UPLC-MS/MS法与ELISA法测定胖大海中黄曲霉毒素的比较研究

Comparative study on determination of aflatoxins in Sterculiae Lychnophorae Semen by UPLC-MS/MS and ELISA

目的采用超高效液相色谱-串联质谱法(UPLC-MS/MS)和酶联免疫吸附法(ELISA)对胖大海中的黄曲霉毒素进行检测,比较两种方法的检测结果以及方法学差异性。方法UPLC-MS/MS法测定:样品采用70%甲醇提取经过免疫亲和柱净化,色谱柱为C_(18)(100 mm×2.1 mm,1.7μm),柱温为40℃,流动相为水含0.1%甲酸(A)-甲醇含0.1%甲酸(B),梯度洗脱,流速为0.3 mL·min^(-1),多反应检测扫描采集模式,正负离子同时扫描进行数据采集。同时采用ELISA法测定,样品采用甲醇提取,酶联免疫试剂盒反应。结果UPLC-MS/MS法测定:黄曲霉毒素B1、B2、G1、G2线性关系良好,检测限分别为0.0135,0.0029,0.0086,0.0036μg·kg^(-1),定量限分别为0.0450,0.0098,0.0286,0.0119μg·kg^(-1),平均回收率分别为98.98%,100.32%,98.53%,99.56%,RSD≤2.42%(n=9)。ELISA法测定:黄曲霉毒素B1和总量的定量限分别为0.16,0.08μg·kg^(-1),检出限分别为0.05,0.02μg·kg^(-1),平均回收率分别为93.07%,89.67%,RSD≤13.99%(n=9)。两种方法测定结果的标准偏差在3.46%~7.63%之间。结论UPLC-MS/MS法的定量限低,检测限较低,加样回收率高,灵敏度和准确度较高,结果可靠。与UPLC-MS/MS法相比,ELISA法测定胖大海中黄曲霉毒素总量和黄曲霉毒素B1结果差异较小,未出现假阳性等情况。

Objective Ultra-high performance liquid chromatography with triple quadrupole mass spectrometry(UPLC-MS/MS)and enzyme-linked immunosorbent assay(ELISA)were used to detect aflatoxins in Sterculiae Lychnophorae Semen,and the test results and methodological of the two methods were compared.Methods UPLC-MS/MS method was used to measure the samples:the Sterculiae Lychnophorae Semen were extracted with 70%methanol and purified by immune affinity column and separated by the C_(18)(100 mm×2.1 mm,1.7μm)column(temperature:40℃)and the mobile phase was water[contains 0.1%formic acid(A)]-methanol[contains 0.1%formic acid(B)]at a flow rate of 0.3 mL·min^(-1).Multiple-reaction monitoring(MRM)was performed to identify,and positive and negative ions were scanned at the same time for data acquisition.Simultaneously,ELISA method was used to measure the same samples,and the samples were extracted by methanol and reacted with enzyme-linked immunoassay kit.Results Good linearity was obtained in UPLC-MS/MS method in detecting the four aflatoxins(B1,B2,G1,G2).The limits of detection(LOD)were 0.0135,0.0029,0.0086,0.0036μg·kg^(-1) and the limits of quantification(LOQ)were 0.0450,0.0098,0.0286,0.0119μg·kg^(-1),respectively.The sample recovery rates were 98.98%,100.32%,98.53%,99.56%,and the RSD was not greater than 2.42%(n=9).The LOQ of aflatoxin B1 and total amount by ELISA were 0.16,0.08μg·kg^(-1),and the LOD were 0.05 and 0.02μg·kg^(-1),respectively.The recoveries were 93.07%and 89.67%,and the RSD was not greater than 13.99%(n=9).Comparing the measurement results of the two methods,the relative deviation of the results of the two methods is between 3.46%and 7.63%.Conclusion The UPLC-MS/MS method has a lower LOQ and high sample recovery rate.It is specific,simple,fast and sensitive and can get reliable results.Compared with the UPLC-MS/MS method,the ELISA method in the determination of the total amount of aflatoxin and aflatoxin B1 in the Sterculiae Lychnophorae Semen showed little difference in the results,and no false positive results occurred.