高效液相色谱同步检测饲料中黄曲霉毒素B_(1)、玉米赤霉烯酮及呕吐毒素的方法

A Method for Simultaneous Determination of Aflatoxin B_(1), Zearalenone andDeoxynivalenol in Feed by High Performance Liquid Chromatography

本研究旨在建立同步检测饲料中黄曲霉毒素B_(1)、玉米赤霉烯酮及呕吐毒素的高效液相色谱(HPLC)法。样品用乙腈-水提取,经涡旋、高速离心,移取2 mL上清液加入28 mL含1%吐温-20的磷酸盐缓冲液(PBS,pH 7.0),过三合一免疫亲和柱净化,用2 mL甲醇洗脱;洗脱液经50℃浓缩氮气吹干后用50%甲醇-水复溶,采用高效液相色谱仪串联光化学衍生器、荧光检测器和紫外检测器进行检测。结果表明:黄曲霉毒素B_(1)标准品溶液浓度在2.5~50.0 ng/mL、玉米赤霉烯酮标准品溶液浓度在50~2500 ng/mL、呕吐毒素标准品溶液浓度在250~5000 ng/mL时线性关系良好,平均加标回收率为81.2%~92.3%,相对标准偏差(RSD)为4.3%~9.5%。采用该方法检测了4种不同饲料样品中的黄曲霉毒素B_(1)、玉米赤霉烯酮及呕吐毒素含量,所有样品均检测出1种以上霉菌毒素,说明饲料中霉菌毒素混合污染较为普遍。由此可见,本试验所建立的方法准确度和精密度高,稳定性好,可作为饲料中黄曲霉毒素B_(1)、玉米赤霉烯酮及呕吐毒素的同步检测方法。

A high performance liquid chromatographic(HPLC)method was developed for simultaneous determination of aflatoxin B_(1),zearalenone and deoxynivalenol in feed.Samples were extracted with acetonitrile-water,after vortexing and high-speed centrifugation,2 mL of supernatant was transferred and mixed with 28 mL phosphate buffer saline(PBS,pH 7.0)containing 1%tween-20.Then the extracted solution was purified with immune-affinity column and eluted with 2 mL of methanol.The eluent was blow-dried by concentrated nitrogen at 50℃and then redissolved in 50%methanol-water.Finally,the reconstituted solution was detected by a high performance liquid chromatography coupled with a photochemical derivatizer,a fluorescence detector and an ultraviolet detector.The results showed that the calibration curves of aflatoxin B_(1)were linear in 2.5 to 50.0 ng/mL,the calibration curves of zearalenone were linear in 50 to 2500 ng/mL,and the calibration curves of deoxynivalenol were linear in 250 to 5000 ng/mL.The average recovery ratio was ranged from of 81.2%to 92.3%,and the relative standard deviation(RSD)was ranged from 4.3%to 9.5%.The established method was then used to determine the contents of aflatoxin B_(1),zearalenone and deoxynivalenol in four kinds of feed samples,it was found that all the detected samples contained more than one mycotoxin,implying that the feed samples were widely contaminated with mycotoxins.In conclusion,the established method has high accuracy,precision and good stability,and it can be used as a simultaneous detection method for aflatoxin B_(1),zearalenone and deoxynivalenol in feed.