A sensitive LC-MS/MS assay for brevisulcenal and brevisulcatic acid toxins produced by the dinoflagellate Karenia brevisulcata

Abstract

A toxic dinoflagellate, Karenia brevisulcata, devastated almost all marine life in Wellington Harbour, New Zealand during the late summer of 1998. Brevisulcatic acids (BSXs) and brevisulcenals (KBTs), both polycyclic ether toxins, have been identified as the causative agents. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the sensitive and specific determination of BSXs and KBTs in culture medium, seawater and shellfish. Acidified algal culture, or seawater, was extracted using reverse phase solid phase extraction cartridges. Shellfish tissue homogenate was blended with methanol-water (9:1) and partitioned with hexane to remove non-polar lipids. This extraction protocol is similar to that used for analysis of lipophilic shellfish toxins. LC-MS/MS (triple quadrupole) was used for quantitative analysis with gradient elution (acidic buffer), positive electrospray ionization and multiple-reaction monitoring. Purified toxins were available for 4 KBTs (KBT-F, -G, -H and -I) and 4 BSXs (-1, -2, -4, and -5), and were used to calibrate the instrument responses. Relative response factors were used for semi-quantitative analysis of BSX-3 and BSX-6, using BSX-1 and BSX-4 respectively. Calibration curves for all toxins monitored were linear over the concentration range tested (5–200 ng mL−1) with r2 values >0.99. The method limit of quantitation was determined to be 2 ng mL−1 for BSXs and KBTs, except KBT-I, which was 5 ng mL−1. Validation data was generated for culture medium and shellfish. Toxin recoveries were typically >70% with relative standard deviations <20% across all of the matrices tested. In addition, toxins specific to K. brevisulcata were able to be detected in seawater at a cell concentration of 10,000 cells L−1, which represents the suggested trigger level for this harmful algal species. This method shows suitable performance characteristics to be regarded a useful tool to monitor toxin levels in a variety of sample matrices during future bloom events.