固相萃取-气相色谱-质谱法检测染毒尿样中芥子气-谷胱甘肽加合物的β-裂解产物

合成并纯化了芥子气-谷胱甘肽的β-裂解产物1,1′-磺酰基二(2-甲巯基)乙烷(SBMTE),纯度为98.9%.建立了固相萃取-气相色谱质谱法检测染毒尿样中SBMTE的方法,对尿样中SBMTE的提取富集、 Oasis HLB固相萃取柱净化条件等因素进行了优化.实验中采用DB-5MS毛细管柱进行分离,以化学电离源(CI)质谱选择离子模式(SIM)进行检测,并通过内标法定量.SBMTE的线性范围为1～100 ng/mL,r=0.9991,回收率为89.0%～92.6%,检出限为0.5 ng/mL.方法满足芥子气中毒人群尿中SBMTE的检测要求.     

高效液相色谱-电喷雾离子阱串联质谱法检测芥子气经皮肤染毒SD大鼠肺中的DNA加合物

运用高效液相色谱-电喷雾离子阱串联质谱(HPLC-ESI-MS/MS)技术,建立了快速、简单、灵敏的SD大鼠肺中N7-(2-羟乙基硫代乙基)鸟嘌呤(N7-HETEG)的检测方法。以N7-苯甲基鸟嘌呤为内标,用甲醇和水为流动相进行梯度洗脱,正离子模式检测,方法的检出限(信噪比(S/N)≥10)为300 pg/mL,定量限(S/N≥20)为850 pg/mL。在300 pg/mL～1.28 μg/mL的质量浓度范围内,N7-HETEG浓度与N7-HETEG和内标的峰面积比呈良好的线性关系(线性相关系数为0.9929)。高、中、低3个添加水平的日内测定精密度(以相对标准偏差(RSD)计)和日间测定精密度均小于10%(n=7),回收率为100%～132%。对SD大鼠背部皮肤染芥子气,剂量分别为5.5、11、22和45 mg/kg,染毒4 d后检测大鼠肺脏中N7-HETEG的含量。各个不同染毒剂量下,每克组织中分别检测到（0.56±0.16）、（0.67±0.12）、（1.36±0.68）和(5.14±0.92) ng N7-HETEG, N7-HETEG的含量随着染毒剂量的增大而增大,表明N7-HETEG可用作芥子气暴露的体内生物标志物。     

超高效液相色谱-串联质谱法同时检测人血浆中6种芥子气染毒标识物

针对芥子气( HD)染毒血浆的溯源性分析需求,采用超高效液相色谱-串联质谱技术( UHPLC-MS/MS),建立了人血浆中6种HD生物标识物( TDG,TDGO,SMO,SBMSE,MSMTESE,SBSNAE)的高灵敏度和专属性检测方法。应用甲醇和乙腈混合有机溶剂沉淀HD染毒血浆中的蛋白,然后用HLB柱固相萃取( SPE)纯化,经超高效液相色谱梯度洗脱分离,在三重四极杆串联质谱正离子多反应监测( MRM)模式下进行定性与定量检测。结果表明,6种目标物的线性范围为0.05~500 ng/mL(R2=0.9840~0.9955),检测限在0.01~1.0 ng/mL之间,各分析物的精密度(n=5)≤5.5%,加标回收率在86.5%~110.8%之间。本方法通过SPE的引入和UHPLC程序的优化,有效地解决了基质背景对分析可靠性的影响问题,并成功应用于国际禁止化学武器组织( OPCW)第5次生物医学样品分析演练HD染毒血浆样品的鉴定。     

尿液中新型合成大麻素及其代谢物的检验研究

利用液相色谱-高分辨质谱（LC-HRMS）建立了尿液中3种新型合成大麻素及5种代谢物的快速检验方法。前处理分别采用沉淀蛋白和固相萃取2种方法,选用Thermo Hypersil GOLD C18(100 mm×2.1 mm,1.9μm)色谱柱进行分离,以含10 mmol/L甲酸铵的0.1%甲酸水溶液和含0.1%甲酸的甲醇溶液进行梯度洗脱;采用正离子扫描模式,一级质谱全扫描/数据依赖二级质谱扫描（Full MS/dd-MS2）进行检测。结果表明,沉淀蛋白法检出限为1～3 ng/mL,定量限为2～5 ng/mL,固相萃取法检出限为0.1～1 ng/mL,定量限为0.5～2 ng/mL,回收率为76.5%～91.7%,基质效应范围为77.6%～97.3%,日内日间相对标准偏差均小于12%。将建立的方法用于检验疑似合成大麻素吸食者的尿液检材并检出了目标物,表明所建立的方法可应用于公安机关对实际案件的检验。     

