Elucidation of the presence and location of t-Boc protecting groups in amines and dipeptides using on-column H/D exchange HPLC/ESI/MS

Abstract

High performance liquid chromatography/mass spectrometry (HPLC/MS) has become a widely used technique for routine analysis of pharmaceutical compounds. The constant search for new drugs requires the development of time-efficient methods that can be employed in high-throughput screening of combinatorial libraries of a variety of compounds, including amines and peptides. Conventional HPLC/MS is a powerful technique that can easily be automated and is suitable for comprehensive screening purposes. However, the unequivocal determination of the presence and location of important carbamoyl protecting groups of amines is often elusive because of their inherent instability under MS conditions. In this study, the use of on-column H/D exchange HPLC/ESI/MS for structure elucidation of t-Boc protecting groups which can often not be detected by MS because of facile McLafferty rearrangement has been examined. We demonstrate that employing a deuterated mobile phase in HPLC/MS analysis provides a convenient tool for the determination of the absence or presence of t-Boc protecting groups in amines and peptides. . This fragmentation pathway may proceed via a labile intermediate carbamoyl acid intermediate 3, which easily eliminates carbon dioxide and is therefore often not observed. Because the unprotected amino alcohol 4 affords fragment 2 after ionization in the MS detector, mass spectrometry does not provide a means to differentiate between 1 and 4, i.e., the determination of the presence of the t-Boc group requires isolation and further spectroscopic analysis or the use of reference samples for a time-consuming comparison of HPLC retention times which is unsuitable for high-throughput screening.Structure elucidation by mass spectrometry has become increasingly successful through the introduction of chemical ionization with ND 3 , D 2 O, and CD 3 OD for exchange of hydrogen for deuterium of organic compounds in the gas phase [2]. This technique can be applied to H/D exchange MS analysis of various classes of compounds including alcohols, carboxylic acids, amines, amides, and thiols [3]. It has also been shown that hydrogens bonded to carbon atoms in aromatic compounds can be replaced by deuterium in the gas phase [4]. The coupling of HPLC and mass spectrometry combines a powerful separation technique with the sensitive and informative MS detection mode, which opens a new venue for H/D exchange MS analysis of complex mixtures containing polar compounds. To date, various MS techniques including chemical ionization detection mass spectrometry (CI/MS) [5], fast atom

On-line H/D exchange LC–MS strategy for structural elucidation of pharmaceutical impurities

“…The early use of deuterium oxide as a mobile phase for ESI LC-MS was demonstrated by Karlsson using a microbore column with L per min flow rates [14]. The use of D 2 O as the mobile phase for regular HPLC at a flow rate 0.2-1.0 mL/min has been demonstrated in several recent publications [4][5][6][7][8][9][10].…”

“…This warrants the development of a convenient method for the fast characterization of its absence or presence in reaction mixtures [9]. LC-MS has become the primary method for such mixture analyses.…”

“…During the past decade, API techniques including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have become the primary tool for pharmaceutical analysis. By using ESI interface, the application of deuterium oxide as a chromatographic LC-MS mobile phase for conducting H/D exchange experiments has recently been demonstrated for a number of drug metabolism studies [4][5][6][7] as well as for identification of pharmaceutical impurities [8][9][10]. The intention of this manuscript is to provide an overview of the premise of on-line H/D exchange LC-MS experimental setup and its applications in identification of pharmaceutical impurities including both literature data and the data collected in the authors' laboratories.…”

Elucidação estrutural de substâncias orgânicas com auxílio de computador: evoluções recentes

“…A elucidação da estrutura é concluída com o auxílio de técnicas de RMN bidimensionais (HSQC, HMBC, 1 H-1 H COSY etc.). O HiTSE ainda pode ser utilizado ou adaptado para uso com outras técnicas, tais como HPLC-EM 69 .…”

Optimization and Application of APCI Hydrogen–Deuterium Exchange Mass Spectrometry (HDX MS) for the Speciation of Nitrogen Compounds

“…Hunt et al reported use of HDX for determining the hydrogens bonded to heteroatoms in alcohols, phenols, carboxylic acids, amines, amides, and mercaptans by CI [23]. Later, the mass spectra of ESI HDX were used to study proteins, peptides, metabolites, and antibiotics [24][25][26][27][28][29]. Recently, atmospheric pressure photoionization (APPI) HDX coupled to MS has been reported [30], and its use for determining the structure of crude oil compounds has been explored [31].…”