Underestimation of tetracycline antibiotic residues in chicken meat: The role of protein binding

Interesting variations in the analyte content were observed in chicken samples contaminated with tetracycline antibiotics (TCs) following pretreatment with various enzymatic hydrolysis before quantification by conventional analytical methods. Compared with untreated samples, the detectable contents of three TCs in protease-treated samples were 1.51 to 2.05 times higher, whereas lipase treatment did not significantly influence the contents. The marked changes following protease treatment confirmed the presence of protein-associated antibiotics. Infrared spectroscopy analysis indicated that the formation of protein-bound antibiotics resulted from non-covalent interactions between TCs and proteins. Further dissociation experiments determined that the intermolecular forces involved hydrogen bonding, hydrophobic interactions, and electrostatic attraction. Molecular docking substantiated these forces and detailed the binding mechanism at the molecular level. Moreover, the masking effect of protein binding on the determination of TCs was also evidenced in an additional 30 positive chicken samples, suggesting that the actual residue levels of TCs in protein-rich foodstuffs are underestimated.