Determination of estrogens in plasma by high-performance liquid chromatography after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole.
A sensitive method for the determination of estrogens by high-performance liquid chromatography with fluorimetric detection was reported. The estrogens were derivatized with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole to their esters in the presence of 4-piperidinopyridine and 1-isopropyl-3-(3-dimethylaminopropyl)carbodiimide perchlorate. The resulting esters were extracted with a Sep-Pak C18 plus cartridge, and then esters were separated on a Wakosil 5C18 column with water-methanol (10:90, v/v) as the mobile phase. The esters were detected by fluorimetric detection (excitation wavelength = 336 nm, emission wavelength = 440 nm). The within-day relative standards deviations (n = 6) for each estrogen (1.0 ng per 100 microliters of plasma) were 8.7-13.0%, and day-to-day relative standard deviations (n = 6) were 8.3-11.8%. The limits of detection for estrogens (estrone, estradiol, estriol, estetrol, ethynyl-estradiol, 2-hydroxyestradiol, 4-hydroxyestradiol) were 0.1-0.2 pg per 100 microliters of plasma (signal-to-noise ratio 3).
Meiji College of Pharmacy, Tokyo, Japan.
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M
Katayama
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H
Taniguchi
D001562:Benzimidazoles; D001565:Benzoates; D002851:Chromatography, High Pressure Liquid; D004967:Estrogens / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D013050:Spectrometry, Fluorescence
C080307:2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole; D001562:Benzimidazoles; D001565:Benzoates; D004967:Estrogens; D007202:Indicators and Reagents