Retention Time and Optimal Collision Energy Advance Structural Annotation Relied on LC–MS/MS: An Application in Metabolite Identification of an Antidementia Agent Namely Echinacoside

Abstract

The structural annotation of metabolites now relies heavily on HR-MS/MS information, resulting in ambiguous identities in most cases. More auxiliary evidence is therefore desired to achieve confirmative identification. Herein, we made an attempt to involve retention time (t R ) along with optimal collision energy (OCE) as the additionally structural clues, and the applicability validation was conducted via confidence-enhanced metabolite characterization of echinacoside, an antidementia drug candidate within clinical trials. Quantitative structure-retention relationships (QSRR) were modeled via assaying 184 authentic compounds on RPLC, HILIC, and serially coupled RPLC and HILIC (RPLC-HILIC). Online energy-resolved MS was developed to yield breakdown graphs for selected ion transitions, and OCE was demonstrated to be superior to CE 50 toward pointedly denoting the bonds-of-interest. Nineteen metabolites (M1−M19) were confidently identified in biological samples from echinacosidetreated rats by analyzing m/z values first to yield empirical formulas and substructures, and t R and OCE subsequently contributed to sift the candidate structures. Structural identification was validated by oral administration of three relevant compounds in parallel and chromatographic purification as well. Above all, the integration of retention and dissociation behaviors enabled promoting one step forward for structural annotation confidences merely relied on HR-MS/MS.

Cistanches Herba, from an endangered species to a big brand of Chinese medicine

“…Because of the insufficient metabolic stability, the bioavailability of both VB and ECH has been demonstrated, as expected, to be quite low, 194,195 merely 0.83% for ECH and 0.12% for VB 196 . As a consequence, many concerns have paid onto the metabolism profiles of these individual components, in particular VB, 197–202 ECH, 190,202–204 poliumoside, 205,206 isoacteoside, 199 and 2′‐acetylacteoside 199 . All the studies concluded that deglycosylation, hydroxytyrosol clavage, decaffeoylation, deacetylation, reduction, acetylation, glucuronidation, and sulfation were primarily responsible for the low bioavailability as well as for the generation of most metabolites.…”

“…Judging from the chemical structure of a given PhG, theoretically, hydrolytic enzymes are able to dissociate both the ester moieties and ether‐type glucosidic bonds between sugar residues as well as between phenethyl aglycone and glucosyl residue, resulting in the extensive occurrences of hydrolyzed products. These hydrolyzed products such as caffeic acid, tyrosol, and hydroxytyrosol, usually exist as the theoretical intermediates because they usually bear more than one labile site, for example, hydroxyl and carboxyl groups to receive subsequent glucuronidation and/or sulfation 190 . So far, a set of publications are available concerning the metabolism of PhGs in vitro and in vivo when administrating single component or the extract.…”

“…Four components, including ECH, VB, isoacteoside and 2′‐acetylacteoside, all of which are also distributed in the original extract were purified from feces by preparative LC. Because ECH can be transferred into VB via losing the outer glucosyl residue, 190 and inter‐transformation has been demonstrated between VB and isoacteoside, 62 it is, therefore, challenging to definitely point out the original sources of these two compounds.…”

Therapeutic potential of phenylethanoid glycosides: A systematic review

“…In addition, acyl-migration and acetylization of verbascoside (M17) produced isoverbascoside (M18) and acetyl-verbascoside (M19). 213 Three metabolites of forsythiaside A (200 mg/kg, po) were identified in rats, with 42 in urine, 22 in plasma, and 15 in feces. 214 In general, forsythiaside A was mainly methylated, dimethylated, sulfated, glucuronidated, diglucuronidated, and cysteine conjugated.…”

“…Afterward, the two intermediates received sulfonation, glucuronidation, methylation, oxidation, reduction, and/or hydrogenation to finally generate M1−M16. In addition, acyl‐migration and acetylization of verbascoside (M17) produced isoverbascoside (M18) and acetyl‐verbascoside (M19) 213 . Three metabolites of forsythiaside A (200 mg/kg, po) were identified in rats, with 42 in urine, 22 in plasma, and 15 in feces 214 .…”