Analysis of amino acids and biogenic amines in biological tissues as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate derivatives by high-performance liquid chromatography. A deproteinization study

Abstract

The extraction of ornithine, lysine, putrescine, cadaverine, 1,7-diaminoheptane, spermidine and spermine from biological tissues was optimized for HPLC quantitation as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate (OPA/ET/FMOC) derivatives. In applying perchloric acid deproteinization two approaches have been followed: (i) deproteinization with subsequent neutralization by potassium hydroxide and lyophilization, and (ii) deproteinization without neutralization and lyophilization. Neutralization and lyophilization resulted in the loss of free biogenic amines. HPLC analysis of ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Dah), spermidine (Spd) and spermine (Spm) content of biological tissues as their OPA/ET/FMOC derivatives was performed in the supernatant of perchloric acid-deproteinized samples (model solutions and tissues) with an average reproducibility of < or =2.6% relative standard deviation (RSD), including recovery of sample treatment and chromatography.