Hatfield Robert G, Turner Andrew D
Centre for Environment, Fisheries and Aquaculture Science, Barrack Rd, The Nothe, Weymouth, Dorset, DT4 8UB, UK.
J AOAC Int. 2012 Jul-Aug;95(4):1089-96. doi: 10.5740/jaoacint.12-005.
The bioaccumulation of paralytic shellfish toxins in mussels, oysters, cockles, hard clams, razors, and king scallops is monitored in England, Scotland, and Wales by AOAC Official Method 2005.06 LC-with fluorescence detection (FLD). One of the commonly perceived disadvantages of using this method is the long turnaround time and low throughput in a busy laboratory environment. The chromatographic analysis of each sample typically utilizes a 15 min cycle time to achieve toxin oxidation product separation and column equilibration prior to subsequent analysis. A standard RP C18 analytical column, used successfully in recent years, achieves good separation with a long column lifetime. The analysis of a 40 sample qualitative screening batch takes approximately 18 h, including blanks, standards, and other QC samples. The availability of superficially porous column technology has offered the potential to reduce analysis time while retaining column performance on existing hardware. In this study, AOAC Official Method 2005.06 with LC-FLD was transferred to two different commercially available superficially porous columns, and the method performance characteristics were evaluated. Both columns separated all toxins adequately with cycle times less than half that of the existing method. Linearity for each toxin was acceptable up to two times the European maximum permitted limit of 800 microg di-HCl saxitoxin equivalent/kg flesh. LOD and LOQ values were substantially improved for the majority of toxins, with gonyautoxin 1&4 and neosaxitoxin showing up to a two- and fourfold improvement, respectively, depending on the column used. Quantification results obtained from parallel analysis of contaminated samples were acceptable on both columns. Comparative screen results gave a slight increase in the occurrence of contaminated samples, which was attributed to the improved detection limit for most toxins. Issues with rapidly increasing back pressure, however, were identified with both columns, with a limit of around 500 injections. This compares to the >3000 cycles routinely obtained with the standard RP-C18 HPLC columns currently in use. Overall, the gain achieved with these columns through shorter analysis time and improved analytical sensitivity is potentially of benefit in a high-throughput environment. For the routine high-throughput screening of shellfish samples, however, an improved column lifetime is desirable.
在英格兰、苏格兰和威尔士，采用美国官方分析化学师协会（AOAC）的2005.06号官方方法——带荧光检测（FLD）的液相色谱法，对贻贝、牡蛎、鸟蛤、硬壳蛤、蛏子和栉孔扇贝中麻痹性贝类毒素的生物累积情况进行监测。使用该方法通常被认为存在的一个缺点是，在繁忙的实验室环境中周转时间长且通量低。每个样品的色谱分析通常利用15分钟的循环时间，以实现毒素氧化产物的分离和柱平衡，然后进行后续分析。近年来成功使用的标准反相C18分析柱，分离效果良好且柱寿命长。分析一批40个样品的定性筛查大约需要18小时，包括空白样品、标准样品和其他质量控制样品。表面多孔柱技术的出现，为减少分析时间同时在现有硬件上保持柱性能提供了可能。在本研究中，将采用液相色谱 - 荧光检测的AOAC官方方法2005.06转移至两种不同的市售表面多孔柱，并对方法性能特征进行评估。两种柱都能充分分离所有毒素，循环时间不到现有方法的一半。每种毒素在高达欧洲最大允许限量（800微克二盐酸石房蛤毒素当量/千克鱼肉）两倍的范围内线性良好。大多数毒素的检测限（LOD）和定量限（LOQ）值有显著改善，根据所使用的柱不同，膝沟藻毒素1&4和新石房蛤毒素的改善倍数分别高达两倍和四倍。对受污染样品进行平行分析得到的定量结果在两种柱上均可接受。比较筛查结果显示受污染样品的发生率略有增加，这归因于大多数毒素检测限的提高。然而，两种柱都出现了背压迅速增加的问题，极限约为500次进样。相比之下，目前使用的标准反相C18高效液相色谱柱通常能实现超过3000次循环。总体而言，这些柱通过缩短分析时间和提高分析灵敏度所带来的收益，在高通量环境中可能是有益的。然而，对于贝类样品的常规高通量筛查，需要提高柱寿命。
