Structural elucidation of in vitro metabolites of emodin by liquid chromatography–tandem mass spectrometry

Abstract

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was employed to investigate the in vitro metabolism of emodin. Emodin was incubated with rat liver microsomes in the presence of a NADPH-generating system, followed by extraction with ethyl acetate. After separation on a reversed-phase C18 analytical column with a linear gradient elution of methanol and 0.1% formic acid in water, negative electrospray ionization tandem mass spectrometry experiments were performed. As a result, the parent drug and its six metabolites were detected from rat liver microsomal incubations. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. Besides three mono-hydroxylated metabolites (omega-hydroxyemodin, 2-hydroxyemodin, 4-hydroxyemodin), three other metabolites were identified, which were emodic acid, 3-carbomethoxy-6-methoxy-1,8-dihydroxyanthraquinone and physcion, respectively.

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“…5) were all 2 Da more than m/z 269 ([M−H] − ion of emodin) and its product ions at m/z 241 and 225. The elucidation could also be demonstrated by our previous study [26]. As for M95, the aglycone ion [M−H-C 6 H 8 O 6 ] − at m/z 299 was 30 Da higher than the deprotonated ion of emodin (Fig.…”

“…271, 254 and 210 (Fig. 5) According to our previous work concentrated on the in vitro metabolism of emodin [26], the aglycone of M95 was unambiguously assigned as emodic acid. M94, M102 and M104 all gave an [M−H] − molecule at m/z 445, and their MS/MS fragmentations were predominated by the elimination of glucuronidyl residue to give aglycone ions at m/z 269 as base peak.…”

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“…Thus it was deduced that the significant variations likely originated from the process of coextraction of RRR and SMRR. A study reported that emodic acid was a metabolite of emodin via an oxidation reaction [27], hence the wide distribution of phenolic acid in SMRR probably led to the reaction in the heating extraction procedure. Consequently, the extraction temperature and time should be strictly controlled for the preparation of intermediate A to obtain relatively stable products.…”

Characterization of emodin metabolites in Raji cells by LC–APCI-MS/MS

“…Identification of the metabolite as emodic acid was ruled out because the retention time of emodic acid was 3. (9,15,16) were prepared from emodin for comparison (Scheme 1). According to the information obtained from the MS/ MS spectrum and the retention times of authentic samples, the compound at 15.0 min was identified as 3-O-methyl-x-hydroxyemodin (9).…”

“…10 The hepatic microsomes derived from various animal species transformed emodin into several anthraquinone metabolites. [11][12][13][14][15] Though emodin is a selective inhibitor of protein kinase, few biotransformation studies of emodin in human cells have been performed. We have previously shown that emodin inhibits Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells, which are derived from the human B-lymphoid cell line.…”