HPLC-fluorescence determination of amino acids in pharmaceuticals after pre-column derivatization with phanquinone.
4,7-Phenanthroline-5,6-dione (phanquinone) was used as a fluorogenic labeling reagent in pre-column derivatization for the quality control of amino acids in pharmaceuticals. The amino acid adducts were efficiently separated by C12 RP high-performance liquid chromatography (HPLC) using a ternary mixture of triethylamine (TEA) phosphate buffer (pH 2.5, 0.05 M)-methanol- tetrahydrofuran (THF) as mobile phase by varying composition gradient elution and detected fluorometrically. The results obtained by the proposed method were compared statistically, by means of the Student's t-test and the variance ratio F-test, with those obtained by a rapid reference method, which involved o-phthaldialdehyde (OPA) as pre-column reagent; no significant difference was found. The stronger derivatization conditions (60 degrees C, pH 8, 60 min) required for the method with phanquinone are compensated by the major stability of derivatives and by the absence of fluorescent degradation products.
Gatti
R
R
Dipartimento di Scienze Farmaceutiche, Università di Bologna, via Belmeloro 6, 40126 Bologna, Italy. rita.gatti2@unibo.it
Gioia
M G
MG
Andreatta
P
P
Pentassuglia
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G
D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004364:Pharmaceutical Preparations / Q000032:analysis*; D010618:Phenanthrolines / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods
D000596:Amino Acids; D004364:Pharmaceutical Preparations; D010618:Phenanthrolines; C004902:phanquinone