A method for simultaneous quantitative determination of ethoxyquin (EQ) and its major metabolite in Atlantic salmon tissues, ethoxyquin dimer (EQ dimer), has been developed. The separation was achieved on tandem coupled phenyl-hexyl and C18 columns by 2-phase gradient elution with acetonitrile-ascorbic acid-acetic acid-diethyl amine organized in a 23.5 min sequence. Compounds were extracted with hexane from samples saponified in ethanol-NaOH and protected from air- and light-mediated oxidation by addition of saturated ethylenediaminetetraacetic acid, ascorbic acid, and pyrogallol. The identity of peaks was confirmed by spiking samples with standards verified by proton nuclear magnetic resonance spectrometry, mass spectrometry, and high-performance liquid chromatography. The detection limit (at 358/433 nm) of matrix-spiked EQ was 0.02 and 0.06 microg/L for EQ dimer, with 0.5 g sample weighed and resuspension in 0.5 mL hexane. Linearity was in the range of 0.2-175 microg/L for EQ and 0.3-5100 microg/L for EQ dimer. Two more ubiquitous compounds were identified as de-ethylated EQ and quinone imine. Totally, 14 peaks sharing spectral properties of EQ were separated in a single run, including a major peak present in all muscle samples, termed unknown metabolite of EQ (UMEQ). The concentrations of EQ, EQ dimer, and de-ethylated EQ, as well as concentrations of UMEQ (in arbitrary units), in the muscle were correlated to the amount of EQ fed to the salmon, thus indicating their possible metabolic origin. The pattern of 14 peaks in the muscle showed high specificity and could be used to discriminate between wild salmon and salmon fed EQ-supplemented feed. This method will be a useful tool for studying EQ metabolism and kinetics, and for the routine surveillance of residual levels of dietary EQ in farmed Atlantic salmon.