Triethylamine buffer— Mix 4mLof triethylamine and 2000mLof water,and adjust with phosphoric acid to a pHof 3.2.

Solution A— Prepare a mixture of Triethylamine buffer,acetonitrile,and tetrahydrofuran (92:7:1),and degas briefly.Make adjustments if necessary (see System SuitabilityunderChromatography á621ñ).

Solution B— Prepare a suitable mixture of Triethylamine buffer,acetonitrile,and tetrahydrofuran (70:29:1),and degas briefly.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Mobile phase— Use variable mixture of Solution Aand Solution Bas directed forChromatographic system.

Resolution solution— Prepare a solution ofUSP Vancomycin Hydrochloride RSin water containing 0.5mg per mL,heat at 65for 48hours,and allow to cool.

Test preparation A— Prepare a solution of Vancomycin Hydrochloride inSolution Acontaining 10mg per mL.

Test preparation B— Transfer 2.0mLof Test preparation Ato a 50-mLvolumetric flask,dilute withSolution Ato volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1,and is programmed to provide,at a flow rate of about 2mLper minute,a variable mixture of Solution Aand Solution B,the percentage of Solution Bbeing 0%at the time the specimen is injected into the chromatograph and remaining at that level for 12minutes,then increasing linearly to 100%over the next 8minutes,remaining at that level for 2minutes,and then rapidly decreasing to 0%in 1minute and remaining at that level for 7minutes before the next injection.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ),changing the acetonitrile proportion in Solution Ato obtain a retention time of 7.5minutes to 10.5minutes for the main vancomycin peak.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the elution order is resolution compound 1,vancomycin B,and resolution compound 2;the resolution,R,between resolution compound 1and vancomycin Bis not less than 3.0;and the column efficiency,calculated from the vancomycin Bpeak,is not less than 1500theoretical plates.Resolution compound 2is eluted at between 3and 6minutes after the start of the period when the percentage of Solution Bis increasing from 0%to 100%.

Procedure— [NOTE—Use peak areas where peak responses are indicated.Where baseline separation is not achieved,peak areas are defined by vertical lines extended from the valleys between peaks to the baseline.]Separately inject equal volumes (about 20µL)of Test preparation AandTest preparation Binto the chromatograph,record the chromatograms,and measure the responses for all of the peaks.[NOTE—Correct any peak observed in the chromatograms obtained from Test preparation Aand Test preparation Bby subtracting the response of any peak observed in the chromatogram of Solution Aat the corresponding elution time.]Calculate the percentage of vancomycin Bin the specimen tested by the formula: in which rBis the corrected response of the main peak obtained in the chromatogram ofTest preparation B;rAis the sum of the corrected responses of all the peaks,other than the main peak,in the chromatogram obtained from Test preparation A:not less than 80.0%of vancomycin Bis found.Calculate the percentage of each other peak taken by the formula: in which rAi is the corrected response of any individual peak,other than the main peak,obtained in the chromatogram ofTest preparation A:not more than 9.0%of any peak other than the main peak is found.