Comparison of CID and EAD fragmentation with automated assignment for small molecule structure elucidation
70th ASMS Conference on Mass Spectrometry. June 2022
Ismael Zamora1; Christopher Kochansky2; Fabien Fontaine1; Kevin P Bateman2; Jason Causon3; Jose Castro-Perez4; Rolf Kern5
1Lead Molecular Design, S.L., Sant Cugat del Valles, Spain; 2Merck & Co., Inc., Kenilworth, NJ; 3SCIEX, Concord, ON; 4Sciex, Framingham, MA; 5SCIEX, Redwood City, CA
Abstract
Introduction
Collisional-induced dissociation (CID) has been the main workhorse for small molecule structure elucidation in drug metabolite identification studies. Software tools, such as Massmetasite, have been developed to assist in the automatic interpretation of CID MS/MS spectra. The challenge with CID is that for many drug metabolites, non-informative fragmentation occurs, resulting in a lack of useful structural assignments for these metabolites. Commercialization of electron activated dissociation (EAD) on a quadrupole time of flight mass spectrometer provides a potentially powerful new tool for small molecule structure elucidation in drug discovery. Automated interpretation of EAD MS/MS spectra using existing algorithms needs to be tested, modified, and implemented. A comparison of CID and EAD fragmentation using automated interpretation will be presented in this work.
Methods
Small molecule drugs were incubated in rat hepatocytes at 1 µM. Time points: 0, 30 and 120 min were pulled from the incubation and quenched with 1 volume of CH3CN. Samples were vortexed, centrifuged, and the supernatant transferred to an HPLC vial for analysis.
LC separation was performed on a Phenomenex Luna Omega Polar C18, 150 mm column using 0.5µL or 5 µL injection volumes. Gradient separation 0.1% formic acid in water and acetonitrile was performed over 4.75 minutes from 5%B to 95%B with a total of 6.5 minutes.
The samples were analyzed using ZenoTOF 7600 CID-IDA(DDA) and EIEIO IDA(DDA). TOFMS was scanned between m/z 100-1000, CID/EAD MS/MS from 60-1000. Data was processed in MassMetaSite with CID and EAD fragmentation.
Preliminary data
EAD is a free electron fragmentation mode available recently introduced to accurate mass LC-MS/MS. It utilizes high energy electrons which allows for the dissociation of singly charged precursors, in this work an electron kinetic energy of 10 eV was utilized. The MS/MS spectra show significant increases in the number of fragment ions observed when going from Zeno CID to Zeno EAD spectra. The larger number of fragment ions makes it even more important for automated assignment using software tools such as Massmetasite. Many of the new fragments are the result of radical bond cleavage driven by electron-impact excitation of ions from organics (EIEIO) mechanism. Typical software algorithms for MS/MS focus on even electron species, typical of CID fragmentation. With EAD and the production of odd electron fragments, modification of the algorithm is required. The results to date show much richer fragmentation spectra using EAD versus CID and that the modified algorithm can assign the new odd electron fragments.
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