Simultaneous Automatic Determination of Catecholamines and Their 3-O-Methyl Metabolites in Rat Plasma by High-Performance Liquid Chromatography Using Peroxyoxalate Chemiluminescence Reaction

A highly specific and sensitive automated high-performance liquid chromatographic method for the simultaneous determination of catecholamines (CAs; norepinephrine, epinephrine, and dopamine) and their 3-O-methyl metabolites (normetanephrine, metanephrine, and 3-methoxytyramine) is described. Automat...

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Bibliographic Details
Published in:Analytical biochemistry 1999-05, Vol.269 (2), p.386-392
Main Authors: Tsunoda, Makoto, Takezawa, Kazuko, Santa, Tomofumi, Imai, Kazuhiro
Format: Article
Language:English
Subjects:
Animals
Blood Chemical Analysis - instrumentation
Blood Chemical Analysis - methods
Blood Chemical Analysis - statistics & numerical data
catecholamines
Catecholamines - blood
chemiluminescence
Chromatography, High Pressure Liquid - instrumentation
Chromatography, High Pressure Liquid - methods
Chromatography, High Pressure Liquid - statistics & numerical data
Fluorescent Dyes
Luminescent Measurements
Male
metanephrine
Metanephrine - blood
normetanephrine
Normetanephrine - blood
Oxalates
Oxidation-Reduction
rat plasma
Rats
Rats, Sprague-Dawley
Reproducibility of Results
Sensitivity and Specificity
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Summary:A highly specific and sensitive automated high-performance liquid chromatographic method for the simultaneous determination of catecholamines (CAs; norepinephrine, epinephrine, and dopamine) and their 3-O-methyl metabolites (normetanephrine, metanephrine, and 3-methoxytyramine) is described. Automated precolumn ion-exchange extraction of diluted plasma is coupled with HPLC separation of CAs and their 3-O-methyl metabolites on an ODS column, postcolumn coulometric oxidation, fluorescence derivatization with ethylenediamine, and finally peroxyoxalate chemiluminescence reaction detection. The detection limits were about 3 fmol for norepinephrine, epinephrine, and dopamine, 5 fmol for normetanephrine, and 10 fmol for metanephrine and 3-methoxytyramine (signal-to-noise ratio of 3). Fifty microliters of rat plasma was used and 4-methoxytyramine was employed as an internal standard. The relative standard deviations for the method (n= 5) were 2.5–7.6% for the intraday assay and 6.3–9.1% for the interday assay. The method was applicable to the determination of normetanephrine and metanephrine in 50 μl of rat plasma.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1999.4043