MECC-DAD和HPLC-FLD测定牛乳中5种黄曲霉毒素的方法比较研究

Comparison of Micellar Electrokinetic Capillary Chromatography with Diode Array Detector(MECC-DAD)and High Performance Liquid Chromatography with Fluorescence Detector(HPLC-FLD)for Detecting Five Aflatoxins in Raw Milk

建立高效胶束电动毛细管色谱耦合二极管阵列检测器(micellar electrokinetic capillary chromatographydiodearraydetector,MECC-DAD)检测牛乳中痕量黄曲霉毒素的新方法,采用高效液相色谱-荧光检测器(high performance liquid chromatography-fluorescence detector,HPLC-FLD)法及MECC-DAD法检测牛乳样品中5种黄曲霉毒素的残留量,考察2种检测技术的特异性。牛乳样品经免疫亲和柱净化后,MECC-DAD法采用未涂层熔融石英毛细管柱进行分离,以7.5 mmol/L四硼酸钠、30 mmol/L十二烷基磺酸钠及体积分数5%乙腈体积比1∶1∶1的混合液(pH 8.5)为缓冲液,检测波长为214 nm;HPLC-FLD法采用C18柱(150 mm×4.6 mm,5μm)进行分离,乙腈-水为流动相进行梯度洗脱,荧光检测器激发波长360 nm、发射波长440 nm。结果表明:MECC-DAD法的色谱图峰形更对称、平滑、尖锐,无拖尾现象;MECC-DAD和HPLC-FLD法的检出限分别为0.182~1.669、0.014~0.058μg/L,MECC-DAD法的检出限虽高于HPLC-FLD法,但也能满足牛乳中黄曲霉毒素的测定要求;HPLC-FLD法的精密度稍高于MECC-DAD法;MECC-DAD法的加标回收率为80.3%~137.0%,HPLC-FLD法的加标回收率为79.6%~134.0%,二者相当;2种方法对市售牛乳样品的定量检测结果偏差为P=0.900>0.05,没有显著性差异。MECC-DAD法分离快速、高效、经济,更适合牛乳样品中黄曲霉毒素残留量的检测。

A new method for detecting five aflatoxin residues in raw milk was presented using micellar electrokinetic capillary chromatography with a diode array detector(MECC-DAD).The specificity of MECC-DAD was evaluated in comparison with that of high-performance liquid chromatography with a fluorescence detector(HPLC-FLD).In the MECC-DAD method,samples were cleaned up by immunoaffinity column chromatography before separation on an uncoated fused-silica capillary column using 7.5 mmol/L borate buffer with 30 mmol/L sodium dodecyl sulfonate(pH 8.5)and 5%acetonitrile and the detection wavelength was set at 214 nm.In the HPLC-FLD method,samples were separated on a C18 column(150 mm×4.6 mm,5μm)by gradient elution using a mobile phase consisting of acetonitrile and water after being cleaned up by immunoaffinity column chromatography.The analytes were detected by a fluorescence detector at 360 nm excitation wavelength and 440 nm emission wavelength.Results showed that MECC-DAD gave smoother and sharper peaks that were more symmetrical without tailing.The limits of detection of MECC-DAD and HPLC-FLD were 0.182–1.669 and 0.014–0.058μg/L,both meeting the requirements for the determination of aflatoxin residues in raw milk.The precision of HPLC-FLD was somewhat higher than that of MECC-DAD.The recoveries of MECC-DAD and HPLC-FLD were 80.3%–137.0%and 79.6%–134.0%,respectively.When the two methods were applied to the same commercial milk samples,the P value for the deviation between the quantitative results obtained was 0.900,greater than 0.05,indicating no significant difference.In conclusion,MECC-DAD was simple,efficient and inexpensive,making it more suitable for the simultaneous determination of five aflatoxins in milk than HPLC-FLD.