Abstract
This paper describes a method for simultaneous extraction and concurrent quantification of metabolites of norepinepohrine (NE) and 5-hydroxytryptamine (5-HT) in human urine using an isocratic HPLC-ECD technique. These metabolites include 4-hydroxy-3-methoxy mandelic acid (VMA), 3-methoxy-4-hydroxyphenylglycol (MHPG), normetanephrine (NMTN), metanephrine (MTN), and 5-hydroxyindoleacetic acid (5-HIAA). The samples of urine were passed through preconditioned alumina-B column and washed with diethylether to remove interfering substances. Metabolites were extracted in ethylacetate.
The extracts were evaporated under nitrogen, reconstituted in water and injected into a 5μ spherical, C
18
, reverse phase column for HPLC-ECD analysis. The analytes were eluted with mobile phase containing citric acid, sodium acetate, sodium octyl sulfate, Na
2
EDTA, methanol 10%, and isopropanol 0.5%. Isoproterenol was used as an internal standard. Electrochemical detection was carried out at a potential of +0.60 V and a flow rate of 0.5 mL/minute. Peaks were characterized by their retention times. Concentrations were determined by the method using ratio of the peak areas of metabolites to that of internal standard.
A linear relationship between the ratio of the areas and concentrations was obtained between 5.0-90 ng/mL of each VMA, MHPG, NMTN, and MTN. The sensitivity for 5-HIAA was found to be ten times higher with a linear relationship between 1.0-8.0 ng/mL. Total elution time for all the metabolites was less than 20 minutes.
