Simultaneous determination of six bioactive components of total flavonoids of Scorzonera austriaca in rat tissues by LC-MS/MS: application to a tissue distribution study

ABSTRACT

ABSTRACT A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of six bioactive constituents including vitexin, orientin, isoorientin, 2"-O-β-D-xylopyranosyl isoorientin, 2"-O-β-D-xylopyranosyl isovitexin, and 6-C-L-α-arabipyranosyl vitexin in rats' various tissues using isoquercitrin as the internal standard. Biological samples were pretreated by protein precipitation with acetonitrile. Chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. All analytes and internal standard were quantitated through electrospray ionization in negative ion selected reaction monitoring mode. The mass transitions were as follows m/z 431 → 311 for vitexin, m/z 447 → 327 for orientin or isoorientin, m/z 579 → 459 for 2"-O-β-D-xylopyranosyl isoorientin, m/z 563 → 293 for 2"-O-β-D-xylopyranosyl isovitexin, m/z 563 → 353 for 6-C-L-α-arabipyranosyl vitexin, and m/z 463 → 300 for the internal standard, respectively. The lower limits of quantification for the six analytes in different tissue homogenates were 0.8-2.2 ng/ml. The validated assay was successfully applied to a tissue distribution study of the six components in rats after intravenous administration of total flavonoids of Scorzonera austriaca Willd; Asteraceae. The results of the tissue distribution study showed that the high concentrations of six components were mainly in the kidney.