基于液相色谱-质谱技术的芦西丁-DNA加合物快速筛查研究  

Rapid Screening of Lucidin-DNA Adducts Based on LC-MS/MS

作　　者：杨静 王慎兴 王丹 吴春勇[3] 黄青 袁耀佐 YANG Jing;WANG Shenxing;WANG Dan;WU Chunyong;HUANG Qing;YUAN Yaozuo(School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Institute for Food and Drug Control,Nanjing 210019,China;School of Pharmacy,China Pharmaceutical University,Nanjing 211198,China)

机构地区：[1]南京中医药大学药学院,南京210023 [2]江苏省食品药品监督检验研究院,南京210019 [3]中国药科大学药学院,南京211198

出　　处：《中国药学杂志》2024年第4期338-345,共8页Chinese Pharmaceutical Journal

基　　金：江苏省市场监督管理局科技计划项目资助(KJ21125050,KJ207558)。

摘　　要：目的建立基于液相色谱-三重四级杆质谱(LC-QQQ-MS)及液相色谱-高分辨质谱(LC-HRMS)技术的DNA加合物通用型“发现-确证”分析策略,研究茜草中潜在遗传毒性物质芦西丁(lucidin,Luc)与3种2′-脱氧核苷的直接反应性和代谢反应性,筛查和确证可能的Luc特异性DNA加合物。方法采用与DNA加合物具有相同质谱碎裂模式的3种2′-脱氧核苷,建立和优化三重四极杆质谱中性丢失和伪中性丢失扫描模式下未知DNA加合物的非靶向筛查方法,以及高分辨数据依赖性扫描模式下的加合物靶向确证方法。将Luc与2′-脱氧核苷在Ⅰ相代谢激活和未激活条件下分别孵育,筛查生成的特异性DNA加合物,再通过特征性质谱碎裂离子进行结构验证。结果优化后的伪中性丢失扫描对脱氧核苷脱糖基的检测灵敏度可达pg·mL^(-1)级别。在Luc与脱氧核苷体外孵育模型中,发现并确证了6种Luc-DNA加合物,包含2种2′-脱氧胞苷(dC)加合物、2种2′-脱氧腺苷(dA)加合物、2种2′-脱氧鸟苷(dG)加合物,并对其结构进行了表征。Luc-DNA加合物的生成随着Luc暴露量、暴露时间的增加而升高,存在显著的剂量-反应、时间-反应关系,且该种结合无需代谢激活即可发生。结论所建立的DNA加合物“发现-确证”策略灵敏度高、准确性好,能够提供分子水平上加合物的结构信息,适用于评估Luc暴露所致的DNA损伤,为其毒性机理研究和再评价提供了有力数据支持,也为中草药中潜在遗传毒性成分快速筛查提供了重要的方法参考。OBJECTIVE To develop a generalized“discovery-confirmation”strategy for DNA adducts based on liquid chromatography-triple quadrupole mass spectrometry(LC-QQQ-MS)and liquid chromatography-high resolution mass spectrometry(LC-HRMS),study the direct and metabolic reactivity of the potentially genotoxic substance lucidin(Luc)with three 2'-deoxynucleosides in Rubia cordifolia L.,and screen and confirm possible Luc-specific DNA adducts.METHODS Three 2'-deoxynucleosides with the same fragmentation pathways with DNA adducts in mass spectrometry were used to develop and optimize a non-targeted screening method for unknown DNA adducts in QQQ-MS neutral loss and pseudo-neutral loss scanning modes,as well as a confirmatory method for adduct targeting in HRMS data-dependent scanning mode.Luc was incubated with 2'-deoxynucleoside under phase I metabolically activated and inactivated conditions,respectively,and the resulting specific DNA adducts were screened and then structurally verified by characteristic MS spectra of fragmentations.RESULTS The optimized pseudo-neutral loss scanning is sensitive to the detection of deoxyribonucleoside deglycosylation at the pg·mL^(-1) level.Six Luc-DNA adducts,containing two 2'-deoxycytidine(dC)adducts,two 2'-deoxyadenosine(dA)adducts,and two 2'-deoxyguanosine(dG)adducts,were identified and confirmed in vitro incubation model of Luc and 2'-deoxynucleoside,and their structures were characterized.Luc-DNA adduct production increased with increasing Luc exposure and exposure time,and there were significant dose-response and time-response relationships,and the binding did not require metabolic activation to occur.CONCLUSION The established discovery-confirmation strategy for DNA adducts is highly sensitive and accurate.It can provide structural information of the adducts at the molecular level.This is suitable for the assessment of DNA damage caused by the presence of Luc and provides strong data support for the study and re-evaluation of its toxicity mechanism.It also offers an important met

关 键 词：DNA加合物 遗传毒性 茜草 芦西丁 筛查 

分 类 号：R917[医药卫生—药物分析学]