全自动免疫亲和在线净化/高效液相色谱法快速高通量测定饲料中黄曲霉毒素

Rapid and High Throughput Analysis of Aflatoxins in Feed Matrix Using On-line Immunoaffinity Column Clean-up and High Performance Liquid Chromatography

建立了全自动免疫亲和在线净化/高效液相色谱快速高通量测定饲料中黄曲霉毒素(Aflatoxins,AFT)的分析方法。饲料样品经乙腈-水(80∶20,体积比)提取,3 g/L Triton X-100水溶液10倍稀释后,用自动进样器注入RIDACREST在线固相萃取系统并流经黄曲霉毒素免疫亲和小柱,以甲醇-水(45∶55,体积比)为流动相,流速为1.0 m L/min,C18色谱柱(150 mm×3.5 mm,5μm)分离,光化学衍生,荧光检测器测定。根据3倍信噪比的峰响应值,确定黄曲霉毒素B1,B2,G1,G2的检出限分别为0.08,0.05,0.18,0.08μg/kg,分别在1~100,0.24~24,0.56~56,0.24~24μg/kg范围内呈线性相关,相关系数(r2)分别为0.999 4,0.999 7,0.999 8和0.999 8;AFT在猪饲料、鸡饲料、宠物饲料和饲料原料4类样品中的加标回收率为72.6%~103%,相对标准偏差为2.5%~4.9%。该方法一次装柱可检测60个样品,液相色谱分析一个样品总的运行时间为15 min,所以1 d可检测70~80个样品,满足饲料中黄曲霉毒素快速高通量准确定量检测的需要。

A rapid analytical method using on-line immunoaffinity column clean-up and high performance liquid chromatography（HPLC） was developed for the determination of aflatoxins（B1,B2,G1,G2） in feed samples.The samples were extracted with acetonitrile-water solution（80∶20,by volumn）.The extraction solution was diluted by 10 times using 3 g/L triton X-100 water solution,then injected into the RIDA-CREST on-line immunoaffinity column clean-up system,and washed out with methanol-water（45∶55,by volume） as mobile phase at a flow rate of 1.0 mL/min.Aflatoxins were analyzed by HPLC/FLD with post-column photochemical derivatizaton.The detection limits of AFB1,AFB2,AFG1 and AFG2 were 0.08,0.05,0.18,0.08 μg/kg,respectively.The linear ranges for AFB1,AFB2,AFG1 and AFG2 were 1-100,0.24-24,0.56-56,0.24-24 μg/kg,with their correlation coefficients（r^2） of 0.999 4,0.999 7,0.999 8 and 0.999 8,respectively.The recoveries for four kinds of feed samples spiked with aflatoxins were in the range of 72.6%-103%,with relative standard deviations of 2.5%-4.9%.Each immunoaffinity colomn could be used for 60 samples cleanup,and the total running time for each HPLC analysis is 15 min,thus 70-80 samples could be detected per day.This method could satisfy the demands for rapid and accurate analysis of aflatoxin in feed.