Automated derivatization and fluorimetric determination of biogenic amines in milk by zone fluidics coupled to liquid chromatography

A novel zone-fluidics/high pressure liquid chromatographic (ZF-HPLC) method is described for the simultaneous determination of six biogenic monoamines, in the presence of hexylamine as internal standard. Automated on-line derivatization of the analytes with naphthalene-2,3-dicarboxaldehyde/cyanide ions was performed using the ZF concept and the derivatives were injected on the HPLC column for separation/detection. The influence of the ZF operation conditions on the derivatization reaction was investigated. The isoindoles formed were separated on a Kromasil C18 column (250 mm × 4 mm i.d., 5 μm), using an isocratic mobile phase of methanol/water (80:20, v/v) at a flow rate of 1.0 mL min−1. Monitoring and quantification was carried out by fluorescence detection at 424/494 nm. The limits of detection were at the pg level with a sample volume of 20 μL. The whole procedure was evaluated and fully validated for the determination of biogenic amines in milk samples.