Oxygen Isotope Exchange Reaction for Untargeted LC–MS Analysis

LC–MS is a key technique for the identification of small molecules in complex samples. Accurate mass, retention time, and fragmentation spectra from LC–MS experiments are compared to reference values for pure chemical standards. However, this information is often unavailable or insufficient, leading to an assignment to a list of candidates instead of a single hit; therefore, additional features are desired to filter candidates. One such promising feature is the number of specific functional groups of a molecule that can be counted via derivatization or isotope-exchange techniques. Hydrogen/deuterium exchange (HDX) is the most widespread implementation of isotope exchange for mass spectrometry, while oxygen ¹⁶O/¹⁸O exchange is not applied as frequently as HDX. Nevertheless, it is known that some functional groups may be selectively exchanged in ¹⁸O enriched media. Here, we propose an implementation of ¹⁶O/¹⁸O isotope exchange to highlight various functional groups. We evaluated the possibility of using the number of exchanged oxygen atoms as a descriptor to filter database candidates in untargeted LC–MS-based workflows. It was shown that ¹⁶O/¹⁸O exchange provides 62% (median, n = 45) search space reduction for a panel of drug molecules. Additionally, it was demonstrated that studying the fragmentation spectra after ¹⁶O/¹⁸O can aid in eliminating false positives and, in some cases, help to annotate fragments formed with water traces in the collisional cell.