Human serum albumin-adduct biomarkers to prove human poisoning with methanethiol

Exposure to methanethiol (MT) presents a significant challenge in public healthcare and could be a concern in the context of terrorist attacks. Therefore, reliable verification procedures for MT intoxication are essential for forensic, toxicological, and clinical purposes. We developed and validated a bioanalytical method for the simultaneous detection and identification of biomarkers indicative of MT exposure. Neat human serum albumin (HSA) and human plasma were incubated with MT to form adducts, which served as references. Subsequently, HSA and human plasma were subjected to proteolysis using two proteases, resulting in the formation of disulfide adducts detected as adducts of the single amino acid cysteine (MT-Cys), the dipeptide cysteine-proline (MT-Cys³⁴Pro), and the tripeptides aspartic acid-isoleucine-cysteine (AspIleCys⁵¹⁴-MT) and cysteine-proline-phenylalanine (MT-Cys³⁴ProPhe). The adducts were analyzed using a sensitive ultra-performance liquid chromatography-quadrupole exactive orbitrap-high resolution mass spectrometry (UPLC-Q Exactive Orbitrap-HRMS) method operating in full scan mass spectrometry (Full MS) and parallel reaction monitoring (PRM) mode. Time- and concentration-dependent adduct formation during exposure was investigated. The limits of detection (LODs) for the adducts ranged from 20 ng/mL to 2 μg/mL, corresponding to the MT concentrations in plasma. Adducts at Cys³⁴ exhibited the lowest LOD (20 ng/mL MT in plasma), the fastest adduct formation (20 min), and superior stability in plasma at 37 °C. The applicability of the method was demonstrated by the successful detection of adducts in sample from MT-poisoned patient, establishing the method as a reliable bioanalytical procedure for forensic and toxicological analysis.