Development and Optimization of Extraction Methodologies for the Determination of Antibiotics in Honey by...

The administration of antibiotics to food-producing animals, including bees, is a common practice employed to prevent or treat bacterial diseases. Such practices may result in the presence of veterinary residues in honey, potentially compromising the safety and health of consumers. In the European Union, maximum residue limits (MRLs) have not been established for pharmacological substances in honey. In this context, the development of accurate and robust analytical methodologies is of paramount importance for appropriate risk assessment.

This study aimed to develop and optimize a multi-class method for the determination of veterinary drug residues from seven classes (β-lactams, lincosamides, macrolides, quinolones, sulfonamides, tetracyclines, and diaminopyrimidines) using ultrahigh-performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC-ToF-MS).

A preliminary assessment was conducted to evaluate the feasibility of solid-liquid extraction, given the complex matrix of compounds present in honey, by analyzing different solvent solutions in acetonitrile, methanol, water, and/or McIlvaine buffer. The subsequent phase of the study involved the optimization of a purification/cleanup method. This was achieved by comparing the efficacy of several solid-phase extraction (SPE) and QuEChERS protocols.

The results indicated that the most efficacious extraction were acetonitrile-based solutions, specifically acetic acid 1% and formic acid 0.1% in ACN-H2O (80:20, v/v). Finally, the method with the most optimal analytical performance in terms of recovery and matrix effect entailed an initial extraction step with formic acid 0.1% in ACN-H2O (80:20, v/v), followed by a modified QuEChERS protocol.

Acidified organic solvents in combination with QuEChERS method revealed to be the most effective for the determination of antibiotics in honey, ensuring optimum extraction with minimum matrix interference.

The developed analytical protocol with further detection by high-resolution mass spectrometry will be important to assess health risks and to add value to the consumption of honey and honey-based products.