采用超高效液相色谱-四极杆-飞行时间质谱(ultra-high performance liquid chromatography-quadrupole-time-of-fight mass spectrometry,UHPLC-Q-TOF-MS)对中成药、保健品中非法添加的降糖及抗疲劳药物进行定性分析。片剂或溶液制剂经过乙腈超声提取,经Phenomenex Kinetex C18(2.1 mm I.D. ×100 mm L., 2.6 μm)色谱柱分离,体积分数0.1%甲酸水溶液、0.1%甲酸乙腈作为流动相梯度洗脱,电喷雾离子化检测。数据采集模式为正离子模式全扫描(full scan MS)和数据依赖采集(data-dependent acquisition,DDA)模式。数据经过LabSolutions Ver 5.96软件解析,精确分子量软件Formula Predictor计算,确认了26种非法添加化合物的一级质谱、一级精确质荷比、同位素丰度比、保留时间、质量数误差等信息。将不同碰撞能量(10、20、30、40和50 V)下的目标化合物的二级质谱图导出,使用ACD/Labs Ver.2012软件建立26种非法添加化合物的二级质谱库,包含了化合物名称、CAS号、一级精确质荷比、二级质谱图等信息,可用于未知样品二级质谱图自动检索评分。此外,方法学考察显示,26种化合物在各自线性范围内线性关系良好,相关系数r均>0.99,平均回收率75.8%~128.4%,检出限(limits of detection, LOD)0~0.010 9 mg/L,定量限(limits of quantitation, LOQ)0.000 1~0.036 1 mg/L。该方法前处理简便、灵敏度高、扩展性强,为降糖类、抗疲劳类中成药、保健品中的非法添加成分提供快速筛查方法。
A rapid screening method for simultaneous determination of 26 illegal chemicals in Chinese traditional formulated medicine and health products was established. Tablets and solution samples were extracted by ultrasonic with acetonitrile. Prepared samples were separated by Phenomenex Kinetex C18 (2.1 mm I.D. ×100 mm L., 2.6 μm) and ionized by electrospray. The mobile phases were 0.1% formic acid water and 0.1% formic acid acetonitrile. Samples were measured in the positive ionization mode using full-scan MS and data-dependent acquisition (DDA) methods. The acquired data were analyzed by Shimadzu LabSolutions and calculated by Formula Predictor software. High resolution m/z, isotopic abundance ratios, retention times and mass errors of 26 illegal chemicals were determined. Production chromatographs of 26 illegal chemicals in different collision energy (10, 20, 30, 40 and 50 V)were exported. ACD/Labs Ver.2012 software was applied to establish an illegal addition library for 26 chemicals, including the information of compounds names, CAS numbers, m/z, and product ion chromatographs The illegal addition library can be applied to match peaks by production chromatographs automatically. In addition, the results of method validation indicated that the linearities of 26 illegal chemicals were good, with the linear correlation coefficient more than 0.99, and the mean recoveries were 75.8% to 128.4%. The limits of detection (LOD) were 0 to 0.010 9 mg/L, and the limits of quantitation (LOQ) were 0.000 1 to 0.036 1 mg/L. The screening method for 26 illegal chemicals has the characteristics of high efficiency, sensitivity and accuracy, which provides a qualitative method for illegal additives in Chinese traditional patent medicines and health products.
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