UPLC-MS/MS法与HPLC-FLD法分析检测中药中的黄曲霉毒素

Determination of aflatoxins in Chinese materia medica by UPLC-MS/MS and HPLC-FLD

目的:建立黄曲霉毒素G2、G1、B2、B1的超高效液相色谱串联三重四极杆质谱法(UPLC-MS/MS法),同时采用高效液相色谱荧光法(HPLC-FLD法)进行测定。方法:采用UPLC-MS/MS测定,待测黄曲霉毒素的中药经过免疫亲和柱净化,采用C18(2.1 mm×50 mm,1.7μm)进行色谱分离,柱温30℃,以含0.1%甲酸的2.0 mmol·L^-1乙酸铵溶液(A)-甲醇(B)为流动相,梯度洗脱,流速0.3 mL·min^-1,用电喷雾正离子多反应监测模式(MRM)扫描;采用HPLC联用柱后衍生荧光检测方法测定同样的样品;并验证检测结果。结果:UPLC-MS/MS黄曲霉毒素质谱检测的线性关系良好,定量下限范围在0.177 8~0.633 3μg·kg^-1,检测下限范围在0.055 1~0.190 0μg·kg^-1,样品测定结果在1.423~1.975μg·kg^-1之间;同时采用HPLC-FLD荧光检测法进行测定(定量下限范围为0.660~0.204μg·kg^-1,检测下限范围为0.059~0.206μg·kg^-1)。同一样品测定结果误差在1.501%~9.377%之间。结论:与柱后衍生化HPLC-FLD法相比,液质联用测定中药材中黄曲霉毒素的方法,专属性强,操作简单,简便快速,灵敏度高,减少实验成本。

Objective:To establish a method for the determination of aflatoxins in Chinese materia medica by an ultra-high performance liquid chromatography with triple quadrupole mass spectrometry(UPLC-MS/MS)and to conduct simultaneous determination by HPLC with fluorescence detection(HPLC-FLD).Methods:The herbal extract was purified by immune affinity column and separated by a C1,(2.1 mm × 50 mm,1.7 μm)column(temperature:30 ℃)and the mobile phase consisted of 2.0 mmol·L^-1 ammonium acetate-0.1%formic water-methanol at a flow rate of 0.3 mL·min^-1.Multiple-reaction monitoring(MRM)was performed to identify and quantify four aflatoxins(G2,G1,B2,B1).In addition,the HPLC coupled with the post-column derivatization fluorescence detection method was conducted as a validated analysis.Results:Good linearity was obtained in UPLC-MS/MS method.The limit of quantification(LOQ)ranged from 0.177 8-0.633 3 μg·kg^-1 and the limit of detection(LOD)ranged from 0.055 1-0.190 0 μg·kg^-1.The determination results were between 1.423 and 1.975 μg·kg^-1.Meanwhile,samples were determined by the fluorescence detection method(LOQ,0.660-0.204μg·kg^-1 LOD,0.059-0.206 μg·kg^-1),and the relative errors of results obtained by the two methods were 1.501%-9.377%.Conclusion:Compared with the HPLC-FLD coupled with the post-column derivatization method,the UPLC-MS/MS method is specific,simple,fast and sensitive,which can reduce the experimentalcost.