Online energy-resolved MS boosts the potential of LC-MS towards metabolite characterization of salidroside and tyrosol.

Although currently serving as the workhorse for metabolite characterization, one of the most challenging tasks for LC-MS is isomeric differentiation because isomers frequently yield identical quasi-molecular ions and fragmented ion species. Our previous studies have demonstrated that online energy-resolved MS (ER-MS) is an orthogonal technique for MS/MS experiments to facilitate isomeric identification. Herein, attempts were made for the in-depth characterization of the metabolic profiles of an effective natural product named salidroside (SA) in rats using LC coupled with three-dimensional mass spectrometry (LC-3D MS) that was configured by MS1, MS2 and online ER-MS as 1(st), 2(nd), and 3(rd) dimensions, respectively. Moreover, the metabolism characterization of its aglycone, namely, tyrosol (Try) was conducted in parallel to aid in proposing metabolic pathways. High-resolution MS1 and MS2 spectra were acquired by IT-TOF-MS, and subsequent data processing provided theoretical formula and sub-structures for each metabolite. Subsequently, online ER-MS was conducted for precursor > product ion transitions-of-interest to offer linkage information among the sub-structures via building breakdown graphs. As a result, ten (M1-10) and nine (M1, M2, and M5-11) metabolites were detected in SA- and Tyr-administrated biological samples, respectively, and their structures were qualitatively identified. Crucial metabolism occurred for either component. SA initially underwent hydrolysis to produce Tyr, and subsequently hydroxylation, oxidation, glucuronidation, and sulfation were observed as the primary metabolic pathways. To summarize, the metabolic fate of SA was understood in depth, and Tyr, as the hydrolytic product, was responsible for the occurrences of most metabolites (M1, M2, and M5-10). More importantly, identification confidences of the metabolites were significantly advanced by LC-3D MS, suggesting that it is eligible to serve as an integral part of the analyst's toolbox.