摘要
基于质谱数据库,建立了超高效液相色谱-静电场轨道阱高分辨质谱(UHPLC-Orbitrap HRMS)快速筛查和确证凉茶中167种非法添加药物的方法。通过调研,选定了解热镇痛药、糖皮质激素、抗菌药、抗组胺药等167种药物,并利用Orbitrap HRMS和TraceFinder软件采集和记录每种药物的信息,建立高分辨质谱数据库。样品以50%(v/v)甲醇水溶液超声提取。样品溶液采用Waters XBrigde BEH C18色谱柱(100 mm×2.1 mm,2.5μm)分离,0.1%(v/v)甲酸水溶液-0.1%(v/v)甲酸乙腈溶液梯度洗脱。Orbitrap HRMS以全扫描/数据依赖二级质谱扫描(Full MS/dd-MS^(2))模式,正、负离子同时切换采集数据。将样品数据导入TraceFinder软件,以母离子精确质量数、保留时间、碎片离子精确质量数等进行数据库自动筛查。若某成分的母离子精确质量数实测值与理论值偏差小于5×10^(-6),保留时间偏差小于20 s,至少一个碎片离子精确质量数实测值与理论值偏差小于5×10^(-6),且二级质谱与数据库收录谱图相似,可判定该成分检出。结果表明,方法特异性好,各成分线性关系良好,相关系数(r)大于0.99。培氟沙星等5种成分存在本底干扰,不宜采用该法进行定量,其余162种成分回收率为66.4%~118.1%,精密度RSD(n=6)为0.1%~16.1%。方法在0.2 mg/kg下可筛查83种成分,1.0 mg/kg下可筛查167种成分。方法应用于245批样品,检出12批问题样品,阳性成分有对乙酰氨基酚、双氯芬酸钠、氯苯那敏等,并检出标准方法外成分金刚烷胺、右美沙芬、溴苯那敏和环丙沙星。方法检测速度快,分析成分多,筛查结果准确,为凉茶非法添加药物高通量筛查提供了新的技术支撑。
An accurate mass database and a method based on ultra high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry(UHPLC-Orbitrap HRMS)were developed.These were applied in the screening and identification of illegally added medicines in herbal tea.Based on investigations,167 medicines were selected to build an accurate MS database;these medicines included antipyretic analgesics,glucocorticoids,antibiotics,and antihistamines,among other categories.The database was established using Orbitrap HRMS and TraceFinder software.The database carried information on all selected compounds,including the molecular formula,accurate mass of precursor ions and fragment ions,retention time,and mass spectra.The samples were ultrasonically extracted with a 50%(v/v)methanol aqueous solution.The extracted solutions were separated using a Waters XBrigde BEH C18 column(100 mm×2.1 mm,2.5μm).As the mobile phases,0.1%(v/v)formic acid aqueous solution and acetonitrile containing 0.1%(v/v)formic acid were used,with gradient elution.The sample solutions were analyzed by Orbitrap HRMS in the full-scan MS and data-dependent MS/MS acquisition modes(Full MS/dd-MS^(2)).Positive and negative polarity data were simultaneously acquired.Some parameters were optimized to increase the peak intensity and sensitivity of all compounds.The resolutions in the full-MS scan and dd-MS^(2)scan were set to 70000 and 17500,respectively.In the full-MS mode,scanning was performed in the range of m/z 100 to 1000.In the MS/MS mode,the normalized collision energy(NEC)was set to 20%,40%,and 60%for each compound.The inclusion list was not used during the measurement,and the dynamic exclusion time was set to 10.0 s.The loop count was set to 5.After acquiring the sample data with these conditions using Orbitrap MS,they were imported into TraceFinder software,through which the sample information was extracted and automatically matched with the information on compounds in the MS database.Screening and identification were conducted by comparing the retention times as well as the exact masses of precursor ions and fragment ions that were experimentally measured.If the errors between the experimentally and theoretically obtained masses of the precursor ions were below 5×10^(-6) and the deviations in retention times were less than 20 s,then suspicious positive compounds might be identified.Furthermore,if such compounds possess more than one similar fragment ion with a mass tolerance below 5×10^(-6),and exhibit similar ion distributions in the MS/MS profiles(compared to those in the database),they could be confirmed to be the same.The validation result showed that all compounds had good linear relationships,with correlation coefficients(r)greater than 0.99.Because pefloxacin,norfloxacin,desloratadine,astemizole and clindamycin had background interference,the method was not suitable for their quantification.Following experiments using three spiked concentrations,the recoveries of the rest 162 compounds were found to be in the range of 66.4%-118.1%,and the relative standard deviations(RSDs,n=6),in the range of 0.1%-16.1%.When the limit of detection(LOD)was 0.2 mg/kg,83 compounds were detected,while when the LOD was 1.0 mg/kg,167 compounds were detected.All compounds were matched successfully to the standard added sample with the MS database in TraceFinder software.To lower the likelihood of false positive and false negative results,a quality control method was recommended.The method was applied to analyze 245 herbal tea samples,among which 12 positive samples were detected.Thirteen positive compounds were found,including acetaminophen,diclofenac sodium,chlorpheniramine,brompheniramine,dexamethasone,dexamethasone 21-acetate,prednisone,prednisone 21-acetate,metronidazole,erythromycin,ciprofloxacin,amantadine,and dextromethorphan.In particular,amantadine,dextromethorphan,brompheniramine,and ciprofloxacin were newly detected,compared to standard methods.The developed method is rapid and accurate,and will be useful in the high-throughput screening of illegally added medicines in herbal tea.
作者
何嘉雯
温家欣
刘亚雄
胡佳哲
曹雅静
赖宇红
HE Jiawen;WEN Jiaxin;LIU Yaxiong;HU Jiazhe;CAO Yajing;LAI Yuhong(Guangdong Institute for Drug Control, Guangzhou 510663, China)
出处
《色谱》
CAS
CSCD
北大核心
2022年第3期253-265,共13页
Chinese Journal of Chromatography
基金
广东省医学科学研究基金项目(B2020196).
关键词
超高效液相色谱
静电场轨道阱高分辨质谱
非法添加药物
凉茶
ultra high performance liquid chromatography(UHPLC)
electrostatic field orbitrap high-resolution mass spectrometry(Orbitrap HRMS)
illegally added medicines
herbal tea
