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. 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 microl of rat plasma.
Tsunoda
M
M
Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Takezawa
K
K
Santa
T
T
Imai
K
K
D000818:Animals; D001774:Blood Chemical Analysis / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D002395:Catecholamines / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D008163:Luminescent Measurements; D008297:Male; D008676:Metanephrine / Q000097:blood; D009647:Normetanephrine / Q000097:blood; D010070:Oxalates; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D002395:Catecholamines; D005456:Fluorescent Dyes; D010070:Oxalates; D009647:Normetanephrine; D008676:Metanephrine; C044622:peroxyoxalate