LC-ESI-MS/MS method for the simultaneous determination of isoformononetin, daidzein, and equol in rat plasma: Application to a preclinical pharmacokinetic study.

Isoformononetin (methoxy isoflavone) is a potent osteogenic isoflavone abundantly present in Butea monosperma, Pisum sativum, Mung bean, Machaerium villosum, Medicago sativa, and Glycine max. In the current study, an LC-ESI-MS/MS method for the simultaneous evaluation of isoformononetin (IFN), daidzein (DZN) and equol (EQL) was developed and validated in rat plasma using biochanin A as an internal standard. IFN, DZN, and EQL separation was achieved by using acetonitrile and acetic acid (0.1%) in the ratio of 90:10 (% v/v) as mobile phase under isocratic conditions at a flow rate of 0.6 mL/min on Atlantis C₁₈ (4.6 × 250 mm, 5.0 μm) column. The achieved method was linear within the concentration range of 0.5-500 ng/mL. The method was effectively applied to investigate the permeability, protein binding estimation and pharmacokinetics studies of IFN in rats. The PAMPA permeability of IFN was found to be high at pH 4.0 and 7.0. The protein binding was found to be about 91% of IFN. The oral bioavailability of IFN was found to be poor (21.6%). IFN was found to have a moderate clearance (2.9 L/h/kg) and a large apparent volume of distribution (12.1 L/kg). The plasma half-life (t_1/2) and maximum attainable concentration (C_max) of IFN at systemic circulation was found to be 1.9 ± 0.6 h and 269.3 ± 0.4 after oral administration.