Development and validation of liquid chromatography–tandem mass spectrometry method for simultaneous determination of six steroidal saponins in rat plasma and its application to a pharmacokinetics study

• Development and validation of an UHPLC–MS/MS method for selected steroidal saponins.
• The method is simple, sensitive and specific, with a short analysis time (10 min).
• Application to rat plasma demonstrates the method is fit-for-purpose.

A specific and reliable liquid chromatography–electrospray ionization-tandem mass spectrometry method was developed for the simultaneous determination of timosaponin H1 (TH1), timosaponin E1 (TE1), timosaponin E (TE), timosaponin B-II (TB-II), timosaponin B-III (TB-III) and anemarrhenasaponin I (AS-I) in rat plasma. After addition of internal standard (IS) ginsenoside Rh1, plasma samples were pretreated by protein precipitation with acetonitrile. Chromatographic separation was performed on a reverse phase ACQUITY™ BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm) using a gradient mobile phase system of acetonitrile–water containing 0.05% formic acid and 5 mM ammonium formate. The triple quadruple mass spectrometer was set in negative electrospray ionization mode and multiple reaction monitoring (MRM) was used for six steroidal saponins quantification. The precursors to produce ion transitions monitored for TH1, TE1, TE, TB-II, TB-III, AS-I and IS were m/z 1211.5 > 1079.6, 935.5 > 773.4, 935.4 > 773.5, 919.6 > 757.4, 901.5 > 739.3, 757.4 > 595.3 and 637.3 > 475.3, respectively. The method validation was conducted over the curve range of 0.5–400 ng/mL for the six saponins. The intra- and inter-day precisions (RSD%) were less than 9.4% and the average extraction recoveries ranged from 82.5% to 97.8% for each analyte. Six steroidal saponins were proved to be stable during sample storage, preparation and analytical procedures. The validated method was successfully applied for the first time to determine the concentrations of six main steroidal saponins in incurred rat plasma samples, after intragastric administration of the extract of Anemarrhena asphodeloides Bge. for a rat pharmacokinetic study.

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