Development of UPLC-MS/MS Method for Determination of Saikosaponin B2 and B4 in Rat Plasma and Application in Pharmacokinetics Study

OBJECTIVE To establish a UPLC-MS/MS method for the determination of saikosaponin B2 and B4 in rat plasma. The pharmacokinetics of saikosaponin B2 and B4 after intravenous administration in rats were studied. METHODS Saikosaponin F was used as internal standard, UPLC BEH C₁₈(2.1 mm×50 mm, 1.7μm) column was used and the column temperature was set at 40℃. The mobile phase was consisted of acetonitrile and water (containing 0.1% formic acid), gradient elution with the flow rate of 0.4 mL·min^-1 for 4 min. Nitrogen was used as desolvation gas (800 L·h^-1) and cone gas (50 L·h^-1). MRM was used to quantitatively analyze saikosaponin B2 m/z 825.4→617.5, saikosaponin B4 m/z 957.6→ 649.6 and saikosaponin F (internal standard) m/z 973.7→119.0. Rat plasma samples were treated by acetonitrile precipitation to remove protein. RESULTS In the range of 5-5 000 ng·mL^-1, the concentrations of saikosaponin B2 and B4 in rat plasma showed good linear relation ship(r>0.995), with the lower limit of quantification of 5 ng mL^-1. Saikosaponin B2 had an intra-day RSD<12% and inter-day RSD<15%. Saikosaponin B4 had an intra-day RSD<14%, inter-day RSD<15%. The accuracy of saikosaponin B2 and B4 ranged from 89.3% to 110.2%. CONCLUSION The method is sensitive, rapid and selective, and successfully applied to the pharmacokinetics study of saikosaponin B2 and B4 after intravenous administration.