Determination of deltonin in rat plasma by using HPLC–MS/MS and the application of this method in pharmacokinetic studies

• This is the first report of determination of deltonin in rat plasma by using HPLC–MS/MS.
• The method is simple, sensitive, and easily reproducible.
• The lower limit of quantification using 80 μL of rat plasma was 2 ng/mL.
• The method can be used to study pharmacokinetics of orally administered deltonin.

Deltonin is a naturally occurring spirostanol glycoside from Dioscorea zingiberensis C.H. Wright, which is used in traditional Chinese medicine. It exerts strong cytotoxic effect on C26 cells, inhibits C26 derived-tumor growth, and prolongs the survival of tumor-bearing mice after its oral administration, indicating its potential for use as an anti-tumor drug. To investigate the pharmacokinetic profiles of deltonin, a rapid, sensitive, and simplified high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay was developed and validated for the determination of deltonin in rat plasma. After acetonitrile-mediated plasma protein precipitation, chromatographic separation of deltonin was achieved using a reversed phase Hypersil Gold column (150 mm × 2.1 mm, 5 μm), with gradient elution using 0.1% formic acid and acetonitrile. Thereafter, deltonin was quantified using MS/MS with electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode. The flow rate of the mobile phase was 200 μL/min, and the retention time was 9.03 min for deltonin and 6.31 min for the internal standard (IS: 20(S)-ginsenoside Rb1). The linear range of the calibration curve was 2–5000 ng/mL (r2 > 0.99), and the limit of detection (LOD) was 0.46 ng/mL. The intra- and inter-day accuracies ranged from −2.8% to 11.1% and precisions (RSD) were within 13.1%. Deltonin was found to be stable under short-term temperature conditions, post-preparative temperature conditions, and after 3 freeze–thaw cycles conditions. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of deltonin (50 and 100 mg/kg). The pharmacokinetics is characterized by high apparent clearance (CL/F) and apparent volume of distribution (Vd/F).