E. Suzuki et al., ASSAY OF ENZYMATIC O-METHYLATION OF CATECHOL ESTROGENS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH COULOMETRIC DETECTION, Journal of chromatography. Biomedical applications, 617(2), 1993, pp. 221-225
A simple and sensitive method for the determination of guaiaicol oestr
ogens enzymically formed from 2- or 4-hydroxyoestradiol, by menas of h
igh-performance liquid chromatography with coulometric detection, has
been developed. Catechol and guaiacol oestrogens were efficiently sepa
rated on a reversed-phase column, using 0. 5% ammonium phosphate buffe
r (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
0-methylation of catechol oestrogens. After 2- or 4-hydroxyoestradiol
had been incubated with rat red blood cells in the presence of S-aden
osylmethionine, the resulting guaiacols and unchanged substrate were p
ercolated through an Extrelut-3 cartridge. The dried eluate was rediss
olved and directly injected. This simple procedure was as sensitive as
the previously reported method using gas chromatography-mass spectrom
etry in a selected ion monitoring mode.