2种HPLC-柱后衍生化-荧光检测法测定陈皮中黄曲霉毒素的比较

目的:对陈皮中黄曲霉毒素测定的2种柱后衍生化方法(碘衍生化和电化学衍生化)进行比较。方法: 样品经过免疫亲和柱净化,HPLC-柱后衍生化-荧光检测器检测。采用Diamonsil ODS C₁₈(5 μm,4.6 mm×200 mm)色谱柱,荧光检测,激发波长360 nm,发射波长450 nm。柱后衍生化系统(1)碘衍生化:以甲醇-乙腈-水(25:18:57)为流动相,流速0.8 mL·min;衍生化溶液为0.05%碘溶液,流速0.3 mL·min,衍生化反应温度70℃;(2)电化学衍生化:以甲醇-乙腈-水(20:20:60)为流动相(每1 L流动相中加入溴化钾120 mg和4 mol·L硝酸350 μL),流速1.0 mL·min。结果: 碘衍生化,黄曲霉毒素G₂、B₂在2~36 pg,黄曲霉毒素G₁、B₁在2~120 pg范围内线性关系良好;电化学衍生化,黄曲霉毒素G₂、B₂在1.2~36 pg,黄曲霉毒素G₁、B₁在2~120 pg范围内线性关系良好,r>0.9997;回收率在60%~101%之间。结论: 2种衍生化方法的测定结果比较接近,且电化学衍生化方法较灵敏,操作简单、分析速度快,也适用于陈皮中黄曲霉毒素的测定。

Objective: To evaluate two post-column derivatization systems(derivatization with iodine,and with electrochemically generated bromine)for HPLC determination of aflatoxins in Citri Reticulatae Pericarpium. Methods: The samples were cleaned up on immunoaffinity columns and analyzed by HPLC with post-column derivatization and fluorescence detection.For chromatographic separation,a Diamonsil ODS C₁₈(5 μm,4.6 mm×200 mm)column was employed.The detection was observed by fluorescence with the excitation wavelength at 360 nm and the emission wavelength at 450 nm.For the post-column derivatization system with iodine(I₂),the separation was carried out on the mobile phase of methanol-acetonitrile-water(25:18:57)with a flow rate of 0.8 mL·min^-1,the iodine 0.05% solution was used as the derivatization reagent which was delivered at a flow rate of 0.3 mL·min^-1 and the reaction coil was maintained at 70℃.For derivatization with electrochemically generated bromine(KOBRA),the mobile phase consisted of a mixture of methanol-acetonitrile-water(20:20:60)with a flow rate of 1.0 mL·min^-1.To each liter of the mobile phase was added 120 mg potassium bromide and 350 μL of 4 mol·L^-1 nitric acid. Results: Good linear relationships were obtained within the ranges of 1.2-36 pg for aflatoxin G₂ and B₂,and 2-120 pg for aflatox in G₁ and B₁,respectively,r>0.9997.The recoveries were between 60%-101%. Conclusion: The results of two post-column derivatization systems are equivalent.However,KOBRA has higher sensitivity,easier operating procedure,and less analytical time,and it is also applicable to the determination of aflatoxins in Citri Reticulatae Pericarpium.