Determination of a trace level enol impurity by on‐line hydrogen/deuterium exchange combined with ultra‐high mass resolution and ultra‐high mass accuracy Fourier transform ion cyclotron resonance mass spectrometry

Zhong, Wendy; Yang, Jinggang

doi:10.1002/rcm.4229

Cited by 7 publications

(4 citation statements)

References 144 publications

(244 reference statements)

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“…23 This complexity, specifically various combinations of natural abundance isotopomers and deuterated isotopomers, is recalcitrant to chromatographic separation. Whereas a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) at a resolving power >375 000 was able to mass resolve small molecule keto and enol isomers after deuterium labeling, 24 higher resolving power is required for (normally larger) peptides. Although such fine structure was not previously resolved, it was shown to influence the peak shape of peptides analyzed by an FTICR MS instruments at resolving power ~100 000 23 and should therefore also affect peak shape for Orbitrap instruments and potentially low mass peptides on modern quadrupole-time-of-flights (Q-TOFs).…”

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confidence: 99%

Resolving Isotopic Fine Structure to Detect and Quantify Natural Abundance- and Hydrogen/Deuterium Exchange-Derived Isotopomers

2013

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Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide’s isotope distribution, and HDX MS data analysis methods were developed accordingly. A level of complexity that is inherent to HDX MS remained unaddressed, namely, various combinations of natural abundance heavy isotopes and exchanged deuterium shared the same nominal mass and overlapped at previous resolving powers. For example, an A + 2 peak is comprised of (among other isotopomers) a two-2H-exchanged/zero-13C isotopomer, a one-2H-exchanged/one-13C isotopomer, and a zero-2H-exchanged/two-13C isotopomer. Notably, such isotopomers differ slightly in mass as a result of the ~3 mDa mass defect between 2H and 13C atoms. Previous HDX MS methods did not resolve these isotopomers, requiring a natural-abundance-only (before HDX or “time zero”) spectrum and data processing to remove its contribution. It is demonstrated here that high-resolution mass spectrometry can be used to detect isotopic fine structure, such as in the A + 2 profile example above, deconvolving the isotopomer species resulting from deuterium incorporation. Resolving isotopic fine structure during HDX MS therefore permits direct monitoring of HDX, which can be calculated as the sum of the fractional peak magnitudes of the deuterium-exchanged isotopomers. This obviates both the need for a time zero spectrum as well as data processing to account for natural abundance heavy isotopes, saving instrument and analysis time.

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confidence: 99%

Resolving Isotopic Fine Structure to Detect and Quantify Natural Abundance- and Hydrogen/Deuterium Exchange-Derived Isotopomers

2013

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“…Typically, a gain of three deuterons would equate to two exchangeable protons and an ionizing deuteron. However, in the case of a cationic molecule, the number of deuterons gained in the H/D exchange experiment corresponds to the number of exchangeable protons [12–14]. Since the NMR data below are consistent with this molecule's cationic nature, in this specific example, the number of deuterons gained corresponds directly to the number of exchangeable protons.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Unique Cationic Impurity in Preladenant™ Using Accurate MS and NMR

Zhong

Hilton

Martin

et al. 2013

Journal of Heterocyclic Chem

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High resolution MS, 1D, and 2D NMR were used to determine the structure of a unique cationic impurity generated during the synthesis of Preladenant™ H/D exchange experiments were performed by both MS and NMR to confirm the acidic nature of the cationic dihydroimidazole proton. The presence of three exchangeable protons was established by MS experiments and the disappearance of the C11 proton in 1H‐NMR spectrum on equilibration with D2O confirmed the acidic nature of the cationic dihydroimidazole proton. A piperazine ring contraction mechanism is proposed for the formation of the cationic dihydroimidazole.

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“…Online H/D exchange liquid chromatography/mass spectrometry (LC/MS) experiments can be readily set up using deuterated mobile phase for LC/MS analysis of mixtures (Ohashi et al, 1998;Liu et al, 2001;Liu and Hop, 2005;Olsen et al, 2000;Lam and Ramanathan, 2002). The combination of LC/MS technique and online H/D exchange experiment is useful in identification of drug metabolite or impurity (Du et al, 2011(Du et al, , 2013Hou et al, 2013;Zhong and Yang, 2009;Wu et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Liquid chromatography/quadrupole time‐of‐flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso‐phenylcyclopentylamine in rat bile

Liu

Wang

Ding

et al. 2014

Biomedical Chromatography

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MS/MS experiment and accurate mass measurement are powerful tools in metabolite identification. However, sometimes these data do not provide enough information to assign an unambiguous structure to a metabolite. In combination with MS techniques, hydrogen/deuterium (H/D) exchange can provide additional information for structural elucidation by determination of the number of exchangeable hydrogen atoms in a structure. In this study, the principal phase I metabolites of iso-phenylcyclopentylamine in rat bile were identified by high-performance liquid chromatography with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). Since N-oxidation may occur because of the existence of the primary amino group in the structure, it was difficult to differentiate the hydroxylated metabolites from N-oxides by ESI-Q-TOF-MS alone. Therefore, online H/D exchange technique was applied to solve this problem. Finally, 25 phase I metabolites were detected and structurally described, in which 11 were confirmed to be N-oxides. This study demonstrated the effectiveness of high-resolution mass spectrometry in combination with an online H/D exchange technique in rapid identification of drug metabolites, especially in discriminating hydroxylated metabolites from N-oxides.

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Determination of a trace level enol impurity by on‐line hydrogen/deuterium exchange combined with ultra‐high mass resolution and ultra‐high mass accuracy Fourier transform ion cyclotron resonance mass spectrometry

Abstract: Figure 3. The MS/MS experiments obtained for both the keto and the enol forms which also provide us the additional information on where the keto and the enol conversions occurred.

Cited by 7 publications

References 144 publications

Resolving Isotopic Fine Structure to Detect and Quantify Natural Abundance- and Hydrogen/Deuterium Exchange-Derived Isotopomers

Resolving Isotopic Fine Structure to Detect and Quantify Natural Abundance- and Hydrogen/Deuterium Exchange-Derived Isotopomers

Identification of a Unique Cationic Impurity in Preladenant™ Using Accurate MS and NMR

Liquid chromatography/quadrupole time‐of‐flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso‐phenylcyclopentylamine in rat bile

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