A sensitive and selective isotope dilution liquid chromatography-electrospray ionization tandem massspectrometry (LC-ESIMS/MS) method is presented for the simultaneous analysis of the metabolitesof four nitrofuran veterinary drugs, that is, furazolidone, furaltadone, nitrofurantoin, and nitrofurazone,in honey samples. The method entails a combined hydrolysis of protein-bound drug metabolites andderivatization of the resulting metabolites with 2-nitrobenzaldehyde (NBA) during an overnightincubation, followed by a liquid-liquid extraction and a cleanup on a polymeric solid-phase extractioncartridge. Mass spectral acquisition is carried out in the positive ion mode by applying multiple reactionmonitoring (MRM) of three diagnostic transition reactions for each analyte under survey. A reliablequantification is obtained by the use of one deuterated analogue per analyte (NBA-
d4 derivative).The method has been validated in honey according to the European Union criteria for the analysisof veterinary drug residues in food. Expressed in underivatized nitrofuran metabolite concentrations,the decision limits (CC
) ranged within 0.07-0.46
g/kg, and the detection capabilities (CC
) werewithin 0.12-0.56
g/kg. The method has been successfully applied in a survey of honeys of variousgeographical origins, showing that furazolidone is the main nitrofuran antibiotic administered to treatbacterial diseases of bees.Keywords: Nitrofuran metabolites; honey; mass spectrometry; 1-aminohydantoin (AH); 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ); 3-amino-2-oxazolidinone (AOZ); semicarbazide (SC); furazolidone; furaltadone; nitrofurantoin; nitrofurazone; veterinary drugs