Countercurrent Chromatographic Separation of Catecholamine Metabolites from Urine

Abstract

Abstract A continuous-flow method is described for achieving high-resolution separations with liquid-liquid partition without the use of an inert support. The instrument consists of a 60-m coil of Teflon tubing (0.85 mm i.d.) mounted planetarily on a centrifuge arm. The coil is filled with the stationary phase and the mobile phase is pumped through the column in a direction opposing the flow of the stationary phase imposed by centrifugal force. With ethyl acetate as the stationary phase and an aqueous solution of NaCl (5 g/100 ml) and acetic acid (10 ml/100 ml) as the mobile phase, (3-methoxy-4-hydroxyphenyl)-hydroxyacetic acid (VMA) and (3-methoxy-4-hydroxyphenyl)-acetic acid (HVA), the major products of catecholamine metabolism, elute in 2 and 7 h, respectively. With a 280 nm-monitor on the effluent, abnormal amounts of these two compounds can be detected in 1 ml of urine. The fractions containing these compounds were analyzed by both gas—liquid and thin-layer chromatographic methods. HVA is essentially completely separated from other compounds. Although VMA is found with 3-8 other unknown compounds, none interferes with any of the colorimetric assays for VMA. Several abnormal urines from patients with various types of neurosecretory tumors were examined, and a distinctive pattern of urinary excretion of these compounds was seen for each type of case.