Sulfur mustard analytical procedures

Recently, sulfur mustard has been shown to alkylate a cysteine residue in human serum albumin (10). The site of alkylation was identified in a tryptic digest of albumin from blood exposed to [14C]sulfur mustard. A sensitive method for its analysis was developed based on Pronase digestion of alkylated albumin to the tripeptide S-[2-[(hydroxyethyl)thio]ethyl-Cys-Pro-Phe, and detection using micro-LC-MS-MS. In vitro exposure of human blood to > 10 nM sulfur mustard could be detected employing this method. The analytical procedure was successfully applied to albumin samples from Iranian casualties of the Iraq-Iran war. 

Wils et al. (25,26) previously reported an entirely different approach to TDG analysis. TDG in urine was converted back to sulfur mustard by treatment with concentrated HC1. The sample treatment is less straightforward than the methods described above, but analysis as sulfur mustard is facile. Urine, plus 2H8-TDG as internal standard, was cleaned up by elution through two C18 cartridges. Concentrated HC1 was added and the sample stirred and heated at 120 °C. Nitrogen was blown over the solution and sulfur mustard isolated from the headspace by adsorption onto Tenax-TA. The method was used to detect TDG in urine from casualties of CW attacks (see below). A disadvantage of this method is that it may convert metabolites other than TDG to sulfur mustard. This is supported by the detection of relatively high levels of analytes in urine from control subjects. Vycudilik (27) used a similar procedure, but recovered the mustard by steam distillation and extraction. 

The analytical procedure for S-[2-[(hydroxyethyl) thio]ethyl-Cys-Pro-Phe was successfully applied to blood samples from nine Iranian casualties of the Iraq-Iran war, all exhibiting skin injuries compatible with exposure to sulfur mustard. The blood samples were collected 8-9 days after the alleged exposure and stored at — 70 °C. The albumin adduct was detected in all cases, at levels estimated as corresponding to those after in vitro exposure of human blood to mustard concentrations ranging from 0.4-1.8 xM. 

Currently, as discussed by the authors, the analytical methods mentioned above, with the exception of cholinesterase inhibition measurements and immunoassays, cannot yet be easily performed in field laboratories. For that reason, the immunochemical procedures for simple analysis of exposure to sulfur mustard described in this chapter may be a significant contribution to the early detection of exposure to this warfare agent. 

Van der Schans, G.P., Mars-Groenendijk R.H, De Jong,L.P.A. Benschop, andNoort, D. (2004) Standard operating procedure for immunoslotblot assay for analysis of DNA/sulfur mustard adducts in human blood and skin. J. Analytical Toxicology 28, 316-319. 

The higher detection limits for sulfur mustard in the EDS tests at Porton Down reflect the GC/MS analytical procedure used by the Defence Evalnatiai and Research Agency in the United Kingdom, which, along with contracted commercial laboratories, performed the neutralent analyses. 

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