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Characterization and quantification of karlotoxins by liquid chromatography-mass spectrometry

机译：液相色谱-质谱法鉴定和定量卡洛毒素

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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 (λ_(max) 225 nm) at lower wavelengths than KmTx 2 karlotoxins (λ_(max) 235 nm). The exact masses and predicted empirical formulae for the karlotoxins (KmTx 1-1, 1308.8210, C_(67)H_(120)O_(24); KmTx 1-3, 1322.8637, and C_(69)H_(126)O_(23); KmTx 2, 1344.7938, C_(67)H_(121)ClO_(24)) 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 (MS~n) 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.
机译：香叶Karlodinium v​​eneficum是世界性的鞭毛藻，在中卤温带水域中分布全球。已与鱼类杀灭事件有关的来自K. veneficum的毒素或karlotoxins（KmTxs）已从单藻培养物中纯化，并显示出具有溶血，细胞毒和鱼腥毒素的活性。从两个不同的北美K. veneficum菌株中分离出了三种karlotoxins（KmTx 1-1，KmTx 1-3和KmTx 2），并使用液相色谱-质谱法（LC-MS）对其进行了表征。与KmTx 2 karlotoxins（λ_（max）235 nm）相比，KmTx 1 karlotoxins在更低的波长处具有最大UV吸收（λ_（max）225 nm）。 karlotoxins的确切质量和预测的经验公式（KmTx 1-1，1308.8210，C_（67）H_（120）O_（24）; KmTx 1-3，1322.8637和C_（69）H_（126）O_（23 ）; KmTx 2，1344.7938，C_（67）H_（121）ClO_（24））使用高分辨率质谱法测定。尽管单个毒素在反相高效液相色谱（HPLC）中产生一个峰，但MS揭示了在每个峰中共洗脱的同类物。这些同类物可以在正相色谱法下分离，并显示单个羟基化是造成质量差异的原因。多级质谱（MS〜n）表明，这三个KmTx及其同源物共享大部分结构，包括相同的907 amu核心片段。这些数据用于开发文化和环境样品中卡洛毒素的定量LC-MS分析。通过MS检测提供的灵敏度与UV吸收相比，在进样到色谱柱上时，毒素定量为0.2 ng。发现卡洛毒素的水溶液可定量地吸附到PTFE和尼龙膜过滤器上。可以浓缩整份培养物或环境样品中的等分试样，并通过吸附到PTFE注射器式过滤器和脱氧核糖核酸（用甲醇洗脱）进行脱盐，以进行LC-MS分析。这种简化的固相净化过程提供了新的数据，表明每种karlotoxin也可能以硫酸盐衍生物形式存在，并且还提供了从环境样品和整个培养物中快速检测karlotoxin的方法。

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