烟草与尿样中的代谢标记物

卷烟烟气进入人体后会对人体的代谢物产生影响,目前研究较多的是稠环芳烃类、烟草特有亚硝胺类和尼古丁的代谢物。文章分别介绍了稠环芳烃类代谢物1-羟基芘,烟草特有亚硝胺类代谢物NNAL和NNAL-Gluc,尼古丁的代谢物可天宁,对这些代谢物的检测方法、体内的含量水平、各种条件的影响进行了综述。     

雷公藤甲素对大鼠尿样代谢物影响的核磁共振波谱研究

本文采用基于核磁共振的代谢组学方法研究了雷公藤甲素急性中毒大鼠尿样的代谢特征。结果表明给药后代表肝毒性生化指纹的creatine 和taurine相对含量升高,代表能量代谢的citrate, succinate, α-oxoglutarate相对含量下降,反应肠道菌群代谢状况的trimethylamine N-oxide,和hippurate等也异常,并且这些变化在的药物作用后16小时达到最大,56小时后大部分代谢物的浓度恢复正常。另外,在0~16小时实验组样品里观察到了被认为是一种新的急性肝中毒的生化标记物2’-deoxycytidine。以上NMR实验结果表明雷公藤甲素可能影响大鼠的肠道菌群和能量代谢,引发急性肝损伤和轻度肾衰竭。对注射组大鼠血浆的生化分析和肝组织的病理学切片也证明了实验大鼠肝功能的异常现象。以上结果说明NMR实验信息有助于进一步从分子水平上阐明雷公藤甲素的毒理学和药理学机制。     

尿样中神经性毒剂代谢产物的HPLC/Q-TOFMS/MS检测方法研究

建立了尿样中甲基膦酸单乙酯(EMPA)、甲基膦酸单异丙酯(IMPA)、甲基膦酸频哪基酯(PMPA)3种神经性毒剂代谢产物的HPLC/Q-TOFMS/MS检测方法。以StrataSi-1型固相萃取小柱对尿样中的3种神经性毒剂代谢产物进行分离,HPLC/Q-TOFESIMS/MS进行测定,内标法定量。该方法对EMPA、IMPA、PMPA的线性范围均为5～320μg/L,相关系数均不低于0.9974;EMPA、IMPA、PMPA的加标回收率分别为57%、98%、81%;检出限(S/N≥3)均为0.1μg/L,定量下限(S/N≥10)均为1μg/L。并将该方法应用于禁化武组织(OPCW)首次生物医学样品分析演练未知尿样的检测,结果满意。     

基于人工智能技术的烟气暴露大鼠代谢生物标志物筛选方法研究

该研究将主成分分析、偏最小二乘判别分析等多元统计分析方法用于烟草血浆、尿液和肺组织代谢组学数据的分析,以揭示暴露于不同烟气中大鼠血浆、尿液和肺组织中内源性生物标志物的整体变化情况,筛选潜在生物标志物;将血样、尿样和肺组织代谢轮廓谱分析得到的生物标志物进行整合,运用神经模糊网络模型对标志物进行缩减,并用人工神经网络评价模型预测能力,确定烟气暴露不同时间(7,14,30 d)以及不同烟气暴露对大鼠内源性代谢物变化影响"因果效应"密切相关的关键生物标志物群,明确不同烟气对大鼠机体损伤机制的异同。     

ICP-MS技术快速测量尿样中的铀同位素

核事故状态下的应急处理要求对环境介质中的放射性核素进行快速分析。尿样中铀同位素测量作为内照射剂量评价的主要手段,其分析效率越高,则对核事故中涉铀人员的安全救治越及时、有效。尿样中其他无机离子的含量是铀含量的106倍,导致ICP-MS测量过程中尿盐堵塞进样毛细管。为降低样品的含盐量并获得较好的检测结果,本文对样品预处理过程进行优化。采用先加热氧化去除有机物,再进行1~10倍稀释后测试样品的铀同位素丰度及浓度。结果表明,将25m L样品稀释至100m L后效果最佳,分析方法相对标准不确定度为5.4%,回收率95%~105%。     

DLLME-GC法同时测定尿样中4种芳烃类物质尿代产物

建立了分散液相微萃取-气相色谱法同时测定尿样中2,5-己二酮、苯酚、邻甲酚和对硝基酚的方法.确定了最佳色谱条件,优化了分散液相微萃取的萃取剂种类及用量、分散剂种类及用量、pH、萃取时间和加盐量等条件,实现了对尿样中四种有害物质尿代产物的同时提取和测定.在最佳实验条件下,建立4种组分的工作曲线,线性范围为0.02～8.3...     

