Determination of phorbol esters in seeds and leaves of Jatropha curcas and in animal tissue by high-performance liquid chromatography tandem mass spectrometry

In order to improve the economic sustainability of the Jatropha-biofuel chain, seed cake detoxification and utilization of 'non toxic' Jatropa curcas accessions are the main activities pursued with the aim of using J. curcas seed cake in animal feed. Given this growing interest, a robust and reliable method for phorbol esters (PEs) determination is necessary. HPLC-UV is a well-established method to detect and quantify the PEs content in Jatropha seeds and related products, but it seems to be unsuitable for more complex matrices like Jatropha leaves and animal tissues, due to the presence of interfering compounds. The objective of this work was to develop and optimize a LC-MS/MS method for the quantitative determination of PEs in seeds and leaves of J. curcas L. plants from Ghana and Mexico and in liver (as an organ with the function of accumulation) from goats fed with PEs in their diet. The HPLC-UV analysis evidenced five chromatographic peaks in the toxic seed kernels corresponding to the factors C1, C2, C3, C6 and C4-C5, respectively, with a PEs concentration of about 5100 μg/g (as TPA equivalent). No PEs related peaks were detected in Mexican kernel seeds while in the case of leaves and liver the analysis was hampered by the presence of interfering compounds. The toxic kernel seed extract was used as a standard solution for the PEs quantitation in leaves and liver samples by LC-MS/MS, with the standard addition method. The most intense MRM transitions used to quantify and qualify the PEs were: 675 → 311, 693 → 311, and 293 → 265 m/z. The LC-MS/MS method with a LOD and a LOQ of 0.07 and 0.21 μg/g, respectively, resulted in more sensitivity and selectivity than the HPLC-UV method. All three MRM transitions were present in Ghanaian toxic kernel seed, while no peaks were present in the supposed non-toxic Mexican kernel seed. PEs concentration in the leaves of toxic Ghanaian accession resulted in about 1/10 of that in the kernel, while no PEs peaks were found in the J. curcas leaves from Mexico and in liver samples.