Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts
We present the forensic analyses of plasma samples of human victims exposed to sulfur mustard (SM) in a crisis region in the Middle East in 2015. A few hours after exposure, poisoned persons showed typical signs and symptoms of percutaneous SM exposure including erythema and later on blisters and hardly healing skin wounds. Blood samples were collected 15 days after poisoning to be analyzed for the presence of long-lived protein-adduct biomarkers to verify SM poisoning. We applied a novel bioanalytical toolbox targeting four human serum albumin-derived biomarkers that were made accessible after plasma proteolysis. These adducts contained the SM-specific hydroxyethylthioethyl moiety either bound to the thiol group of a cysteine residue (C34*) or to the side-chain carboxylic group of a glutamic acid residue (E230*). Peptide biomarkers were produced from plasma of the victims using proteinase K (C34*PF), pronase (C34*P) and pepsin (AE230*VSKL and LQQC34*PFEDHVKL) for enzymatic protein cleavage. Separation and detection were carried out by selective micro-liquid chromatography–electrospray ionization high-resolution tandem mass spectrometry (µLC–ESI MS/HR MS). In addition to this site-specific adduct detection, a general approach after alkaline hydrolysis of the plasma protein fraction was applied. Liberated thiodiglycol (TDG) was derivatized with heptafluorobutyric anhydride and detected by gas chromatography–electron ionization mass spectrometry (GC–EI MS). The different bioanalytical methods yielded congruent results confirming SM poisoning for all patients who showed clinical signs and symptoms. This is the first time that real cases of SM poisoning were confirmed and presented by such a broad compilation of protein-derived biomarkers.
KeywordsBiomarker Hydroxyethylthioethyl moiety LC–ESI MS/HRMS Protein adducts Vesicant
Chemical warfare agents
Chemical weapons convention
Triple deuterated atropine
- GC–EI MS
Gas chromatography–electron ionization mass spectrometry
Human serum albumin
- µLC–ESI MS/HRMS
Micro-liquid chromatography–electrospray ionization high-resolution tandem mass spectrometry
Selected ion monitoring
We thank Erwin Wagner (InstPharmToxBw) for his excellent technical assistance.
Compliances with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Medically examined patients consented to subsequent blood sample analysis and publication of photographic pictures.
- OPCW (2015). https://www.opcw.org/sites/default/files/documents/2018/11/s-1320-2015_e_.pdf. Accessed May 2019
- OPCW (2017a) OPCW fact-finding mission confirms use of chemical weapons in Khan Shaykhun on 4 April 2017. https://www.opcw.org/media-centre/news/2017/06/opcw-fact-finding-mission-confirms-use-chemical-weapons-khan-shaykhun-4. Accessed 15 Dec 2018
- OPCW (2017b) Statement by H.E. Ambassador Kenneth D. ward permanent representative of the United States of America to the OPCW at the eighty-sixth session of the executive council, https://www.opcw.org/sites/default/files/documents/EC/86/en/ec86nat06_e_.pdf. Accessed 15 Dec 2018
- OPCW (2018a) In response to persistent allegations of chemical weapon attacks in Syria, the OPCW Fact Finding Mission (FFM) was set up in 2014 “to establish facts surrounding allegations of the use of toxic chemicals, reportedly chlorine, for hostile purposes in the Syrian Arab Republic”. https://www.opcw.org/fact-finding-mission. Accessed 15 Dec 2018
- OPCW (2018b) OPCW issues report on technical assistance requested by the United Kingdom, April 12, 2018. https://www.opcw.org/media-centre/news/2018/04/opcw-issues-report-technical-assistance-requested-united-kingdom. Accessed 15 Dec 2018
- OPCW (2018c) Work instruction for the reporting of the results of the OPCW biomedical proficiency tests, quality management system document no. QDOC/LAB/WI/BioPT04. Accessed 4 Jan 2018Google Scholar