Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching high-performance liquid chromatography with fluorescence detection.
An HPLC method for the direct and simultaneous determination of estriol 3- and 16-glucuronides in pregnancy urine is described. The method is based on direct derivatization of the glucuronic acid moiety in estriol glucuronides in urine with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution (or urine sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at 37 degrees C. The resulting fluorescent derivatives were separated by column-switching chromatography using a first column (YMC-Pack C4) for clean-up of the derivatives and a second column (YMC Pack Ph) for the complete separation of the derivatives. The derivatives were detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise ratio=3) for estriol 3- and 16-glucuronides were 150 and 180 fmol in a 5 microl of urine (14 and 17 ng ml(-1) urine), respectively. The present method is highly sensitive and simple without any clean-up such as conventional solid-phase extraction.
Iwata
T
T
Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
Hirose
T
T
Yamaguchi
M
M
D002851:Chromatography, High Pressure Liquid; D004964:Estriol / Q000031:analogs & derivatives* / Q000652:urine; D004966:Estrogens, Conjugated (USP) / Q000652:urine*; D005260:Female; D006801:Humans; D011247:Pregnancy / Q000652:urine*; D012680:Sensitivity and Specificity
D004966:Estrogens, Conjugated (USP); C012961:estriol-16 alpha-(beta-D-glucuronide); C022810:estriol 3-glucuronide; D004964:Estriol