Sites of metabolic substitution: investigating metabolite structures utilising ion mobility and molecular modelling

Drug metabolism is an integral part of the drug development and drug discovery process. It is required to validate the toxicity of metabolites in support of safety testing and in particular provide information on the potential to form pharmacologically active or toxic metabolites. The current methodologies of choice for metabolite structural elucidation are liquid chromatography/tandem mass spectrometry (LC/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. There are, in certain cases, examples of metabolites whose sites of metabolism cannot be unequivocally identified by MS/MS alone. Utilising commercially available molecular dynamics packages and known quantum chemistry basis sets, an ensemble of lowest energy structures were generated for a group of aromatic hydroxylated metabolites of the model compound ondansetron. Theoretical collision cross-sections were calculated for each structure. Travelling-wave ion mobility (IMS) measurements were also performed on the compounds, thus enabling experimentally derived collision cross-sections to be calculated. A comparison of the theoretical and experimentally derived collision cross-sections were utilised for the accurate assignment of isomeric drug metabolites. The UPLC/IMS-MS method, described herein, demonstrates the ability to measure reproducibly by ion mobility, metabolite structural isomers, which differ in collision cross-section, both theoretical and experimentally derived, by less than 1 Å(2). This application has the potential to supplement and/or complement current methods of metabolite structural characterisation.