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 μl 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 20 mg/kg of genistein, and its oral bioavailability was ∼23.4%.