Characterization and quantification of karlotoxins by liquid chromatography–mass spectrometry

Karlodinium veneficum is a cosmopolitan dinoflagellate with a worldwide distribution in mesohaline temperate waters. The toxins from K. veneficum, or karlotoxins (KmTxs), which have been implicated in fish kill events, have been purified from monoalgal cultures, and shown to possess hemolytic, cytotoxic and ichthyotoxic activities. Three karlotoxins (KmTx 1–1, KmTx 1–3 and KmTx 2) have been isolated from two different North American strains of K. veneficum and characterized using liquid chromatography–mass spectrometry (LC–MS). KmTx 1 karlotoxins have a UV absorption maximum (λmax 225 nm) at lower wavelengths than KmTx 2 karlotoxins (λmax 235 nm). The exact masses and predicted empirical formulae for the karlotoxins (KmTx 1–1, 1308.8210, C67H120O24; KmTx 1–3, 1322.8637, and C69H126O23; KmTx 2, 1344.7938, C67H121ClO24) were determined using high resolution mass spectrometry. Although the individual toxins produce a single peak in reverse phase high performance liquid chromatography (HPLC), MS revealed congeners co-eluting within each peak. These congeners could be separated under normal phase chromatography and revealed a single hydroxylation being responsible for the mass differences. Multistage MS (MSn) showed that the three KmTxs and their congeners share a large portion of their structures including an identical 907 amu core fragment.These data were used to develop a quantitative LC–MS assay for karlotoxins from cultures and environmental samples. The sensitivity afforded by MS detection compared to UV absorbance allowed toxin quantification at 0.2 ng when injected on column. Aqueous solutions of karlotoxins were found to quantitatively adsorb to PTFE and nylon membrane filters. Aliquots from whole cultures or environmental samples could be concentrated and desalted by adsorption to PTFE syringe filters and karlotoxins eluted with methanol for analysis by LC–MS. This simplified solid phase cleanup afforded new data indicating that each karlotoxin may also exist as sulfated derivatives and also provided a rapid detection method for karlotoxin from environmental samples and whole cultures.