SYNTHESIS AND MASS-SPECTROMETRIC IDENTIFICATION OF THE MAJOR AMINO-ACID ADDUCTS FORMED BETWEEN SULFUR MUSTARD AND HEMOGLOBIN IN HUMAN BLOOD

As part of a program to develop methods for the verification of allege d exposure to sulphur mustard, we synthesized and characterized three amino acid adducts presumably formed by alkylation of haemoglobin: 4-( 2-hydroxyethylthioethyl)-L-aspartate, 5-(2-hydroxyethylthioethyl)-L-gl utamate and N1- and N3-(2-hydroxyethylthioethyl)-L-histidine. Suitable derivatization methods for GC/MS analysis were developed for these ad ducts as well as for the cysteine and the N-terminal valine adduct. In cubation of human blood with [S-35]sulphur mustard in vitro followed b y acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three major radioactive peaks upon HPLC analysis, one of whi ch coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethy lthioethyl)-L-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal v aline could be tentatively identified and quantitated. Final identific ation was obtained from GC/MS analysis. The most abundant adduct, N1/N 3-(2-hydroxyethylthioethyl)-L-histidine, could not be sensitively anal ysed by GC/MS. A convenient LC-tandem MS procedure was developed for t his compound, enabling the detection of exposure of human blood to 10 mu M sulphur mustard in vitro.