Simultaneous determination of genistein and its four phase II metabolites in blood by a sensitive and robust UPLC-MS/MS method: Application to an oral bioavailability study of genistein in mice.

The purpose of this research was to develop a sensitive and reproducible UPLC-MS/MS method to simultaneously quantify genistein, genistein-7-O-glucuronide (G-7-G), genistein-4'-O-glucuronide (G-4'-G), genistein-4'-O-sulfate (G-4'-S) and genistein-7-O-sulfate (G-7-S) in mouse blood samples. After the method was fully validated over a wide linear range, it was applied to quantify the levels of genistein and its metabolites in a mouse bioavailability study. The linear response range was 19.5-10,000 nM for genistein, 12.5-3200 nM for G-7-G, 20-1280 nM for G-4'-G, 1.95-2000 nM for G-4'-S, and 1.56-3200 nM for G-7-S, respectively. The lower limit of quantification (LLOQ) was 4.88, 6.25, 5, 0.98 and 0.78 nM for genistein, G-7-G, G-4'-G, G-4'-S and G-7-S, respectively. Only 20 microl mouse blood sample from i.v. and p.o. administration were needed for analysis because of the high sensitivity of the method. The intra- and inter-day variance is less than 15% and accuracy is within 85-115%. The analysis was finished within 4.5 min. The applicability of this assay was demonstrated and successfully applied for bioavailability study in FVB mouse after i.v. and p.o. administration of 20mg/kg of genistein, and its oral bioavailability was approximately 23.4%.