HPLC-ED determination of catecholamines and their metabolites in urine

High performance liquid chromatography (HPLC) with electrochemical detection (ED) was applied for the monitoring of catecholamines (epinephrine - E, norepinephrine - NE, dopamine - DA) and their main metabolites (homovanillic acid - HVA, vanillylmandelic acid - VMA) in urine samples of patients with the pheochromocytoma diagnosis. Both amperometric and colorimetric detectors were tested for the clinical applications and the results were compared. The optimization of separation conditions was worked out, especially the effects of mobile phase compositions (pH, ionic strength, organic modifier and ion-pair agent concentrations). Dependences of capacity ratio values k' on all optimized components of the mobile phases were evaluated. Chromatographic conditions were optimized for the suitable chromatographic resolution (Rij > 1.25). Extraction recoveries for liquid-liquid and solid-phase extractions have been worked-out. Detection limits for catecholamines in urine were in the range of 0.3-0.6 ng/ml and 10-20 ng/ml for HVA, VMA using coulometric detector.