Biological Monitoring Of Exposure To Sulfur Mustard—the Determination Of Glutathione Adducts In Rabbit Urine By Solid Phase Extraction-Gas Chromatography-Mass Spectrometry

Sulfur mustard (SM), belonging to a kind of vesicant agent, can cause skin, respiratory and mucous membrane injury, and produces pathological changes such as inflammation, necrosis etc. As a bifunctional alkylating agent, SM reacts rapidly with nucleophiles in physiological conditions. These reactions in the living body include the alkylation of DNA, proteins and glutathione. The adducts formed with glutathione can be clearaged byβ-lyase leading to the formation of many nontoxic metabolites, such as SBMTE, MSMTESE and SBMSE that are excreted in urine. There is no glutathione adducts existed in urine sample from natural individual, so these compounds can be chosen as biomarker to validate exposure to SM. The research for the detection of the biomarkers is helpful for diagnosis and treatment of the individuals after accidental exposure.In this thesis, SPE and SLE pretreatment technique and gas chromatography-mass spectrometric technique were performed for quantitative determination of the glutathione adducts. This thesis consists of five chapters.The first chapter is the introduction. Metabolic processes of individual exposure to SM were introduced. The analytical methods and samples preparation for adducts from every metabolic pathway were reviewed. Then the research objects in the thesis are concluded and pointed out.In chapter 2, based on SPE and SLE separation and purification principles, three pretreatment methods of theβ-lyase metabolites of glutathione adducts, such as SBMTE, MSMTESE and SBMSE spiked in urine sample were developed. The first one was SPE(PEP), which was suited to concentrate and purify the urine samples only spiked SBMTE artificially. The second one would be applied to the pretreatment of the urine samples containing SBMTE, MSMTESE and SBMSE, in which the latter two compounds could be reduced as SBMTE by titanium trichloride (TiCl3) in acidic solution. Because of large amounts of precipitates produced from the reaction of NaOH with TiCl3, it brought much more difficulty in pretreatment process. We tried two ways to load samples on the SLE cartridges, the direct loading of suspension solution - SLE(I) and the supernatant solution loading - SLE(II). The advantage of the former was high recovery with above 90%, which was applied to the purification of the artificial urine, and the latter had more availably purification efficiency, which was applied to dealing with the complicated real urine samples. The third method was SPE(HLB), the experiment results showed which was the most efficient purification one in spite of low recovery. Finally, the optimized pretreatment protocol with high sensitivity and less interference has been achieved effectively. This method can be applied to concentration and purification of real urine samples with low concentration glutathione adducts.In chapter 3, combined with different pretreatment methods above established, three GC-MS methods, such as GC-MS(EI), GC-MS(CI) and GC-MS/MS(EI) for the determination of SBMTE in urine were developed. GC-MS(EI) method with SPE(PEP) as pretreatment protocol only fitted to determine SBMTE in the urine samples without need of reduction. The limit of detection for SBMTE was 0.1 ng/mL in 0.5 mL of urine. The methods with SLE(I) or SPE(HLB) as pretreatment protocol could all be used to determine SBMTE in the complicated real urine samples with LODs of 0.5 ng/mL, in which the former had the high extracted recovery for SBMTE, the latter could make the samples more purified. GC-MS(CI) method with SLE(II)and SPE(HLB) as pretreatment protocol showed poor sensitivity, LODs of 25 ng/mL and 5 ng/mL respectively. GC-MS/MS (EI) method with SPE(HLB) as pretreatment method presented a high sensitive determination of SBMTE in the complicated real urine samples reduced by TiCl3. The linear calibration curve was ranged from 0.1 to 1000 ng/mL with a coefficient (r) of 0.9995, LOQ was 0.1 ng/mL, LOD was 0.02 ng/mL.The chapter 4 was the application of the analysis method of SBMTE in the research of in vivo metabolism behavior of glutathione adducts. Rabbits were administered with high, middle and low dose of SM via percutaneous exposure way. The experiment results showed that the glutathione adducts were eliminated fast, the contents of SBMTE were maintained at higher level in the first two days after exposure, then rapidly descended about 30% in the third day, and less than 4% in the seventh day. The SBMTE could be obviously detected until four weeks later. The contents of SBMTE eliminated in the first week were about 99% of the total contents of SBMTE in four weeks. On day 28 after the exposure, the contents of glutathione adducts with a level of three times higher than the LOQ were detected in the rabbits urine at the high dose group. On day 24 and 21 after the exposure, only a level of the LOD was detected in the rabbits urine at the middle and low dose groups, respectively. This is very important for the retrospective analysis of SM exposure.The chapter 5 was the application of the analysis method of SBMTE in four individuals after accidental exposure, which happened in HeBei province on March 20th. Urine samples from four individuals were analyzed to confirm that the symptoms were caused by HD exposure. On day 8 after the exposure, patients had SBMTE levels of LOD. The event comfirmed that the analysis method of SBMTE we established had the practical application value.