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 beta-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 C(18) 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 microL 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 <13.4%. The extraction recovery of each analyte and internal standard was 70-80%. In the present study, we have investigated the pharmacokinetic profiles of isorhamnetin after oral application in rats equipped with a jugular catheter. After oral dosing of isorhamnetin, the mean values (n = 10) of C(max) were 57.8, 64.8 and 75.2 ng . mL(-1) which were achieved at a T(max) of 8.0, 6.4 and 7.2 h for oral doses of 0.25, 0.5 and 1.0 mg . kg(-1) body weight, respectively. The corresponding mean values for isorhamnetin area under the curver (AUC) from 0 to 60 h were 838.2, 1262.8, 1623.4 ng . h . mL(-1). Our results further demonstrated that the samples analyzed showed isorhamnetin could not be transformed into quercetin or kaempferol in rats, indicating that the demethylation of the 3'-oxymethyl group of isorhamnetin does not occur in Wistar rats.