The identification of metabolites, whether from in vitro or in vivo studies, is an ongoing challenge for drug discovery and development. Metabolite identification typically uses an array of chromatographic and mass spectrometric methods, and may require multiple injections of the same sample. This is to ensure that enough information has been collected to detect all metabolites and to have sufficient fragmentation information available to elucidate structures.
We describe in this application note a workflow that enables the collection of both parent and fragment information from a single injection (Figure 1). This MSE data acquisition uses two interwoven scan functions: the first (low collision energy, or low CE) function contains data from the intact metabolites, and the second (high collision energy, or high CE) function contains data from the fragment ions.
The high throughput screening (HTS) analysis was performed using ACQUITY UPLC® and SYNAPT™ Mass Spectrometry systems. The resulting LC/MS data were obtained with mass accuracies typically in the sub-2 ppm range.
Since all data are collected in one run, post-acquisition processing of multiple fragment ions is possible. With this approach, the entire dataset is mined post-acquisition for specific metabolite masses, precursor and product ions, and neutral losses because all the necessary data has been collected simultaneously. Selectivity for biotransformation of the parent drug is achieved through exact mass measurement.