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Archives of Toxicology

, Volume 93, Issue 7, pp 1881–1891 | Cite as

Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts

  • Harald JohnEmail author
  • Marianne Koller
  • Franz Worek
  • Horst Thiermann
  • Markus Siegert
Toxicokinetics and Metabolism

Abstract

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.

Keywords

Biomarker Hydroxyethylthioethyl moiety LC–ESI MS/HRMS Protein adducts Vesicant 

Abbreviations

ACN

Acetonitrile

CWA

Chemical warfare agents

CWC

Chemical weapons convention

d3-Atr

Triple deuterated atropine

FA

Formic acid

GC–EI MS

Gas chromatography–electron ionization mass spectrometry

HSA

Human serum albumin

HETE

Hydroxyethylthioethyl

HFBA

Heptafluorobutyric anhydride

iPrOH

Iso-propanol

TDP

Thiodipropanol

µLC–ESI MS/HRMS

Micro-liquid chromatography–electrospray ionization high-resolution tandem mass spectrometry

SIM

Selected ion monitoring

SM

Sulfur mustard

SPE

Solid-phase extraction

TDG

Thiodiglycol

TOF

Time-of-flight

UF

Ultrafiltration

Notes

Acknowledgements

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.

Informed consent

Medically examined patients consented to subsequent blood sample analysis and publication of photographic pictures.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Harald John
    • 1
    Email author
  • Marianne Koller
    • 1
  • Franz Worek
    • 1
  • Horst Thiermann
    • 1
  • Markus Siegert
    • 1
    • 2
  1. 1.Bundeswehr Institute of Pharmacology and Toxicology (InstPharmToxBw)MunichGermany
  2. 2.Department of ChemistryHumboldt-Universität zu BerlinBerlinGermany

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