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

• Analyze using GC-MS with methane negative ion chemical ionization. 2 (Exhibit 22-3 continues)Medical Aspects of Chemical WarfareEXHIBIT 22-3SAMPLE PREPARATION METHODS FOR THE Gas Chromatographic-Mass SpectrometricANALYSIS OF SULFUR MUSTARD ADDUCTS TO BLOOD BIOMOLECULESProcedure of Fidder et al for sulfur mustard adduct to N-terminal valine of hemoglobin*:• Isolate globin from blood using following procedure:- Centrifuge blood and remove plasma layer.- Wash RBCs with saline solution.- Lyse RBCs with water and place solution into ice water bath.- Centrifuge and transfer supernatant into tube containing HCl/acetone.- Wash the precipitate with HCl/acetone, acetone, and ether.- Dry precipitate.• Mix isolated globin from blood sample with internal standard (globin isolated from blood that was previouslyexposed to deuterated sulfur mustard).• Dissolve globin in formamide.• Add pyridine and pentafluorophenyl isothiocyanate to solution.• Incubate solution at 60°C for 2 hours.• Add toluene, mix, centrifuge, and freeze samples in liquid nitrogen.• Remove toluene layer and wash with water, aqueous Na 2CO 3, and water.• Dry toluene with MgSO 4, evaporate to dryness, and dissolve in toluene.• Filter solution through a preconditioned Florisil solid phase extraction cartridge.• Wash cartridge with dichloromethane.• Elute with methanol/dichloromethane.• Evaporate to dryness and dissolve in toluene.• Add heptafluorobutyryl imidazole and heat solution.• After cooling, wash with water, aqueous Na 2CO 3, and water.• Dry toluene with MgSO 4and concentrate solution.• Analyze using GC-MS with methane negative ion chemical ionization. 1Procedure of Capacio et al for sulfur mustard adducts to aspartic and glutamic acid residues of blood proteins:• Precipitate blood proteins:- For plasma samples, use acetone.- For whole blood or RBCs, use HCl/acetone.• Centrifuge solution and discard supernatant.• Wash protein pellet with acetone and ether.• Centrifuge solution and discard supernatant.• Dry protein at room temperature.• Add dried protein to NaOH solution.• Heat solution at 70°C for 1.5 hours.• Neutralize solution with HCl and dry with sodium sulfate.• Add ethyl acetate, mix, and remove ethyl acetate layer.• Add internal standard (deuterated thiodiglycol) to ethyl acetate and dry with sodium sulfate.• Add pyridine and pentafluorobenzoyl chloride.• After 10 minutes, add water and sodium bicarbonate.• Remove ethyl acetate layer and dry with sodium sulfate.• Pass ethyl acetate through a preconditioned silica solid-phase–extraction cartridge and collect the filteredsolution.• Pass additional ethyl acetate through cartridge, collect, and combine the two fractions.724