title: Optimized verification method for detection of an albumin-sulfur mustard adduct at Cys(34) using a hybrid quadrupole time-of-flight tandem mass spectrometer after direct plasma proteolysis.
abstract: The vesicant sulfur mustard (SM) is a banned chemical warfare agent that is controlled by the Organisation for the Prohibition of Chemical Weapons (OPCW). Bioanalytical procedures are mandatory for proving an alleged use and incorporation of SM into the body. We herein present the development and application of a novel optimized procedure suitable for qualitative verification analysis of plasma targeting the SM-adduct of human serum albumin (HSA) alkylated at the cysteine(34) residue. Diluted human plasma is directly mixed with pronase in an ultrafiltration device (10kDa cut-off) for proteolysis (4h, 37°C). Following ultrafiltration the filtrate is diluted and analyzed by microbore liquid chromatography-electrospray ionization high resolution tandem-mass spectrometry (μLC-ESI HR MS/MS) targeting the alkylated dipeptide hydroxyethylthioethyl-CysPro (HETE-CP). A hybrid quadrupole time-of-flight mass spectrometer provided high mass spectrometric resolution in the MS/MS mode enabling highest selectivity and sensitivity (lower limit of detection corresponding to 9.8nM SM in plasma). Kinetics of HETE-CP formation from heparin-, citrate-, and EDTA-plasma as well as serum are presented and the influence of different EDTA and pronase concentrations was characterized. The novel procedure was applied to plasma samples provided by the OPCW as well as to patientś plasma derived from real cases of SM-poisoning.
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, D-80937 Munich, Germany. Electronic address: HaraldJohn@bundeswehr.org.
authors.forename: Harald
authors.initials: H
authors.lastname: John
authors.affiliation: Department of Chemistry, Ludwig-Maximilians University of Munich, Butenandtstrasse 5-13, D-81377 Munich, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Siegert
authors.affiliation: Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, D-12489 Berlin, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Gandor
authors.affiliation: Hamm-Lippstadt University of Applied Sciences, Biomedical Engineering, Marker Allee 76-78, D-59063 Hamm, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Gawlik
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, D-80937 Munich, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kranawetvogl
authors.affiliation: Department of Chemistry, Ludwig-Maximilians University of Munich, Butenandtstrasse 5-13, D-81377 Munich, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Karaghiosoff
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, D-80937 Munich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
mesh_terms: D002619:Chemical Warfare Agents / Q000378:metabolism* / Q000506:poisoning; D002853:Chromatography, Liquid; D003545:Cysteine; D006801:Humans; D007509:Irritants / Q000378:metabolism* / Q000633:toxicity; D007700:Kinetics; D009151:Mustard Gas / Q000378:metabolism* / Q000506:poisoning; D011041:Poisoning / Q000097:blood / Q000175:diagnosis; D011402:Pronase / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis; D012709:Serum Albumin / Q000378:metabolism*; D000075462:Serum Albumin, Human; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014462:Ultrafiltration
chemical_list: C422455:ALB protein, human; D002619:Chemical Warfare Agents; D007509:Irritants; D012709:Serum Albumin; D011402:Pronase; D003545:Cysteine; D009151:Mustard Gas; D000075462:Serum Albumin, Human
keywords: Albumin-adducts; High resolution mass spectrometry; Hydroxyethylthioethyl; Pronase; Verification analysis; Vesicant