Simultaneous UFLC–ESI–MS/MS determination of piperine and piperlonguminine in rat plasma after oral administration of alkaloids from Piper longum L.: Application to pharmacokinetic studies in rats

The alkaloids from Piper longum L. showed protective effects on Parkinsonʹs disease models in our previous study and piperine and piperlonguminine were the two main constituents in the alkaloids. The present study aimed at developing a rapid, sensitive, and accurate UFLC–ESI–MS/MS method and validating it for the simultaneous determination of piperine and piperlonguminine in rat plasma using terfenadine as the internal standard. The analytes and internal standard (IS) were extracted from rat plasma using a simple protein precipitation by adding methanol/acetonitrile (1:1, v/v). A Phenomenex Gemini 3 u C18 column (20 mm × 2.00 mm, 3 μm) was used to separate the analytes and IS using a gradient mode system with a mobile phase consisting of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) at a flow rate of 0.4 mL/min and an operating column temperature of 25 °C. The total analytical run time was 4 min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with transitions at m/z 286.1–201.1 for piperine, m/z 274.0–201.1 for piperlonguminine, and m/z 472.4–436.4 for the IS. The calibration curves were both linear (r > 0.995) over a concentration range of 1.0 to 1000 ng/mL; the lower limit of quantification (LLOQ) was 1.0 ng/mL for both piperine and piperlonguminine. The intra-day and inter-day precisions (RSD %) were <12.1%, accuracies ranged from 86.6 to 120%, and recoveries ranged from 90.4 to 108%. The analytes were proven stable in the short-term, long-term, and after three freeze–thaw cycles. The method was successfully applied to pharmacokinetic studies of piperine and piperlonguminine in rats after oral administration of alkaloids from P. longum L.