Vancomycin Activity Assay Using Microbiological Methods

INTRODUCTION. Vancomycin is a tricyclic glycopeptide antibiotic produced by Amycolatopsis orientalis. To determine the activity of antibiotics obtained by biosynthesis, in particular vancomycin, chemical or physicochemical identification of the active compound is not available; instead, two pharmacopeial methods are used: the agar diffusion and the turbidimetric method. Both methods are based on inhibiting the growth of the test microorganism under the appropriate experimental conditions. Turbidimetric method has several advantages over the agar diffusion method for parenteral preparations used in the form of solutions, as it is more sensitive to low concentrations, more cost-effective and takes less time to obtain the results. The State Pharmacopoeia of the Russian Federation (SP RF) provides one method for determining the activity of antibiotics — the agar diffusion method. Previously, turbidimetric method was developed to determine vancomycin activity.

AIM. This study aimed to evaluate the determination results of vancomycin activity obtained using turbidimetric method in order to use it as an alternative to the agar diffusion method.

MATERIALS AND METHODS. The test conditions for the method using agar diffusion corresponded to SP RF: test-microorganism, seed dose: Bacillus subtilis NCTC 10400/ ATCC 6633 (Becton Dickinson France S.A.S.), 20×10⁷ spores per ml medium; purified sterile water for the preparation of basic solutions, sterile buffer No. 4 for process solutions. The turbidimetric method included Staphylococcus aureus NCTC 7447/ ATCC 6538P (Becton Dickinson France S.A.S), purified sterile water, and solvent for process solutions — sterile buffer solution, pH 8.0 (adapted from the European Pharmacopoeia). A culture medium developed at the Scientific Centre for Expert Evaluation of Medicinal Products was used to determine the activity of aminoglycosides. The activity was determined by comparing with a reference standard of vancomycin hydrochloride EP CRS banth 4 for microbiological analyses. For each test (agar diffusion and turbidimetry), five accurately weighed samples were taken with an accuracy of 0.0001 g of the test sample; each sample was analyzed in three replicates.

RESULTS. The calculated main parameters of the analysis of variance, Non-parallelism and Regression of the results of the test and reference solutions, confirmed the adequacy of the data obtained using both methods. Comparison of the sample variances of the two methods (turbidimetry and agar diffusion) according to Fisher’s test and Bartlett’s test, the calculated value χ² (P=95%, f=1), and comparison of the mean values according to the Student’s t-test confirmed that both samples belonged to the same general sample. The systematic error and uncertainty for the turbidimetric method was significantly higher than the agar diffusion: ε 1.5% and ε 0.77%, δ=4.7% and δ=0.97%, respectively.

CONCLUSIONS. The turbidimetric method can be deemed as an alternative for determining vancomycin activity. However, in order to minimize the influence of the human factor and the external study conditions, a preliminary standardization of conditions, with a subsequent implementation control is warranted for the turbidimetric method.

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