Objective: Catecholamines and their metabolites are used for diagnosis of pheochromocytoma. Our communication deals with validation of methods for determination of normetanephrine (NMN) and metanephrine (MN) in plasma and urine. Settings: Institute of Clinical Biochemistry and Diagnostics, Medical Faculty Hospital, Charles University, Hradec Kralové, Czech Republic Material and Methods: HPLC method with electrochemical detection (ED) was used. Separation conditions was as follows: mobile phase 1 mol/l NAH2PO4,0.34 mmol/l 1-octansulfonic acid sodium salt, 0.13 mmol/l EDTA, 75 ml/l acetonitrile, C18 RP analytical column, flow rate 1 ml/min. Detection conditions (electrochemical detector ESA Coulochem II): conditioning cell with potential 370 mV, analytical cell 150 and -390 mV. Sample preparation: addition of 4-hydroxy-3- -methoxy-benzylammoniumchloride as internal standard, isolation and purification were performed by solid-phase extraction (Oasis MCX, Waters). Sorbent was conditioned with methanol and water and washed by 0.1 mol/l HCl and methanol. For sample elution 5% ammonium hydroxide in methanol was used. Results: Average of repeatability 7.4% and reproducibility 8%. Recovery values ranged from 94–111%. We also calculated inter-in reference intervals. Plasma values were: 0.1–0.61 nmol/l for NMN and 0.06–0.31 nmol/l for MN. Urine values: 0.480–2.400 μmol/24 h for NMN and 0.264–1.440 μmol/24 h for MN. Conclusion: Rapid and precise method for determination of catecholamine metabolites in plasma and urine was successfully validated.

        Key words: metanephrine, normetanephrine, pheochromocytoma, HPLC-ED.