§ 436.366 - High-performance liquid chromatography assay for determining chromatographic purity of vancomycin.

(a) Apparatus. A suitable high-performance liquid chromatograph equipped with:

(1) A suitable ultraviolet detection system operating at a wavelength of 254 nanometers or preferably 280 nanometers;

(2) A suitable recording device of at least 25-centimeter deflection;

(3) A suitable chromatographic data managing system; and

(4) A 25-centimeter analytical column having an inside diameter of 4.6 millimeters and packed with octadecyl silane chemically bonded to porous silica or ceramic microparticles; 5 micrometers in diameter.

(b) Reagents.—(1) 0.2 percent triethylammonium phosphate buffer. To 2,000 milliliters of distilled water, either add 4 milliliters of triethylamine or 4 grams of triethylammonium chloride. Adjust the pH to 3.2 with phosphoric acid.

(2) Sample solvents. (i) Vancomycin hydrochloride: Mobile Phase A.

(ii) Vancomycin base: 5 milliliters Mobile Phase A; add 0.1N HCl dropwise with swirling until sample dissolves: dilute to volume with Mobile Phase A.

(c) Mobile Phases—(1) Mobile Phase A. Add 70 milliliters of acetonitrile and 10 milliliters of tetrahydrofuran to 920 milliliters of 0.2 percent triethylammonium phosphate buffer and mix well. Filter the mobile phase through a suitable glass fiber filter or equivalent that is capable of removing particulate contamination to 1 micron in diameter. Degas the mobile phase, briefly, just prior to its introduction into the chromatographic pumping system.

(2) Mobile Phase B. Add 290 milliliters of acetonitrile and 10 milliliters of tetrahydofuran to 700 milliliters of 0.2 percent triethylammonium phosphate buffer and mix well. Filter the mobile phase through a suitable glass fiber filter or equivalent that is capable of removing particulate contamination to 1 micron in diameter. Degas the mobile phase, briefly, just prior to its introduction into the chromatographic pumping system.

(d) Operating conditions. Perform the assay at ambient temperature with a typical flow rate of about 2.0 milliliters per minute. Use a detector sensitivity setting that gives a peak height for the main peak (Vancomycin B) that is at least 50 percent of scale. The run time is 30 minutes per injection and the gradient conditions are as follows: (0, 12, 12.5, 8, 0, 2)

Time (minutes)Mobile phase A (percent)Mobile phase B (percent)Gradient condition0 100 0 Initial conditions.12 100 0 Isocratic region.20 0 100 Linear ramp.22 0 100 Isocratic region.23 100 0 Return to initial.30 100 0 Reequilibration.

(e) Preparation of resolution and sample solutions—(1) Resolution solution. Prepare a solution of vancomycin hydrochloride reference standard in water containing 0.5 milligram per milliliter. Heat at 65 °C for 24 hours and allow to cool. This procedure generates two desamido-vancomycin isomers. The first desamido isomer elutes during the isocratic period and before the vancomycin B peak; the second desamido isomer elutes during the gradient ramp and is used to demonstrate the effective performance of this stage.

(2) Sample preparation. In a volumetric flask either dissolve a representative sample or dilute a representative portion with sample solvent to give a sample preparation containing approximately 10 milligrams per milliliter. Pipet 2 milliliters of this sample solution into a separate 50-milliliter volumetric flask and dilute to volume with sample solvent to give a diluted sample preparation containing approximately 0.4 milligram per milliliter.

(f) Procedure. Optimize chromatographic conditions under isocratic conditions by equilibrating the system while pumping 100 percent mobile phase A through the column. Inject 20 microliters of the resolution solution onto the column and record the chromatogram. Adjust the acetonitrile concentration of mobile phase A as needed to provide a retention time for vancomycin B of 7.5 to 10.5 minutes. Use the resolution solution to perform the system suitability tests. The elution order is resolution compound 1, vancomycin B, resolution compound 2. Return the system to the initial gradient operating conditions. Separately inject 20 milliliters of each diluted (0.4 milligram per milliliter) and concentrated (10 milligrams per milliliter) sample solution onto the column and record each chromatogram.

(g) System suitability test. Using the resolution solution described in paragraph (e)(1) of this section, test the performance of the chromatographic system as follows:

(1) Asymmetry factor. Calculate the asymmetry factor (A), measured at a point that is 10 percent of the vancomycin B peak height from the baseline, as follows:

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(2) Efficiency of the column. From the number of theoretical plates (n) calculated as described in § 436.216(c)(2) calculate the reduced plate height (hr) for the vancomycin B peak as follows:

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(3) Resolution. The resolution (R) between the vancomycin B peak and the peak for resolution compound 1 is not less than 3.0. Resolution compound 2 is eluted between 3 and 6 minutes after the start of the period when the percentage of mobile phase B is increasing from 0 percent to 100 percent.

(4) Coefficient of variation (relative standard deviation). The coefficient of variation (SR in percent) of five replicate injections of the resolution solution is calculated as described in § 436.216(c)(4) is satisfactory if it is not more than 2.0 percent.

(5) Capacity factor (k). Calculate the capacity factor (k) for vancomycin B as follows:

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(h) Calculations. (1) Calculate the percentage of vancomycin B in the specimen as follows:

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(2) Calculate the percentage of each other peak as follows:

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