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Ann Clin Biochem. 2014 May;51(Pt 3):360-7. doi: 10.1177/0004563213501478. Epub 2013 Oct 1.

A rapid direct assay for the routine measurement of oestradiol and oestrone by liquid chromatography tandem mass spectrometry.

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Biochemistry Department, University Hospital of South Manchester, UK.



Liquid chromatography tandem mass spectrometry (LC-MS/MS) is rapidly becoming the technology of choice for measuring steroid hormones. We have developed a rapid LC-MS/MS assay for the routine analysis of serum oestradiol and oestrone. The assay uses a relatively small volume and has a rapid run time.


Supported liquid extraction was performed on 250 µL of sample using methyl tertiary butyl ether. The extract was dried and reconstituted with 100 µL of 40% methanol. Online automated solid phase extraction was performed on 75 µL of extract using C18 cartridges on a Waters OSM coupled to a Waters TQS mass spectrometer. Serum samples (n = 197) were analysed by LC-MS/MS and a commercial immunoassay.


The lower limit of quantitation for oestradiol and oestrone was 10 and 6 pmol/L, respectively. The coefficient of variation (CV) of the assay for oestradiol and oestrone concentrations of 125 pmol/L was <7%. The assay had a CV of 10% at 22 pmol/L for oestradiol and 5% at 16 pmol/L for oestrone. The average recovery for oestradiol was 102% and oestrone 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.


We have developed a rapid assay for the LC-MS/MS measurement of oestradiol and oestrone which does not require derivatization 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.


Serum oestradiol; liquid chromatography tandem mass spectrometry; oestrone

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