Low level exposure to sulfur mustard: Development of a SOP for analysis of albumin adducts and of a system for non-invasive diagnosis on skin

The present research program aims at 1. Development of a mass spectrometric or fluorescence-based method for retrospective detection of exposure to low doses of sulfur mustard, based on improvement of analysis of the adducted tripeptide cysteineS-2-hydroxyethylthioethyl-proline-phenylalanine S-HETECys-Pro-Phe in albumin and of adducts to histidine in hemoglobin and albumin. 2. Development of immunochemical assays for quantitation of adduct levels of sulfur mustard to hemoglobin, albumin and keratin. The procedure for isolation of albumin from human blood could be substantially shortened by using affinity chromatography. The lowest detectable exposure level was improved by one order of magnitude 1 nM by work-up of 20 mg amounts of albumin. Furthermore, the use of an internal standard has been worked out. After i.v. administration of sulfur mustard to the marmoset, the specific S-HETECys-Pro-Phe adduct could be analyzed after pronase digestion of albumin, for at least 28 days after the challenge. A work-up procedure was developed for rapid isolation 6f the histidine adducts from amino acid mixtures resulting from acidic hydrolysis of globin or albumin. Derivatization of the histidine adducts with trifluoroacetic acid anhydride for GC-MS analysis resulted in the formation of a tristrifluoroacetyl derivative with favorable mass spectrometric properties. For detection with laser-induced fluorescence, various derivatization methods have been explored and several fluorescent derivatives of the histidine adducts have been prepared. An immunoslotblot assay was developed for detection of sulfur mustard adducts to keratin. The lower detection limit of the immunoslotblot assay for detection of sulfur mustard adducts to keratin was at 25 fmol adducted sulfur mustard using 0.5 mug keratinslot, corresponding to an adduct level of 1 sulfur mustard adduct5 x lOsub 7 unadducted amino acids.