In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization of metabolites

Abstract

A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D 2 O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H 2 O or D 2 O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site uring the process of drug discovery, it is highly desirable to increase the number of successful drug candidates for preclinical, clinical and commercial development. Therefore, the drug discovery process is constantly scrutinized and improved [1]. Adding to this pressure is the generation of vast numbers of new chemical entities resulting from combinatorial chemistry technology [2]. Drug metabolism plays an important role in the drug discovery process [3]. Specifically, the identification of metabolites during the early stage of development can be helpful to medicinal chemists trying to block some of the metabolic hot spots and produce an appropriate drug that is less susceptible to metabolism and increase the half-life of the drug. Therefore, rapid identification of drug metabolites is imperative for drug development [4,5].Hydrogen/deuterium exchange is a well-established technique for studying structure, stability, folding dynamics, and intermolecular interactions in proteins in solution [6]. During solution phase H/D exchange, labile protons in the side chains and amide hydrogens, which are not protected from solution generally exchange rapidly. Exchanges of these unprotected hydrogens occur on the order of a few to a few tenths per second under the experimental conditions described in the aforementioned studies. If however, amide or side chain hydrogens are protected from solution (e.g., when they are hydrogen-bonded in structurally stable secondary-structure elements), the exchange rates can be considerably reduced. Methods in which H/D exchange experiments are combined with either nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry are also well-established [7,8]. NMR methods, when coupled with H/D exchange are the ideal choice for monitoring individual residues or each amide hydrogen; however, these methods are limited to highly purified proteins or metabolites that are soluble at high concentrations, thus eliminating the possibility of determining structural features of drugs and metabolites that are i...

Deuterium-exchange metabolomics identifies N-methyl lyso phosphatidylethanolamines as abundant lipids in acidophilic mixed microbial communities

“…3. Similar isotope exchange techniques have been used in past metabolomics experiments (Liu et al 2001(Liu et al , 2007Lam and Ramanathan 2002). To lessen the amount of labeled D 2 O needed, these prior experiments often introduced D 2 O in the gas phase at the ion source, which results in less complete D/H exchange than the method used in the current study (Fig.…”

CE hydrogen deuterium exchange‐MS in peptide analysis

“…ACN rather than methanol was employed in this work as it is not a protic solvent and its use therefore eliminates the need to employ an expensive deuterated organic solvent in the deuterium exchange studies. The use of deuterated mobile phases in HPLC-MS produces more efficient (H/D) exchange compared with postcolumn addition techniques [16]. This observation has been exploited in this study by employing both deuterated BGE and sheath buffers in CE-MS.…”

“…The lower sensitivity of CE-H/D-MS technique may be due to the increased viscosity of D 2 O reducing the efficiency of the solvent evaporation process in ESI [16]. The LODs (36S/N) for leucine enkephalin using CE-MS and CE-H/D-MS were 0.4 and 2 mg/ mL, respectively in SIM mode.…”

Structural characterization of sulfadiazine metabolites using H/D exchange combined with various MS/MS experiments

“…Surprisingly, this method has infrequently been applied for characterization of metabolites° [25][26][27][28][29][30][31][32][33].°The°higher°cost°of°D 2 O could be considered a certain drawback, but the consumption of D 2 O can be significantly reduced by using smaller columns and smaller solvent bottles. Alternatively, fractions can be collected and redissolved in D 2 O containing solvents.…”