In Vitro Adduct Formation of Phosgene with Albumin and Hemoglobin in Human Blood

The development of procedures for retrospective detection and quantitation of exposure to phosgene, based on adducts to hemoglobin and albumin, is described. Upon incubation of human blood with [(14)C]phosgene (0-750 microM), a significant part of radioactivity (0-13%) became associated with globin and albumin. Upon Pronase digestion of globin, one of the adducts was identified as the pentapeptide O=C-(V-L)-S-P-A, representing amino acid residues 1-5 of alpha-globin, with a hydantoin function between N-terminal valine and leucine. Micro-LC/tandem MS analyses of tryptic as well as V8 protease digests identified one of the adducts to albumin as a urea resulting from intramolecular bridging of lysine residues 195 and 199. The adducted tryptic fragment could be sensitively analyzed by means of micro-LC/tandem MS with multiple-reaction monitoring (MRM), enabling the detection in human blood of an in vitro exposure level of >/=1 microM phosgene.