A simple U-HPLC-MS/MS method for the determination of liensinine and isoliensinine in rat plasma.

An ultra-high performance liquid chromatography tandem mass spectrometry (U-HPLC-MS/MS) method was developed and validated to determine liensinine and isoliensinine in rat plasma simultaneously. Plasma samples were prepared using protein precipitation with acetonitrile. The two analytes and the internal standard pirfenidone were separated on an Acquity U-HPLC BEH C18 column with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40mL/min. Both liensinine and isoliensinine were eluted at 0.63 and 0.82min, respectively. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 611.6 → 206.2 for liensinine and m/z 611.4 → 192.2 for isoliensinine. The linearity of this method was found to be within the concentration range of 5-700ng/mL for liensinine and isoliensinine in rat plasma. The lower limits of quantification (LLOQ) were all 5ng/mL for liensinine and isoliensinine. The relative standard deviations (RSD) of intra and inter precision were less than 10% for both liensinine and isoliensinine. The method was also successfully applied to the pharmacokinetic study of liensinine and isoliensinine in rats.