James K J, Furey A, Sherlock I R, Stack M A, Twohig M, Caudwell F B, Skulberg O M
Chemistry Department, Cork RTC, Bishopstown, Ireland.
J Chromatogr A. 1998 Mar 6;798(1-2):147-57. doi: 10.1016/s0021-9673(97)01207-7.
Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC-FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2-84.9% at concentrations of 10 micrograms/l. The R.S.D. values were 1.7-3.9% (n = 8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.
蓝藻神经毒素与饮用受污染水导致的动物死亡有关。之前曾使用高效液相色谱法（HPLC）结合紫外检测对类毒素-a（AN）和高类毒素-a（HMAN）进行分析，但该方法灵敏度不足且易受干扰。最近开发了一种灵敏的荧光（FL）方法，使用4-氟-7-硝基-2,1,3-苯并恶二唑（NBD-F）进行衍生化来测定AN，该方法已应用于同时测定AN、HMAN及其环氧和二氢降解产物。采用微量合成法由AN和HMAN制备二氢和环氧衍生物。通过电喷雾质谱和其苯并恶二唑衍生物的HPLC-FL证实，这些化合物产率很高。使用等度反相HPLC可轻松分离所有六种NBD衍生物。使用弱阳离子交换（WCX）固相萃取（SPE）从加标水样中回收这些化合物，在浓度为10微克/升时回收率为83.2-84.9%。相对标准偏差值为1.7-3.9%（n = 8），所有六种化合物的检测限均优于10纳克/升，表明该方法具有高灵敏度。该方法成功应用于天然样品中毒素降解产物的分析。从爱尔兰卡拉赫湖的底栖颤藻水华中分离出二氢类毒素-a（0.8毫克/克），发现其含有两种异构体，但其比例与合成材料中的不同。
