免疫磁珠高通量自动净化-超高效液相色谱法测定粮食中玉米赤霉烯酮

Determination of Zearalenone in Grains by High-throughput Automatic Purification with Immunomagnetic Beads Coupled with Ultra-high Performance Liquid Chromatography

基于免疫磁珠高通量自动净化的前处理方法,使用超高效液相色谱仪对粮食中玉米赤霉烯酮的含量进行测定。首先将免疫磁珠与玉米赤霉烯酮的反应时间、样品提取液和不同粮食基质的净化效果进行优化。经方法验证,超高效液相色谱的检出限和定量限分别为3.5μg/kg和12.0μg/kg。在优化条件下,全麦粉和玉米全粉阴性样品的3个添加水平的加标回收率在99.04%~109.26%之间,变异系数不大于6.88%。玉米全粉、全麦粉、糙米粉、小麦粉等粮食基质中玉米赤霉烯酮成分的国家有证标准物质或质控样品的测定结果在标准值及其扩展不确定度范围内,变异系数不大于3.54%,测定结果较为满意。采用Bland-Altman法对免疫磁珠净化法和免疫亲和柱净化法之间的差异进行分析,结果显示这2种净化方法的差异在可接受范围内,因此,这2种方法在净化和富集粮食中玉米赤霉烯酮时可互相代替使用。免疫磁珠净化方法配套使用的真菌毒素全自动净化仪可同时净化10~24个样品,平均每个样品的净化时间约为2~3 min,实现粮食中玉米赤霉烯酮的高通量自动净化。超高效液相色谱分析速度快、灵敏度高、准确性高,可用于检测粮食中玉米赤霉烯酮的含量。

In this work,ultra-high performance liquid chromatography(UPLC)with sample pretreatment by high-throughput automatic purification using immunomagnetic beads was used to accurately determine the content of zearalenone in grains.The reaction time of immunomagnetic beads with zearalenone and the extraction solvent composition were optimized,and the purification effects of immunomagnetic beads on different grain matrices were evaluated.The detection limit and quantitation limit of UPLC were 3.5 and 12.0μg/kg,respectively.Under the optimized conditions,the recoveries of spiked negative whole wheat and corn flour samples were 99.04%–109.26%,with coefficients of variation not greater than 6.88%.The results of the UPLC method for zearalenone in the national certified reference materials or quality control samples for whole corn flour,whole wheat flour,brown rice flour and wheat flour agreed with the certified values within the extended uncertainty range with coefficients of variation not greater than 3.54%.Analysis by the Bland-Altman method indicated that the difference between immunomagnetic bead purification and immunoaffinity column purification was acceptable.Thus,the two methods could be used interchangeably with each other to purify and enrich zearalenone from grains.The automatic mycotoxin purification system based on immunomagnetic beads,allowing the high-throughput automatic purification of zearalenone from grains,could purify 10-24 samples simultaneously,and it averagely took 2-3 minutes to purify one sample.In conclusion,UPLC has the advantages of rapidity,high sensitivity and high accuracy and can be used for the determination of zearalenone in grains.