Harwood D Tim, Shi Feng, Satake Masayuki, Holland Patrick T
Cawthron Institute, Private Bag 2, Nelson 7010, New Zealand.
Cawthron Institute, Private Bag 2, Nelson 7010, New Zealand.
Toxicon. 2014 Jun;84:19-27. doi: 10.1016/j.toxicon.2014.03.004. Epub 2014 Mar 27.
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 r(2) 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.
1998年夏末，一种有毒的双鞭毛藻——短沟凯伦藻，几乎摧毁了新西兰惠灵顿港的所有海洋生物。短沟凯伦藻酸（BSXs）和短沟凯伦烯醛（KBTs）这两种多环醚毒素，已被确定为致病因子。已开发并验证了一种液相色谱串联质谱（LC-MS/MS）方法，用于灵敏且特异的测定培养基、海水和贝类中的BSXs和KBTs。酸化的藻类培养物或海水，使用反相固相萃取柱进行萃取。贝类组织匀浆与甲醇-水（9:1）混合，并用己烷进行分配以去除非极性脂质。该萃取方案类似于用于分析亲脂性贝类毒素的方案。LC-MS/MS（三重四极杆）用于梯度洗脱（酸性缓冲液）、正电喷雾电离和多反应监测的定量分析。有4种KBTs（KBT-F、-G、-H和-I）和4种BSXs（-1、-2、-4和-5）的纯化毒素，用于校准仪器响应。相对响应因子分别用于以BSX-1和BSX-4对BSX-3和BSX-6进行半定量分析。所监测的所有毒素的校准曲线在测试浓度范围（$5 - 200 ng mL^{-1}$）内呈线性，$r^{2}$值>0.99。除KBT-I的定量限为$5 ng mL^{-1}$外，BSXs和KBTs的方法定量限确定为$2 ng mL^{-1}$。针对培养基和贝类生成了验证数据。在所有测试基质中，毒素回收率通常>70%，相对标准偏差<20%。此外，在细胞浓度为$10,000 cells L^{-1}$的海水中能够检测到短沟凯伦藻特有的毒素，这代表了该有害藻华物种的建议触发水平。该方法显示出合适的性能特征，可被视为在未来藻华事件期间监测各种样品基质中毒素水平的有用工具。
