液相色谱串联质谱法同时检测DNA中3_甲基腺嘌呤和3_乙基腺嘌呤

田永峰等
摘要利用阳离子交换固相萃取柱(Waters Oasis MCX)富集净化DNA样品,建立了液相色谱串联质谱(LCMS/MS)同时检测DNA中3甲基腺嘌呤(N3MeA)和3乙基腺嘌呤(N3EtA)的方法。采用氘代3甲基腺嘌呤(d3N3MeA)和氘代3乙基腺嘌呤(d5N3EtA)为内标; 进样量3 μL,分析时间为13 min;亲水相互作用色谱柱(Waters XBridge HILIC)进行液相分离,流动相为10 mmol/L甲酸铵乙腈溶液(5∶95, V/V, pH=4.0),流速250 μL/min;质谱条件:电喷雾离子源,多反应监测正离子扫描方式;电喷雾电压:5500 V, 雾化气: 369 Pa, 气帘气:185 Pa, 电离温度: 400 ℃,驻留时间: 40 ms。本方法对N3MeA和N3EtA的检出限分别为0.043和0.007 μg/L,方法回收率为87.8%~103.0%。采用本方法检测了卷烟烟气粒相物暴露的DNA中N3MeA和N3EtA含量。结果表明,卷烟烟气粒相物暴露后的小牛胸腺DNA中3甲基腺嘌呤和3乙基腺嘌呤可被本方法定量检出。1引言
卷烟烟气中包含超过5000种化学物质\[1,2\],其中包含致突变并可导致DNA发生烷基化损伤的物质\[3\]。这些物质可与DNA反应形成烷化类的DNA加合物,已经成为多学科的研究热点\[4~7\]。烟气中致癌性的烷化试剂与DNA反应的主要位点是鸟嘌呤的N7位和腺嘌呤的N3位,DNA反应后形成7烷化鸟嘌呤和3烷化腺嘌呤\[8,9\]。3烷化腺嘌呤中比较重要的是3甲基腺嘌呤(N3methyladenine,N3MeA)\[10\]、和3乙基腺嘌呤(N3ethyladenine,N3EtA)\[8\],其结构式见图1。3甲基腺嘌呤在3烷化腺嘌呤中突变性最强,并且具有基因毒性,3乙基腺嘌呤虽然不具有基因毒性,但是在经体内烷基糖基酶的修复后会在DNA中产生脱嘌呤的位点,这些位点被认为是引起基因突变并导致癌症的主要因素\[11~16\]。因此,DNA中的3甲基腺嘌呤,3乙基腺嘌呤被认为是卷烟烟气接触后DNA发生烷基化损伤的标志物\[17,18\]。准确测定DNA中3甲基腺嘌呤和3乙基腺嘌呤,可用于评估DNA受到的破坏程度及可能的致癌风险\[19\]。
[TS(]图13甲基腺嘌呤和3乙基腺嘌呤结构式
Fig.1Structures of N3methyladenine (N3MeA) and N3ethyladenine (N3EtA) [HT5][TS)]
目前,DNA中3烷化腺嘌呤的检测方法有气相色谱质谱法(GCMS)\[8\],该方法前处理比较繁琐,需要化学衍生化,并且灵敏度低和专一性不强;酶联免疫法(ELISA)解决了3烷化腺嘌呤检测中的专一性问题,但是操作过程繁琐,结果稳定性较差\[20\]。液相色谱串联质谱技术因其具有灵敏度高、选择性好、操作简便、分析结果稳定可靠的特点,成为DNA加合物研究中的理想方法\[21,22\]。Hu 等首次报导了一种在线固相萃取结合液相色谱串联质谱同时分析DNA中7甲基鸟嘌呤、3甲基腺嘌呤和O6甲基鸟嘌呤的方法\[23\],该方法检测7甲基鸟嘌呤、3甲基腺嘌呤和O6甲基鸟嘌呤的运行时间为12min。目前,同时检测DNA中3甲基腺嘌呤和3乙基腺嘌呤的液相色谱串联质谱方法还未见报道。
本实验建立了一种同时测定DNA中3甲基腺嘌呤、3乙基腺嘌呤的液相色谱串联质谱方法。本方法灵敏度高,稳定性好。利用本方法研究了卷烟烟气粒相物暴露后,DNA中3甲基腺嘌呤和3乙基腺嘌呤的含量。本方法可用于临床上评估烷基化试剂暴露后DNA的损伤程度。
2实验部分
2.1仪器、试剂与材料
1200快速液相色谱仪(美国安捷伦公司)配备G1367D自动进样器、G1312B二元溶剂泵、G1316B柱温箱; API 5500 三重四级杆串联质谱仪(美国加州应用生物系统公司),配备电喷雾电离源(ESI); 数据采集与处理在Analyst 1.5.1软件上实现。RH20吸烟机(德国伯格瓦特公司)。
氘代3甲基腺嘌呤(d3N3MeA)、氘代3乙基腺嘌呤(d5N3EtA)、3甲基腺嘌呤、3乙基腺嘌呤(纯度≥98%,加拿大Toronto Research Chemical公司); 甲醇、乙腈、氨水、乙酸乙酯和甲酸铵(美国TEDIA有限公司),DMSO(上海安普公司),所有的试剂均为色谱纯,实验中所用的水为超纯水。OASIS MCX 6cc固相萃取小柱(500 mg,6 mL,Waters公司)。3R4F标准参比卷烟购自美国肯塔基大学。
2.2.3标准工作溶液的配制分别准确称10 mg的3甲基腺嘌呤、3乙基腺嘌呤、氘代3甲基腺嘌呤和氘代3乙基腺嘌呤固体于5个10 mL容量瓶中,用乙腈定容,分别得到1 g/L的3甲基腺嘌呤、3乙基腺嘌呤、氘代3甲基腺嘌呤和氘代3乙基腺嘌呤储备液。然后用储备液稀释配制不同浓度的标准溶液和工作溶液。
3结果和讨论
3.1质谱条件的选择
