High‐throughput quantification of isoflavones, biochanin A and genistein, and their conjugates in female rat plasma using LC‐ESI‐MS/MS: Application in pharmacokinetic study

Isoflavones containing foods and dietary supplements are widely consumed for putative health benefits ( e.g . cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). This paper describes the development and validation of a sensitive high throughput LC‐ESI‐MS/MS method for quantifying biochanin A (BCA) and genistein (GEN), and their conjugates in rat plasma. The analytes were separated on a Supelco Discovery C18 (4.6×50 mm, 5.0 μm) column under isocratic condition using acetonitrile/methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 v/v as a mobile phase. The intra‐ and inter‐day assay precision ranged from 2.66 to 8.34% and 4.40 to 8.10% (RSD %), respectively, and intra‐ and inter‐day assay accuracy was between 90.67–109.25% and 95.86–106.32%, respectively, for both the analytes. The lowest quantitation limit for BCA and GEN was 0.5 ng/mL in 0.1 mL of rat plasma. The method was successfully applied to the estimation of BCA, GEN and their conjugates in rat plasma following oral administration of BCA. Circulating conjugates (glucuronides/sulfates) of BCA and GEN were quantified using enzymatic hydrolysis of plasma samples. The levels of isoflavones glucuronides/sulfates were found to be much greater than the corresponding aglycones.