Assay of enzymic O-methylation of catechol oestrogens by high-performance liquid chromatography with coulometric detection.
A simple and sensitive method for the determination of guaiaicol oestrogens enzymatically formed from 2- or 4-hydroxyoestradiol, by means of high-performance liquid chromatography with coulometric detection, has been developed. Catechol and guaiacol oestrogens were efficiently separated on a reversed-phase column, using 0.5% ammonium phosphate buffer (pH 3.0)-acetonitrile (59:41, v/v) as the mobile phase, and detected coulometrically in a screening-oxidation mode at +0.10 V and +0.35 V, respectively. The method was applied to the assay of in vitro enzymic O-methylation of catechol oestrogens. After 2- or 4-hydroxyoestradiol had been incubated with rat red blood cells in the presence of S-adenosylmethionine, the resulting guaiacols and unchanged substrate were percolated through an Extrelut-3 cartridge. The dried eluate was redissolved and directly injected. This simple procedure was as sensitive as the previously reported method using gas chromatography-mass spectrometry in a selected ion monitoring mode.
Suzuki
E
E
Hatano Research Institute, Food and Drug Safety Center, Kanagawa, Japan.
Saegusa
K
K
Matsuki
Y
Y
Nambara
T
T
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D004912:Erythrocytes / Q000737:chemistry; D002393:Estrogens, Catechol / Q000097:blood*; D008297:Male; D008745:Methylation; D051381:Rats; D011918:Rats, Inbred SHR; D012680:Sensitivity and Specificity
D002393:Estrogens, Catechol