Low level exposure to sulfur mustard: Development of a SOP for analysis of albumin adducts

The need for retrospective detection procedures for exposure to low levels of chemical warfare agents has been urgently illustrated by the conflicts in the Gulf Area. Furthermore, in the case of a terrorist attack with CWA, rapid and reliable diagnosis of the exposure is essential. The present research aims at development of a mass spectrometric method for retrospective detection of exposure to low doses of sulfur mustard, based on improvement of analysis of an adducted tripeptide in albumin. The albumin assay is based on our previous finding that upon pronase treatment of sulfur mustard alkylated albumin, a tripeptide Cysteine(S-2-hydroxyethylthioethyl)-Proline-Phenylalanine ((S-HETE)Cys-Pro-Phe) results which has favorable mass spectrometric properties. The procedure for isolation of albumin from human blood could be substantially shortened by using affinity chromatography. The entire procedure, i.e., albumin isolation, pronase digestion and mass spectrometric analysis can be performed within 3 h. After exposure of rats (0.3 mg/kg, i.v.), the tripeptide adduct (S-HETE)Cys-Pro-Tyr could be determined until 7 days after the exposure; the observed half-life time of sulfur mustard – modified rat albumin was 2 days, which is in good correspondence with literature values for native rat albumin. In the corresponding globin samples of these animals, the N-terminal valine adduct could still be determined after 28 days after the exposure. Remarkably, the maximum adduct level was reached after 2-3 days, implicating the presence of intact sulfur mustard in the animal during the first 2-3 days after the exposure. A tentative SOP has been drafted, which has been demonstrated to U.S. Army Medical Research Institute of Chemical Defense in a satisfactory way. The presented method has the potential to become a generic assay for diagnosis of exposure to a wide array of alkylating agents, which is highly relevant within the field of operational toxicology.