Pre-Column Fluorescent Derivatization for High Pressure Liquid Chromatography with o-Phthalaldehyde: Separation of Urinary Catecholamines

Abstract

High-pressure liquid chromatography was used to separate the flourescent adducts formed from the reaction of o-phthalaldehyde with primary biogenic amines. Using precolumn derivatization and isocratic elution techniques fluorescent o-phthalaldehyde adducts can be detected and quantified with fluorometry in the low picogram range. The relative retention values of the o-phthalaldehyde adducts strongly depend on the pH of the eluent and percent organic solvent in the mobile phase. The method was applied to the analysis of free norepinephrine in pooled urine samples and in small-volume (2-h) urine collections obtained from thermally stressed subjects. Samples were treated with alumina, and the catecholamines, including internal standard 3,4-dihydroxy-benzylamine, eluted from it were reacted with o-phthalaldehyde prior to injection onto a reverse-phase column (octadecyl-silica stationary phase) with methanol/0.08 mol/liter acetic acid (50/50 by vol) as the mobile phase. Assay of pooled urine specimens (n = 10) for norepinephrine gave within-run and day-to-day coefficients of variation of 4.3 and 5.6% respectively. The use of o-phthalaldehyde as a pre-column derivatization agent for fluorometric determination of primary amines is rapid, sensitive and specific and applicable to many important biogenic amines.