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.