title: Applying human and pig hepatic in vitro experiments for sulfur mustard study: screening and identification of metabolites by liquid chromatography/tandem mass spectrometry.
abstract: BACKGROUND
  Sulfur mustard is a chemical warfare agent (CWA) with high toxicity and complex metabolism. This study aimed at identification of new metabolic biomarkers for sulfur mustard using in in vitro exposures and various mass spectrometric techniques.
  
  
  METHODS
  Human and pig liver subcellular fractions were used as biocatalysts. Metabolites were screened by liquid chromatography and tandem mass spectrometry (LC/MS/MS) using positive electrospray ionization (ESI). For structural identification, product ion scans (MS/MS, MS(3) ) and accurate mass measurements using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) were acquired.
  
  
  RESULTS
  Sulfur mustard is metabolized in vitro by S-oxidation and glutathione (GSH) conjugations. One S-oxidized metabolite, bis(2-chloroethyl) sulfoxide (m/z 175), was formed in both species only when liver microsomes were present in incubations, and it was the main metabolite if GSH was not added into the reaction mixture. However, conjugation with GSH was found to be a spontaneous reaction in physiological pH and buffered solution. Three GSH conjugates of sulfur mustard were detected and identified, among which two were novel; 2-((2-(S-glutathionyl)ethyl)thio)ethanol (m/z 412) and 2-((2-(S-glutathionyl)ethyl)thio)ethyl phosphate (m/z 492).
  
  
  CONCLUSIONS
  To our knowledge, this was the first time that S-oxidized metabolites and GSH conjugates of sulfur mustard have been detected and identified from human samples in vitro by LC/MS/MS. The usefulness of the GSH conjugates to serve as biomarkers for sulfur mustard exposure in human samples requires further studies.
authors.affiliation: VERIFIN, Finnish Institute for Verification of the Chemical Weapons Convention, Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014, Finland.
authors.forename: Mia
authors.initials: M
authors.lastname: Halme
authors.affiliation: Research and Development, Centre for Military Medicine, Finnish Defence Forces, P.O. Box 50, FI-00301, Helsinki, Finland.|School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland.
authors.forename: Maija
authors.initials: M
authors.lastname: Pesonen
authors.affiliation: VERIFIN, Finnish Institute for Verification of the Chemical Weapons Convention, Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014, Finland.
authors.forename: Ullastiina
authors.initials: U
authors.lastname: Hakala
authors.affiliation: School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Pasanen
authors.affiliation: School of Pharmacy/Toxicology, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211, Kuopio, Finland.
authors.forename: Kirsi
authors.initials: K
authors.lastname: Vähäkangas
authors.affiliation: VERIFIN, Finnish Institute for Verification of the Chemical Weapons Convention, Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014, Finland.
authors.forename: Paula
authors.initials: P
authors.lastname: Vanninen
mesh_terms: D000818:Animals; D002619:Chemical Warfare Agents / Q000378:metabolism* / Q000633:toxicity; D002853:Chromatography, Liquid; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D009151:Mustard Gas / Q000378:metabolism* / Q000633:toxicity; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization; D013454:Sulfoxides / Q000378:metabolism; D013552:Swine; D053719:Tandem Mass Spectrometry
chemical_list: D002619:Chemical Warfare Agents; D013454:Sulfoxides; D005978:Glutathione; D009151:Mustard Gas