A high-throughput colorimetric-based bioassay method for determination of fosmidomycin in plasma and urine and application for pharmacokinetic study.

摘要

INTRODUCTION: Fosmidomycin is a phosphonic acid derivative originally isolated as a natural antibiotic from Streptomyces lavendulae. It was originally used as an antibiotic, and later on as an antimalarial drug. A simple, sensitive, selective and reproducible bioassay based on colorimetric method was developed for the determination of fosmidomycin in human plasma and urine. METHODS AND RESULTS: Enterobacter cloacae ATCC 23355 strain was used as a test organism. Inhibition of bacterial growth was assessed using MTT assay. Calibration curves were prepared from concentration response curves in plasma (0, 0.5, 1, 2.5, 5, 12.5, 25, 50 ng/microl) and urine (0, 0.5, 1, 2.5, 5, 12.5, 25, 50 and 100 ng/microl) and were all linear with correlation coefficients better than 0.990. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 10% (% coefficient of variations: % C.V.) Good accuracy was observed for both the intra-day and inter-day assays, as indicated by the minimal deviation of mean values found between the measured samples and the theoretical values (below +/-10%). Limit of quantification (L.O.Q.) was accepted as 0.5 ng using 20 microl plasma or 10 microl urine sample. The mean recovery for fosmidomycin was greater than 99%. The method was free from interference from other commonly used antibiotics. DISCUSSION: The analytical method established in this study meets the criteria of high sensitivity, accuracy and reproducibility for routine use in pharmacokinetic studies. The method has advantages over the recently developed bioassay in its simplicity, accuracy, short analysis time, good sensitivity, and the requirement for smaller volumes of samples. The method appears to be robust and has been applied to a pharmacokinetic study to determine the plasma and urinary excretion of fosmidomycin in a total of 24 Thai patients with acute uncomplicated falciparum malaria following oral doses of 1800 fosmidomycin given every 10 h for 3 days.