Measurement of catecholamines in biological fluids by high-performance liquid chromatography: a comparison of...

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 favour of the use of a molar excess of PH 8.5 Tris--HCl buffer. A novel lyophilisation step serves to concentrate the catechols and by reconstituting in mobile phase, chromatography disturbances are minimised. The addition of 2 mM octanesulphonic acid to citrate--phosphate mobile phase at pH 6.0 gave optimal resolution and sensitivity. That HPLC separation can improve the specificity of the trihydroxyindole reaction, to the extent of providing a reliable analytical method, has been demonstrated and validated by the technique of HPLC with electrochemical detection. A correlation coefficient of 0.98 was obtained between the two techniques as applied to the measurement of urinary catecholamines. The HPLC--fluorimetric method was sensitive enough to measure 0.1 ng/ml of noradrenaline or adrenaline 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.