Evaluation of high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA) and particle concentration fluorescence immunoassay (PCFIA) methods for the screening, quantitation and pharmacokinetic study of furosemide in horses

Abstract

Equine plasma and urine samples were analyzed by using a high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA) and particle concentration fluorescence assay (PCFIA). Although ELISA and PCFIA were rapid, simple and sensitive for the screening of furosemide, they did not give reproducible quantitative results. The HPLC method, which required relatively longer analysis time, provided simple and reproducible quantitative analysis of furosemide in plasma and urine. The performance of the three methods was compared for the quantitation of furosemide in plasma obtained from thoroughbred mares dosed intravenously with furosemide (500 μg/kg ( n = 7) and 1.0 mg/kg ( n = 5)). Although the plasma furosemide profiles determined by ELISA, PCFIA and HPLC were similar, ELISA and PCFIA methods exhibited considerable variation in values. At high furosemide concentrations, the PCFIA method gave better quantitative values than ELISA. However, at trace furosemide concentrations the PCFIA method gave false positive values which were not confirmed by HPLC or ELISA. The pharmacokinetic values obtained from the HPLC data and the pharmacokinetic values obtained previously from the gas chromatographic data [4,7] were comparable. The data obtained by ELISA and PCFIA were not suitable for the pharmacokinetic calculations.