HPLC法同时测定尿样中芳香族化合物的6种代谢产物

建立了同时测定尿样中反,反-粘康酸、马尿酸、苯乙醇酸、苯乙醛酸、对氨基酚和对硝基酚的高效液相色谱法(HPLC).优化了色谱分离及检测条件,并采用时间程序波长检测.确定了最佳样品预处理条件:以5 mL二氯甲烷-异丙醇(7∶3)为萃取溶剂;样品用量5 mL;NaCI加入量0.75 g;萃取时间2 min,分别在酸性和中性条...     

薄层色谱-荧光检测法测定尿样中的黄蝶呤

系统研究了在展开剂V(正丙醇)∶V(浓氨水)=2∶3中利用薄层层析将尿液中的黄蝶呤和其它蝶呤类化合物分离,然后利用薄层荧光扫描法测定黄蝶呤。黄蝶呤在0.01～0.08μg范围内,其荧光斑点的积分面积与黄蝶呤的检测量呈良好的线性关系,从而建立了黄蝶呤的新的检测方法。方法可应用于尿样中的黄蝶呤测定。     

大鼠体内复方清开灵代谢物的分析

建立了复方中药清开灵代谢物的液相色谱-质谱/质谱分析方法。采用Phenomenex Luna C18色谱柱,以0.1%甲酸(A)及V(甲醇)∶和V(乙腈)=4∶1混合液(B)作流动相,采用梯度洗脱(0 min,B为0%;33min,B为60%;66 min,B为88%;75 min,B为100%),流速:0.5 mL/min,离子阱质谱负离子模式进行检测。通过差谱方法对可能的代谢物进行快速搜索。对发现的重要的代谢物根据分子量以及多级质谱的碎片离子数据,结合体内代谢反应规律,进行指认和结构鉴定。共鉴定了3个主要的代谢物,其中一个为黄芩苷的代谢物,另两个为绿原酸的代谢物,后两个代谢物未见文献报道。本实验为研究复方中药复杂体系体内的代谢产物提供了一个有效而且快捷的模式。     

电喷雾解吸电离质谱法用于临床尿样的直接分析

将电喷雾解吸电离质谱(DESI-MS)用于临床尿样的分析, 优化了电喷雾溶剂流速、电喷雾电压和喷雾锥距离等重要参数. 采用普通滤纸作为样品载体, 在不需要样品预处理的前提下同时快速测定了临床尿样中的钾、钠、尿素、尿酸、丙酮酸和肌苷等多种成分, 并对各种成分的主要离子进行了串联质谱鉴定. DESI-MS在进行多组分同时测定时不需要进行样品预处理, 缩短了测定时间, 单个样品的分析时间不到1 min. 同时, 采用内标法对所测定组分进行了半定量分析.     

Rapid quantitation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in rat urine using ultra-fast liquid chromatography mass spectrometry (UFLC/MS/MS)

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a component of tobacco smoke and is rapidly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Limited information is available on the relative systemic exposures resulting from NNK administration via the oral, intraperitoneal injection, and inhalation routes. Moreover, there is a need for a rapid method for simultaneous quantitative analyses of NNK and NNAL in rat urine. We developed a method based on Ultra Fast Liquid Chromatography Mass Spectrometry (UFLC/MS/MS) for the extraction and analysis of the potent lung carcinogens NNK and NNAL. Following addition of synthetic labeled internal standards, urine was introduced to 96 well plate Evolute^® Express CX 30?mg solid phase extraction system. The eluates were dried under vacuum and reconstituted in mobile phase before injecting to the LC system. The use of UFLC allowed for a 7.1?min run time. The precision and accuracy of the samples was 1.2-6.6% relative standard deviation (%RSD) and 91-113% of the concentration added, respectively. The limits of detection for NNK and NNAL were 70 and 3.0?pg/mL, respectively. The selectivity and sensitivity of this method improves the ability to measure these compounds at low concentrations and greatly facilitate toxicological studies of the NNK and NNAL.     

