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The use of deuterium oxide as a mobile phase in the routine analysis of pharmaceutical compounds was investigated. The deuterium exchange of labile hydrogen atoms aids in structural confirmation and elucidation of unknown impurities and degradation products. Although deuterium oxide as a mobile phase does in some cases change the retention times, the changes in retention times do not interfere with the analysis. A study of the high-performance liquid chromatography system shows that equilibration times for the deuterium-containing mobile phases are similar to equilibration times with changes of other mobile phases. The use of this technique in the analysis of pharmaceutical compounds and other small molecules is presented.

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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