UPLC-MS/MS法快速测定减肥类中成药及保健食品中非法添加15种化学药的研究

Rapid determination of 15 chemical drugs illegally added into slimming Chinese patent medicines and health foods by UPLC-MS/MS

目的建立一种快速、准确检测减肥类中成药及保健食品中非法添加15种化学药的方法。方法采用UPLC-MS/MS法,以Waters Acquity BEH-C18柱（100 mm×2.1 mm,1.7μm）为色谱柱,以0.1%甲酸甲醇溶液（A）-0.1%甲酸水溶液（B）为流动相,梯度洗脱：0-3 min,33%-45%A;3-5 min,45%-55%A;5-7 min,55%-70%A;7-9 min,70%-80%A;9-10 min,80%-90%A;10-11 min,90%-33%A;11-13 min,33%A;体积流量0.2 m L/min;柱温40℃。选择ESI离子源、正离子扫描、多反应监测（MRM）模式测定15种临床常用的化学减肥药,通过比较MRM通道中样品峰与对照品峰的分子离子峰、二级碎片离子峰、色谱保留时间等信息确定添加的化学药物,并根据外标法以质谱峰面积计算添加药物的准确量。结果在上述色谱及质谱条件下,硫酸特布他林、盐酸麻黄碱、茶碱、咖啡因、多索茶碱、盐酸克仑特罗、盐酸妥洛特罗、盐酸班布特罗、盐酸芬氟拉明、呋塞米、吲达帕胺、酚酞、盐酸西布曲明、盐酸N-单去甲基西布曲明、盐酸N,N-双去甲基西布曲明等15种化学药物的分离良好,方法检测限（LOD）均在0.1-5.0 ng/g,定量限（LOQ）均在0.3-15.0ng/g,标准加样回收率均在91.8%-110.8%。在86批样品（包括胶囊、颗粒剂等不同基质类型）中检出74批添加了化学药,阳性率为86.0%,样品中主要检出盐酸西布曲明（39批）、呋塞米（20批）、酚酞（23批）、茶碱（1批）、咖啡因（15批）,其中检出2种的22批,3种的1批。相较而言,未明确标示生产厂家的产品中非法添加更严重。结论方法简便、准确,灵敏度高,可作为减肥类中成药及保健食品中非法添加15种化学药的定性定量方法。

Objective To establish a rapid and accurate method for the determination of 15 chemical drugs which were illegally added into the slimming Chinese patent medicines（CPM） and health foods. Methods The UPLC-MS/MS method was adopted. The samples were extracted with methanol by ultrasonic processing and separated on a Waters Acquity BEH C18 column（100 mm × 2.1 mm, 1.7 μm） with 0.1% formic acid methanol（A）- 0.1% formic acid water（B） as mobile phase by gradient elution（0—3 min, 33%—45% A; 3—5 min, 45%—55% A; 5—7 min, 55%—70% A; 7—9 min, 70%—80% A; 9—10 min, 80%—90% A; 10—11 min, 90%—33% A; 11—13 min, 33% A at a flow rate of 0.2 m L/min, and the column temperature was 40 ℃. A positive-ion（ESI＋） source and an MRM mode were used to separate and quantitatively determine 15 chemical drugs. The obtained molecular ions, fragment ions, and retention time for MRM channels were used to identify the 15 kinds of drugs by comparison with those of reference substances. The obtained peak areas were used to determine the accurate contents of chemical drugs in CPM and the health foods. Results A good resolution of 15 kinds of chemical drugs, including terbutaline hydrochloride, ephedrine hydrochloride, theophylline, caffeine, doxofylline, clenbuterol hydrochloride, tulobuterol hydrochloride, bambuterol hydrochloride, fenfluramine hydrochloride, furosemide, indapamide, phenolphthalein, sibutramine hydrochloride, N-demethylated sibutramine hydrochloride, and hydrochloric acid N,N-dinor sibutraminel, was obtained under this UPLC and MS/MS condition. The limits of qualitation and quantitation were in the range of 0.1—5.0 ng/g and 0.3—15.0 ng/g. The standard addition recoveries were in the range of 91.8%—110.8%. In the 86 batches of samples（including capsules, granules, and other different matrix types） were detected in the 74 batches of added chemicals, the positive rate was 86.0%. Sibutramine hydrochloride（39 batches）, furosemide（20 batches）, phenolphthalein（23 batches）, theophylline（1 batch）, and caffeine（15 batches） were checked out in the samples, 22 batches of which two kinds were checked out, one batch of which three kinds were checked out. By contrast, the products which were not clearly marked manufacturer illegally added more seriously. Conclusion The method is simple, accurate, and highly sensitive, which can be used for the determination of illegally added chemical drugs in slimming CPM and health foods.