用蠕动泵以10 μL/min的流速连续注射,分别将50 μg/L的3甲基腺嘌呤、3乙基腺嘌呤、氘代3甲基腺嘌呤和氘代3乙基腺嘌呤标准溶液注入ESI离子源中,在正离子模式下进行一级质谱扫描,得到相应的准分子离子峰\[M+H\]+,然后对准分子离子峰进行子离子扫描,得到碎片离子信息。选取丰度较强、干扰较小的作为定量离子对。为了达到更高的灵敏度,对碰撞能量、去簇电压等参数进行了优化。在多反应监测扫描模式下,3甲基腺嘌呤优化结果为定量离子对m/z 150/123,定性离子对为m/z 150/108,内标离子对为m/z 153/126; 3乙基腺嘌呤优化结果为:定量离子对m/z 164/136,定性离子对为m/z 164/119,内标离子对为m/z 168/137。
3.3方法学考察
分别吸取0.1,0.2,0.5,1,2,5和10 μg/L 的3乙基腺嘌呤,1,2,5,10,20和40 μg/L的3甲基腺嘌呤标准溶液各3 μL,注入LCMS/MS系统进行分离分析。3甲基腺嘌呤和3乙基腺嘌呤的线性回归方程分别为y=0.25x+0.0238和y=0.0145x-0.00135 (y为峰面积,x为浓度),线性良好,相关系数r均大于 0.999。利用加标稀释得到方法的检出限,3倍信噪比对应的浓度为检出限,3甲基腺嘌呤和3乙基腺嘌呤的检出限分别为0.043 和0.007 μg/L。采用样本加标的方法获得方法回收率,总共选取了3个添加水平,每个添加水平重复测定4次,得到方法的回收率和重复性,结果见表1。3甲基腺嘌呤和3乙基腺嘌呤的回收率分别为88.0%~102.3%和98.0%~102.0%,方法精密度小于10%,证明方法的准确性和重复性结果较好。
3.4方法应用
使用本方法对卷烟烟气暴露的DNA中3甲基腺嘌呤和3乙基腺嘌呤进行分析,结果表明,3甲基腺嘌呤含量为1.17~3.78μg/L, 未检出3乙基腺嘌呤。 由此可见,本方法对DNA样本检测灵敏度高,选择性好,适用于卷烟烟气暴露的DNA烷基化损伤研究以及环境中烷基化试剂对DNA暴露后的3甲基腺嘌呤、3乙基腺嘌呤的生物监测。
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AbstractA liquid chromatographytandem mass spectrometry (LCMSMS) method has been developed for the simultaneous determination of N3methyladenine (N3MeA) and N3ethyladenine (N3EtA) in calf thymus DNA. The DNA samples has been purified and enriched by cation exchange cartridge (Waters Oasis MCX). d3N3MeA and d5N3EtA were used as isotope internal standard. The DNA samples were injected with autosampler. The injected volume was 3 μL and analysis time was 13 min. The sample separation was carried out on hydrophilic interaction chromatograph (Waters XBridge HILIC) with 10 mmol/L ammonium formateacetonitrile (5∶92, V/V, pH=4.0) as mobile phase. The flow rate was set at 250 μL/min. Mass spectrometry was performed by electrospray ionization (ESI) with multireactions monitoring (MRM). The optimized operation conditions of MS were as follows: nebulizer gas 369 Pa; curtain gas 185 Pa, turbo ionspray temperature 400 ℃, ionspray voltage 5500 V, dwell time 40 ms. The limits of detection were 0.043 and 0.007 μg/L for N3MeA and N3EtA, respectively. The recoveries were between 87.8% and 103.0% for N3MeA and N3EtA. This method was successfully applied to the determination of N3MeA and N3EtA in calf thymus DNA by cigarette smoke condensate (CSC) exposure. This method is appropriate for routine analysis and accurate quantification of N3MeA and N3EtA by CSC exposure.
