COMPARATIVE ASSAY OF AMOXICILLIN BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND MICROBIOLOGICAL METHODS FOR PHARMACOKINETIC STUDIES IN CALVES

An HPLC method and a microbiological assay developed for measuring amoxicillin in calf plasma were used in pharmacokinetic studies of the beta-lactam antibiotic following intramuscular administration of the test compound. Collected samples were concomitantly assayed by the two methods. The HPLC technique included protein precipitation of plasma samples with trichloroacetic acid, isocratic elution on a reversed-phase column (LiChrospher RP 18 end-capped, 5-mu-m) and fluorimetric detection after post-column derivatization with fluorescamine. The microbiological agar gel diffusion method used Sarcina lutea ATCC 9341 as the test organism, paper disks being impregnated with plasma followed by 1 h diffusion and 16 h incubation. Both methods show full selectivity with regard to endogenous compounds and a coadministered antibiotic (i.e. colistin). A similar limit of quantitation (0.1-mu-g of amoxicillin per ml of plasma) was attained with good reproducibility (relative standard deviation values were, respectively, 1.4 and 7.3% for HPLC and microbiological techniques). Linearity was in the range of 0.1-5-mu-g ml-1 (HPLC) and 0.1-1.6-mu-g ml-1 (microbiological assay). Results obtained by HPLC and the microbiological assay were compared by a statistical evaluation test which showed a good correlation between the two methods.