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

In this work, a fast and effective dispersive liquid-liquid microextraction was developed for the isolation and preconcentration of free 17 β-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 600 μL mixture of tetrachloromethane as extraction solvent and ethanol as dispersive solvent (1:5, v/v) into 2 mL of urine containing 8% NaCl and following centrifugation at 10 000 rpm, 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 = 280 nm) using a C-18 column and methanol/water (60:40, v/v) as the mobile phase. The method was linear within the concentration range 1.0-250.0 mg/L (r = 0.9997) and provided a limit of detection of 0.25 mg/L. The proposed method was applied to the determination of free estradiol in real human pregnancy urine.