Oxygen Isotope Exchange Reaction for Untargeted LC–MS Analysis

Abstract

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.

PyFragMS─A Web Tool for the Investigation of the Collision-Induced Fragmentation Pathways

“…Additional improvement of the accuracy of identification can be achieved by considering the MS/MS spectra of the compounds after isotope exchange. Recently, we demonstrated that for the given MS/MS spectrum of an unknown molecule after the 16 O/ 18 O exchange reaction, the number of possible candidates with a known structure but unknown MS/MS spectra can be reduced via the substructure mapping based on the molecular formula of MS/MS fragments and predicted position of 18 O labels. , It is obvious that the accurate consideration of the fragmentation pathways may improve the quality of the identification even more.…”

Carbon and Oxygen Isotopic Ratio Analysis by FT ICR MS for Natural Vanillin Authentication

“…Oxygen isotope exchange of vanillin was carried out in the same manner, using 3 μL of vanillin at 100 μg/mL, was freeze-dried and resuspended in 50 μL of H 2 18 O, and again freeze-dried. This procedure was repeated two times and finally resuspended in 100 μL of H 2 18 O before FT-ICR-MS measurements and following the process outlined by Opisenko et al with minor modifications . Briefly, 50 μL of vanillin at 100 μg/mL was evaporated under vacuum, and then 75 μL of acetonitrile and 75 μL of water- 18 O (H 2 18 O) together with 0.1% formic acid were added.…”

“…As recently outlined by Opisenko et al, the reaction is based on reversible hydration of the electrophilic carbon, and hydroxyl groups of phenolic compounds are not exchangeable even when heating the solution at 95 °C. 20 Notably, the 18 O exchange of formyl O atom was observed only when vanillin was placed at 37 °C for 24 h with H 2 18 O. No 18 O exchange was observed when the sample was freeze-dried with H 2 18 O twice, suggesting that vanillin enrichment with 18 O could not occur during sample preparation.…”

“…This procedure was repeated two times and finally resuspended in 100 μL of H 2 18 O before FT-ICR-MS measurements and following the process outlined by Opisenko et al with minor modifications. 20 Briefly, 50 μL of vanillin at 100 μg/mL was evaporated under vacuum, and then 75 μL of acetonitrile and 75 μL of water- 18 O (H 2 18 O) together with 0.1% formic acid were added. The solution was incubated for 24 h at 37 °C.…”

Analysis of 16O/18O and H/D Exchange Reactions between Carbohydrates and Heavy Water Using High-Resolution Mass Spectrometry

“…16 O/ 18 O isotope exchange.To perform oxygen isotope ( 16 O/ 18 O) exchange, we followed the procedure described by Samuel and Silver [ 5 ].This method was also used by us in previous studies [ 28 , 29 ]. The target compound was dissolved in H 2 18 O.The solution was incubated at 95 °C for either 24 or 48 h in a sealed glass vial.…”