A new liquid chromatography/mass spectrometry method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine

4‐(Methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is a carcinogenic nitrosamine produced upon curing tobacco. It is present in tobacco smoke and undergoes metabolism to 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL) in the lungs. NNAL undergoes further uridine diphosphate glucuronosyltransferase (UGT)‐mediated metabolism to give N‐ and O‐glucuronide metabolites, which together with free (non‐conjugated) NNAL are then excreted in the urine. The ability to conduct validated analyses of free and conjugated NNAL in human urine is important in order to assess inter‐individual differences in lung cancer risk from exposure to cigarette smoke. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography/multiple reaction monitoring/mass spectrometry (LC/MRM‐MS) provides the highest bioanalytical specificity possible for such analyses. We describe a novel derivatization procedure, which results in the formation of a pre‐ionized N‐propyl‐NNAL derivative. The increased LC/MS sensitivity arising from this derivative then makes it possible to analyze free NNAL in only 0.25 mL urine. This substantial reduction in urine volume when compared with other methods that have been developed will help preserve the limited amounts of stored urine samples that are available from on‐going longitudinal biomarker studies. The new high sensitivity SID LC/MRM‐MS assay was employed to determine free and conjugated NNAL concentrations in urine samples from 60 individual disease‐free smokers. Effects of inter‐individual differences in urinary creatinine clearance on NNAL concentrations were then assessed and three metabolizer phenotypes were identified in the 60 subjects from the ratio of urinary NNAL glucuronides/free NNAL. Poor metabolizers (PMs, 14 subjects) with a ratio of NNAL glucuronides/free NNAL <2 (mean = 1.3), intermediate metabolizers (IMs, 36 subjects) with a ratio between 2 and 5 (mean = 3.4), and extensive metabolizers (EMs, 10 subjects) with a ratio >5 (mean = 11.1). Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolic study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone to the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in vitro in human bronchial epithelial cells using chiral capillary electrophoresis

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) with one chiral center at the carbinol is a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). As tobacco specific N-nitrosamines (TSNAs), NNK and NNAL are the most pulmonary carcinogens in tobacco products and smoke. In this paper, a chiral CE method modified with highly sulfated β-cyclodextrin (S-β-CD) was developed to investigate the stereoselective formation of NNAL from NNK in vitro in normal human bronchial epithelial (NHBE) cells. Combined with solid phase extraction (SPE) of the cell samples, NNK and NNAL enantiomers were baseline separated under the proposed CE conditions, with satisfactory recoveries (72.5-113% for NNK and (±)-NNAL) and low limits of detection (LOD, 2.5-3 μg/mL for NNK and (±)-NNAL). The cytotoxicity of NNK in NHBE cells was investigated through the cell counting kit (CCK) assay and proved to be highly dependent on the NNK's concentration. The metabolic results obtained from CE analysis demonstrated that NNK was preferentially metabolized to (+)-NNAL through carbonyl reduction. Meanwhile, the ratio of [(+)-NNAL]/[(-)-NNAL] was independent of NHBE cells' incubation time with NNK, but could be changed according to the original incubation concentration of NNK. This chiral CE method could be useful for the study on toxicology and metabolic transformations of related TSNAs.     

固定化β-葡萄糖醛酸酶反应器的制备及其在4-甲基亚硝胺基-1-(3-吡啶)-1-丁醇的O-糖苷化合物分析中的应用

采用溶胶-凝胶法,以丙烯酰胺作为单体,制备了基于有机-硅胶杂化整体柱的β-葡萄糖醛酸酶反应器。优化了硅酸甲酯和γ-(甲基丙烯酰氧)丙基三甲氧基硅的物质的量的比、丙烯酰胺和聚乙二醇的用量,以及水浴温度等制备条件,获得了孔隙均匀、通透性良好、机械强度高的有机-硅胶杂化整体柱。采用光学显微镜和扫描电镜表征杂化整体柱。进一步将β-葡萄糖醛酸酶共价键合在整体柱上,以4-甲基亚硝胺基-1-(3-吡啶)-1-丁醇(NNAL)的O-糖苷化合物(NNAL-O-Gluc)为底物研究酶反应器的水解效果,实验结果证明酶反应器在室温条件下的水解效率大大提高,实现了NNAL-O-Gluc高效水解与分析,解决了目前NNAL-O-Gluc分析中前处理水解效率低的问题。     

