An HPLC–MS/MS method for the simultaneous determination of luteolin and its major metabolites in rat plasma and its application to a pharmacokinetic study

A selective and accurate HPLC-MS/MS method was established to simultaneously quantify luteolin and its active metabolites (diosmetin, chryseriol, and luteolin-7-O-glucuronide) in rat plasma. The analytes were separated on a C₁₈ column with a mobile phase of water containing 0.5% formic acid and acetonitrile under gradient elution to shorten the total chromatographic run time and increase the resolution of diosmetin and chryseriol. A triple quadruple mass spectrometer coupled with an electrospray ionization source in the negative ion mode was used to detect the analytes. The multiple reaction monitoring transitions were of m/z 284.9→132.9 for luteolin, m/z 298.9→283.9 for diosmetin and chryseriol, m/z 461.1→284.9 for luteolin-7-O-glucuronide, and m/z 300.9→150.9 for the internal standard. The method was linear within the concentration ranges of 0.06-90 μg/mL for luteolin, 0.03-12 μg/mL for diosmetin, 0.015-4.8 μg/mL for chryseriol, and 0.06-60 μg/mL for luteolin-7-O-glucuronide. The intra- and interday precisions were all within 6.0%. Accuracy ranged from -3.2 to 6.4%. The matrix effect and instability were not observed during bioanalysis. This method was used to study the pharmacokinetic characteristics of luteolin and its metabolites in rats after treatment with luteolin.