LC/MS analysis of brevetoxin metabolites in the Eastern oyster ( Crassostrea virginica)

Brevetoxin (PbTx) metabolism was examined in the Eastern oyster ( Crassostrea virginica) following exposure to a Karenia brevis red tide, by using LC/MS(/MS) and cytotoxicity assay. Metabolites observed in field-exposed oysters were confirmed in oysters exposed to K. brevis cultures in the laboratory. Previously, we identified a cysteine conjugate and its sulfoxide (MH +: m/z 1018 and 1034) as metabolites of the brevetoxin congener PbTx-2. In the present study, we found a cysteine conjugate and its sulfoxide with A-type brevetoxin backbone structure (MH +: m/z 990 and 1006), as probable derivatives of PbTx-1. We also found glycine-cysteine–PbTx ( m/z 1047 and 1075), γ-glutamyl-cysteine–PbTx ( m/z 1147), and glutathione–PbTx ( m/z 1176 and 1204) conjugates with A- and B-type backbone structures. Amino acid–PbTx conjugates react with fatty acids through amide linkage to form a series of fatty acid–amino acid–PbTx conjugates. These fatty acid conjugates are major contributors to the composite cytototoxic responses obtained in extracts of brevetoxin-contaminated oysters. Other brevetoxin derivatives found in oysters are consistent with hydrolytic ring-opening and oxidation/reduction reactions.