Synthesis and mass spectrometric identification of the major amino acid adducts formed between sulphur mustard and haemoglobin in human blood.
As part of a program to develop methods for the verification of alleged 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-glutamate and N1- and N3-(2-hydroxyethylthioethyl)-L-histidine. Suitable derivatization methods for GC/MS analysis were developed for these adducts as well as for the cysteine and the N-terminal valine adduct. Incubation of human blood with [35S]sulfur mustard in vitro followed by acidic hydrolysis of isolated globin and derivatization with Fmoc-Cl afforded three radioactive peaks upon HPLC analysis, one of which coeluted with the synthetic Fmoc derivative of N1/N3-(2-hydroxyethylthioethyl)-L-histidine. After pronase digestion of globin the adducts of histidine, glutamic acid, aspartic acid, cysteine and N-terminal valine could be tentatively identified and quantitated. Final identification was obtained from GC/MS analysis. The most abundant adduct, N1/N3-(2-hydroxyethylthioethyl)-L-histidine, could not be sensitively analysed by GC/MS. A convenient LC-tandem MS procedure was developed for this compound, enabling the detection of exposure of human blood to 10 microM sulphur mustard in vitro.
Noort
D
D
Department of Chemical Toxicology, TNO Prins Maurits Laboratory, Rijswijk, The Netherlands.
Hulst
A G
AG
Trap
H C
HC
de Jong
L P
LP
Benschop
H P
HP
D000596:Amino Acids / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*; D002619:Chemical Warfare Agents / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D005914:Globins / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000737:chemistry; D007202:Indicators and Reagents; D009151:Mustard Gas / Q000737:chemistry*; D013462:Sulfur Radioisotopes
D000596:Amino Acids; D002619:Chemical Warfare Agents; D006454:Hemoglobins; D006893:Hydroxyeicosatetraenoic Acids; D007202:Indicators and Reagents; D013462:Sulfur Radioisotopes; D005914:Globins; D009151:Mustard Gas