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.