Comparison of Information-Dependent Acquisition, SWATH, and MSAll 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 MSAll (also called MSE) 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 MSAll) 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 MSAll method. However, the quality of the acquired MS2 spectra decreased in the order of IDA, SWATH, and MSAll 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 MSAll 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 MSAll method given its better quality of MS2 spectra with an identical MS2 acquisition hit rate.