KeywordsN3Methyladenine; N3Ethyladenine; Liquid chromatographytandem mass spectrometry; Deoxyribonucleic acid
24Chen H, Lin C, Jiang X Y, Pang Y Q, Tang G L, Hou H W, Jiang J H, Hu Q Y. Food. Chem. Toxicol., 2012, 50(3): 612-618
AbstractA liquid chromatographytandem mass spectrometry (LCMSMS) method has been developed for the simultaneous determination of N3methyladenine (N3MeA) and N3ethyladenine (N3EtA) in calf thymus DNA. The DNA samples has been purified and enriched by cation exchange cartridge (Waters Oasis MCX). d3N3MeA and d5N3EtA were used as isotope internal standard. The DNA samples were injected with autosampler. The injected volume was 3 μL and analysis time was 13 min. The sample separation was carried out on hydrophilic interaction chromatograph (Waters XBridge HILIC) with 10 mmol/L ammonium formateacetonitrile (5∶92, V/V, pH=4.0) as mobile phase. The flow rate was set at 250 μL/min. Mass spectrometry was performed by electrospray ionization (ESI) with multireactions monitoring (MRM). The optimized operation conditions of MS were as follows: nebulizer gas 369 Pa; curtain gas 185 Pa, turbo ionspray temperature 400 ℃, ionspray voltage 5500 V, dwell time 40 ms. The limits of detection were 0.043 and 0.007 μg/L for N3MeA and N3EtA, respectively. The recoveries were between 87.8% and 103.0% for N3MeA and N3EtA. This method was successfully applied to the determination of N3MeA and N3EtA in calf thymus DNA by cigarette smoke condensate (CSC) exposure. This method is appropriate for routine analysis and accurate quantification of N3MeA and N3EtA by CSC exposure.
KeywordsN3Methyladenine; N3Ethyladenine; Liquid chromatographytandem mass spectrometry; Deoxyribonucleic acid
24Chen H, Lin C, Jiang X Y, Pang Y Q, Tang G L, Hou H W, Jiang J H, Hu Q Y. Food. Chem. Toxicol., 2012, 50(3): 612-618
AbstractA liquid chromatographytandem mass spectrometry (LCMSMS) method has been developed for the simultaneous determination of N3methyladenine (N3MeA) and N3ethyladenine (N3EtA) in calf thymus DNA. The DNA samples has been purified and enriched by cation exchange cartridge (Waters Oasis MCX). d3N3MeA and d5N3EtA were used as isotope internal standard. The DNA samples were injected with autosampler. The injected volume was 3 μL and analysis time was 13 min. The sample separation was carried out on hydrophilic interaction chromatograph (Waters XBridge HILIC) with 10 mmol/L ammonium formateacetonitrile (5∶92, V/V, pH=4.0) as mobile phase. The flow rate was set at 250 μL/min. Mass spectrometry was performed by electrospray ionization (ESI) with multireactions monitoring (MRM). The optimized operation conditions of MS were as follows: nebulizer gas 369 Pa; curtain gas 185 Pa, turbo ionspray temperature 400 ℃, ionspray voltage 5500 V, dwell time 40 ms. The limits of detection were 0.043 and 0.007 μg/L for N3MeA and N3EtA, respectively. The recoveries were between 87.8% and 103.0% for N3MeA and N3EtA. This method was successfully applied to the determination of N3MeA and N3EtA in calf thymus DNA by cigarette smoke condensate (CSC) exposure. This method is appropriate for routine analysis and accurate quantification of N3MeA and N3EtA by CSC exposure.
KeywordsN3Methyladenine; N3Ethyladenine; Liquid chromatographytandem mass spectrometry; Deoxyribonucleic acid
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