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

J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Jul 5;774(1):33-8. doi: 10.1016/s1570-0232(02)00188-5.

Abstract

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.

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid / methods*
  • Estradiol / metabolism*
  • Glucuronides / metabolism*
  • Humans
  • Kinetics
  • Microsomes, Liver / metabolism*
  • Rats
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence / methods*

Substances

  • Glucuronides
  • Estradiol