An Isocratic HPLC-ECD Assay of Urinary Normetanephrine and Metanephrine

Abstract

This report describes a procedure to quantify simultaneously urinary normetanephrine (NMTN) and metanephrine (MTN) using an isocratic HPLC-ECD methodology. An aliquot of urine (5ml) was adjusted to pH of 1.0, spiked with a known concentration of internal standard, (3-methoxy-4-hydroxy benzylamine, MHBA) and the two metabolites were hydrolyzed in a boiling water bath. The metabolites were adsorbed on a Biorex-70 column and eluted with ammonium hydroxide. Final extraction was carried out in a mixture of ethyl acetate and acetone (2:1, v/v). After drying the extract under nitrogen, it was dissolved in mobile phase, filtered through 0.2μ filters, and injected into a 4 μ Nova-Pak, C₁₈ column of the HPLC system. Mobil phase for elution contained citric acid, sodium acetate, EDTA-Na₂, sod. octyl sulfate, dibutylamine, methanol 2% and isopropanol 2%. Peaks were detected by the electrochemical detector at a potential of + 0.55V and characterized using the retention times obtained from HPLC profiles of the standards. Calibration of HPLC was performed by spiking 5 ml of metabolite free urine (MFU) with known amount of standards of NMTN, MTN and MHBA and injecting the extract to obtain chromatographic profile. Concentrations of the metabolites were calculated on a pre-programmed data module using ratio of the areas of the analytes to that of the internal standard. This assay showed a linear relationship between 5–80 ng/ml for NMTN and 5–90 ng/ml MTN. Sensitivity of the method was below 5.0 ng/ml. The total elution time was 15 min. Six to eight urine samples could be extracted and assayed in one day.