Tetracyclines in Processed Animal Proteins: A Monitoring Study on Their Occurrence and Antimicrobial Activity

Abstract

In 2013, the European Union (EU) lifted the feed ban restriction, authorizing the use of non-ruminant (NR) processed animal proteins (PAPs) as ingredient in aquafeed. A further relaxation is soon expected, and NR PAPs will be allowed in next future in poultry and pig feed, avoiding cannibalism. Other potential hazards linked to PAPs as raw material should be evaluated. Antibiotics administered along the lifecycle of animals may leave residue in tissues and bones and still be present in PAPs. This monitoring study aimed to determine tetracyclines (TCLs), known to cumulate in bones, in PAPs and their possible residual antibiotic activity (RAC). A sensitive Liquid Chromatography coupled to Tandem Mass Spectrometry (LC-MS/MS) method for the quantification of TCLs in PAPs was developed and applied to 55 PAPs from EU manufactures. Most PAP samples (n = 40) contained TCLs (concentrations 25.59 ÷ 456.84 µg kg−1). Among samples containing more than 25 µg kg−1 for at least three TCLs, three PAPs were chosen for RAC test before and after TCLs extraction procedure applying an in vitro acidic digestion: in two out of those three samples, RAC was observed after in vitro digestion. TCLs were determined in the digested PAPs (concentrations 26.07 ÷ 64.55 µg kg−1). The detection of TCLs in PAPs should promptly target the risk assessments of this unconsidered way of exposure to antibiotic residues.

Optimization and Validation of a Quick and Responsive LC-ESI-MS/MS Method to Evaluate Tetracyclines Residues in Processed Animal Proteins (PAPs)

“…Preliminary data [24] reveal a residual antimicrobial activity in PAPs, which represents a possible involvement of this raw material in the development of antimicrobial resistance. Since Regulation (CE) n° 1372/2021 allows the use of insect PAPs, gelatine and collagen from non-ruminant, a validation study should be carried out to extend the proposed method to the assessment of the risk associated with these matrixes as well.…”

“…It is well known that TCL, OTC and CTC are not stable and can undergo epimerization to 4-epiTCL, 4-epiOTC, 4-epiCTC during extraction procedure, especially due to pH change from 3 to 5, while DOC seems not to epimerize under analytical condition described (Fig. 1a-d) [24]. The chromatographic condition and the LC column allow the separation of TCLs and their 4-epimers while the correct identi cation and quantitation is carried out thanks to the difference in retention time and in ion ratio between product ions.…”

“…To perform matrix calibration for TCLs quanti cation, ve aliquots (4.0 ± 0.5 g) of blank sample were spiked with working solution (40 ng µL − 1 ) at concentration of 25, 50, 100, 250, 500 µg Kg − 1 . The calibration levels and real samples were treated as already reported in previous research [24].…”

“…The 5500 QTRAP system (AB Sciex, Milan, Italy) operated in ESI positive ion mode with the enhanced source parameters byMorello et al 2021. The quantitative analyses were carried out in Multiple Reaction Monitoring (MRM).…”