Endocrine Abstracts

Oestradiol (E₂) and oestrone (E₁) are implicated in many diseases and drive cell proliferation in breast, ovarian, and endometrial cancer. Inactive sulphated oestrogens (E₂S and E₁S) represent a circulating reservoir forming active de-sulphated oestrogens. Therefore, new methods that accurately measure both sulphated and non-sulphated oestrogen concentrations have potential value for understanding oestrogen-related cancer. We present here a novel mass spectrometry method that extracts and quantifies both oestrogens and their sulphates together.

The method used was a development of a method Owen et al. (2013). Samples with representative internal standards were extracted with methanol using Isolute solid phase extraction columns and analysed by Waters Xevo LC–MS/MS in negative ion mode with 0.3 mM ammonium fluoride (aqueous phase). Separation of these four steroids was further optimised using MUSCLE software. The MUSCLE software (Multi-objective Unbiased optimisation of Spectrometry via Closed Loop Experimentation) has recently been developed by Bradbury et al. and enables robust, objective and automated optimisation of targeted LC–MS/MS analyses, as described at www.muscleproject.org

The MUSCLE software optimised method analysed E₁, E₂, E₁S and E₂S over the linear range 0.5–500 ng/ml with a run time of 6.5 min. For E₁ and E₂ the CV at 18 nmol/l was 5 and 15% respectively and at 554 nmol/l 5 and 4% respectively. For E₁S and E₂S this assay demonstrated a CV at 14 nmol/l of 9 and 14% respectively and at 427 nmol/l 9 and 8% respectively. The average recovery for all oestrogens was ~100%.

This novel method involves rapid extraction and analysis to accurately quantify E₁, E₂ and their sulphates and represents a significant improvement on previous methodologies. This method can be applied to both cell culture medium and serum based research, and will be a significant benefit to future oestrogen-related research.