Assay of urinary free catecholamines: Routine use in a clinical chemistry laboratory

Summary: The Bio-Rad HPLC method for the assay of urinary free catecholamines was employed during a period of 13 months, on samples of 720 subjects which were all normal but one with a pheochromocytoma diagnosis. The non-parametric reference limits were in accordance with those of other authors. Preliminary clean-up was carried out both in a standard urine and in the sample urine by means of a disposable micro-cation column prefilled with Bio-Rex 70 and ammonium acetate buffer which retain the weakly basic amines. Next epinephrine (E), norepinephrine (NE), dopamine (DA) and an internal standard (IS, N-methildopamine) were removed using ammonium pentaborate, a highly specific eluant. The final eluate was separated by HPLC on a Bio-Rad cation-exchange column, using 5% methanol in 0.06 M phosphate buffer (pH 5.2) as the mobile phase. Lyphochek normal and abnormal urine controls were elaborated with Lyphline Quality Control Program based on Westgard’s rules.Many amines may be retained on a carboxylic acid resin. However, very few compounds possess a cis-diol structure. In the absence of drug therapy, only three compounds are commonly seen in HPLC chromatograms in addition to E, NE, DA and IS. Two unidentified compounds elute before E. Alpha-methyldopa (Aldomet TM) is a common anti-hypertensive drug which forms two metabolites closely related to biogenic amines.Alpha-methylnorepinephrine elutes between E and NE. Alpha-methyldopamine coelutes with dopamine.The easy and quick method, giving good reproducibility and excellent clinic agreement, is suitable to pheochromocytoma differential diagnosis in clinical chemestry laboratories.