Rapid assay for catechol-O-methyltransferase activity by high-performance liquid chromatography-fluorescence detection.
A rapid assay for measuring the activities of catechol-O-methyltransferase (COMT) is described. The method is based on high-performance liquid chromatography (HPLC)-fluorescence detection, and includes on-line extraction of catecholamines with a precolumn, separation of norepinephrine (NE) and normetanephrine (NMN) on an ODS column, electrochemical oxidation, and post-column fluorogenic derivatization using ethylenediamine. The method took less than 25 min for one sample, which is half that of the previous method and the sensitivity was similar. The intra-day assay precisions were 0.52-1.6%, and the inter-day assay precisions were 3.6-5.8% for rat liver and cerebral cortex (n = 5). The method is suitable for the rapid measurement of COMT activities of many biological samples.
Hirano
Yoko
Y
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Tokyo 113-0033, Japan.
Tsunoda
Makoto
M
Funatsu
Takashi
T
Imai
Kazuhiro
K
D000818:Animals; D002394:Catechol O-Methyltransferase / Q000032:analysis* / Q000302:isolation & purification; D002540:Cerebral Cortex / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D008566:Membranes / Q000201:enzymology; D009638:Norepinephrine / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods
D002394:Catechol O-Methyltransferase; D009638:Norepinephrine