Simultaneous Determination of Isorhamnetin,Quercetin and Kaempferol in Beagle Dog Plasma Using UPLC-MS

In this paper,a rapid and sensitive UPLC-MS method was developed and validated for the identification and quantification of isorhamnetin,quercetin and kaempferol in Beagle dog plasma.The analysis was performed by using UPLC-MS on a Waters UPLC column(Acquity UPLC BEH C_(18) 1.7 μm,2.1 mm×50 mm) using the mobile phase consisting of 0.1%formic acid in water and acetonitrile at a flow rate of 0.4 mL/min.Full assay validation was implemented including specificity,linearity,accuracy,precision,recovery and stability.All samples had no interference from endogenous substances at the retention times of either the three analytes or the IS.The linear concentration ranges of the calibration curves for quercetin,isorhamnetin and kaempferol ranged from 3—900,2—600 and 2—600 ng/mL.The lower limit of quantification was 3 ng/mL for quercetin,2 ng/mL for isorhamnetin and kaempferol,respectively.The RSD values of the intra-and inter-day precisions were less than 5.2%at each concentration.A simple,rapid and specific UPLC-MS method for the quantification of isorhamnetin,quercetin and kaempferol in beagle dog plasma was established,which could benefit pharmacokinetic and clinical research studies of the three flavonoids.