【作者】 姚莉；

【导师】 关亚风； 杨俊；

【作者基本信息】 中国科学技术大学 ， 分析化学， 2014， 博士

【摘要】 卷烟烟气中含有上千种物质,其中六十多种是致癌物质,可能诱发肺癌、膀胱癌、胰腺癌、肝癌和喉癌等癌症。目前中国烟民人数已超过3亿,占全世界吸烟总人数的三分之一,中国每天约有3000人死于与吸烟相关的疾病。其中肺癌的死亡率居各种恶性肿瘤的首位,发病率呈明显上升趋势,且吸烟是诱发肺癌的重要原因。烟气中烟草特有亚硝胺和多环芳烃与肺癌发生有显著的相关性。研究烟气有害成分4-甲基亚硝胺-1-(3-毗啶)-1-丁酮(NNK)和苯并[a]芘(BaP)在人体内代谢标记物,能够更加直接判断卷烟烟气对人体的危害性。NNK和BaP在人体内代谢标记物4-甲基亚硝胺-1-(3-吡啶)-1-丁醇(NNAL)和3-羟基-苯并芘(3-OHBaP)的含量均为pg/mL数量级,因此对分析检测工作提出很高的挑战。为了实现高灵敏度的分析目标,本文开展烟气致癌成分人体代谢标记物分离分析新技术的应用研究。在充分调研文献的基础上,主要进行了以下几部分的研究工作：1.尿液中NNAL分子印迹固相萃取分析新技术研究选择分子印迹柱(MIP)净化和富集尿液中NNAL,并与Waters Oasis HLB柱和Waters Oasis MCX柱对比。MIP柱的选择性最好,净化后基质干扰最小,离子信号抑制率为38%。选择高分离能力的超高效液相色谱柱(UPLC),提高目标物NNAL与干扰基体组分的分离度；以同位素13C6-NNAL为内标,建立准确检测尿样中生物标记物NNAL的超高效液相色谱串联质谱(UPLC-MS/MS)分析方法。NNAL在5-1200pg/mL范围内具有良好的线性关系,相关系数(r2)为0.9953。方法的加标回收率为88.5%-93.7%。批内精密度为3.6%-7.4%,批间精密度为5.4%-9.7%,方法的检出限为0.4pg/mL。2.尿液中游离和糖苷结合态NNAL的五氟苯基柱(PFP)同时分离和分析新技术研究建立了LC-MS/MS法同时检测吸烟者尿液中NNAL及其N、O-糖苷化合物(NNAL-N-Gluc和NNAL-O-Gluc)的分析方法。比较Agilent UPLC XDB-C18柱(50mm×4.6mm i.d.,1.8μm)、Agilent Eclipse Plus-C18柱(100mm×4.6mm i.d.,3.5μm)和Phenomenex Kinetex PFP柱(100mmx4.6mm i.d.,2.6μm)对NNAL、 NNAL-N-Gluc和NNAL-O-Gluc的分离效果,三种物质在6分钟的色谱运行时间内即在PFP柱上实现基线分离。通过对比常用的C18柱、HLB柱及混合型阳离子交换柱MCX,优选出适合同时净化三种代谢物的商品化固相萃取小柱MCX。该分析方法样品前处理步骤简单、快速,不需要酶解或碱消解步骤,大大提高了方法的准确度和分析效率。NNAL.NNAL-N-Gluc和NNAL-O-Gluc的检出限分别为1.5、15和20pg/mL。3.头发中NNAL分散液液微萃取富集的分离分析新技术研究建立了基于LC-MS/MS法检测头发中NNAL的分析方法。将两步固相萃取和反相分散液液微萃取结合用于前处理,极大提高了对样品的净化和富集作用。实验优化了反相超声辅助分散液液微萃取的参数：以80μL1.0%甲酸水溶液为萃取剂,超声时间4.0min。在优化的实验条件下,NNAL富集倍数为20。方法的加标回收率为87.3%-107.7%,批内精密度为4.1%-8.5%,批间精密度为6.9%-11.3%,检出限为0.08pg/mg。最后,将该方法应用于分析吸烟者头发中NNAL,NNAL含量为0.27-0.67pg/mg。4.尿液中3-OHBaP丹磺酰氯衍生化分析新技术研究建立了分析尿液中超痕量3-OHBaP准确灵敏的分析方法。以β-葡萄糖苷酸酶／芳基硫酸酯酶酶解尿样中3-OHBaP葡萄糖醛酸和芳基硫酸结合物为游离态3-OHBaP。用固相萃取净化和富集3-OHBaP,再以丹磺酰氯衍生,最后以LC-MS/MS电喷雾离子化正离子模式检测3-OHBaP丹磺酰氯衍生物(3-OHBaP-Dansyl)。通过将3-OHBaP与丹磺酰氯衍生增加目标物的电喷雾离子化效率和在碰撞池中产生碎片离子的效率,从而显著提高目标物在LC-MS/MS上的检测灵敏度。在优化的质谱条件下以LC-MS/MS检测相同浓度3-OHBaP-Dansyl和3-OHBaP,3-OHBaP-Dansyl的峰高是3-OHBaP的30倍。该方法的线性范围为0.25-40.0pg/mL,相关系数(r2)为0.9924。加标回收率为87.7%-108.2%,批内精密度为4.6%-9.6%,批间精密度为7.2%-11.3%,方法的检出限为0.1pg/mL。5.研究中国人体内对烟气致癌物NNK的解毒能力通过问卷和自愿取样方式,获得75份24小时尿液样品(包括男性主动吸烟者、女性主动吸烟者,烤烟型卷烟抽吸者和混合型卷烟抽吸者),分别测定各尿液样品中游离NNAL、总NNAL、尼古丁、可天宁和肌酐含量。在分析和统计样品检测数据的基础上,建立中式卷烟消费者尿液中可天宁和NNAL的相关关系(r=0.66,p<0.0001)。尿液中可天宁和总NNAL浓度均随着吸烟剂量增大而增大。以尿液中NNAL-Glucs与游离NNAL浓度比值反映人体对NNK代谢解毒能力,比较文献报道的黑人和白人体内NNAL-Glucs和NNAL浓度比值,结果发现对于男性,白种人和黑种人解毒能力较强。对于女性,黄种人与白种人解毒能力均比黑种人高。更多还原

【Abstract】 Cigarette smoke contains more than sixty chemical carcinogens that cause cancers of various types. Cigarette smoking is widely regarded as the leading cause of lung cancer and is also causally associated with laryngeal, bladder, pancreatic cancer and other preventable diseases. So far there are more than300million Chinese smokers, accounting for one-third of the total population of worldwide smokers. Almost3000Chinese people die from tobacco-related diseases each day. In particular, lung cancer is the leading incident cancer and cigarette smoking causes about90%of lung cancer cases. The tobacco-specific nitrosamines and polycyclic aromatic hydrocarbons are regarded as the major aetiological factors in lung cancer. Analysis of the