Stereospecific analysis of flurbiprofen and its major metabolites in plasma and urine by chiral-phase liquid chromatography

Abstract

Direct chirol-phase HPLC methods have been developed for the determination of flurbiprofen and its major metabolites, namely 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen, in biological fluids using a derivatized amylose chiral stationary phase (CSP; Chiral-pak AD). Quantification of all three analytes, both free and conjugated, in urine was carried out following liquid-liquid extraction using tandem ultraviolet (UV) and fluorescence detection. Determination of flurbiprofen and the 4′-hydroxy-metabolite in plasma utilized the same CSP but required modificotion in the mobile phase composition and sole use of fluorescence detection. The urine assay was linear (r > 0.998) between 0.05-10 μg mL-1, 0.1-20 μg mL-1 and 0.01-2 μg mL-1 for the enantiomers of flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. The plasma assay was linear (r > 0.997) between 0.1-6 μg mL-1 and 0.01-0.6 μg mL-1 for the enantiomers of flurbiprofen and 4′-hydroxy-flurbiprofen respectively. Both assays, typically yielded within- and between-day imprecision and accuracy values less than 10% for the enantiomers of the different analytes. Initial volunteer studies suggest that the disposition of flurbiprofen displays modest enantioselectivity in humans.