A sensitive assay for palytoxins, ovatoxins and ostreocins using LC-MS/MS analysis of cleavage fragments from micro-scale oxidation

Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 mu g kg(-1) for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 mu g kg(-1)) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of <= 18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL(-1) and the LOQ 4 ng mL(-1), which corresponds to 10 mu g kg(-1) in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required. (C) 2012 Published by Elsevier Ltd.