分散液液微萃取-上浮溶剂固化/高效液相色谱法测定沉积物中的十溴联苯醚

建立了沉积物中痕量十溴联苯醚的分散液液微萃取-上浮溶剂固化-高效液相色谱-紫外法(DLLME-SFO-HPLC-UV)。以正交试验数据为训练样本,采用BP(Back propagation)神经网络模型优化了分散液液微萃取-上浮溶剂固化条件:分散剂为1.00mL甲醇、萃取剂为35.0μL十二醇、NaCl质量浓度为10.00%、萃取时间10min和pH=5,其萃取率(ER)可达62.22%。方法的线性范围为3.5～1400ng/g(r=0.9960),检出限(LOD)和定量限(LOQ)分别为2.3pg/g(S/N=2)和5.6pg/g(S/N=5),实际样品的加标回收率为97.7%～104.2%。本方法集萃取、富集、分离步骤于一体,简化了沉积物中十溴联苯醚的前处理过程。     

血液与尿液中铅形态化合物的HPLC-ICP-MS分析

建立了血液和尿液中2种铅形态化合物(三甲基铅、三乙基铅)的高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)分析方法。利用苯溶液萃取铅形态化合物,超声离心后以硫代硫酸钠溶液进行反萃取。以0.1 mol/L乙酸铵-0.1 mol/L乙酸与甲醇为流动相进行等度洗脱,Agilent Zorbax Plus C_(18)(4.6 mm×100 mm,3.5μm)对提取物进行分离,ICP-MS分析样品中铅形态化合物。结果表明,血液和尿液中2种铅形态化合物的线性范围分别为3～200 ng/mL和5～400 ng/mL,检出限为0.85～1.31 ng/mL,定量下限为3.00～5.00 ng/mL,日内及日间相对标准偏差(RSD)为1.8%～10%,提取回收率为85.3%～104%,基质效应为88.3%～117%。该方法样品前处理简单,检出限低,准确度高,适用于人体血液和尿液中三甲基铅、三乙基铅的测定。     

同位素稀释超高效液相色谱-串联质谱法测定人血清中孕酮

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)测定人血清中孕酮的分析方法。血清样品经乙酸乙酯、正己烷液液萃取(LLE)后,采用Acquity UPLC BEH C_(18)色谱柱(2.1 mm×100 mm,1.7μm)进行梯度分离,色谱运行时间为5 min,采用电喷雾(ESI)正离子电离模式和多反应监测(MRM)扫描模式,同位素内标法定量。考察了两步萃取法对孕酮的提取效果,不同流动相的分离效果以及样品稳定性,结果表明以甲醇-0.1%氨水溶液为流动相时分离效果较好。优化条件下,孕酮在10～10 000pg/mL范围内线性关系良好(r~2=0.999 8),方法检出限和定量下限分别为5、10 pg/mL;平均加标回收率为91.5%～106%,日内相对标准偏差(RSD)为1.2%～8.2%,日间RSD为4.1%～9.1%。采用该方法对30个真实血清样品进行测定,孕酮质量浓度为0.050 2～1.363 5 ng/mL,均在正常生理范围内。该方法灵敏度高、准确可靠,可用于临床血清样品中孕酮生理水平的检测。     

高效液相色谱-三重四极杆/复合离子阱质谱测定大鼠血浆中11种合成大麻素

建立并验证了一种用于定量检测大鼠血浆中11种合成大麻素(SCs)的高效液相色谱-串联质谱(HPLC-MS/MS)方法。选择Waters UPLC HSS T3(150 mm×2.1 mm,1.8μm)色谱柱,以乙腈为蛋白沉淀剂(血浆∶乙腈=1∶9,V/V),实现了11种目标物质的色谱分离和检测。方法学验证数据表明,本方法在0.05～10 ng/mL范围内具有良好线性(R²>0.991),基质效应为69.5%～119.0%,回收率为43.8%～131.5%,相对标准偏差为4.0%～28.3%,定量限为0.05～1 ng/mL。本方法适用于血浆中SCs的定性定量检测。     

超高效液相色谱-串联质谱法同时测定血液中115种农药

建立了超高效液相色谱-串联质谱法(UPLC-MS/MS)一针进样同时测定血液中115种农药的方法。血液样品与提取剂甲醇按1∶5体积比进行蛋白沉淀,涡旋振荡2 min,以12 000 r/min离心10 min,取上清液进行UPLC-MS/MS分析。质谱分析采用电喷雾离子源(ESI),正离子模式和负离子模式同时切换采集。结果表明,115种目标农药在1～200 ng/mL质量浓度范围内线性关系良好(r0.99),13%的目标农药检出限(S/N≥3)为0.2 ng/mL,其余目标物均为0.1 ng/mL,所有目标农药的定量下限(S/N≥10)均为1 ng/mL。在10、50、100 ng/mL加标水平下,方法的基质效应为81.8%～115%,目标农药的回收率为80.4%～109%,日内精密度(Intra-RSD)为1.6%～11%,日间精密度(Inter-RSD)为1.9%～13%。该方法简便快速、灵敏度高、稳定性好,适用于血液样品中多农药的定性定量分析。     

