A: Infrared Absorption á197Kñ.

B: The retention time of the major peak in the chromatogram of theAssay preparation corresponds to that in the chromatogram of theStandard preparation,as obtained in theAssay.

Test solution: 5mg per mL,in a mixture of chloroform and methanol (9:1).

Change to read:

Buffer solution— Prepare as directed in theAssay.

Solution A— Prepare a filtered and degassed mixture ofBuffer solution and acetonitrile (80:20).

Solution B— Prepare a filtered and degassed mixture of acetonitrile andBuffer solution (60:40).

Mobile phase— Use variable mixtures ofSolution AandSolution Bas directed underChromatographic system.Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ).

Diluting solution— Prepare a filtered mixture of 0.02Msodium acetate,previously adjusted with acetic acid to a pHof 4.0,and acetonitrile (70:30).

Standard solution— Dissolve an accurately weighed quantity of USP Etoposide RSinDiluting solution to obtain a Standard stock solution having a known concentration of about 2.0mg per mL.Dilute this Standard stock solution quantitatively and stepwise withDiluting solution to obtain a solution having a known concentration of about 10µg per mL.

System suitability solution— Transfer about 20mg ofn-propylparaben,accurately weighed,to a 100-mLvolumetric flask,and dissolve in and dilute withDiluting solution to volume.Transfer 5mLof this solution and 5mLof the Standard stock solution to a 50-mLvolumetric flask,and dilute withDiluting solution to volume.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,dilute withDiluting solution to volume,and mix.

Test solution— Transfer about 100mg of Etoposide,accurately weighed,to a 50-mLvolumetric flask,and dissolve in and dilute withDiluting solution to volume.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×15-cm column that contains packing L11having a diameter of less than 5µm.The flow rate is about 1.5mLper minute.Chromatograph theSystem suitability solution usingSolution A,and record the peak responses as directed forProcedure:the relative retention times are about 0.20for lignan P,1.0for etoposide,and 1.43for picroetoposide;and the resolution,R,between propylparaben and etoposide is not less than 1.1.The chromatograph is programmed forProcedure as follows.

Procedure— Separately inject equal volumes (about 25µL)of theStandard solution and theTest solution into the chromatograph,record the chromatograms for at least 40minutes,and measure the peak responses.Calculate the percentages of lignan Pand picroetoposide in the portion of Etoposide taken by the formula: USP28in whichCis the concentration,in mg per mL,of USP Etoposide RSin theStandard solution;Wis the weight,in mg,of Etoposide taken to prepare the Test solution;USP28riis the peak response for each related compound obtained from theTest solution;and rSis the peak response for etoposide obtained from theStandard solution:not more than 0.5%of lignan Pand 1.0%of picroetoposide is found.Calculate the quantity of any other impurity observed in the chromatogram of theTest solution by the same formula:not more than 2.0%of all related compounds and other impurities is found.

Buffer solution— Dissolve 5.44g of sodium acetate in 2000mLof water,adjust with glacial acetic acid to a pHof 4.0,and filter.

Mobile phase— Prepare a filtered and degassed mixture ofBuffer solution and acetonitrile (74:26).Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Etoposide RSin acetonitrile to obtain a Standard stock solution having a known concentration of about 2.0mg per mL.Transfer 5.0mLof this Standard stock solution to a 50-mLvolumetric flask,dilute withMobile phase to volume,and mix.

System suitability solution— Dissolve an accurately weighed quantity of USP Etoposide Resolution Mixture RSin Mobile phaseto obtain a solution having a known concentration of 0.3mg per mL.

Assay preparation— Transfer about 100mg of Etoposide,accurately weighed,to a 50-mLvolumetric flask,dissolve in and dilute with acetonitrile to volume,and mix.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute withMobile phase to volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L11.The flow rate is about 1mLper minute.Chromatograph theSystem suitability solution,and record the peak responses as directed for Procedure:the resolution,R,between the etoposide and a-etoposide peaks is not less than 1.35.Chromatograph the Standard preparation,and record the peak responses as directed forProcedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of theStandard preparation and theAssay preparation into the chromatograph,and allow theAssay preparation to elute for not less than 1.5times the retention time of etoposide.Record the chromatograms,and measure the responses for all the peaks.Calculate the quantity,in mg,of C29H32O13in the portion of Etoposide taken by the formula: in whichCis the concentration,in mg per mL,of USP Etoposide RSin theStandard preparation;and rUand rSare the etoposide peak responses obtained from theAssay preparation and theStandard preparation,respectively.