Simultaneous determinations of 17 marker compounds in Xiao–Chai–Hu–Tang by LC–MS/MS: Application to its pharmacokinetic studies in mice

• An UPLC–MS/MS method to quantify 17 compounds in Xiao–Chai–Hu–Tang in blood was developed.
• The sensitivity and robust method was validated.
• The sensitivity and robust method was applied for pharmacokinetic study in mice.

The purpose of this study is to develop and validate an UPLC–MS/MS method to quantify different marker compounds from Xiao–Chai–Hu–Tang (XCHT, a Chinese traditional herbal) in biological samples and apply the method to pharmacokinetic study. A Waters BEH C18 UPLC column was used with acetonitrile/0.1% formic acid mobile phases. The mass analysis was performed in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode. A one-step protein precipitation by methanol was used to extract the analytes from blood. Seventeen commercially available compounds from the different compositing herbals were selected as markers. The results revealed that all of the calibration curves showed good linear regression (r2 > 0.9918). The intra-day and inter-day precisions (RSD) of all of these markers at three different levels were less than 15.0% and the bias of the accuracies ranged from −13.5% to 16.6%.The extraction recoveries of all of these 17 markers were from 70.8% to 113.7% and the matrix effects ranged from 71.8% to 114.8%. The stabilities of these compounds in blood were evaluated by analyzing three replicates of QC samples at three different concentrations following storage at 25 °C for 6 h, 4 °C for 24 h, and −80 °C for 30 days. All the samples displayed 85–115% precision and accuracy after various stability tests. The validated method was successfully applied to pharmacokinetic study in A/J mouse with oral administration of XCHT. All of these markers were detected and the pharmacokinetic parameters of 8 compounds were able to be calculated. This method is sensitive and reproducible that can be used for XCHT’s in vivo study.