Study On The Application Of New Technology To Separation And Analysis Of Metabolism Biomarkers Of Tobacco Smoke Carcinogens In Human

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.