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

Medical DiagnosticsExhibit 22-3 continued*The lower limit of detection for the assay was determined using in vitro exposures of sulfur mustard in human whole blood and wasdetermined to be equivalent to a 100 nM exposure level. 1,3 Following the administration of a single dose of sulfur mustard to a marmoset(4.1 mg/kg; intravenous), the valine adduct was still detected in blood taken 94 days later. 4 Intact hemoglobin with the sulfurmustard adducts attached have been examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,but to date the technique has only been utilized for in vitro experiments at relatively high concentrations of sulfur mustard. 5GC: gas chromatographyHCl: hydrochloric acidMS: mass spectrometryRBC: red blood cellData sources: (1) Fidder A, Noort D, de Jong AL, Trap HC, de Jong LPA, Benschop HP. Monitoring of in vitro and in vivo exposureto sulfur mustard by GC/MS determination of the N-terminal valine adduct in hemoglobin after a modified Edman degradation.Chem Res Toxicol. 1996;9:788–792. (2) Capacio BR, Smith JR, DeLion MT, et al. Monitoring sulfur mustard exposure by gas chromatography-massspectrometry analysis of thiodiglycol cleaved from blood proteins. J Anal Toxicol. 2004;28:306–310. (3) Noort D, Fidder A,Benschop HP, de Jong LP, Smith JR. Procedure for monitoring exposure to sulfur mustard based on modified edman degradation ofglobin. J Anal Toxicol. 2004;28:311–315. (4) Noort D, Benschop HP, Black RM. Biomonitoring of exposure to chemical warfare agents: areview. Tox Appl Pharmacol. 2002;184:116–126. (5) Price EO, Smith JR, Clark CR, Schlager JJ, Shih ML. MALDI-ToF/MS as a diagnostictool for the confirmation sulfur mustard exposure. J Appl Toxicol. 2000;20(suppl 1):S193–S197.blood samples that are collected and made availablefor verifying sulfur mustard exposure are from a singletime point after the exposure. In this instance, the patientwith more severe injuries (patient D1, see above)had blood and urine collected almost daily for the first10 days after exposure and then again on days 29, 35,and 42. The incident also provided an opportunityto examine blood and urine metabolite levels fromindividuals with very different levels of injury. PatientD1 had extensive vesication of the arm and leg, whilepatient D2 developed only a single small blister. (Theurinary metabolite results were detailed earlier in thissection.) As expected, the observed concentrations ofboth urine hydrolysis metabolites and GSH reactionproducts were much greater in patient D1. Sulfurmustard metabolite concentrations in the blood werealso much greater in patient D1. Blood metaboliteswere assayed using two different methods. The firstassay targeted the sulfur mustard adduct to cysteine-34 of albumin using pronase digestion of the proteinfollowed by LC-MS-MS analysis. 116 Based on in-vitroexposures to sulfur mustard in human whole blood,concentrations of albumin adducts found in the plasmaof patient D1 were 350 nM on day 2 after the exposureand had decreased by 74% (90 nM) on day 42. 105 Therate of decrease over that time was consistent withthe reported half-life of human albumin of 21 days.Albumin adduct concentrations for patient D2 over thesample collection period of 2 to 7 days after exposureremained stable and ranged between 16 and 18 nM.The second assay targeted plasma protein adducts bycleaving the adduct with base, followed by analyzingthe derivatized adduct using negative ion chemicalionization GC-MS. 118 This was the first reported use ofthis assay in the verification of a human exposure tosulfur mustard. Concentrations of the plasma proteinadducts were 97 nM on day 2 and decreased by 76% (23nM) by day 42, based on in-vitro exposures to sulfurmustard in human plasma instead of whole blood. 122The assay could not detect plasma protein adducts inpatient D2. The assay was modified slightly to lowerthe reported lower limit of detection of 25 nM, butthe limited amount of plasma received did not permitabcprotein COOH + S(CH 2 CH 2 Cl) 2proteinCOOCH 2 CH 2 SCH 2 CH 2 OHNaOHprotein + S(CH 2 CH 2 OH) 22 C 6 F 5 COClS(CH 2 CH 2 OCOC 6 F 5 ) 2Fig. 22-11. Analytical approach of Capacio et al. (a) Reactionof protein carboxylic acid groups with sulfur mustard. (b)Hydrolysis of the ester groups to release thiodiglycol. (c)Derivatization of thiodiglycol using pentafluorobenzoylchloride.Data source: Capacio BR, Smith JR, DeLion MT, et al. Monitoringsulfur mustard exposure by gas chromatography-massspectrometry analysis of thiodiglycol cleaved from bloodproteins. J Anal Toxicol. 2004;28:306–310.725