Metabolism of cyadox by the intestinal mucosa microsomes and gut flora of swine, and identification of metabolites by high‐performance liquid chromatography combined with ion trap/time‐of‐flight mass spectrometry

Abstract

Cyadox (CYX), 2-formylquinoxaline-1,4-dioxide cyanoacetylhydrazone, is an antimicrobial and growth-promoting feed additive for food-producing animals. To reveal biotransformation of CYX in swine intestine, CYX was incubated with swine intestinal microsomes and mucosa in the presence of an NADPH-generating system and swine ileal flora and colonic flora, respectively. The metabolites of CYX were identified using high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry (LC/MS-ITTOF). Structural elucidation of the metabolites was precisely performed by comparing their changes in molecular mass, full scan MS/MS spectra and accurate mass measurements with those of the parent drug. Finally, seven metabolites were identified as follows: three reduced metabolites (cyadox 1-monoxide (Cy1), cyadox 4-monoxide (Cy2) and bisdesoxycyadox (Cy4)); hydroxylation metabolite (3-hydroxylcyadox 1-monoxide (Cy3)); hydrolysis metabolite of the amide bond (N-decyanoacetyl cyadox (Cy5)); a hydrogenation metabolite (11,12-dihydro-bisdesoxycyadox (Cy6)) and a side-chain cleavage metabolite (2-hydromethylquinoxaline (Cy7)). Only one metabolite (Cy1) was found in intestinal microsomes. Cy1, Cy2 and Cy4 were detected in intestinal mucosa, ileal and colonic flora. In addition, Cy3 and Cy5 were only obtained from ileal flora, and Cy6 and Cy7 alone were observed in colonic bacteria. The results indicated that N→O group reduction was the main metabolic pathway of CYX metabolism in swine ileal flora, intestinal microsomes and mucosa. New metabolic profiles of hydrogenation and cleavage on the side chain were found in colonic bacteria. Among the identified metabolites, two new metabolites (Cy6, Cy7) were detected for the first time. These studies will contribute to clarify comprehensively the metabolism of CYX in animals, and provide evidence to explain the pharmacology and toxicology effects of CYX in animals.

In vitro metabolism of cyadox in rat, chicken and swine using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry

“…7(C)). The results suggested that the prevailing metabolic pathways of CYX observed in intestinal microflora were the reduction of the N→O group, hydroxylation, hydrolysis and double-bond-reduced reactions, which were consistent with the metabolism study of CYX in vitro by Xu et al [11]. Furthermore, the metabolites detected in intestinal microflora were almost polar-decreased and low molecular weight metabolites, conjecturing that drug effect and toxicity might be strengthened by intestinal microflora.…”

“…Among the identified metabolites, fourteen metabolites of CYX were detected by intestinal microflora of swine, eight of which were different from the report by Xu et al [11], with seven new metabolites (Cy15, Cy16, Cy19, Cy20, Cy21, Cy23, Cy24) detected for the first time. The possible metabolic pathways of CYX by rat, chicken and swine intestinal microflora were proposed (Fig.…”

“…Previous researches demonstrated that CYX could be converted to different metabolites, such as monocyadox, bidesoxycyadox, quinoxaline-2-carboxylic acid [8,9]. Two recently published papers have investigated the metabolism of CYX in the in vitro systems of animals and more novel metabolites recognized [10,11]. For better elucidation of the metabolic pathways and their relation to the toxicity and food safety, more comprehensive metabolism studies in animals need to be carried out.…”

Estimation of residue depletion of cyadox and its marker residue in edible tissues of pigs using physiologically based pharmacokinetic modelling

“…Once absorbed, CYA is extensively metabolized in the liver and intestine, producing a number of metabolites, with BDCYA (1,4-bisdesoxycyadox) being the main metabolite and designated as the marker residue in some jurisdictions (Liu et al, 2009;Xu et al, 2012;Wu et al, 2012). BDCYA, the N-oxide reduction product of CYA, is formed by reducing oxygen on both the N1 and N4 sites on the benzene moiety which would lead to DNA damage by intermediate radical production Badham et al, 2010).…”

The critical role of oxidative stress in the toxicity and metabolism of quinoxaline 1,4-di-N-oxides in vitro and in vivo

“…Xu et al revealed that when CYA was incubated with swine intestinal microsomes and mucosa in the presence of an NADPH-generating system and swine ileal flora and colonic flora, respectively, three N!O group reduced metabolites (detected in intestinal mucosa, ileal and colonic flora), one hydroxylation metabolite (detected in ileal flora), one hydrolysis metabolite of the amide bond (detected in ileal flora), a new hydrogenation metabolite (detected in colonic bacteria) and a new side-chain cleavage metabolite (detected in colonic bacteria) were identified, indicating that N!O group reduction was the main metabolic pathway of CYA metabolism in swine ileal flora, intestinal microsomes and mucosa (Xu et al, 2011).…”