多次进样循环使用流动相HPLC-DAD法的建立及在快速筛查消渴类中成药中非法添加化学成分中的应用

Development of HPLC-DAD method based on recycling mobile phase for mutiple sample injections and application of the method for rapid screening of chemicals illegally adulterated in traditional Chinese medicine

目的：建立无需更换流动相而可用于中成药中非法添加化学成分现场快速筛查的HPLC-DAD法。方法：采用岛津Shim-pack VP-ODS色谱柱,使用2种等组成的流动相：Ⅰ为乙腈-0.02 mol.L-1甲酸铵缓冲液（pH3.8）（50∶50）;Ⅱ为甲醇-0.01 mol.L-1乙酸铵（35∶65）。检测波长229 nm,参比波长550 nm,DAD波长扫描范围190~600 nm。测定时先将样品检出峰的保留时间与对照品比较,找出保留时间一致的疑似物;再将疑似物的光谱与相应的对照品比较。结果：建立的色谱条件可以分离消渴类中成药中常见的10种非法化学添加成分;循环使用流动相进样150次后,除基线稍有提高外,色谱峰分离度和理论塔板数均未明显变化;采用循环使用150次后的流动相成功筛查出未知样品中的非法添加物格列本脲,并得到了LC-MS的确认。结论：将流动相循环使用检测多个样本是可行的,适合现场采用车载或便携式HPLC仪进行快速药品筛查的需要;本文建立的色谱条件可以用于快速筛查消渴类中成药中的非法添加化学成分。

Objective：To develop an HPLC-DAD method without the change of mobile-phase for the rapid screening of chemicals illegally adulterated in traditional Chinese medicine.Method：HPLC was performed on Shim-pack VP-ODS column by two kinds of composition-constant mobile-phases with different modifiers：mobile phase Ⅰ,acetonitrile-0.02 mol·L-1ammonium formate solution（pH 3.8）（50∶50）;mobile phase Ⅱ,methanol-0.01 mol·L-1 ammonium acetate（35∶65）.The detection wavelength was 229 nm with the reference wavelength of 550 nm,and the scanning range of DAD was 190-600 nm.Firstly,comparison of peak retention time of a sample chromatogram with that of references possiblely adulterated was employed to screen suspicious substances in the sample;then the suspicious substance was confirmed by using the DAD spectrum of the reference corresponding to the suspicious substance for the comparison.Results：The HPLC conditions developed here are able to well separate 10 chemicals commonly detected in anti-diabetic traditonal Chinese medicines;and it was found that peak resolution and theoretical plate number has no obivious change after recycling the mobile phase for 150 sample injections,except for a little increase of baseline;glibenclamide was successfully detected in an unknown sample by using the mobile-phase which has been used for 150 sample injections,and the result was confirmed by LC-MS method.Conclusions：It is feasible to recycle mobile-phase for mutiple sample analysis,enabling the rapid on-site screening of chemicals illegally adulterated in traditional Chinese medicine by using the onboard or portable HPLC instrument.The HPLC-DAD conditions developed in this report are suitable for the rapid screening of chemiclas in anti-diabetic traditonal Chinese medicines.