Characterization and identification of the metabolites of dihydromethysticin by ultra‐high‐performance liquid chromatography orbitrap high‐resolution mass spectrometry

Abstract

Dihydromethysticin, a natural component from Piper methysticum Forst, has been reported to display pharmacological effects in mental disorders and some malignant tumors. However, the metabolism of this component remained unknown. The goal of this work was conducted to discover the metabolic profiles of dihydromethysticin. The in vitro incubation was performed by incubating dihydromethysticin with rat, monkey, and human liver microsomes and hepatocytes. An analytical assay of ultra‐high performance liquid chromatography combined with Orbitrap high‐resolution mass spectrometry was utilized to detect and identify the metabolites. With high resolution mass spectrometric determination, the accurate mass, elemental composition, and product ions of the metabolites were determined, which enabled structural characterization to become easy. Under the present conditions, four phase‐I metabolites, as well as six phase‐II metabolites, were detected and their tentative structures were characterized by mass spectra. M4 was found as the most abundant metabolite both in liver microsomes and hepatocytes. Cytochrome P450 1A2, 2C9, and 3A4 contributed to the formation of this metabolite by using human recombinant P450 enzymes. M4 can be oxidized into reactive ortho‐quinone intermediate followed by conjugating with glutathione. M4 was also subject to glucuronidation (M1 and M2) and methylation (M5). Demethylenation, oxidation, hydroxylation, glucuronidation, glutathionylation, and methylation were the primary metabolic pathways of dihydromethysticin. This study provides in vitro metabolism data of dihydromethysticin, which is indispensable for understanding the disposition of this compound.

Performance in (Ultra‐)high‐performance liquid chromatography—How to qualify and optimize instruments in practice

“…particularly in pharmacy [2][3][4][5][6][7], for example, in the recent analysis of traditional Chinese medicine [8][9][10][11][12][13][14][15][16][17][18][19][20][21].…”

“…This allows even higher theoretical plate numbers and even faster separations. Therefore, UHPLC is a popular technique where highly complex separations are required, particularly in pharmacy [2–7], for example, in the recent analysis of traditional Chinese medicine [8–21].…”

Rapid separation and characterization of the in vitro metabolites of moscatilin by ultra‐high performance liquid chromatography coupled to hybrid quadrupole orbitrap tandem mass spectrometry

“…Therefore, it is better to know the metabolite profile of a drug candidate in different species. With the development of MS, especially high-resolution MS, identification of drug metabolites becomes much easier as high-resolution MS can provide the formulas and fragment ions of the metabolites [15,16].…”