Non-targeted workflow for identification of antimicrobial compounds in animal feed using bioassay-directed screening in combination with liquid chromatography-high resolution mass spectrometry

Abstract

A non-targeted workflow is reported for the isolation and identification of antimicrobial active compounds using bioassay-directed screening and LC coupled to high-resolution MS. Suspect samples are extracted using a generic protocol and fractionated using two different LC conditions (A and B). The behaviour of the bioactive compound under these different conditions yields information about the physicochemical properties of the compound and introduces variations in co-eluting compounds in the fractions, which is essential for peak picking and identification. The fractions containing the active compound(s) obtained with conditions A and B are selected using a microbiological effect-based bioassay. The selected bioactive fractions from A and B are analysed using LC combined with high-resolution MS. Selection of relevant signals is automatically carried out by selecting all signals present in both bioactive fractions A and B, yielding tremendous data reduction. The method was assessed using two spiked feed samples and subsequently applied to two feed samples containing an unidentified compound showing microbial growth inhibition. In all cases, the identity of the compound causing microbiological inhibition was successfully confirmed. ARTICLE HISTORY

Bioassay-directed analysis-based identification of relevant pyrrolizidine alkaloids

“…Bioassay-directed identification approaches have been applied to identify chemicals with divergent bioactivity, such as endocrine activity [e.g. estrogenic activity (Nielen et al 2004 ) and anti-androgenic activity (Rostkowski et al 2011 )], and antimicrobial activity (Wegh et al 2017 ). Furthermore, application of a strategy based on broad screening and bioassay-directed identification with liquid chromatography high-resolution mass spectrometry (LC–HRMS) has been proposed to prevent intoxications and identify toxins and toxicants relevant for food and feed safety (Gerssen et al 2019 ).…”

Triclosan is the Predominant Antibacterial Compound in Ontario Sewage Sludge

“…EDA has been widely used to identify toxic contaminants binding to nuclear receptors − or exerting oxidative stress . This strategy has been largely expanded through the development of high-resolution mass spectrometry-based nontargeted analysis. , A recent study applied EDA to identify antimicrobials in animal feed, wherein didecyldimethylammonium chloride was identified and confirmed by an authentic standard . However, the EDA strategy is prone to high false discovery rates due to the coelution of hundreds of compounds in the same fractions.…”

“…20,22 A recent study applied EDA to identify antimicrobials in animal feed, wherein didecyldimethylammonium chloride was identified and confirmed by an authentic standard. 24 However, the EDA strategy is prone to high false discovery rates due to the coelution of hundreds of compounds in the same fractions. A previous study from our group proposed an alternative workflow involving a protein affinity pulldown system combined with untargeted chemical analysis (PUCA) to directly isolate and identify bioactive chemicals using His-tagged proteins.…”