Identification of key metabolites of tectorigenin in rat urine by HPLC-MS(n)

This is a report about the identification of key metabolites of tectorigenin in rat urine using high-performance liquid chromatography-electrospray ionization ion trap tandem mass spectrometric method (HPLC-ESI-MS(n)). Six healthy rats were administered a single dose (80 mg/kg) of tectorigenin by oral gavage. Urine was sampled for 0-24 h and centrifuged at 12,000 rpm for 10 min to obtain the supernatants, then the supernatants were purified by solid-phase extraction with a C(18) cartridge. The chromatographic separation was carried out on a reversed-phase C(18) column with a gradient elution program whereas acetonitrile-0.1% formic acid water was used as mobile phase. Mass spectra were acquired in negative ionization mode and a data-dependant scan was used for the identification of the key metabolites of tectorigenin in the urine samples. As a result, four phase II metabolites and the parent drug tectorigenin were found and identified in rat urine for the first time.     

在线液相-气相二维色谱高灵敏测定卷烟主流烟气中的4-（N-甲基亚硝胺基）-1-（3-吡啶基）-1-丁酮

建立了在线液相-气相二维色谱测定卷烟主流烟气中4-(N-甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)的方法。 NNK 的分析在在线凝胶气质联用仪上进行,采用自行装填的微型碱性氧化铝柱,并把仪器上的凝胶柱换成氧化铝柱,用于 NNK 的分析。捕集有主流烟气总粒相物的剑桥滤片用二氯甲烷提取,以 D4-NNK 为内标,提取液经微型氧化铝柱分离,含 NNK 的部位切割进入气相色谱,排干溶剂后启动气相色谱升温经毛细管柱进行分离,用质谱检测。本方法将烟气国标方法 NNK 测定中的氧化铝柱色谱净化和气相色谱-质谱分析在线连接起来,可不经样品前处理净化直接进样分析;每次进样可达40μL,是常规气相色谱-质谱分析最大进样量(2.0μL)的20倍,显著提高了分析灵敏度。方法线性范围达1.2~120 ng/ mL,相关系数为r=0.9998,回收率为93.9%~96.0%;检出限和定量限分别为0.25 ng/ mL 和0.9 ng/ mL,样品分析结果与中国烟草总公司企业标准方法进行对比,结果相符合。     

1-(2-氯-4-吡啶基)-3-苯基脲的合成

氯氨基吡啶;氯吡啶基苯基脲;1-(2-氯-4-吡啶基)-3-苯基脲的合成     

Simultaneous determination of nicotine and 3-vinylpyridine in single cigarette tobacco smoke and in indoor air using direct extraction to solid phase

The aim of the present study was to develop a new analytical method of chromatographic determination of two important markers of ETS exposure: nicotine and 3-vinylpyridine (3-ethenylpyridine, 3-EP) in mainstream (MS) and sidestream (SS) smoke of one single cigarette and in indoor air using direct solid phase extraction combined with gas chromatography. The method can be utilised for both nicotine and 3-EP determination in SS and MS of one single cigarette as well as it allows for a precise determination of compound distribution in indoor air. The application of the same analytical method for both kinds of samples allows anticipating indoor air distribution of both analysed compounds in a very precise way. The precision of the method (calculated as a relative standard deviation) was 9.78% for nicotine and 2.67% for 3-EP; whereas the accuracy (evaluated by a recovery study conducted at three different levels) was 70.1 and 87.3%, respectively. The limit of detection was 0.06 µg per cigarette for both nicotine and 3-EP. The method was evaluated by determining the compounds of interest in two commercially available brands of cigarettes as well as in the reference cigarettes 3R4F and also in indoor air polluted with tobacco smoke. Determined levels of compounds of interest in MS varied from 586 to 772 (nicotine) µg per cigarette and from 3.5 to 10.7 (3-EP) µg per cigarette. In SS smoke the level varied from 14,370 to 22,590 (nicotine) µg per cigarette and from 185 to 550 (3-EP) µg per cigarette, whereas levels in indoor air polluted with tobacco smoke varied from 50.1 to 157.3 (nicotine) µg m^?3and from 7.7 to 20.8 (3-EP) µg m^?3.