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| Analysis of Urinary Biomarkers for Diagnosis of Patients Exposed to Sulfur Mustard |
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| DOI： |
| KeyWord:sulfur mustard urine sample ultra performance liquid chromatography-tandem mass spectrometry biomarker biomarker profile |
| Author | Institution |
| ZHANG Ya-jiao,XU Bin,XU Hua,WU Jian-feng,CHEN Jia,WANG Chun-yan,GUO Lei,QIU Ze-wu,XIE Jian-wei |
1.State Key Laboratory of Toxicology and Medical Countermeasures,and Laboratory of Toxicant Analysis,Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Beijing,China;2.Fifth Medical Center of Chinese PLA,Beijing,China |
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| Abstract: |
| Sulfur mustard(SM) is a powerful vesicant chemical warfare agent,which is also a predominant agent found in the abandoned chemical weapons in China by Japanese imperialist army after World War Ⅱ.As a bifunctional alkylating agent,SM reacts rapidly with numerous nucleophiles,eg.water,glutathione,DNA and proteins under physiological conditions via the inter mediate episulfonium ion.Currently,some major metabolites of SM have been used as biomarkers for research and development of toxicity and drug.To address the challenge of early clinical diagnosis and verification of SM exposure,a retrospective method towards exposed urinary biomarkers was developed and applied in this work.A novel ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) combined with two steps of solid phase extraction(SPE) was established for simultaneous quantification of seven free metabolites in urine,with limits of detection(LOD) of 5 pg/mL-1 ng/mL and limits of quantitation(LOQ) of 10 pg/mL-5 ng/mL,respectively.The urine matrix interference was reduced effectively due to the established two step SPE procedure(MCX-HLB SPE),leading to the improvement of mass spectrum response to analytes.Especially for thiodiglycol(TDG),a high polarity metabolite,the mass spectrometry response was increased by 64 times after the clean up step with MCX SPE.Therefore,better recoveries of 720%-117% and matrix effects of 853%-117% of the method were achieved.Meanwhile,by the established isotope dilution UPLC-MS/MS for the determination of four urinary SM-nucleobase adducts,an overall analysis of the sulfur mustard urinary biomarkers was performed for a male patient with suspected sulfur mustard exposure with burn degree of Ⅱ° and 1%,who was hospitalized on the 3rd day and discharged on the 7th day after exposure.Three hydrolysis/oxidation products,three glutathione(GSH) adduct β lyase metabolites and four SM-nucleobase adducts were successfully found in the urine samples,including mustard sulfoxide reported as free urinary biomarkers in the first time,which could verify that the patient was indeed suffered from a sulfur mustard exposure.The concentration-time profile demonstrated that the biomarkers reached maximum concentration at 3 to 4 days except for TDG,then decreased and still detectable until the 7th day after exposure.In the 10 detected biomarkers,the hydrolysis products of SM are endogenous substances with background values in vivo as 0-8 ng/mL,meaning that they were not suitable biomarkers for SM exposure in this work.SMO,the direct oxidative metabolite of SM,was identified to be an exogenous substance and could be used as a direct evidence for SM exposure.In addition,similar with SMO,GSH adduct β lyase metabolites are only produced upon exposure to SM.Considering that the concentration of GSH adduct β lyase metabolites was relatively higher than those of hydrolysis/oxidation products and SM-nucleobase adducts,they would be important for early diagnosis and medical treatment monitoring of sulfur mustard exposure. |
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