基质分散固相萃取-超高效液相色谱-串联高分辨质谱法同时测定干血斑样品中22种镇定催眠类药物

构建了干血斑样品中22种镇定催眠类药物的超高效液相色谱-串联高分辨质谱法定性、定量检测方法。干血斑样品经过乙腈提取后,应用基质分散固相萃取法对提取液进行净化,考察了不同种类净化剂以及用量对净化效果的影响,采用C18色谱柱进行分离,对色谱条件和质谱条件进行优化,使用同位素内标法进行定量分析。结果表明：22种被测物质在5 ng～800 ng/mL范围内线性表现良好(r²>0.99),检出限为2 ng/mL～8 ng/mL,定量限为5 ng/mL～20 ng/mL,被测物质在低、中、高三个添加水平下回收率为80.2%～113.5%,相对标准偏差为0.9%～14.3%。本研究样本存储方便,保存时间长,前处理操作简单,应用精确质量数进行定性和定量,极大降低了假阳性的发生,为临床药物研究和法医毒物分析提供了新的技术支持。     

Monitoring urinary metabolites resulting from sulfur mustard exposure in rabbits,using highly sensitive isotope-dilution gas chromatography–mass spectrometry

A highly sensitive method for the determination of sulfur mustard (SM) metabolites thiodiglycol (TDG) and thiodiglycol sulfoxide (TDGO) in urine was established and validated using isotope-dilution negative-ion chemical ionization (NICI) gas chromatography–mass spectrometry (GC–MS). TDGO in the samples was reduced with TiCl₃, and then determined together with TDG as a single analyte. The sample preparation procedures, including two solid-phase-extraction (SPE) clean-up steps, were optimized to improve the sensitivity of the method. The limits of detection (LOD) for both TDG and TDG plus TDGO (TDG + TDGO) were 0.1 ng mL^?1, and the limits of quantitation (LOQ) for both were 0.3 ng mL^?1. The method was used in a rabbit cutaneous SM exposure model. Domestic rabbits were exposed to neat liquid SM at three dosage levels (0.02, 0.05, and 0.15 LD₅₀), and the urinary excretion of four species of hydrolysis metabolites, namely free TDG, free plus conjugated TDG (total TDG), free TDG + TDGO, and free plus conjugated TDG + TDGO (total TDG + TDGO), was evaluated to investigate the metabolic processes. The total urinary excretion profiles of the metabolites, including the peak time, time window, and dose–response and time–response relationships, were clarified. The results revealed that the concentrations of TDG and TDG + TDGO in the urine increased quickly and then decreased rapidly in the first two days after SM exposure. The cumulative amount of total TDG + TDGO excreted in urine during the first five days accounted for 0.5–1 % of the applied dose of SM. It is also concluded that TDG and TDGO in urine existed mainly in free form, the levels of glucuronide and of sulfate conjugates of TDG or TDGO were very low, and most hydrolysis metabolites were present in the oxidized form (TDGO). The study indicates that the abnormal increase of TDG and TDGO excretion levels can be used as a diagnostic indicator and establishes a reference time-window for retrospective analysis and sampling after SM exposure.     

Extraction of thiodiglycol from soil using pressurised liquid extraction

Thiodiglycol (TDG) is the predominant hydrolysis product of the chemical warfare agent sulfur mustard. The extraction of TDG was investigated using pressurised liquid extraction and the results compared for a variety of different solvents and soils. TDG was analysed underivatised by gas chromatography with flame photometric detection. A mixture of methanol-water (9:1), proved to be the most efficient extracting solvent for TDG at a temperature of 150 degrees C and 10 MPa.     

多环芳烃暴露的生物标志物——尿中羟基多环芳烃

多环芳烃(PAHs) 是典型的持久性有机污染物,在职业高PAHs 暴露环境下,容易诱发肺癌、皮肤癌等癌症。对PAHs 的暴露评价可为流行病学研究和污染物风险评价等提供有效的数据。由于暴露途径的复杂化,采用尿样中PAHs 的代谢产物———羟基多环芳烃作为标志物来综合评价人体对PAHs 的内暴露情况已经成为研究的热点。本文系统介绍了多环芳烃的吸收、代谢、尿中PAHs 代谢产物的主要存在形式、主要的生物标志物以及它们的主要影响因素。     

Determination of thiodiglycol, a mustard gas hydrolysis product by gas chromatography-mass spectrometry after tert-butyldimethylsilylation

