Comparison of Information-Dependent Acquisition, SWATH, and MS^All Techniques in Metabolite Identification Study Employing Ultrahigh-Performance Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

Sensitive and selective liquid chromatography–mass spectrometry (LC–MS) analysis is a powerful and essential tool for metabolite identification in drug discovery and development. An MS² (or tandem, MS/MS) mass spectrum is acquired from the fragmentation of a precursor ion by multiple methods including information-dependent acquisition (IDA), SWATH (sequential window acquisition of all theoretical fragment-ion spectra), and MS^All (also called MS^E) techniques. We compared these three techniques in their capabilities to produce comprehensive MS² data by assessing both metabolite MS² acquisition hit rate and the quality of MS² spectra. Rat liver microsomal incubations from eight test compounds were analyzed with four methods (IDA, MMDF (multiple mass defect filters)-IDA, SWATH, or MS^All) using an ultrahigh-performance liquid chromatography–qudrupole time-of-flight mass spectrometry (UHPLC–Q-TOF MS) platform. A combined total of 227 drug-related materials (DRM) were detected from all eight test article incubations, and among those, 5% and 4% of DRM were not triggered for MS² acquisition with IDA and MMDF-IDA methods, respectively. When the same samples were spiked to an equal volume of blank rat urine (urine sample), the DRM without MS² acquisition increased to 29% and 18%, correspondingly. In contrast, 100% of DRM in both matrixes were subjected to MS² acquisition with either the SWATH or MS^All method. However, the quality of the acquired MS² spectra decreased in the order of IDA, SWATH, and MS^All methods. An average of 10, 9, and 6 out of 10 most abundant ions in MS² spectra were the real product ions of DRM detected in microsomal samples from IDA, SWATH, and MS^All methods, respectively. The corresponding numbers declined to 9, 6, and 3 in the urine samples. Overall, IDA-based methods acquired qualitatively better MS² spectra but with a lower MS² acquisition hit rate than the other two methods. SWATH outperformed the MS^All method given its better quality of MS² spectra with an identical MS² acquisition hit rate