Quantitative determination of isorhamnetin, quercetin and kaempferol in rat plasma by liquid chromatography with electrospray ionization tandem mass spectrometry and its application to the pharmacokinetic study of isorhamnetin

A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of quercetin, kaempferol and isorhamnetin in rat plasma. After being treated with β‐glucuronidase and sulfatase, the analytes were extracted by liquid/liquid extraction with the internal standard (IS; baicalein). The chromatographic separation was performed on a Diamonsil C18 column with a mobile phase consisting of 2% formic acid/methanol (10:90, v/v) at a flow rate of 1.00 mL/min, with a split of 200 µL to the mass spectrometer. Validation results indicated that the lower limit of quantification (LLOQ) was 1 ng · mL−1. The assay exhibited a linear range of 1–200 ng · mL−1 and gave a correlation coefficient of 0.9980 or better for each analyte. Quality control samples (1, 5, 20 and 100 ng · mL−1) in six replicates from each of three different runs demonstrated an intra‐assay precision (RSD) of 1.1–8.9%, an inter‐assay precision of 1.6–10.8%, and an overall accuracy (bias) of