Simultaneous determination of pimpinellin, isopimpinellin and phellopterin in rat plasma by a validated UPLC-MS/MS and its application to a pharmacokinetic study after administration of Toddalia asiatica extract.

A rapid and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for simultaneous determination of three bioactive coumarins of Toddalia asiatica extract including pimpinellin, isopimpinellin and phellopterin in rat plasma for the first time. Phenacetin was used as the internal standard (IS). Plasma samples were extracted by liquid-liquid extraction with methyl tert-butyl ether. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH C₁₈ column with an isocratic mobile phase consisting of methanol-5 mmol/L ammonium acetate (65:35, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization (ESI) source with positive ionization mode. The method was linear for all analytes over investigated range with all correlation coefficients greater than 0.9942. The lower limits of quantification (LLOQ) were 25.0 ng/mL for pimpinellin, 10.0 ng/mL for isopimpinellin and 5.00 ng/mL for phellopterin. The intra- and inter-day precision (RSD%) was within 12% and the accuracy (RE%) ranged from -2.3% to 5.5%. The rapid and sensitive method was fully validated and successfully applied to the pharmacokinetic study of pimpinellin, isopimpinellin and phellopterin in rats following oral administration of Toddalia asiatica extract.