Assay for dopamine beta-hydroxylase in rat serum and adrenal medulla by high-performance liquid chromatography with...

A highly sensitive method for the assay of dopamine beta-hydroxylase in rat serum and in sample solution prepared from rat adrenal medulla is described which employs high-performance liquid chromatography with fluorescence detection. Octopamine, formed enzymatically from the substrate tyramine, is separated by Dowex 50/-X4 column chromatography and oxidized with periodate to p-hydroxybenzaldehyde, which is then converted into a fluorescent compound with 2,2'-dithiobis(1-aminonaphthalene). The derivative, after extraction with n-hexane--chloroform, is separated by normal-phase chromatography on Alox T. The limit of detection for octopamine formed enzymatically is 10 pmol. This method requires as little as 5 microliter of rat serum.