An improved assay of urinary catecholamine metabolites, 3-methoxy-4-hydroxyphenylglycol and vanillylmandelic acid, using high performance liquid chromatography with electrochemical detection

An improved method for the measurement of norepinephrine metabolites, 3-methoxy-4-hydroxyphenylglycol (MHPG) and vanillylmandelic acid (VMA), in urine using High Performance liquid Chromatography with Electrochemical Detector is described. An aliquot of urine was charged with an internal standard, dihydroxybenzylamine (DHBA), and the conjugated metabolites were hydrolyzed enzymatically. The hydrolysed and the free metabolites were extracted in ethyl acetate, dried, and dissolved in an aqueous buffer. An aliquot of appropriately diluted extract was injected into reverse phase C₁₈ Nova-Pack, 4u column. Metabolites were eluted with a mobile phase containing citric acid-acetate buffer, sodium octyl sulfate and 5% methanol. Peaks were detected with an electrochemical detector. Integration and calculations were performed by a preprogrammed data module. The concentrations were determined using the ratio of the areas of peaks of metabolites to that of internal standard. A linear relationship was observed between of 20-100 ng VMA and 30-90 ng of MHPG, an intraassay coefficient of variance (%CV) was 8.6% and 3.2 for VMA and MHPG respectively. Interassay coefficient of variance was 10.3% for VMA, and 4.7% for MHPG. A total run time for each urine extract, including integration and calculations is less than 20 minutes. The method is specific, sensitive and therefore, can be used for assaying a number of samples for urinary metabolites of catecholamine.