Medical Aspects of Chemical Warfare (2008) - The Black Vault

Medical Aspects of Chemical Warfarethe Iran-Iraq war, a small number of samples from individualsexposed to sulfur mustard in laboratory andfield situations were collected, stored, and analyzedusing more recently developed methods.Some of sulfur mustard’s physical properties andbiochemical reactions are addressed in this volume(see Chapter 8, Vesicants) and have been reviewedextensively elsewhere. 78 Of primary importance inthe development of assays for sulfur mustard is theformation of a highly reactive sulfonium ion that isproduced following cyclization of an ethylene group ofsulfur mustard. The sulfonium ion readily reacts withnucleophiles, such as water, or combines with a varietyof nucleophilic sites in macromolecules. The resultingchemical reactions are able to produce a number of freemetabolites and stable adducts that can be exploitedfor analysis in blood, urine, and tissue samples. 7,79,80This section focuses primarily on metabolites thathave been identified in biomedical samples from sulfurmustard casualties.Before the early 1990s analytical methods for verifyingexposure to sulfur mustard consisted of assaysfor the unmetabolized compound or the hydrolysisproduct TDG. Since 1995 a number of significantadvances have occurred. Many new metabolites havebeen identified from specimens of sulfur-mustard–exposedindividuals, and instrument advances, suchas the ability to interface LC with MS and the use oftandem MS, have resulted in significant increases intest sensitivity and selectivity. Most newer methodsof verifying exposure to sulfur mustard require extensivesample processing prior to introduction intothe analytical system. The use of MS has enabled theincorporation of isotopically labeled forms of analytesfor use as internal standards during the earliest stagesof sample preparation. This has resulted in greaterreproducibility of assays and made them more amenableto quantitative analysis. Although the laboratorymethods presented in this section are not consideredroutine or standard, the efforts by a small number oflaboratories worldwide that are active in this area ofresearch have made the methods more attainable to awider range of laboratories.Analysis of Urine SamplesThere are currently five urinary metabolites of primaryinterest in verifying exposure to sulfur mustard.Two of the metabolites, TDG and thiodiglycol sulfoxide(TDG-sulfoxide), are derived from chemical hydrolysisreactions (Figure 22-6). The other three productsare formed following sulfur mustard’s reaction withglutathione (GSH). Each of the five analytes has beenidentified in the urine of sulfur-mustard–exposedindividuals.Analytical MethodsEfforts to analyze specific biomarkers of sulfur mustardexposure in urine samples prior to 1995 targetedeither unmetabolized sulfur mustard or TDG (Table22-6). Vycudilik prepared urine samples by initiallysaturating them with sodium chloride followed byorganic extraction using diethylether. The organic portionwas evaporated under nitrogen and reconstitutedwith methylene chloride. After the addition of silicagel, the methylene chloride was evaporated undernitrogen, reconstituted with solvent, and analyzedusing GC-MS. 81 Vycudilik later modified the methodto isolate possible conjugates of sulfur mustard. 82 Theprimary difference in the latter study was the additionof strong acid to the urine samples. Urine sampleswere mixed with an equal amount of concentratedhydrochloric acid and saturated with sodium chloride.Using steam distillation, the distillate was collectedin ether. Following sodium chloride saturation ofthe aqueous layer, the ether layer was dried and theresidue dissolved in methylene chloride and silica gel.Samples were analyzed using GC and high-resolutionMS. Vycudilik reported that the methods could not distinguishbetween sulfur mustard and its hydroxyethylmetabolites present in the urine samples. 82Wils et al treated urine with concentrated hydrochloricacid to convert TDG back to sulfur mustard. 83,84Two methods were reported, only the later methodis described here. The urine was passed through twoC18 solid phase extraction cartridges. Next, a solutionCH 2 CH 2 ClCH 2 CH 2 OHCH 2 CH 2 OHO CH 2 CH 2 OHSSOSSCH 2 CH 2 ClSulfur Mustard (HD)CH 2 CH 2 OHThiodiglycol (TDG)CH 2 CH 2 OHTDG sulfoxideO CH 2 CH 2 OHTDG sulfoneFig. 22-6. Hydrolysis of sulfur mustard to produce thiodiglycol, followed by oxidation reactions.HD: sulfur mustardTDG: thiodiglycol708