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 (t1/2) and maximum attainable concentration (Cmax) of IFN at systemic circulation was found to be 1.9 ±â€¯0.6 h and 269.3 ±â€¯0.4 after oral administration.

In vivo prediction of CYP-mediated metabolic interaction potential of formononetin and biochanin A using in vitro human and rat CYP450 inhibition data.

Formononetin (FMN) and Biochanin A (BCA) are the principal isoflavones present in commercially available extracts of red clover that are widely been consumed for various health benefits. We investigated the in vitro effects of FMN and BCA on catalytic activity of human/rat cytochrome P450 enzymes to assess the drug interaction potential of red clover. IC50 and Ki values of FMN and BCA for CYPs were determined in human/rat liver microsomes. FMN and BCA showed concentration-dependent inhibition of CYP1A2 activity with IC50 values of 13.42 and 24.98µM in human liver microsomes and 38.57 and 11.86µM in rat liver microsomes, respectively. The mode of inhibition of human CYP1A2 by FMN was found to be competitive with apparent Ki value of 10.13±1.96µM. FMN also inhibited human CYP2D6. BCA exerted moderately inhibitory effects on human CYP2C9. The predicted in vivo inhibition for CYP1A2 was insignificant (R value <1.1) at hepatic level while at intestinal level, it was significant (R value >11). The inhibitory effects on other CYPs were found to be minimal. Red clover may be considered safe to be consumed along with co-prescribed medications; however, precaution must be taken while co-administering it with CYP1A2 substrates.

LC-ESI-MS/MS method for bioanalytical determination of osteogenic phytoalexin, medicarpin, and its application to preliminary pharmacokinetic studies in rats.

Medicarpin is the active phytoalexin found in the stem bark of Butea monosperma having potent osteogenic activity. An LC-ESI-MS/MS was developed and validated for quantification of medicarpin in rat plasma using liquid-liquid extraction technique and diethyl ether as the extraction solvent. Medicarpin was separated on RP18 column (4.6mm×50mm, 5.0µm) using methanol and 10mM ammonium acetate (pH 4.0) in the ratio of 80:20 (v/v) as mobile phase. The method was linear within the concentration range of 1-500ng/mL and its sensitivity was 1ng/mL. The precision value for intra- and inter-day assays and stability assays was within 0.88-14.22% while the accuracy ranged between 87.46-116.0% at all four QC levels. The validated method was successfully applied to study the preclinical pharmacokinetics of medicarpin in rats. Medicarpin showed multiple peak phenomenon upon oral administration. Its oral bioavailability was 17.43%. It was found to be a rapidly absorbed (Tmax=15min), 81.61% protein bound and pH stable compound. The present study provides important information regarding preliminary pharmacokinetics of medicarpin for its further exploration as a potential therapeutic agent.