MECC-DAD和HPLC-FLD测定牛乳中5种黄曲霉毒素的方法比较研究

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 chromatographydiode array detector，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，5 μm）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、0.014～0.058 μg/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。

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

A new method for detecting five aflatoxin residues in raw milk was presented using micellar electrokinetic capillarychromatography with a diode array detector (MECC-DAD). The specificity of MECC-DAD was evaluated in comparisonwith that of high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). In the MECC-DADmethod, samples were cleaned up by immunoaffinity column chromatography before separation on an uncoated fused-silicacapillary column using 7.5 mmol/L borate buffer with 30 mmol/L sodium dodecyl sulfonate (pH 8.5) and 5% acetonitrileand 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, 5 μm) by gradient elution using a mobile phase consisting of acetonitrile and water after being cleanedup by immunoaffinity column chromatography. The analytes were detected by a fluorescence detector at 360 nm excitationwavelength and 440 nm emission wavelength. Results showed that MECC-DAD gave smoother and sharper peaks that were moresymmetrical without tailing. The limits of detection of MECC-DAD and HPLC-FLD were 0.182–1.669 and 0.014–0.058 μg/L,both meeting the requirements for the determination of aflatoxin residues in raw milk. The precision of HPLC-FLD wassomewhat higher than that of MECC-DAD. The recoveries of MECC-DAD and HPLC-FLD were 80.3%–137.0% and79.6%–134.0%, respectively. When the two methods were applied to the same commercial milk samples, the P value forthe deviation between the quantitative results obtained was 0.900, greater than 0.05, indicating no significant difference. Inconclusion, MECC-DAD was simple, efficient and inexpensive, making it more suitable for the simultaneous determinationof five aflatoxins in milk than HPLC-FLD.