| Summary: | Monitoring of biogenic amines in food is important for quality control, in terms of freshness evaluation and even more for food safety. A novel and cost-effective method was developed and validated for the determination of the main biogenic amines: histamine, putrescine, cadaverine, spermidine and spermine in fish tissues. The method includes extraction of amines with perchloric acid, pre-column derivatization with Pyrene Sulfonyl Chloride (PSCl), extraction of derivatives with toluene, back-dissolution in ACN after evaporation and determination by reversed phase high performance liquid chromatography with UV and intramolecular excimer fluorescence detection. The structure of the pyrene-derivatives was confirmed by liquid chromatography–mass spectrometry with electrospray ionization. The standard addition technique was applied for the quantitation due to significant matrix effect, while the use of 1,7-diaminoheptane as internal standard offered an additional confirmation tool for the identification of the analytes. Method repeatability expressed as %RSD ranged between 7.4–14% for the different amines and recovery ranged from 67% for histamine up to 114% for spermine. The limits of detection ranged between 0.1–1.4 mg kg<sup>−1</sup> and the limits of quantification between 0.3–4.2 mg kg<sup>−1</sup>. The method was applied to canned fish samples and the concentrations of the individual biogenic amines were below the detection limit up to 40.1 mg kg<sup>−1</sup>, while their sum was within the range 4.1–49.6 mg kg<sup>−1</sup>.
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