A method for determining thiodiglycol (TDG), a mustard gas hydrolysis product in water, serum and urine samples using gas chromatography-mass spectrometry (GC-MS) after tert-butyldimethylsilylation (TBDMS) is described. Quantitation of TDG was performed by measuring the respective peak area on the extracted ion chromatogram of m/z 293, using an internal standard, the TDG homologue, thiodipropanol, peak area of which was measured as m/z 321. The presence of salts in the sample solution not only suppressed the loss of TDG by vaporization during the evaporation of water, but also facilitated the rate of production of di-silylated derivative, bis(tert-butyldimethylsilyoxylethyl)sulfide (TDG-(TBDMS)2). Under the pretreatment conditions used, in which 0.5 ml of water sample supplemented with 100 microM potassium chloride was evaporated to dryness under reduced pressure, followed by reaction with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide at 60 degrees C for 1 h, TDG-(TBDMS)2 was reproducibly detected with about a 55% recovery and a limit of detection (LOD, scan mode, S/N = 3) of 5.4 ng/ml. TDG was also determined by GC-MS from a 0.5 ml serum sample (after perchloric acid deproteinization) and from a 0.1 ml urine sample, after TBDMS derivatization. The LOD was determined to be 7.0 and 110 ng/ml for serum and urine, respectively.     

Determination of the sulfur mustard hydrolysis product thiodiglycol by microcolumn liquid chromatography coupled on-line with sulfur flame photometric detection using large-volume injections and peak compression.

A selective, direct and relatively rapid method has been developed for the determination of thiodiglycol (TDG) in aqueous samples. TDG is the main hydrolysis product of the chemical warfare agent sulfur mustard. The method of analysis is based on the on-line coupling of reversed-phase microcolumn liquid chromatography and sulfur-selective flame photometric detection. To improve sensitivity and efficiency, peak compression by displacement was used in combination with large-volume injections. A concentration of 1% n-propanol was added to the sample to obtain the best sensitivity and efficiency after a 10 microliters injection. Detection limits of 0.25 microgram/ml were achieved with efficiencies of 4.10(5) plates per meter. The method was successfully applied during the Fourth Official Proficiency Test organized by the Technical Secretariat of the Organization for Prohibition of Chemical Weapons for the determination of TDG in a soil sample.     

Quantitative analysis of the sulfur mustard hydrolysis product thiodiglycol (2,2'-sulfobisethanol) in in vivo microdialysates using gas chromatography coupled with pulsed flame photometric detection

An analytical method employing gas chromatography is presented for assessing the concentrations of the sulfur mustard hydrolysis product thiodiglycol (TDG) in cutaneous in vivo microdialysates. The use of a pulsed flame photometric detector allows for selective detection of the analyte following solvent exchange and derivatization with heptafluorobutyric anhydride. Quantitative assessment is performed using thiodipropanol (TDP) as a surrogate internal standard. A linear relationship and a very significant correlation (r2 = 0.9982) between the ratio of TDG and TDP concentrations and the ratio of the square root of peak heights is demonstrated. The suitability of the analytical method is verified by the evaluation of blank in vivo microdialysates spiked with known amounts of TDG. The limit of detection in microdialysates is 0.200 nmol/mL (24.4 ng/mL) and the limit of quantitation was 0.364 nmol/mL (44.4 ng/mL). The presented method provides selective, sensitive, rapid, and high-throughput analysis of microdialysates containing TDG, providing an efficient alternative for high-performance liquid chromatography and capillary electrophoresis techniques.     

Synthesis,Characterization and Mass Spectrometric Analysis of Cysteine and Valine Adducts of Sulphur Mustard

The synthetic standards for cysteine and valine adducts of sulphur mustard have been synthesized using 2-(2-chloroethylthio) ethanol (half-mustard). These adducts have been recognized as biomarkers for exposure to sulphur mustard. The adducts have been fully characterized using 1 H and 13 C NMR spectroscopy and mass spectrometric techniques.     

人体邻苯二甲酸酯暴露的尿液生物标志物分析方法

邻苯二甲酸酯（phthalates，PAEs）是一类典型的环境内分泌干扰物。近年来，由于PAEs产量和使用量的增加，其对人体健康的危害尤其是生殖发育毒性受到了人们高度关注。由于PAEs在环境、食物（材）中广泛存在，导致人体不可避免地长期暴露于PAEs化合物，因此很有必要开展人体PAEs暴露评估。对人体尿液样品中的PAEs代谢物进行筛选和定量是评价PAEs暴露的重要手段，而建立它们准确、可靠的分析方法是重要前提。目前，邻苯二甲酸单酯和次级代谢物分别是短链和长链PAEs暴露最为常用的生物标志物。离线或在线固相萃取与高效液相色谱-串联质谱联用已成为测定PAEs代谢物的首选方法。本文主要综述了人体PAEs暴露的尿液生物标志物的分析方法，并讨论了这些方法在实际应用中的优点、局限性及挑战。