Direct measurement of free estradiol in human serum by equilibrium dialysis-liquid chromatography-tandem mass spectrometry and reference intervals of free estradiol in women.

ABSTRACT

BACKGROUND: Measurement of free estradiol offers a better representation of the bioactive fraction of the hormone. We describe a direct equilibrium dialysis-liquid chromatography-tandem mass spectrometry (ED-LC-MS/MS) method for serum free estradiol. METHODS: Two hundred fifty microliter aliquots of serum were dialyzed for 22h followed by liquid-liquid extraction and derivatization with dansyl chloride. Free estradiol was measured using LC-MS/MS with an AB SCIEX 5500 mass spectrometer in positive ion and multiple reaction monitoring (MRM) mode. RESULTS: The limits of detection and quantification for free estradiol were 0.25 and 0.5pg/ml (0.9 and 1.8pmol/l) respectively. Total imprecision was less than 10%. Results of method comparison showed 3 times overestimation using indirect methods of measurement. Reference intervals in pre-menopausal women in follicular, mid-cycle, and luteal phases of cycle were <2.4, <3.1 and <2.6pg/ml (8.8, 11.4, 9.5pmol/l) respectively; in post menopausal women the concentrations were ≤0.5pg/ml (1.8pmol/l). CONCLUSIONS: ED-LC-MS/MS is a direct method for accurately measuring free estradiol, independent of total estradiol or sex hormone binding globulin concentrations. Imprecision and sensitivity of the method are adequate for clinical diagnostic applications. The degree of variation observed in the method comparison reinforces the relevance of method specific reference ranges.