芥子气中毒人员尿液标记物谱分析及其临床诊断应用

该研究基于暴露生物标记物溯源思路,应用于解决芥子气(SM)临床早期诊断、溯源确证难题。建立了芥子气中毒患者尿液中7种游离代谢产物的两步固相萃取/超高效液相色谱-串联质谱(SPE/UPLC-MS/MS)同时定量方法,检出限为5 pg/mL~1 ng/mL,定量下限为10pg/mL~5 ng/mL;结合前期建立的4种游离碱基加合物的同位素稀释-UPLC-MS/MS定量方法,对1例疑似芥子气中毒人员尿液中可能赋存的生物标记物进行了全筛查分析。尿液中共检出3类10种生物标记物,包括首次报道的游离代谢产物芥子亚砜,可确证患者为芥子气中毒;除硫二甘醇外,标记物含量均在暴露后3~4 d达到峰值,随后降低,至7 d仍可检出,其中谷胱甘肽加合物的β裂解产物含量相对较高,可作为芥子气中毒早期诊断与疗效评估的重要指标。

Analysis of Urinary Biomarkers for Diagnosis of Patients Exposed to Sulfur Mustard

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 WarⅡ.As a bifunctional alkylating agent,SM reacts rapidly with numerous nucleophiles,e.g.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 72.0%-117%and matrix effects of 85.3%-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 ofⅡ°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-βlyase 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-βlyase metabolites are only produced upon exposure to SM.Considering that the concentration of GSH adduct-βlyase 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.