Post-column hydrogen–deuterium exchange technique to assist in the identification of small organic molecules by mass spectrometry

Abstract

To improve the certainty that a specific small organic molecule has been detected in a given sample by high-resolution mass spectrometry, other techniques that give conclusive evidence about the chemical structure of a compound like nuclear magnetic resonance or complementary information on its composition such as hydrogen–deuterium exchange (HDX) are often necessary. This study presents a systematic investigation that aims to improve the applicability of post-column HDX for those purposes. Key parameters like mobile-phase flow rates, volume percentage of H2O in the mobile phase, and D2O addition flow rates were optimized to provide an isotopic pattern that allows the accurate determination of the number of exchangeable hydrogen atoms in small organic molecules. A loop injection setup was used to emulate chromatographic conditions in the optimization process, and trimethoprim, a widely used anti-infective, was used as test compounds for the experiments. As expected, results showed that deuteration percentage decreased with a higher mobile-phase flow rate and increased with higher D2O flow rate. The post-column HDX technique was then validated with extracts of samples of river water and plants separated by liquid chromatography in hydrophilic interaction or reversed-phase modes. Mass spectra showed a completely visible isotopic pattern that allowed assessing correctly and unambiguously the number of exchangeable hydrogens in the compounds of interest. This study shows that post-column HDX can be used as a complementary technique to identify unknown small organic molecules in complex matrices. The current paper proposes an efficient, cost-effective, versatile technique of HDX that is helpful to assign a unique structure to a given high-resolution mass spectrometry signal.

Application of Spectral Accuracy to Improve the Identification of Organic Compounds in Environmental Analysis

“…Upon extraction, samples were thawed at room temperature and buffered to pH 7 with phosphoric acid monobasic and phosphoric acid dibasic. Water samples were then extracted using a previously reported method . Briefly, samples were loaded in polymeric Strata-X solid-phase extraction cartridges (Phenomenex, USA) and then eluted with 2 × 3 mL of an ACN-MeOH (1:1, v/v) solution.…”

“…Water samples were then extracted using a previously reported method. 4 Briefly, samples were loaded in polymeric Strata-X solid-phase extraction cartridges (Phenomenex, USA) and then eluted with 2 × 3 mL of an ACN-MeOH (1:1, v/v) solution. Eluates were evaporated under a nitrogen flow and reconstituted to 10 mL with MeOH spiked at the same concentrations as the previous solutions (80 and 300 μg L −1 ).…”

“…Using tandem mass spectrometry (MS/MS) databases, such as mzCloud or Mass Bank, may help to reach confidence level 2 by searching library spectrum matches, however it is difficult to perform such confirmation for less known organic micropollutants or their transformation products that might be absent from those databases. For that reason, complementary techniques have been recently developed to obtain more information on the composition and structure of unknowns such as postcolumn hydrogen–deuterium exchange and comparison of data acquired in the positive and negative ionization mode. , The former could be used as diagnostic evidence to obtain more information about the presence of specific functional groups (e.g., exchangeable hydrogens present in alcohol and amine groups) on a molecule, while the latter can be employed to confirm the presence of certain compounds. Dual ionization has been used for metabolomic profiling in the past to broaden the range of detection of MS methods.…”

Hydrogen/deuterium exchange mass spectrometry in the world of small molecules

“…Such protons are considered labile or mobilizable and can readily (on a millisecond timescale) be replaced by deuterons when exposed to a deuteration agent such as D 2 O or MeOD. Solution‐phase HDXs are very simple to implement and can be done mainly in three ways (Figure 3A): (1) by reconstitution of a dried compound, fraction, or extract, with any protic deuteriated donor solvent (e.g., D 2 O) before sample infusion‐MS analysis, (2) by using protic deuteration agent as mobile phase for LC/MS analysis, or (3) by post‐column addition of D 2 O or other deuteration agent during the LC/MS acquisition process (Chen et al, 2007; Eysseric et al, 2016; D. Q. Liu et al, 2007; Nassar, 2003; Olsen et al, 2000).…”

Electrochemistry-High Resolution Mass Spectrometry to Study Oxidation Products of Trimethoprim

“…Since tandem mass (MS/MS) spectra of oxidation products of trimethoprim are not yet stored in spectral libraries and standards are rare or not commercially available, diverse techniques were used to improve the identification level of the accurate mass data obtained by the high-resolution mass spectrometer. Those complementary techniques were: hydrogen-deuterium exchange (HDX) [30], which reveals the number of exchangeable hydrogen atoms such as those present in alcohol or amine functional groups; MS/MS experiments and comparison with MS/MS data found in the literature; in-silico fragmentation analysis of precursor ions based on the Mass Frontier software from HighChem and spectral accuracy determined by MassWorks software from Cerno Bioscience. Spectral accuracy measures the similarity between mathematically transformed experimental isotopic pattern of an ion and the theoretical isotopic pattern corresponding to a given molecular formula [31].…”