Measurement of catecholamines in biological fluids by high-performance liquid chromatography: a comparison of fluorimetric with electrochemical detection
Causon, R.C.; Carruthers, M.E.
Journal of Chromatography 229(2): 301-309
1982
ISSN/ISBN: 0021-9673 PMID: 7096468 Document Number: 186892
An improved method for the determination of catecholamines in biological fluids, by reversed-phase high-performance liquid chromatography (HPLC) with fluorimetric detection is presented. The pH titration previously employed in the alumina extraction was abandoned in favor of the use of a molar excess of pH 8.5 Tris.sbd.HCl buffer. A novel lyophilization step serves to concentrate the catechols and by reconstituting in mobile phase, chromatography disturbances are minimized. The addition of 2 mM octanesulphonic acid to a citrate.sbd.phosphate mobile phase at pH 6.0 gave optimal resolution and sensitivity. The HPLC separation can improve the specificity of the trihydroxyindole reaction, to the extent of providing a reliable analytical method, was demonstrated and validated by the technique of HPLC with electrochemical detection. A correlation coefficient of 0.98 was obtained between the 2 techniques as applied to the measurement of urinary catecholamines. The HPLC-fluorimetric method was sensitive enough to measure 0.1 ng/ml of noradrenaline at a signal-to-noise ratio of 2.0. Application of the method to the quantitative determination of catecholamines in human urine, plasma and rat brain homogenates is demonstrated.