Sensitive liquid chromatographic method using fluorescence detection for the determination of estradiol 3- and 17-glucuronides in rat and human liver microsomal incubations: formation kinetics.
We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20-4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis-Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.
Alkharfy
Khalid M
KM
Department of Pharmaceutical Sciences, University of Pittsburgh, 807 Salk Hall, Pittsburgh, PA 15261, USA.
Frye
Reginald F
RF
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004958:Estradiol / Q000378:metabolism*; D020719:Glucuronides / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D020719:Glucuronides; D004958:Estradiol