Analysis of long-lived sulfur mustard-human hemoglobin adducts in blood samples by red blood cells lysis and on-line coupling of digestion on an immobilized-trypsin reactor with liquid chromatography-tandem mass spectrometry

As a highly alkylating chemical warfare agent, sulfur mustard reacts with blood proteins such as hemoglobin to form long-lived hydroxyethylthioethyl adducts that can be used as biomarkers of exposure. An optimized method was developed for the extraction of hemoglobin from blood samples. This procedure, involving the hemolysis of the red blood cells by freezing at -80 degrees C in two cycles of 1 h, followed by the purification of the lysate by ultrafiltration on 100 and 50 kDa cutoffcentrifugal devices, was then applied to the extraction of hemoglobin from blood samples spiked with sulfur mustard at different concentrations (ranging from 0.014 to 28 mu g mL(-1)). More than 75% of the protein was extracted from the blood samples and the method demonstrated a satisfying repeatability, with a RSD of 12.6%. The extracted hemoglobin was then digested on-line on a laboratory-made trypsin IMER coupled with the analysis by liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) of the resulting alkylated peptides. A linear response was observed for the 13 alkylated peptides targeted for the sulfur mustard concentration range studied, with RSD down to 0.1% for the digestion repeatabilty. The limit of quantification of the method was estimated to be 0.4 ng mL(-1) as concentration of exposure to sulfur mustard in whole blood. Finally, a variation of the alkylation rates of hemoglobin was observed between the biological matrix and pure sample, since the preferential adduction sites in blood were the residues beta-His(97) and beta-Val(98), both located on the alkylated peptide beta-T-11, while for purified hemoglobin in water, the residue beta-His(77) was the main adduction site. Thus, even though blood samples require an additional sample treatment step compared to pure standards, carrying out the study with whole blood allowed to collect information that are more representative of the phenomena occurring in the organism upon exposure to sulfur mustard. (C) 2022 Elsevier B.V. All rights reserved.

Automated summary

An optimized method for extracting hemoglobin from blood samples spiked with sulfur mustard was developed and showed satisfactory repeatability. The study conducted with whole blood provided more representative information of the phenomena occurring in the organism upon exposure to sulfur mustard.