Evaluation of Free and Released Sulfur Mustard Diols in Human Blood and Urine Using Multi-ionization GC-QQQ and GC-Q-TOF.

Exposures to blister agents such as sulfur mustard (HD) have been documented in both military and civilian settings, underscoring the need for reliable clinical assays and biomarkers for confirming human exposure. Accordingly, the Organisation for the Prohibition of Chemical Weapons (OPCW) designates sulfur mustard hydrolysis products (diols) as biomarkers for exposure verification. Because confirmatory analyses in public health and forensic contexts often rely on qualitative identification rather than quantitation alone, we evaluated sensitive qualitative approaches for detecting the diol hydrolysis products of eight of the nine sulfur mustard analogs listed in Schedule 1.A.04 of the Chemical Weapons Convention (CWC) Annex on Chemicals. Human serum spiked with sulfur mustard analogs and plasma and urine spiked with corresponding diols underwent strong base treatment, solid-phase extraction (SPE), derivatization with N-heptafluorobutyrylimidazole (HFBI), and liquid-liquid extraction (LLE). The resulting diol bis-HFB derivatives were analyzed by gas chromatography-triple quadrupole mass spectrometry (GC-QQQ) using electron ionization (EI) and positive chemical ionization (PCI) with methane or ammonia reagent gases. Derivatized diols were detected at concentrations as low as 1.00 ng/mL across all matrices, with PCI-methane yielding signal-to-noise ratios (S/N) > 15. Gas chromatography-quadrupole time-of-flight (GC-Q-TOF) analysis provided high-resolution MS and MS/MS spectra to support molecular formula confirmation of precursor and product ions. By combining EI and PCI with high-resolution mass spectrometry for structural and molecular formula confirmation, this approach enables sensitive, matrix-independent detection, with PCI(CH4) providing the most effective balance between structural information and analytical performance for GC-MS/MS implementation.