Tandem mass spectrometry (MS/MS) is widely used for the identification of metabolites at all stages of the pharmaceutical discovery and development process. The assignment of ions in the product ion spectra can be time-consuming and hence delay feedback of results that may influence the direction of a project. A deeper understanding of the processes involved in generation of the product ions formed via collision-induced dissociation may allow development of chemically intelligent software to aid spectral interpretation. Current commercially available spectral interpretation software takes a mainly arithmetical approach resulting in extensive lists of numerically plausible ions, many of which may not be chemically feasible. In this study, high-resolution MS/MS spectra were obtained for maraviroc and two of its synthetic metabolites, and structures for the product ions proposed. Density functional theory (DFT) based on in silico modelling was undertaken to investigate whether the fragmentation observed was potentially a result of bond lengthening (and hence weakening) as a consequence of protonation of the molecule at the most thermodynamically stable site(s). It was determined that for all three compounds, where the product ions resulted from simple bond cleavages (not rearrangements), the bonds that cleaved had been calculated to elongate after protonation. It was also noted that the protonated molecule may represent a mixture of singly charged protonated species and that the most basic sites in the molecule may not necessarily be the most thermodynamically stable for protonation. Copyright # 2010 John Wiley & Sons, Ltd.The identification of metabolites is an integral part of the drug discovery and development process. Structural elucidation of metabolites is required at the discovery stage to flag up any potentially active or toxic metabolites and direct chemical synthesis towards compounds that give the required pharmacokinetic properties. For example, a site of metabolism may be blocked, perhaps by the insertion of a fluorine atom, to reduce the metabolic clearance of compounds in a series. In development, metabolites need to be fully characterised for the regulatory submission and to ensure that all the metabolites observed in humans are also present in the toxicology species.Atmospheric pressure ionisation mass spectrometry is currently the technique of choice for identifying metabolites. Although not as definitive as NMR (nuclear magnetic resonance) when assigning structure, mass spectrometry has the advantages of high sensitivity and ease of hyphenation to chromatographic systems. Chromatography is required as the metabolites are present in a complex mixture of biological matrix and other drug-related components. Protein precipitation is often used for sample preparation rather than analyte extraction in order to avoid loss of polar metabolites. Protein precipitation leads to a particularly high endogenous background making the detection of drugrelated material difficult and hence the use of a separation ...