Simultaneous determination of timosaponin B-II and A-III in rat plasma by LC–MS/MS and its application to pharmacokinetic study

• We developed a novel LC–MS/MS method for determination of TB-II and TA-III in plasma.
• The method was validated to be rapid, specific, accurate and sensitive.
• The pharmacokinetics of TB-II and TA-III in timosaponins was investigated in rats.
• Huangbai alkaloids can affect the pharmacokinetics of TB-II and TA-III in timosaponins.

A rapid, specific and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed and validated for the simultaneous determination of timosaponin B-II (TB-II) and A-III (TA-III) in rat plasma. Plasma samples were pretreated via simple protein precipitation with acetonitrile and ginsenoside Rg2 was used as internal standard. Chromatographic separation was carried out on an Agilent XDB-C8 (150 mm × 2.1 mm i.d., 5 μm) column by isocratic elution with acetonitrile–2 mmol/L ammonium acetate (55:45, v/v). The detection was performed on a Sciex API 4000+ triple-quadrupole tandem mass spectrometer with TurboIonSpray ionization (ESI) inlet via the negative ion multiple reaction monitoring (MRM) mode. The results showed that the calibration curve was linear in the concentration range of 3–3000 ng/mL for TB-II and 0.3–3000 ng/mL for TA-III, respectively. The intra- and inter-day precisions were less than 13.25%, and the accuracy ranged from 100.88% to 104.07% at three QC levels for both. The pharmacokinetic profiles of TB-II and TA-III in timosaponins (total timosaponin) at three dose levels (TB-II 150, 300, 600 mg/kg and TA-III 0.59, 1.17, 2.34 mg/kg, respectively) and in timosaponins–Huangbai alkaloids mixtures (1:1, 1:3, w/w, TB-II 300 mg/kg and TA-III 1.17 mg/kg) were studied for the first time in rats by this LC–MS/MS method. After single oral administration of timosaponins, mean Cmax and AUC0−t of TB-II and TA-III increased but non-proportional to the oral doses. When timosaponins–Huangbai alkaloids (1:1, 1:3, w/w) mixtures were administered, Cmax and AUC0−t of TB-II in the mixtures were obviously higher than the corresponding values in timosaponins at the same dose level.