Beyond the library ceiling: an accumulation-time rule for electron activated dissociation in data-dependent metabolomics, and the structural information it adds for conjugates, isomers and unknowns in human plasma and urine

Electron activated dissociation (EAD) reveals what collision-induced dissociation (CID) destroys or ignores: intact conjugates, radical backbone cleavages, and double-bond positions. In untargeted data-dependent acquisition, however, generic settings yield empty spectra, and the few EAD libraries available do not yet cover them. We quantified both limits on a ZenoTOF 8600 with a reversed-phase method in human plasma and urine. A 3 × 3 grid of accumulation time (35 to 210 ms) and candidates per cycle (5 to 20), replicated in a second plasma pool and in urine, shows that, at fixed electron energy, the accumulation time decides the outcome: 2.7% informative spectra with the generic 10 ms setting, 70 to 78% at 35 ms and 96 to 99% at 105 and 210 ms. The rule: the accumulation must cover the 30 ms electron reaction. At 105 ms and 10 candidates (1.4 s cycle), 4,200 precursors per injection receive an informative spectrum, 80% of whose fragments CID lacks. Against the public CID library, the plasma pools yielded 124 metabolites with CID but 55 with EAD, although 92% of the former carried an EAD spectrum: the ceiling is the library, not the acquisition. Beyond that ceiling, EAD read what the library could not. Of 36 urinary glucuronides of steroids, isoflavones, indoles, and bile acids, 32 kept the conjugate in EAD where CID gave the aglycone and the library named none correctly; methylxanthine, methylguanine, and dimethyluric acid isomers with identical CID spectra were separated; and 17 unannotated urinary ions received an identity or class, among them the glycation product MG-H1, named Bisphenol A by the library. Leucine and isoleucine remain unresolved at 12 eV, and EAD-cell background ions must be removed before interpreting low-mass ions. The duty-cycle rule, ion-count criteria, and annotated spectra are the starting material for EAD libraries of biofluid metabolites.