Optimization of blood collection card method/enzyme-linked immunoassay for monitoring exposure of bottlenose dolphin to brevetoxin-producing red tides

Blood collection cards have been successfully used as a tool to monitor brevetoxin (PbTx) exposure in several species, including fish, mice, and rats. Previous methanolic methods used for extracting brevetoxin from blood collection cards have shown dolphin blood to have matrix difficulties in several biological assays. To better biomonitor protected marine mammal species in the Florida area, which is historically prone to unusual mortality events caused by brevetoxin exposure, we have modified the previous extraction method to consistently recover brevetoxin with a known efficiency from dolphin blood collection card samples with minimal matrix interference. A combination of phosphate-buffered saline (PBS) with 6% MeOH and 100% acetonitrile was used to elute blood from the cellulose card and precipitate proteins, respectively. Analysis was performed using a newly developed direct enzyme-linked immunoassay (ELISA), which yields a sample limit of quantification of 1 ng PbTx-3 equiv/mL. This extraction method allowed for linear recovery of PbTx-3 spiked into dolphin blood (1-30 ng/mL) with a consistent recovery rate of 58% and has subsequently been used to monitor brevetoxins in dolphins, as well as sea turtles and manatees, in regions endemic to red tides. In addition, two known metabolites of PbTx-2 were isolated and also found to be detectable using the ELISA. The cysteine conjugate (m/z 1018) and cysteine sulfoxide conjugate (m/z 1034) were found to have linear recoveries of 87% and 66%, respectively. In summary, this method of extracting brevetoxins and their metabolites from blood collection cards, in conjunction with the ELISA detection method, is a simple and reliable way to biomonitor physiologically relevant toxin levels in protected marine animals.