ESI–MS/MS Characterisation of Phenolic Reaction Products of Nitrogen Mustards and N , N ‐Dialkylaminoethyl‐2‐Chlorides and Their Rapid Screening by LC–MS/MS in Neutral Loss Scan Mode

RATIONALE: Nitrogen mustards (HN1, HN2 and HN3) and N,N-dialkylaminoethyl-2-chlorides (where alkyl = methyl, ethyl, isopropyl, or propyl) contain reactive chloride bonds that react with phenol in environmental samples to form phenolic reaction products. Identifying these products is crucial for the Chemical Weapons Convention (CWC) verification, as they serve as markers for the presence of scheduled chemicals. METHODS: ions (1-13) were collected at varied collision energies. Isomers were separated via LC with an Agilent Eclipse AAA column and gradient elution. A targeted LC-MS/MS in neutral-loss scan (94 u) method was developed and validated for rapid screening of compounds 1-13 in a complex aqueous sample. RESULTS: Distinct product ions observed in the MS/MS spectra of 1-13 enabled structural characterisation and differentiation between isomers. Major fragmentation involved loss of a phenol (94 u) and C-C bond cleavages, which were influenced by the structure and size of the alkyl groups. A neutral loss scan LC-MS/MS method was developed for rapid screening of 1-13 at low levels (LOD ~ 10 ppb; LOQ ~ 30 ppb), even within complex matrices. CONCLUSIONS: Phenolic reaction products of nitrogen mustards and N,N-dialkylaminoethyl-2-chlorides were comprehensively characterised, including isomer differentiation, through their ESI-MS/MS product ion spectra. The validated LC-MS/MS neutral-loss scan method enabled efficient screening of these markers in complex samples. These LC-MS/MS methods are highly valuable for OPCW proficiency testing and CWC verification programs.