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

Abstract

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 MS(2) (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 MS(2) data by assessing both metabolite MS(2) acquisition hit rate and the quality of MS(2) 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 MS(2) 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 MS(2) acquisition increased to 29% and 18%, correspondingly. In contrast, 100% of DRM in both matrixes were subjected to MS(2) acquisition with either the SWATH or MS(All) method. However, the quality of the acquired MS(2) 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 MS(2) 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 MS(2) spectra but with a lower MS(2) acquisition hit rate than the other two methods. SWATH outperformed the MS(All) method given its better quality of MS(2) spectra with an identical MS(2) acquisition hit rate.

Comparison of Information-Dependent Acquisition on a Tandem Quadrupole TOF vs a Triple Quadrupole Linear Ion Trap Mass Spectrometer for Broad-Spectrum Drug Screening

“…In addition, alternative ways to collect product ions have been proposed [e.g., sequential window acquisition of all theoretical fragment-ion spectra (SWATH) and all-ions fragmentation]. These tech- niques have been shown to collect more product ion spectra than IDA; however, the quality of spectra is worse, since spectra are contaminated by coeluting compounds (31 ). A "contaminated" spectrum may not be detrimental to compound identification by targeted analysis, but it is unclear how it will affect the identification of unexpected or unknown compounds, which is the main selling point of HRMS.…”

Stepped collisional energy MSAll: an analytical approach for optimal MS/MS acquisition of complex mixture with diverse physicochemical properties

“…The advent of sub-2 μm 'core-shell' separation on ultra performance liquid chromatography (UPLC) systems has partially alleviated the problem by improving separation efficiency, facilitating the application of DIA in characterization of complex mixtures by providing clean MS/MS spectra for efficiently resolved individual components. [16] Nevertheless, the components present in Qili extract are so complex that co-elution of several species still exist (Fig. 7A) despite the fact that the UPLC conditions (i.e., mobile phases, gradient profile, column temperature) have all been optimized.…”

“…'elevated' CE of 45 V (as normally set in DIA analysis), [16] where the fragment ions at m/z 161.02, 179.03 and 197.05 belong to rosmarinic acid and the ions at m/z 799.49 and 961.54 were produced from 20-O-glucopyranosyl Ginsenoside Rf.…”

Nested data independent MS/MS acquisition

“…However, currently available instrumentation does not permit the unit mass isolation of all precursor ions of interest in order to produce a set of truly comprehensive MS/ MS data. Targeted techniques must still be used if unit mass isolation is required [11]. Currently available data independent acquisition (DIA) works with mass isolation windows that are clearly wider than unit mass.…”