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苯并芘DNA加合物anti-BPDE-N2-dG四种立体异构体的合成及色谱分离
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作者单位
赵旭霞,冯蕊,郭娅男,郑速进,张加玲,阎小青 山西医科大学公共卫生学院卫生检验教研室 
基金项目:国家自然科学基金(81502851);中国博士后科学基金(2014M561208);山西医科大学博士启动基金(055235)
中文摘要:体外合成了苯并芘DNA加合物—邻二醇环氧苯并芘-脱氧鸟苷加合物(anti-BPDE-N2-dG)四种立体异构体(两对手性异构体)。通过优化体外反应条件,anti-BPDE-N2-dG四种异构体的合成产量较现有合成方法提高了2倍多,为定量检测生物体中anti-BPDE-N2-dG提供了标准品。并首次将五氟苯基色谱柱应用于该立体异构体的色谱分离提纯,通过优化色谱条件,采用常规的五氟苯基色谱柱(250 mm×4.6 mm,5 μm),以乙腈-0.1%甲酸水(22.5∶77.5)为流动相,流速1.2 mL/min条件下,45 min内即可分离提纯四种立体异构体。该方法与常规C18柱(250 mm×4.6 mm,5 μm)需要160 min,苯基柱(250 mm×4.6 mm,5 μm)需要85~100 min才能将四种立体异构体实现色谱分离相比,缩短了分离时间,提高了提纯效率。通过紫外吸收光谱、质谱、圆二色谱对四种立体异构体进行表征,确定出峰顺序为trans(-)、trans(+)、cis(+)、cis(-)-anti-BPDE-N2-dG。此外,利用高效液相色谱-串联质谱(HPLC-MS/MS)检测anti-BPDE-N2-dG四种立体异构体标准品时,使用常规的五氟苯基色谱柱可在30 min内完成分离检测,与相同规格的苯基柱需要60 min相比提高了检测效率。
中文关键词:苯并芘DNA加合物;邻二醇环氧苯并芘-脱氧鸟苷加合物;五氟苯基色谱柱  高效液相色谱(HPLC)  串联质谱
 
Synthesis and Chromatographic Separation of Four Stereoisomers of Anti benzo[a]pyrene Diol Epoxide-deoxyguanosine Adducts
Abstract:Four stereoisomers of anti benzo[a]pyrene diol epoxide deoxyguanosine adduct(anti-BPDE-N2-dG) were synthesized in vitro.By optimizing the reaction conditions,the yields of four isomers were two times higher than those of the reported synthesis methods.The synthesis provided standard substance for the quantitative detection of anti BPDE N2 dG in vivo.The reaction mixture of anti-BPDE-N2-dG stereoisomers were further separated and purified by reversed phase high performance liquid chromatography(HPLC),in which a pentafluorophenyl silica gel column(PFP column,250 mm×4.6 mm,5 μm) was introduced for the first time.It was found that the four isomers could be separated completely on the PFP column within 45 min,using acetonitrile-0.1% formic acid water(225∶775) as mobile phase at a flow rate of 1.2 mL/min.Compared with the conventional C18 column(250 mm×4.6 mm,5 μm) taking 160 min or silicone polymer coating phenyl column(250 mm×4.6 mm,5 μm) needing 85-100 min for separation of the four isomers,the reported method greatly shortened the separation and purification time.The four stereoisomers of the anti BPDE N2 dG were identified and characterized by a combination of HPLC-MS/MS,UV and circular dichroism(CD) spectra.The order of the peaks in chromatograms were determined as trans(-),trans(+),cis(+) and cis(-)-anti-BPDE-N2-dG.In addition,in the analysis of HPLC-MS/MS in MRM mode,the four stereoisomers were rapidly and well separated on the PFPP column within 30 min using a mobile phase consisted of acetonitrile-0.1% formic acid(28∶72) at a flow rate of 0.5 mL/min.This work demonstrates that the introduction of the PFPP column is very helpful for the separation,purification and detection of anti-BPDE-N2-dG isomers.
Key Words:benzo[a]pyrene DNA adducts  anti benzo[a]pyrene diol epoxide deoxyguanosine adducts(anti-BPDE-N2-dG)  pentafluorophenyl silica gel column  high performance liquid chromatography(HPLC)  MS/MS
引用本文:赵旭霞,冯蕊,郭娅男,郑速进,张加玲,阎小青.苯并芘DNA加合物anti-BPDE-N2-dG四种立体异构体的合成及色谱分离[J].分析测试学报,2020,39(3):315-322.
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