Improved assay for catechol-O-methyltransferase activity utilizing norepinephrine as an enzymatic substrate and reversed-phase high-performance liquid chromatography with fluorescence detection.
We have previously established a rapid catechol-O-methyltransferase (COMT) assay using norepinephrine (NE) as a natural substrate and flow-injection analysis. In this study, the method is improved for screening of COMT inhibitors or activators using reversed-phase high-performance liquid chromatographic separation with fluorescence detection. The excess substrate, NE, was removed by the addition of borate in the eluent for HPLC to make an ionic complex with NE, which was eluted faster than the enzymatic product, normetanephrine. The method had good precision and accuracy, and was able to assay one sample in 5 min, showing the usability for screening of COMT inhibitors or activators.
Aoyama
Nozomi
N
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Tsunoda
Makoto
M
Imai
Kazuhiro
K
D000818:Animals; D002394:Catechol O-Methyltransferase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D008099:Liver / Q000201:enzymology; D009638:Norepinephrine / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D002394:Catechol O-Methyltransferase; D009638:Norepinephrine