335 zyxwvut Journal of Chrvmatogmphy, 225 (1981) 335-345 Biomedical Applications Ekevier Scientific Publishing Company, Amsterdam -Printed in The Netherlands cHRoMmo_ 972 THE USE OF C&KFYL COLUMNS FOR THE ANALYSIS OF CATECHOLAMINE S BY ION-PAIR REVERSED-PHASE LIQUID CHROMATOGRAPHY WITH AMPEROMETRIC DETECTION _ RICHARD L. MICHAUD, MICHAEL J_ BANNON and ROBERT H_ ROTH* Departments of Pharmacology and Prychiairy, Yale University School of Medicine, New Haven. CT 06510 (USA_) (First received February 6th, 1981; revised manuscript received May 21st, 1981) SUMMARY The chromatographic behavior of norepinephrine (NE), epinephrine, dopamine, 3,4- dihydroxypheny!alanine, and 3,4_dihydroxyphenylacetic acid on octylsilane (C,) 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 3.1 M NaH,PO,, 0.1 m&f EDTA, and O-2 m&f l-octanesulfonate, admixed with a volume of methanol equal to 4% of the aqueous volume, the performance of the Cl, columns compares favorably to that of the more widely used C,, columns_ The column eluates were monitored with an amperometric detector utilizing a glassycarbon 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 per- mitting quantitation of 5-pg levels or more in the injected samples_ By coupling the liquid chromatographic system to a procedure which eliminates noncatechol 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 zyxwvutsrqponmlkjihgfedcbaZ plasma. INTRCDUCTION : Ionexchange [l-6] and reversed-phase [6-121 high-performance liquid chnkatographic (HPLC) techniques have been used successfuliy to resolve the catecholamines and their related metabolites in extracts of tissues and in body fluids. Reversed-phase chromatography appears to- be most useful in the analysis of mixtures of such substances which possess b&h acidic and basic organic functional groups [S] _ The most widely used reversed-phase methods 0378/~347/81/0000-9000/$02.50 0 1981 Elsevier Scientific Publishing Company