Optimisation of a reversed-phase high-performance liquid chromatographic method for the determination of flavone acetic acid and its major human metabolites in plasma and urine.

A high-performance liquid chromatographic method for the determination of flavone acetic acid (FAA) and its major human metabolites in plasma and urine is described. Two factors were identified as being the key to resolving the metabolites; pH and buffer ionic strength. Run at optimal conditions of 10 mM ammonium acetate, pH 5.5-propan-2-ol (80:20) and a column temperature of 40 degrees C on a muBondapak C18 10 microns particle column (30 cm X 3.8 mm I.D.), two major metabolites were identified [FAA, retention time (tR) 6.02 min +/- 0.5% coefficient of variation (C.V.); metabolite 1, tR 4.13 min +/- 1.1% C.V.; metabolite 2, tR 5.10 min +/- 0.5% C.V. and hesperidin, internal standard, tR 4.69 min +/- 1.6% C.V.]. A solid-phase technique using Bond Elut C2 40-microns particles is described which extracts FAA, metabolites and internal standard with efficiencies in excess of 90%. Considerable attention has to be paid to sample preparation: FAA has poor aqueous solubility at acidic pH and the metabolites degrade back to FAA via intermediates at alkaline pH. Both problems can be avoided by buffering and diluting samples with 10 mM ammonium acetate, pH 5.5.