Validated HPLC–MS/MS Method for Simultaneous Determination of Curcumin and Piperine in Human Plasma

Purpose: To develop a sensitive and rapid method for the simultaneous determination of curcumin and piperine in human plasma. Methods: The method was based on high-performance liquid chromatography (HPLC) with electrospray ionization tandem mass spectrometer (ESI-MS/MS) detection in positive ionization mode. The analytes and internal standard were isolated from acidified plasma using liquid–liquid extraction (LLE). The organic extracts were evaporated, reconstituted in mobile phase and injected into the HPLC-MS/MS system. The analytes were chromatographed on a XB-C8 analytical column and MS-MS detection was performed on an electrospray ionization tandem mass spectrometer operated in multiple reaction monitoring (MRM) mode. Precursor→product combinations of m/z 369.9→177.0, 286.3→201.1 and 285.6→193.1 were used to quantify curcumin, piperine and the internal standard (IS), respectively. The assay was validated in the concentration range of 1.0 – 100.0 ng/ml for curcumin and 0.5 – 800.0 ng/ml for piperine using 0.5 ml of plasma. Results: The lowest limit of quantification (LLOQ) for curcumin and piperine was 1.00 and 0.50 ng/ml, respectively. The precision of the assay (expressed as coefficient of variation, CV) was < 12.6 % at all concentrations within the standard curve range, with adequate assay accuracy. Stability data revealed that the drugs were stable in plasma under various test conditions. Conclusion: The method is highly selective and rugged for the estimation of curcumin and piperine in human plasma and would be applicable to toxicokinetic, pharmacokinetic, bioavailability, and bioequivalence studies.