Assay of Biogenic Monoamines in Milk by Fluorescence-LC Following Derivatization with Naphthalene-2,3-dicarboxaldehyde
Maria Notou and Anastasia ZotouAffiliation:
Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, GR – 541 24 Thessaloniki, Greece.
AbstractA 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.
Biogenic amines, derivatization, fluorescence, HPLC, milk, naphthalene-2, 3-dicarboxaldehyde.
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