Sulfur mustards analytical methods

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. 

This paper will review the known metabolic pathways of CW agents, excretion profiles where these have been measured, and methods for the analysis of metabolites in urine or blood. Examples are provided of detection in cases of human exposure. The review focuses mainly on sulfur mustard and nerve agents that represent the greatest global CW threat, and for which most analytical methods have been developed. 

Analytical methods have been reported for unchanged agent and six of the urinary excretion products described above. These are TDG, TDGO, the bis A-acetylcysteine conjugate (1), two 3-lyase metabolites (2) and (3), and the guanine adduct (6). These methods have been applied to animal and/or human exposures to sulfur mustard. 

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. 

Marianne Roller was born in Bavaria, Germany, in 1958. She did her diploma in chemistry at the Technical University of Munich in 1984 and received her Ph.D. with magna cum laude under Prof. Ivar Ugi in 1989. During the following years, she specialized on instrumental analytics. Since 1995, she has been responsible for the development of GC-MS and LC-MS methods for the detection of chemical warfare agents in biomedical samples (verification) at the German Forces. Sulfur mustard, whose homologues can be used as educts in the synthesis of sulfur macrocycles, is one of her objects of interest. 

Analytical Methods for Urine. Efforts to analyze for specific biomarkers in urine of sulfur mustard exposure prior to 1995 targeted either unmetabolized sulfur mustard or TDG. Vycudihk (1985) prepared urine samples using organic extraction with diethylether and analyzed them using GC-MS. The method was later modified (Vycudihk, 1987) by the addition of a strong acid to the urine samples to isolate possible conjugates of sulfur mustard. Vycudihk indicated that the methods could not distinguish between sulftir mustard and its hydroxyethyl metabohtes that were present in the urine samples. 

The final sulfur mustard urinary biomarker to be discussed is also a reaction product of sulfur mustard with glutathione 1,1 -sulfonyl-bis-[2-S-(V-acetylcystein T)ethane. Using solid-phase extraction for sample cleanup and analyte concentration followed by analysis with negative ion electrospray LC-MS-MS, Read and Black (2004b) were able to achieve detection limits of 0.5-1.0 ng/mL. Methods for the analysis of urine samples for sulfur mustard verification are summarized in Table 19.5. 

Assays for several other potential urinary analytes have been developed, but the analytes have yet to be confirmed in human exposed samples. N7-(2-hydroxyethylthioethyl) guanine is a breakdown product from alkylated DNA that has been observed in animal studies. Fidder et al. (1996a) developed both a GC-MS method that requires derivatization of the analyte and also a LC-MS-MS method that can analyze the compound directly. Other possible urinary analytes are an imidazole derivative formed from the reaction of sulfur mustard with protein histidine residues (Sandelowsky et al., 1992) and sulfur mustard adducts to metallothionien (Price et al., 2000). 

Analytical Methods Used for the Verification of Exposure to Sulfur Mustard in Biomedical Samples... 

Analytical Methods for Blood. Methods to measure sulfur mustard adducts to DNA in white blood cells have been developed using LC with fluorescence detection (Ludlum et al., 1994) and using an enzyme-linked immunosorbent assay (ELISA) (van der Schans et al., 1994, 2004). The DNA adduct that appears to be the most abundant results from sulfur mustard attachment to the N7 position of deoxyguaninosine (Fidder et al., 1994). The immunochemical method developed by van der Schans used monoclonal antibodies that were raised against N7-(2-hydroxyethylthioethyl)-guanosine-5 -phosphate. 

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. 

A recent study published in the Chinese Journal of Instrumental Analysis, Fenxi Ceshi Xuebao, showed a detection limit of 500 ng of Sulfur Mustard (HD) by using accelerated solvent extraction-gas chromatography (ASE-GC) coupled with a flame photometric detector (EPD) in the sulfur mode, in soil. In this case, the study showed evidence that ASE results in better recoveries and sensitivity than liquid solid extraction (LSE) [50]. In 1996, a paper was published on a method for the analysis of Lewisite through derivatization of the compound before introduction into a gas chromatograph. In order to simplify the derivatization process, a tube packed with absorbent was used for collection of airborne vapors. If a positive response occurs when screening analytes using a GC coupled with an FPD, then the same sample can be analysed using a GC equipped with an AED for confirmation based on the elemental response to arsenic (in the case of Lewisite) and sulfur (in the case of Sulfur Mustard) within the appropriate GC retention time window [54]. 

A number of modifications/improvements to methods for the analysis of metabolites of sulfur and nitrogen mustards, and hydrolysis products of nerve agents, have been reported in a special issue of the Journal of Analytical Toxicology, 28 (5) (2004) pp. 305-392. 

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