Triethylamine buffer— Mix 4 mL of triethylamine and 2000 mL of water, and adjust with phosphoric acid to a pH of 3.2.

Solution A— Prepare a mixture of Triethylamine buffer, acetonitrile, and tetrahydrofuran (92:7:1), and degas briefly.

Solution B— Prepare a suitable mixture of Triethylamine buffer, acetonitrile, and tetrahydrofuran (70:29:1), and degas briefly.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621), changing the acetonitrile proportion in Solution A to obtain a retention time of 7.5 to 10.5 minutes for the main vancomycin peak.

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

Test preparation A— Prepare a solution of Vancomycin Hydrochloride in Solution A containing 10 mg per mL.

Test preparation B— Transfer 2.0 mL of Test preparation A to a 50-mL volumetric flask, dilute with Solution A to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 2 mL per minute. The chromatograph is programmed as follows. 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 1 and vancomycin B is not less than 3.0; and the column efficiency, calculated from the vancomycin B peak, is not less than 1500 theoretical plates. Resolution compound 2 is eluted at between 3 and 6 minutes after the start of the period when the percentage of Solution B is increasing from 0% to 100%.

Procedure— [NOTE—Where baseline separation is not achieved, peak areas are defined by vertical lines extended from the valleys between peaks to the baseline. The main component peak may include a fronting shoulder, which is attributed to monodechlorovancomycin. This shoulder should not be integrated separately.] Separately inject equal volumes (about 20 µL) of Test preparation A and Test preparation B into the chromatograph, record the chromatograms, and measure the area responses for all of the peaks. [NOTE—Correct any peak observed in the chromatograms obtained from Test preparation A and Test preparation B by subtracting the area response of any peak observed in the chromatogram of Solution A at the corresponding elution time.] Calculate the percentage of vancomycin B in the specimen tested by the formula: in which rB is the corrected area response of the main peak obtained in the chromatogram of Test preparation B; and rA is the sum of the corrected area 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 B is found. Calculate the percentage of each other peak taken by the formula: in which rAi is the corrected area response of any individual peak, other than the main peak, obtained in the chromatogram of Test preparation A: not more than 9.0% of any peak other than the main peak is found.

(Official January 1, 2007)