Simultaneous Determination of Homovanillic and Vanillylmandelic Acid by HPLC Using a Coulometric Detector with Renewable Glassy Carbon Microbeads Based Working Electrode

Homovanilic (HVA) and vanillylmandelic (VMA) acid are catecholamines metabolites commonly used as cancer biomarkers. New method for simultaneous determination of both these metabolites is presented, using recently developed flow-through coulometric detector based on glassy carbon microbeads. This application presents the potential opportunities of the above mentioned coulometric detector for the simultaneous determination of mixture of substances in complicated matrix in combination with gradient chromatography. Optimum conditions were set as follows: mobile phase consisting of acetonitrile (eluent A) and ten time diluted B-R buffer pH 3 (eluent B) with gradient program 5 % A (2 min), 5 % A to 25 % A (8 min), 25 % A (4 min), 25 % A to 95 % A (1 min), and 95 % A (4 min); flow rate of mobile phase 0.7 mL min⁻¹, detection potential +1.3 V, and injection volume 20 μL. Calibration dependences of both metabolites were linear from concentration 100 μmol L⁻¹ to their quantitation limits (i.e. 0.6 μmol L⁻¹ for HVA and 1.3 μmol L⁻¹ for VMA, respectively). Standard addition method was applied for the determination of these analytes in human urine. Obtained results are in agreement with results of HPLC method with UV detector.