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Total Unknown Screening

MS(n) data interpretation

One of the challenges in analytical monitoring of the extremely toxic "V"-type chemical weapons [O-alkyl S-(2-dialkylamino) ethyl alkylphosphonothiolates] is to distinguish and identify compounds of similar structure. Mass spectrometry analysis of these compounds reveals mostly fragment/ product ions representing the amine-containing residue. H​ence, isomers or derivatives with the same amine residue exhibit similar mass spectral patterns in both classical electron-ionization mass spectrometry (EI/MS) and electrospray ionization-mass spectrometry (ESI/MS), leading to unavoidable ambiguity in the identification of the phosphonate moiety. Employing MS3 experiments enhanced the selectivity for those low-abundance product ions, and provided spectra indicative of the different phosphonate groups. Study of the fragmentation pathways, revealing some less-expected structures, was carried out and allowed the formulation of mechanistic rules and the determination of sets of ions typical of specific groups.​

Data processing and databases

Screening for unknown toxic xenobiotics in the body is of great importance in forensic and clinical toxicology. Most of the compounds of interest (xenobiotics and their metabolites) may be present in the body only as residues, and are hidden in heavily-loaded matrices such as urine, blood etc. In order to identify these unknown xenobiotics, samples are usually analyzed by high resolution MS instruments such as LC-QTOF / Orbitrap, utilizing their accurate mass measurement capability together with isotopic peak ratios. The identification of "real", "total" unknowns in LC-MS/MS analysis is an extremely challenging task. No preliminary guiding information as to the nature of the unknown exists, such as possible elements in the compound, sub-structures or any analogies to known compounds (unlike in metabolomics studies), nor an MS/MS library spectrum. Therefore, the identification process must be very general, and take into account various possible molecular formulas and structures. We studied the capability and limitations of identification based on accurate mass measurements. For that purpose, we have chosen 15 model compounds with a broad range of chemical and physical properties, spiked in urine. We developed a sample preparation procedure and a LC-MS/MS (QTOF) analysis method, applying a broad LC-gradient, large mass range (including very low m/z values), automat​ed MS/MS routine​s and a peak digging strategy.​