Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis Zografou, 157 71 Athens, Greece.
Anal Bioanal Chem. 2010 Jul;397(6):2245-52. doi: 10.1007/s00216-010-3727-3. Epub 2010 May 2.
Anatoxin-a is a potent alkaloid neurotoxin produced by a number of cyanobacterial species and released in freshwaters during cyanobacterial blooms. Its high toxicity is responsible for several incidents of lethal intoxications of birds and mammals around the world; therefore anatoxin-a has to be regarded as a health risk and its concentration in lakes and water reservoirs should be monitored. Phenylalanine is a natural amino acid, also present in freshwaters, isobaric to anatoxin-a, with a very similar fragmentation pattern and LC retention. Since misidentification of phenylalanine as anatoxin-a has been reported in forensic investigations, special care must be taken in order to selectively determine traces of anatoxin-a in the presence of naturally occurring phenylalanine. A fast LC tandem MS method was developed by using a 1.8 microm 50 x 2.1 mm C18 column for the separation of anatoxin-a and phenylalanine, achieving a 3-min analysis time. Isotopically labelled phenylalanine-d(5) was employed as internal standard to compensate for electrospray ion suppression and sample preconcentration losses. Both compounds were preconcentrated 1,000-fold on a porous graphitic carbon solid-phase extraction (SPE) cartridge after adjustment of sample pH to 10.5. The method was validated by using lake water spiked at four different levels from 0.01 to 1 microg L(-1). Anatoxin-a recovery ranged from 73 to 97%, intra-day precision (RSD%) ranged from 4.2 to 5.9, while inter-day precision (RSD%) ranged from 4.2 to 9.1%. Limits of detection and quantification were 0.65 and 1.96 ng L(-1) respectively. The method was successfully applied for the detection of anatoxin-a in Greek lakes at concentrations ranging from less than 0.6 to 9.1 ng L(-1).
石房蛤毒素-a 是一种由多种蓝藻产生的强效生物碱神经毒素，在蓝藻水华期间释放到淡水中。其高毒性导致了世界各地鸟类和哺乳动物发生了几起致命中毒事件；因此，石房蛤毒素-a 必须被视为健康风险，其在湖泊和水库中的浓度应进行监测。苯丙氨酸是一种天然存在于淡水中的氨基酸，与石房蛤毒素-a 等重，具有非常相似的碎片模式和 LC 保留。由于在法医调查中报告了将苯丙氨酸错误识别为石房蛤毒素-a 的情况，因此必须特别小心，以便在存在天然苯丙氨酸的情况下选择性地确定痕量的石房蛤毒素-a。通过使用 1.8 微米 50 x 2.1 毫米 C18 柱分离石房蛤毒素-a 和苯丙氨酸，开发了一种快速 LC 串联 MS 方法，实现了 3 分钟的分析时间。同位素标记的苯丙氨酸-d(5)被用作内标，以补偿电喷雾离子抑制和样品预浓缩损失。在将样品 pH 调节至 10.5 后，将两种化合物在多孔石墨碳固相萃取 (SPE) 小柱上预浓缩 1000 倍。通过在四个不同水平（0.01 至 1 μg L(-1)）对湖水进行加标验证了该方法。石房蛤毒素-a 的回收率范围为 73%至 97%，日内精密度（RSD%）范围为 4.2%至 5.9%，而日间精密度（RSD%）范围为 4.2%至 9.1%。检测限和定量限分别为 0.65 和 1.96 ng L(-1)。该方法成功应用于检测希腊湖泊中的石房蛤毒素-a，浓度范围为 0.6 至 9.1 ng L(-1)。
