Endocrine Abstracts

Introduction: Most clinical laboratories use immunoassays to measure estradiol despite limitations such as poor specificity, poor sensitivity and wide variability between different manufacturers’ assays. LC–MS/MS assays overcome the issues of sensitivity and specificity however the methods reported in the literature often involve complex sample preparation and lengthy run times. We describe a simple, rapid assay for the simultaneous measurement of serum estradiol and estrone.

Methods: Sample (250 μl) was diluted with water after the addition of internal standards. After mixing, the diluted samples are transferred to the wells of a Biotage SLE+ plate. After extraction with MTBE, the ether is dried then extract is reconstituted with 100 μl of 40% methanol. Extract was extracted further using on-line solid phase extraction on a C18 cartridge by a Waters Acquity/OSM followed by a Waters TQS tandem mass spectrometer.

Results: The lower limits of quantitation for estradiol and estrone were 10 and 6 pmol/l respectively. The CV of the assay for estradiol and estrone concentrations of 125 pmol/l was < 7%. Further the estradiol assay demonstrated a CV of 10% at 22 pmol/l and the estrone assay had a CV of 5% at 16 pmol/l. The average recovery for estradiol was 102% and estrone was 106%. The comparison with a commercial immunoassay gave the following equation: Immunoassay =0.94×LC–MS/MS +21 pmol/l. The run time was 4.5 min per sample.

Discussion: We have developed a rapid assay for the LC-MS/MS measurement of estradiol and estrone which does not require derivatisation in the sample preparation. The assay is suitable for routine clinical use or for clinical trials. The assay demonstrated superior performance compared to immunoassays at lower concentrations making it more suitable for use in males and patients on aromatase inhibitors.