Identification of Erythromycin and Clarithromycin Metabolites Formed in Chicken Liver Microsomes Using Liquid Chromatography–High-Resolution Mass Spectrometry

Abstract

Nontargeted analysis can be used for the rapid screening and confirmatory analysis of veterinary drugs and their metabolites, which are important for the comprehensive safety evaluation of animal-derived foods. Here, a novel nontargeted screening approach based on liquid chromatography coupled with electrospray ionization–high-resolution mass spectrometry (LC/ESI–HR-MS) was developed to determine erythromycin, clarithromycin, and their metabolites in chicken liver microsomes. Erythromycin and clarithromycin were incubated in vitro in the presence of NADPH for 60 min to generate metabolites in chicken liver microsomes. After the incubation, the supernatant was extracted using ultrasonic shaking, orbital shaking, and centrifugation before analysis using LC/ESI-HR-MS in positive ion mode on an Agilent Eclipse Plus C18 column (100 mm × 2.1 mm; i.d. 3.5 µm) with 0.1 percent formic acid-water and acetonitrile as the mobile phases for gradient elution at 0.4 mL/min. The results show that erythromycin can produce N-desmethyl-erythromycin A in chicken liver microsomes, but clarithromycin cannot produce N-desmethyl-clarithromycin in chicken liver microsomes. The N-desmethyl-erythromycin A and N-desmethyl-clarithromycin were tentatively identified in chicken liver microsomes using the established quick analytic method, which will provide a theoretical foundation for future research on pharmacokinetics and drug elimination in poultry.

Quantitative analysis of erythromycin, its major metabolite and clarithromycin in chicken tissues and eggs via QuEChERS extraction coupled with ultrahigh-performance liquid chromatography-tandem mass spectrometry

“…Various analytical methods, including HPLC–MS/MS ( Campanharo et al, 2023 ), HPLC with UV diode array detection (DAD) ( Cañadas et al, 2022 ), electrochemical sensors ( Lai et al, 2023 , Li et al, 2022 ), TLC densitometric methods ( Sharkawi et al, 2023 ), and UHPLC–MS/MS ( Du et al, 2021 ), have been used for identifying ERY, CLA and other MACs in fish, milk, honey, egg and animal tissues. In previous studies, Wang et al (2021 ) developed an LC with a time-of-flight (ToF)-MS/MS method to verify whether ERY and CLA can produce N -desmethyl metabolites in chicken liver microsomes. The results showed that ERY produced N - d -ERY in chicken liver microsomes after incubation for 60 min and after an NADPH cofactor was added.…”

“…Both erythromycin A (ERY) and clarithromycin (CLA), two MACs that share comparable antibacterial processes and have an impact on both gram-positive and gram-negative bacteria, are members of the same class ( Juan et al, 2010 ). In our previous study, the major metabolite of ERY in chicken liver microsomes was determined to be N -desmethyl-erythromycin A ( N - d -ERY) ( Wang et al, 2021 ). Reports have indicated that N - d -ERY does not possess antibacterial activity; however, N - d -ERY represents a potential risk to individuals ( Lundquist et al, 2014 , Sun et al, 2022 ).…”

Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site

“…At least 500 kinds of macrolide antibiotics are known, most of which are derived from Streptomyces species. The frequently used macrolide antibiotics are erythromycin [ 1 ], oleandomycin [ 2 , 3 ], josamycin [ 4 , 5 ], midecamycin [ 4 , 6 ], spiramycin [ 7 , 8 ], roxithromycin [ 9 , 10 ], azithromycin [ 11 , 12 ], and clarithromycin [ 13 , 14 ]. Macrolides exhibit high activity against Gram-positive bacteria, and are thus used widely as excellent bacteriostatic agents due to their low toxicity and broad-spectrum activities.…”