Development of an LC-MS/MS method for quantification of two pairs of isomeric flavonoid glycosides and other ones in rat plasma: Application to pharmacokinetic studies.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of six flavonoid glycosides - isoorientin (1), orientin (2), 2″-O-β-d-xylopyranosyl isoorientin (3), 2″-O-β-d-xylopyranosyl isovitexin (4), 6-C-l-α-arabipyranosyl vitexin (5) and vitexin (6) - in rat plasma using isoquercitrin as the internal standard (IS). Plasma samples were prepared by a one-step protein precipitation with acetonitrile. Chromatographic analysis was carried out on a 25 cm C18 column with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. Six analytes and IS were detected through electrospray ionization in negative-ion selection reaction monitoring mode. The mass transitions were as follows: m/z 447.2 → 327.0 for 1, m/z 447.2 → 327.0 for 2, m/z 579.3 → 458.9 for 3, m/z 563.0 → 293.1 for 4, m/z 563.0 → 353.0 for 5, m/z 431.1 → 311.1 for 6, and m/z 463.1 → 300.2 for IS. Calibration curves exhibited good linearity (r2  > 0.9908) over a wide concentration range for all compounds. Intra- and inter-day precision (RSD, %) at four different levels were both <14.2% and the accuracy (RE, %) ranged from -11.9 to 12.0%. The extraction recoveries of the six components ranged from 88.2 to 103.6%. The validated assay was successfully applied to the pharmacokinetic studies of the six components in male rat plasma after intravenous administration of total flavonoids of Scorzonera austriaca Wild.