Assessment of the stability of antimicrobials and resistance genes during short- and long-term storage condition: accounting for uncertainties in bioanalytical workflows

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Unravelling complexities in antimicrobial agent–microbe interactions in the context of antimicrobial resistance (AMR) requires robust analytical workflows accounting for all uncertainties. Temporal storage of wastewater samples under refrigerated or frozen conditions prior to chemical and biological analysis is widely used to facilitate laboratory routine but may affect stability of analytes over time. Yet, little knowledge exists regarding stability of biological and chemical determinants in environmental samples, which hampers validity of research outputs. This study examines, for the first time, the stability of 32 antimicrobials (AAs) including commonly used classes of antibiotics and their representative metabolites and variation of 5 antibiotic resistance genes (ARGs) (ermB, sul1, tetW, blaCTX-M, qnrS), as well as intI1 and 16S rRNA genes in a reference wastewater sample stored under freezing condition for up to 1 year. Ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) and quantitative PCR (qPCR) techniques were adopted to measure concentration of AAs and ARGs, respectively. Results suggested that parent compounds are less affected by freezing storage compared to the metabolites. β-Lactams, clindamycin, and N-desmethyl clindamycin are the most-affected compounds which were poorly recovered (34–67%) from the starting concentration. By contrast, sulfonamides, macrolides, quinolones, and azoles are generally stable under freezing condition. No consistent differences were observed in gene copies between fresh and frozen samples, and ermB and tetW showed the highest variabilities at 30% under freezing condition. Overall, this study adds to the current knowledge on environmental AMR monitoring and emphasises the need for standardised protocols for AMR monitoring in the wastewater samples. Graphical Abstract: [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)6027-6038
Number of pages12
JournalAnalytical and Bioanalytical Chemistry
Issue number24
Early online date1 Aug 2023
Publication statusPublished - 31 Oct 2023

Bibliographical note

Funding Information:
The support of Engineering and Wessex Water Services Ltd and EPSRC Impact Acceleration Account (Project number: EP/R51164X/1) and Wessex Water Services Ltd. Innovative Pathways Control Project (IPC Project) is greatly appreciated.

Funding Information:
This study was funded by the Medical Research Council and Newton Fund through a UK-CHINA AMR Partnership Hub Award (MR/S013717/1).


  • Antibiotic resistance genes
  • Antimicrobials
  • Freezing storage
  • Stability

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry


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