Stroka Joerg, von Holst Christoph, Anklam Elke, Reutter Matthias
European Commission, Directorate General Joint Research Centre, Institute for Reference Materials and Measurements, Retieseweg, B-2440 Geel, Belgium.
J AOAC Int. 2003 Nov-Dec;86(6):1179-86.
A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B1 in cattle feed at a possible future European regulatory limit (1 ng/g). The test portion was extracted with acetone-water (85 + 15), filtered, diluted with water, and applied to an immunoaffinity column. The column was washed with water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase liquid chromatography (RP-LC) and detected by fluorescence after post column derivatization (PCD) involving bromination. PCD was achieved with either pyridinium hydrobromide perbromide (PBPB), used by 14 laboratories, or an electrochemical cell and addition of bromide to the mobile phase, used by 7 laboratories. Both derivatization techniques were not significantly different when compared by the t-test; the method was statistically evaluated for all laboratories together (bromination and PBPB). The cattle feed samples, both spiked and naturally contaminated with aflatoxin B1, were sent to 21 laboratories in 14 different countries (United States, Japan, and Europe). Test portions were spiked at levels of 1.2 and 3.6 ng/g for aflatoxin B1. Recoveries ranged from 74 to 157%. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the relative standard deviation for repeatability (RSDr) ranged from 5.9 to 8.7%. The relative standard deviation for reproducibility (RSDR) ranged from 17.5 to 19.6%. The method showed acceptable within- and between-laboratory precision for this matrix, as evidenced by HORRAT values, at the target levels of determination for aflatoxin B1. No major differences in RSD were observed, showing that the composition of the feeds was not a factor for the samples tested and that the method was applicable for all materials used.
开展了一项合作研究，以评估免疫亲和柱净化液相色谱（LC）法在未来欧洲可能的监管限量（1 ng/g）下测定牛饲料中黄曲霉毒素B1的有效性。将测试部分用丙酮 - 水（85 + 15）萃取，过滤，用水稀释，然后应用于免疫亲和柱。用水冲洗柱子以去除干扰化合物，并用甲醇洗脱纯化的黄曲霉毒素B1。通过反相液相色谱（RP - LC）分离并测定黄曲霉毒素B1，并在涉及溴化的柱后衍生化（PCD）后通过荧光检测。14个实验室使用过溴化吡啶鎓（PBPB）实现PCD，7个实验室使用过电化学池并向流动相中添加溴化物实现PCD。通过t检验比较时，两种衍生化技术没有显著差异；对所有实验室（溴化和PBPB）的方法进行了统计评估。将添加了黄曲霉毒素B1以及天然受污染的牛饲料样品发送到14个不同国家（美国、日本和欧洲）的21个实验室。黄曲霉毒素B1的测试部分添加水平为1.2和3.6 ng/g。回收率范围为74%至157%。基于添加样品（2个水平的盲样对）以及天然受污染样品（3个水平的盲样对）的结果，重复性相对标准偏差（RSDr）范围为5.9%至8.7%。再现性相对标准偏差（RSDR）范围为17.5%至19.6%。如HORRAT值所示，在黄曲霉毒素B1的目标测定水平下，该方法在实验室内和实验室间均显示出可接受的精密度。未观察到RSD有重大差异，表明饲料成分不是测试样品的影响因素，并且该方法适用于所有使用的材料。
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