James K J, Bishop A G, Healy B M, Roden C, Sherlock I R, Twohig M, Draisci R, Giannetti L, Lucentini L
Chemistry Department, Cork Institute of Technology, Bishopstown, Cork, Ireland.
Toxicon. 1999 Feb;37(2):343-57. doi: 10.1016/s0041-0101(98)00184-6.
The rare diarrhoeic shellfish poisoning (DSP) toxin, dinophysistoxin-2 (DTX-2), which is an okadaic acid (OA) isomer, has been isolated from a marine phytoplankton biomass that consisted mainly of Dinophysis acuta. Using a large double plankton net (length 5.9 m), bulk phytoplankton samples were collected off the south-west coast of Ireland and extracted with methanol and chloroform. Liquid chromatography coupled with ionspray mass spectrometry and tandem mass spectrometry (LC-MS, LC-MS-MS) showed the sample contained DTX-2 and OA, at a concentration of 80 pg/cell and 60 pg/cell, respectively. Flash chromatography using silica, sephadex LH20 and C18-silica, followed by preparative reversed-phase LC, separated DTX-2 from OA. The efficiency of the separation procedures was substantially improved by the use of a bioscreen to detect DSP toxins in eluate fractions and the application of a new derivatisation procedure for the chromatographic elucidation of toxin profiles with fluorimetric detection (LC-FLD). Thus, 1/1000th aliquots of eluate fractions were assayed using protein phosphatase-2A for the presence of inhibitory compounds. Positive fractions were further analysed for DSP toxins by LC-FLD following derivatisation using the hydrazine reagent, luminarine-3. The identity and purity of the free isolated DTX-2 was confirmed using flow injection analysis (FIA) and liquid chromatography (FIA-MS, LC-MS and LC-MS-MS).
罕见的腹泻性贝类中毒（DSP）毒素——鳍藻毒素-2（DTX-2），是冈田酸（OA）的一种异构体，已从主要由尖刺鳍藻组成的海洋浮游植物生物质中分离出来。使用大型双浮游生物网（长度5.9米），在爱尔兰西南海岸采集大量浮游植物样本，并用甲醇和氯仿进行提取。液相色谱结合离子喷雾质谱和串联质谱（LC-MS、LC-MS-MS）分析表明，样本中含有DTX-2和OA，浓度分别为80皮克/细胞和60皮克/细胞。采用硅胶、葡聚糖LH20和C18硅胶进行快速色谱分离，随后进行制备型反相液相色谱，将DTX-2与OA分离。通过使用生物筛选法检测洗脱液组分中的DSP毒素，并应用一种新的衍生化程序结合荧光检测（LC-FLD）对毒素谱进行色谱解析，大大提高了分离程序的效率。因此，使用蛋白磷酸酶-2A对洗脱液组分的千分之一等分试样进行检测，以确定是否存在抑制性化合物。阳性组分在使用肼试剂鲁米那林-3进行衍生化后，通过LC-FLD进一步分析DSP毒素。使用流动注射分析（FIA）和液相色谱（FIA-MS、LC-MS和LC-MS-MS）确认了游离分离的DTX-2的身份和纯度。
