3.5%of all individual related substances,other than rifampin quinone,having retention times of up to 3in relation to the retention time of rifampin is found.

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.

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 water,acetonitrile,1.0Mdibasic potassium phosphate,1.0Mmonobasic potassium phosphate,and 1.0Mcitric acid (640:250:77:23:10).

Standard preparation— Transfer about 40mg of USP Rifampin RS,accurately weighed,to a 200-mLvolumetric flask.Dissolve in and dilute with acetonitrile to volume,and mix.Sonicate for about 30seconds,if necessary,to ensure dissolution.[NOTE—Use this solution within 5hours.]Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with Solvent mixtureto volume,and mix.[NOTE—Prepare this final dilution immediately prior to injection into the chromatograph.]

Assay preparation— Using Rifampin,proceed as directed for Standard preparation.

Resolution solution— Dissolve suitable quantities of USP Rifampin RSand USP Rifampin Quinone RSin acetonitrile to obtain a solution containing about 0.1mg of each per mL.Transfer 1.0mLof this solution to a 10-mLvolumetric flask,dilute with Solvent mixtureto 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 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 column efficiency determined from the rifampin peak is not less than 1000theoretical plates,and 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.The relative retention times are about 0.6for rifampin quinone and 1.0for rifampin.Calculate the quantity,in mg,of rifampin (C43H58N4O12)in the portion of Rifampin taken to prepare the Assay preparationby the formula: in which Cis the concentration,in mg per mL,calculated on the dried basis,of USP Rifampin RSin the Standard preparation,and rUand rSare the area responses of the rifampin peaks obtained from the Assay preparationand the Standard preparation,respectively.