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

Abstract

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-Osulfate (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 were 19.5–10,000 nM for genistein, 12.5–3,200 nM for G-7-G, 20–1280 nM for G-4’-G, 1.95–2,000 nM for G-4’-S, and 1.56–3,200 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 ~24%.

A simple and robust high-performance liquid chromatography coupled to a diode-array detector method for the analysis of genistein in mouse tissues

“…In this case, the previously validated method by Supko in mouse plasma is comparable to the here-developed method [26]. However, no data regarding HPLC-UV validation methods in mouse tissues have been found, being the present study only comparable to already published HPLC GEN quantification methods coupled to a MS detector [23,31]. In these cases, LLOQ values of 0.1 ng/mL [19] and 1 ng/mL [21] in human serum, 8.5 ng/mL in human plasma [20] were reached.…”

“…The analyte was considered stable in the extracts of each biological matrix if 85-115% of the reference concentration was obtained. No further stability experiments were performed because the conditions had already been tested in human serum [21] and mouse plasma [26,31].…”

“…However, Supko achieved mean recovery values of almost 100% in mouse plasma after an exhaustive extraction method [26] when compared to ours. Solid-phase extraction methods without containing enzymes for the quantification of GEN and other isoflavone metabolites have shown recoveries around 78.8% in human plasma [31].…”

Dietary Phytochemicals as Cancer Preventive Agents: Efficacy and Mechanisms

“…A previous rodent study has indicate that more than 80% of genistein is converted to glucuronides and sulfates after both intravenous and oral administrations at 20 mg/ kg and that the absolute bioavailability is 23.4% [135]. Pharmacokinetic studies have shown that genistein has very long half-life (46 h) after oral administration and that different dose of genistein show no correlation of AUC, suggesting that there may be unknown mechanism of elimination and recycling system [135,136]. In human, genistein is extensively metabolized and the plasma level of unconjugated form is very low [137,138].…”

Risk assessment for peri- and post-menopausal women taking food supplements containing isolated isoflavones

“…In this study the bioavailability at these three doses (estimated from AUCs after oral and after intravenous dosing) was 21.9 %, 33.5 % and 19.0%, respectively. Mice Yang et al (2010) investigated the systemic availability after i.v. and oral dosing of 20 mg/kg genistein in FVB mice.…”