High performance liquid chromatography method for rapid quantification of phorbol esters in Jatropha curcas seed

Jatropha curcas seeds are a promising source of oil for biodiesel production. The presence of toxic phorbol esters in oil, biodiesel byproducts/co-products (e.g., seedcake and seed meal) and different plant parts however, raises both occupational and eco-toxicological concerns. Due to toxicity issues, there is a need to develop a rapid, sensitive, and accurate method for determining phorbol ester content. An HPLC/UV-based rapid determination of phorbol esters using a C18 column (20, 50, and 75 mm columns) was devised and validated. Vortex extraction was found to be the best method for extracting phorbol esters. The separation of Jatropha phorbol esters was best achieved with increased column lengths and run times (e.g., run times of 6, 8, and 12.5 min for 20, 50, and 75 mm columns, respectively). The limit of detection and limit of quantification for Jatropha phorbol esters (phorbol 12-myristate 13-acetate equivalent) ranged from 26 to 68 ng and 107-200 ng using three columns, respectively. The finalized method was rapid (≤12.5 min run time) and able to detect low concentrations (up to 26 ng) of phorbol esters when compared to conventional methods (>35 min run time and 400-800 ng detection limit). The analyzed phorbol ester concentrations for different Jatropha samples (kernel, kernel meal, seedcake, seed meal, and oil) were within the acceptable range of reported values. The developed method was shown to be a rapid, sensitive, accurate, and cost-effective approach, which would find diverse applications ranging from value-added co-product development risk assessment to agricultural research.