Liquid chromatographic fluorescence determination of amino acids in plasma and urine after derivatization with phanquinone

Phanquinone (4,7-phenanthroline-5,6-dione) as a pre-column derivatization fluorogenic reagent for liquid chromatographic determination of primary amino acids in biological samples has been investigated. The derivatization reaction was carried out at 68°C both in presence of aqueous phosphate buffer (pH 8) for 30 min and without buffer for 60 min to allow the determination of basic amino acids (Orn, Lys, Arg). The resulting derivatives were separated under reversed-phase HPLC. The proposed method was validated and applied to the determination of a variety of amino acids directly in urine and after deproteinization with 5-sulphosalicilyc acid in plasma samples. The detection and quantitation limits were found in the range of 10-450 fmol and 35-1400 fmol, respectively.