title: Evidence of Sulfur Mustard Exposure in Human Plasma by LC-ESI-MS-MS Detection of the Albumin-Derived Alkylated HETE-CP Dipeptide and Chromatographic Investigation of Its Cis/Trans Isomerism.
abstract: Sulfur mustard (SM) is a chemical warfare agent that causes painful blisters and chemically modifies endogenous biomacromolecules by alkylation to hydroxyethylthioethyl (HETE) adducts representing valuable long-term markers for post-exposure analysis. The albumin adduct formed in human plasma in vitro (HETE bound to the side chain of cysteine 34) was isolated and cleaved by current lots of pronase primarily generating the internal modified dipeptide (HETE-cysteine-proline, HETE-CP) instead of the formerly reported HETE-CPF tripeptide. The analyte was detected by liquid chromatography-electrospray ionization tandem-mass spectrometry (LC-ESI-MS-MS). In principle, HETE-CP undergoes a dynamic on-column equilibrium of cis-trans isomerism thus requiring separation at 50°C to obtain one narrow peak. Accordingly, we developed both a novel longer lasting but more sensitive microbore (1 mm i.d., flow 30 µL/min, cycle time 60 min, LOD 50 nM) and a faster, less sensitive narrowbore (2.1 mm i.d., 200 µL/min, cycle time 16 min, LOD 100 nM, both on Atlantis T3 material at 50°C) LC-ESI-MS-MS method suitable for verification analysis. The corresponding tri- and tetrapeptide, Q(HETE)-CPF were monitored simultaneously. HETE-CP peak areas were directly proportional to SM concentrations added to plasma in vitro (0.05-100 µM). Albumin adducts formed by deuterated SM (d8-SM) served as internal standard.
authors.affiliation: Department of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Gandor
authors.affiliation: Hamm-Lippstadt University of Applied Sciences, Biomedical Engineering, Hamm, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Gawlik
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany haraldjohn@bundeswehr.org.
authors.forename: Harald
authors.initials: H
authors.lastname: John
mesh_terms: D000478:Alkylation; D015415:Biomarkers / Q000097:blood; D002619:Chemical Warfare Agents / Q000737:chemistry / Q000506:poisoning*; D002853:Chromatography, Liquid / Q000379:methods*; D004151:Dipeptides / Q000097:blood* / Q000737:chemistry; D006801:Humans; D006900:Hydroxylation; D057230:Limit of Detection; D009151:Mustard Gas / Q000737:chemistry / Q000506:poisoning*; D011041:Poisoning / Q000097:blood; D011402:Pronase / Q000737:chemistry; D011485:Protein Binding; D059748:Proteolysis; D012709:Serum Albumin / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015415:Biomarkers; D002619:Chemical Warfare Agents; D004151:Dipeptides; D012709:Serum Albumin; C028962:cysteinylproline; D011402:Pronase; D009151:Mustard Gas