The Development and Validation of an LC-MS/MS Method for the Determination of Glyphosate, AMPA, and Glufosinate in Honey Following FMOC-Cl Derivatization: Application to Italian Samples

Glyphosate-based herbicides are among the most widely used pesticides worldwide, but data on their occurrence in food products, particularly honey, remain limited. The analytical determination of glyphosate, its primary metabolite aminomethylphosphonic acid (AMPA), and glufosinate ammonium is technically challenging due to their high polarity and distinctive physicochemical properties, requiring the development of dedicated single-residue analytic methods. In this study, honey samples were prepared by derivatizing the target analytes with 9-fluorenylmethyl chloroformate (FMOC-Cl), followed by solid-phase extraction (SPE) using hydrophilic-lipophilic balanced (HLB) cartridges to improve matrix clean-up and enhance analytical sensitivity. Quantification was performed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The optimized method was validated according to the SANTE/11312/2021 guidelines, with all parameters, including sensitivity, linearity, mean recovery (accuracy), precision (RSDr), and limit of quantification (LOQ), meeting the required performance criteria. The validated method was applied to 126 honey samples of various botanical origins, representative of Italian production. The results indicated a frequent detection of glyphosate residues, although concentrations were generally low and remain below levels of regulatory concern.