A method validated based on the European Union (EU) regulations for determining the presence of furazolidone, furaltadone,
nitrofurazone, nitrofurantoin and their corresponding metabolites AOZ, AMOZ, SC and AH in fish muscle was developed. Samples
were acid-hydrolyzed, treated with 2-nitrobenzaldehyde and extracted with ethyl acetate. The extracts were dried in a N2 stream
and redissolved in methanol:water (50:50, v/v). Analysis was performed by LC/ESI/MS/MS. This developed method carried limits
of quantification lower than 10 μg/kg for the nitrofurans and 1.0 μg/kg for the metabolites. It was observed that the decision limit
(CCα) ranged from 2.93 to 5.01 μg/kg for the nitrofurans and 0.19 to 0.43 μg/kg for the metabolites. The detection capability (CCβ)
was between 3.62 and 6.20 μg/kg for the nitrofurans and between 0.23 and 0.54 μg/kg for the metabolites. The linear calibration
curve parameters in the fortified fish muscle were between 1.0-100.0 μg/kg and 0.1-10.0 μg/kg for the nitrofurans and metabolites,
respectively.