Determination Of Daphnetin And Its 8-O-Methylated Metabolite In Rat Plasma By UFLC-MS/MS:Application To A Pharmacokinetic Study

Objective:A rapid,sensitive and reliable ultrafast liquid chromatography tandem mass spectrometry(UFLC-MS/MS)method was developed and validated to determine daphnetin and daphnetin-Me simultaneously in rat plasma after intragastric administration and the method was applied to a pharmacokinetic study of daphnetin and daphnetin-Me in rats.Methods:50μL rat plasma sample was treated with precipitated protein and liquid-liquid extraction,added 3-aminocoumarin as the internal standard,using cetonitrile and ultramure water containing 0.1% formic acid as the mobile phase.A Shim-pack C18 column was utilized for chromatographic separation.The MS quantification for daphnetin,daphnetin-Me and 3-aminocoumarin(internal standard,IS)was carried out on a triple quadrupole mass spectrometer with positive multiple reaction monitoring mode(daphnetin: m/z 179.15→51.10;daphnetin-Me: m/z 193.30→150.05;IS: m/z 162.00→106.20).Results:The method exhibited a linear range of 1–2000 ng/m L.The intraand inter-assay precisions(RSD%)were ≤ 8.29% with the accuracies(RME%)within ± 5.95%.This method was successfully applied to a pharmacokinetic study of daphnetin after a single dose of 20 mg/kg in rats.Daphnetin and daphnetin-Me peaked almost at the same time.Compared with that of daphnetin,a 2.1-fold higher area under the concentration-time curve(AUC)for daphnetin-Me were observed.These results would be beneficial in facilitating further investigation of pharmacological mechanisms,as well as the rational application of daphnetin and daphnetin-containing drug preparations and may lay a foundation for the research and development of new drugs containing daphnetin.Conclusion:A rapid,sensitive and reliable ultrafast liquid chromatography tandem mass spectrometry(UFLC-MS/MS)method was developed and validated to simultaneously determine daphnetin and daphnetin-Me in rat plasma.The method was applied to a pharmacokinetic study of daphnetin and daphnetin-Me in rats.