The use of c8-octyl columns for the analysis of catecholamines by ion-pair reversed-phase liquid chromatography with...

The chromatographic behavior of norepinephrine (NE), epinephrine, dopamine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylacetic acid on octylsilane (C8) reversed-phase high-performance liquid chromatography columns was observed under various mobile phase conditions including manipulations of pH, pairing ion and methanol concentrations. The optimum isocratic conditions permitting quantitative resolution of these substances in minimum time and with maximum detector response were determined. Employing a pH 3.0-3.2 mobile phase comprising an aqueous buffer solution containing 0.1 M NaH2PO4, 0.1 mM EDTA, and 0.2 mM 1-octanesulfonate, admixed with a volume of methanol equal to 4% of the aqueous volume, the performances of the C8 columns compares favorably to that of the more widely used C18 columns. The column eluates were monitored with an amperometric detector utilizing a glassy-carbon flow-cell electrode. The detector response for Ne was 1.5-2.0 nA/ng and the baseline noise was as little as 0.002 nA thereby permitting quantitation of 5-pg levels or more in the injected samples. By coupling the liquid chromatographic system to a procedure which eliminates non-catechol contaminants from the neuronal and body fluid specimens by alumina adsorption of the catechols, a sensitive and dependable method was developed and employed for the determination of catechol levels in discrete regions of rat brain, cat spinal cord, and in human plasma.