An isocratic HPLC-ECD assay of urinary normetanephrine and metanephrine

Adarsh Kumar, Mahendra Kumar, B. J. Fernandez, K. Goodkin, Neil Schneiderman, C. Eisdorfer

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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 (5 ml) 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 0.2 μ filters, and injected into a 4 μ Nova-Pak, C18 column of the HPLC system. Mobile 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 +0.55 V 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 as 15 min. Six to eight urine samples could be extracted and assayed in one day.

Original languageEnglish
Pages (from-to)2257-2269
Number of pages13
JournalJournal of Liquid Chromatography
Volume18
Issue number11
StatePublished - Jan 1 1995

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ASJC Scopus subject areas

  • Molecular Medicine

Cite this

Kumar, A., Kumar, M., Fernandez, B. J., Goodkin, K., Schneiderman, N., & Eisdorfer, C. (1995). An isocratic HPLC-ECD assay of urinary normetanephrine and metanephrine. Journal of Liquid Chromatography, 18(11), 2257-2269.