Identification of polycavernoside A as the causative agent of the fatal food poisoning resulting from ingestion of the red alga Gracilaria edulis in the Philippines

Outbreaks of seaweed poisonings are widely spread over the pacific area. Fatal glycosidic macrolides, polycavernosides, and potent tumor promoters, aplysiatoxins, have been previously isolated from edible seaweed. During 2002-2003, three fatal poisoning incidents occurred resulting from ingestion of two edible red alga, Acanthophora specifera and Gracilaria edulis, in Philippines causing eight deaths among 36 patients. Analytical methods for polycavernosides and aplysiatoxins were first developed, and the causative toxin from G. edulis, collected during the second poisoning event on December 2, 2002, was then investigated. The semipurified toxic fraction obtained from this alga based on mouse bioassay was applied to LC-diode array detection (LC-DAD) and LC/electrospray-MS (LC/ESI-MS) analyses. Both LC-DAD and LC/ MS chromatograms of this fraction suggested the presence of polycavernoside A (PA) by comparison with the authentic PA. The amount of PA in the alga was estimated as 84 and 72 nmol/kg, using the standard calibration curves for LC-DAD and for LC/ESI-MS in single ion monitoring (SIM) mode, respectively. Other polycavernoside congeners, A2, A3, and B2, and aplysiatoxin and debromoaplysiatoxin were less than the detection limit (2 nmol/kg alga, signal-to-noise ratio: 3) by LC/ESI-MS SIM analysis. In ESI-MS/MS, authentic polycavernosides showed the daughter ions corresponding to a sequential loss of fucosylxylose residues. These fragmentations were applied to LC/ESI-MS/MS polycavernosides in selective reaction monitoring (SRM) mode. On SRM mass chromatograms, the toxic fraction from the alga showed the peaks corresponding to PA, supporting the identification of PA as the cause of poisoning of G. edulis in Philippines.