metabolites of carcinogenic4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) in humans can be useful in toxicology and epidemiologic study of cancer risk evaluation in relation to exposure to the carcinogens in tobacco and tobacco smoke. Urinary biomarkers concentrations of NNK and BaP are usually in the pg/mL range, so it presents a considerable challenge for the analysis.In this study, some new technology in separation and analysis was applied for the study of ultra-trace metabolism biomarkers of tobacco smoke carcinogens in human. The main work of this thesis is summarized as follows:1. Study on a MISPE method for the analysis of urinary NNALAn effective analytical method based on molecularly imprinted solid-phase extraction (MISPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established for the determination of NNAL in human urine. The extraction performances of NNAL on three different polymeric SPE sorbents HLB, MCX and NNAL-MIP were evaluated. The MISPE sorbent was found to give the highest extraction recovery and the lowest ion suppression ratio for NNAL compared with the MCX and HLB sorbent The Chromatographic separation was achieved on a reversed-phase Agilent UPLC XDB-C18analytical column of50mm×4.6mm with1.8μm particle size. Combined with the use of isotope internal standard13C6-NNAL, this method achieves good accuracy and precision. Good linearity relationship was obtained in the range of5-1200pg/mL with a correlation coefficient of0.9953. The accuracy ranged from88.5%to93.7%. The intra-and inter-day relative standard deviations varied from3.6%to7.4%and from5.4%to9.7%, respectively. The limit of detection (LOD) was0.4pg/mL.2. Study on a PFP chromatographic column separation technology for the simultaneous determination of NNAL and its N-and O-glucuronides in human urineA direct measurement method for simultaneous determination of urinary NNAL, NNAL-N-Gluc and NNAL-O-Gluc by LC-MS/MS in a single run was developed for the first time. Three different types of HPLC columns including Agilent UPLC XDB-C18column (50mm×4.6mm i.d.,1.8μm), Agilent Eclipse Plus-C18column (100mm×4.6mm i.d.,3.5μm) and Phenomenex Kinetex PFP column (100mm×4.6mm i.d.,2.6μm) were evaluated for the separation of NNAL, NNAL-N-Gluc and NNAL-O-Gluc. Chromatographic separation was achieved on the PFP column within6min run time. The extraction performances of the analytes on three different SPE sorbents including C18, HLB and MCX were evaluated. The analytes were analyzed by LC-MS/MS operated in electrospray positive ionization mode with multiple reaction monitoring data acquisition. Compared with the previously reported studies, the proposed method was more accurate and more rapid without the need for time-consuming and laborious enzymatic or base hydrolysis steps. The LODs were1.5,15and20pg/mL for NNAL, NNAL-N-Gluc and NNAL-O-Gluc, respectively.3. Study on the dispersive liquid-liquid microextraction technology for the enrichment and analysis of NNAL in human hairIn this section, two-step SPE combined with reverse-phase ultrasound-assisted dispersive liquid-liquid microextraction (USA-DLLME) as a sample preparation technique was proposed for the sensitive determination of NNAL in human hair samples. The samples were digested with NaOH solution, extracted using C18SPE and molecularly imprinted SPE procedure followed by USA-DLLME procedure for further purification and enrichment before LC-MS/MS analysis. The parameters which affect the extract efficiency were optimized. Under the optimized conditions, an enrichment factor of20was obtained. Accuracies ranged between87.3%and107.7%. Intra-and inter-day relative standard deviations varied from4.1%to8.5%and from6.9%to11.3%, respectively. The LOD was0.08pg/mg. Finally, the developed method was applied for the analysis of NNAL in smokers’ hair. The level of NNAL was between0.27and0.67pg/mg. 4. Study on the dansyl chloride derivatization technology for the quantification of urinary3-hydroxybenzo[a]pyreneIn this work, a sensitive and selective LC-MS/MS method for the determination of urinary3-OHBaP was developed. Following enzymatic hydrolysis of the glucuronide and sulfate conjugates, the metabolite was enriched and cleaned up by SPE and then derivatized with dansyl chloride. The derivatization of3-OHBaP introducing a dansyl group into the molecule greatly enhanced the detection sensitivity by improving both the efficiency of electrospray ionization in the positive ion mode and collision-induced dissociation in the collision cell. Injecting the same concentration of3-OHBaP-Dansyl and3-OHBaP, an increase in the response (peak height) of approximate30-fold over the underivatized3-OHBaP was observed. Good linear relationship was obtained over the concentration range of0.25-40.0pg/mL with a correlation coefficient (r2) of0.9924. Accuracy ranged from87.7%to108.2%. Intra-and inter-day relative standard deviations varied from4.6%to9.6%and7.2%to11.3%, respectively. The LOD was0.1pg/mL. The proposed analytical method was successfully applied to analyze3-OHBaP in human urine from smokers and nonsmokers for biomonitoring the exposure to carcinogenic PAHs.5. Study on the metabolites of a tobacco-specific lung carcinogen in the urine of Chinese smokersThe study subjects consisted of75smokers including50men and15women smoking cured cigarettes and10men smoking blended cigarettes. Concentrations of urinary metabolites including free NNAL, total NNAL and cotinine were analyzed and expressed as milligram creatinine to normalize for urinary concentration. Urinary cotinine levels correlated with total NNAL levels (r=0.66, p<0.000). Urinary cotinine and total NNAL measurements increased steadily with increased smoking cigarettes per day. Furthermore, the differences in the exposure and metabolism of NNK in Chinese smokers were compared with those in the blacks and the whites reported in the literature. The NNAL glucuronidation ratio was greater in Chinese women than that in black women, whereas there was no significant difference in the NNAL-Glucs:NNAL ratio between Chinese women and white women. Conversely, in men, the mean ratio of NNAL-Glucs:NNAL in the yellows was lower than that in both the whites and the blacks.更多还原