Dispersive liquid–liquid microextraction as an effective preanalytical step for the determination of estradiol in human urine

Abstract

In this work, a fast and effective dispersive liquid-liquid microextraction was developed for the isolation and preconcentration of free 17β-estradiol, the main human estrogen, from real human urine samples. To optimize the extraction technique, few important parameters such as type and volume of extraction and dispersive solvents, centrifugation conditions, effect of salt addition, and extraction time were studied. Optimal conditions were obtained when injecting 600μL mixture of tetrachloromethane as extraction solvent and ethanol as dispersive solvent (1:5, v/v) into 2mL of urine containing 8% NaCl and following centrifugation at 10000rpm, thus reaching enrichment factor 28 and extraction recovery 98% for estradiol. Procedure was evaluated by means of high-performance liquid chromatography with UV detection (λ=280nm) using a C-18 column and methanol/water (60:40, v/v) as the mobile phase. The method was linear within the concentration range 1.0-250.0mg/L (r=0.9997) and provided a limit of detection of 0.25mg/L. The proposed method was applied to the determination of free estradiol in real human pregnancy urine.