Simultaneous determination of six isoflavonoids in rat plasma after administration of total flavonoid from Gegen by ultra‐HPLC‐MS/MS

A simple, specific, and sensitive ultra‐performance liquid chromatography‐tandem mass spectrometry method was developed for the simultaneous determination of 3′‐hydroxypuerarin, 6′′‐ O ‐xylosylpuerarin, mirificin, puerarin, 3′‐methoxypuerarin and daidzin in rat plasma. After the addition of methanol containing 0.1% formic acid and 10% ascorbic acid, the analytes and rutoside were obtained by protein precipitation, then separated on a Thermo Syncronis C18 column (2.1 mm × 10 cm, 1.7 μm) by gradient elution and monitored using an electrospray ionization interface operating in positive ion and selective reaction monitoring acquisition mode. The calibration curves of these analytes showed good linearity ( r > 0.99) within the test ranges. The lower limit of quantification was 0.0200 μg/mL for 3′‐hydroxypuerarin, 0.0101 μg/mL for 6′′‐ O ‐xylosylpuerarin, 0.0100 μg/mL for mirificin and puerarin, 0.0098 μg/mL for 3′‐methoxypuerarin, and 0.0090 μg/mL for daidzin. The intraday and interday precision and accuracy were all within 15%. The extraction recoveries were from 74.0 to 95.8%. The validated method was successfully applied to pharmacokinetic studies of the six isoflavonoids in rat plasma after intravenous administration of total flavonoids from Gegen.