Assay of Biogenic Monoamines in Milk by Fluorescence-LC Following Derivatization with Naphthalene-2,3-dicarboxaldehyde

A new, simple HPLC method is described for the isocratic separation of biogenic monoamines, in milk samples, in the presence of hexylamine as the internal standard, after pre-column derivatization with naphthalene-2,3- dicarboxaldehyde /cyanide ions. The derivatization reaction variables including buffer pH and concentration, reaction time, naphthalene-2,3-dicarboxaldehyde (NDA) and cyanide concentrations were optimized. The isoindoles formed were eluted by means of methanol/water (80:20, v/v), at a flow rate of 1 mL min-1, on an Inertsil ODS-3 column (250 x 4 mm i.d., 5 µm), in less than 30 min. Fluorometric detection was used, at excitation and emission wavelengths of 424 and 494 nm, respectively. The limits of detection were in the pg level (7 – 80 pg) using a 10 µL-loop. The whole procedure was evaluated and fully validated for the determination of biogenic amines in milk samples, after ultrasound-assisted liquidliquid extraction. The intra-assay precision and accuracy were calculated in samples spiked with different levels of biogenic amines and yielded RSD and recovery values in the range 0.3 – 4.2% and 82.5 – 103.8% respectively.