Characterization of the metabolic fate of sinapic acid in rats.

Sinapic acid (SA) is ubiquitously distributed in the plant kingdom as a free organic acid and more frequently as a biosynthetic pioneer for SA derivatives, e.g., SA esters. Broad biological and pharmacological activities have been disclosed for SA. Because of the metabolism lability property, metabolites instead of the parent compound should be the primary forms after oral treatment of SA, and those metabolites should also be rapidly observed from SA following administration of SA derivative. Hence, the metabolites might provide a primary contribution to the pharmacological properties of SA; however, the metabolite profile remains unclear. Here, our efforts were devoted to addressing this issue through deploying online energy-resolved mass spectrometry (ER-MS) to accomplish isomer identification which is the key issue hindering metabolite identification, notably those conjugated metabolites. After recording breakdown graphs of concerned fragment ions with online ER-MS, the positive correlations between optimal collision energy (OCE) and bond dissociation energy (BDE) were applied to assign candidate structures to isomeric signals. Moreover, in vitro metabolism with liver cellular subfractions, UV-triggered cis-/trans-configuration transformation, and wet-chemistry hydrogenation were carried out to justify the structures. As a result, sixteen metabolites (M1-M16) were found and confirmatively identified in rat plasma and urine following SA administration, and sulfation, glucuronidation, demethylation, reduction, and dihydroxylation served as the primary metabolic channels. Noteworthily, greater distribution occurred for sulfation and glucuronidation products while inferior distributions were observed for phase I metabolites, and the half-life (T1/2) of most metabolites was greater than that of SA. This study provides a comprehensive insight into the metabolic fate of SA. More importantly, the fortification of online ER-MS and quantum structure calculation to the conventional LC-MS program is eligible to achieve unambiguous identification of isomeric metabolites.

MS/MS fingerprint comparison between adjacent generations enables substructure identification: Flavonoid glycosides as cases.

MS/MS spectrum matching currently serves as a favored means to identify the concerned metabolites attributing to the accessibility of several famous databases. However, the rule that takes the entire structure into account frequently leads to "0 hit" when inquiring MS/MS (usually MS2) spectrum in the databases. Conjugation plays an important role for the high-level structural diversity of metabolites in all organisms, and a given conjugate usually consists of two or more substructures. If MS3 spectra participate in database retrieval, the structural annotation potential of those databases should be dramatically expanded via identifying substructures. Attributing to the ubiquitous distribution pattern, flavonoid glycosides were deployed as the representative family to justify whether the primary fragment ion termed as Y0+, resulted from neutral loss of glycosyl residue(s), generated identical MS3 spectrum with MS2 spectrum of the aglycone cation namely [A+H]+. Because of owning unique ability to measure MS/MS spectrum with the exactly desired exciting energy, linear ion trap chamber of Qtrap-MS was responsible for generating the desired MS3 and MS2 spectra. When taking both m/z and ion intensity features into consideration, the findings included: 1) glycosides sharing identical aglycones produced the same MS3 spectra for Y0+; 2) different MS3 spectra for Y0+ occurred amongst glycosides bearing distinct, even isomeric, aglycones; 3) isomeric aglycones generated different MS2 spectra; and 4) MS3 spectra for Y0+ agreed with MS2 spectra of [A+H]+ when comparing paired glycoside and aglycone. Together, fingerprint comparison between MS3 and MS2 spectra could structurally annotate the substructures and further advance MS/MS spectrum matching towards the identification of, but not limited to, aglycones for flavonoid glycosides.