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Sulfur mustard (SM, bis[2‐chloroethyl]‐sulfide) is a banned chemical warfare agent that was frequently used in recent years and led to numerous poisoned victims who developed painful erythema and blisters. Post‐exposure analysis of SM incorporation can be performed by the detection of human serum albumin (HSA)‐derived peptides. HSA alkylated by SM contains a hydroxyethylthioethyl (HETE)‐moiety bound to the cysteine residue C34 yielding the dipeptide biomarker C(‐HETE)P after pronase‐catalyzed proteolysis. We herein present a novel procedure for the selective precolumn nicotinylation of its N‐terminus using 1‐nicotinoyloxy‐succinimide. The reaction was carried out for 2 h at ambient temperature with a yield of 81%. The derivative NA‐C(‐HETE)P was analyzed by micro liquid chromatography‐electrospray ionization tandem‐mass spectrometry working in the selected reaction monitoring mode (μLC‐ESI MS/MS SRM). The derivative was shown to be stable in the autosampler at 15°C for at least 24 h. The single protonated precursor ion (m/z 428.1) was subjected to collision‐induced dissociation yielding product ions at m/z 116.1, m/z 137.0, and m/z 105.0 used for selective monitoring without any plasma‐derived interferences. NA‐C(‐HETE)P showed a mass spectrometric response superior to the non‐derivatized dipeptide thus yielding larger peak areas (factor 1.3 ± 0.2). The lower limit of identification corresponded to 80 nM SM spiked to plasma in vitro. The presented procedure was applied to real case plasma samples from 2015 collected in the Middle East confirming SM poisoning. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM) is a banned chemical warfare agent recently used in the Syrian Arab Republic conflict causing erythema and blisters characterized by complicated and delayed wound healing. For medical and legal reasons, the proof of exposure to SM is of high toxicological and forensic relevance. SM reacts with endogenous human serum albumin (HSA adducts) alkylating the thiol group of the cysteine residue C34, thus causing the addition of the hydroxyethylthioethyl (HETE) moiety. Following proteolysis with pronase, the biomarker dipeptide C(-HETE)P is produced. To expand the possibilities for verification of exposure, we herein introduce a novel biomarker produced from that alkylated dipeptide by derivatization with propionic anhydride inducing the selective propionylation of the N-terminus yielding PA-C(-HETE)P. Quantitative derivatization is carried out at room temperature in aqueous buffer within 10 s. The biomarker was found to be stable in the autosampler at 15 °C for at least 24 h, thus documenting its suitability even for larger sets of samples. Selective and sensitive detection is done by micro liquid chromatography-electrospray ionization tandem-mass spectrometry (μLC-ESI MS/MS) operating in the selected reaction monitoring (SRM) mode detecting product ions of the single protonated PA-C(-HETE)P (m/z 379.1) at m/z 116.1, m/z 137.0, and m/z 105.0. The lower limit of detection corresponds to 32 nM SM in plasma in vitro and the limit of identification to 160 nM. The applicability to real exposure scenarios was proven by analyzing samples from the Middle East confirming poisoning with SM. [ABSTRACT FROM AUTHOR]

Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys34 residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: "hydroxyethylthioethylthioethyl." Targeting both peptide markers from plasma, a micro liquid chromatography–electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (μLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys34. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys34 allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts. [ABSTRACT FROM AUTHOR]