Endocrine Abstracts

Aims: Develop and validate a LC–MS/MS assay for the measurement of serum 17-hydroxyprogesterone (17-OHP) and to establish reference ranges for levels observed in the follicular and luteal phases of the menstrual cycle.

Method: Serum samples (200 μl) underwent liquid extraction using di-ethyl ether (1 ml). A deuterated internal standard was used. 20 μl extract was injected onto a Waters Atlantis C18 column using a Waters 2795 Alliance HPLC separations module coupled to a Waters Premier tandem mass spectrometer. Samples were also analysed using RIA (Coat-A-Count, DPC). Serum samples were obtained from patients during the follicular phase of the menstrual cycle (days 1–3) and luteal phase (day 21).

Results: Measurement of 17-OHP was linear to 100 nmol/l (R²=0.99), lower limit of quantitation was determined as 0.3 nmol/l and inter-assay CV were <20%. Correlation with RIA was poor with results up to 40% lower by LC–MS/MS (mean bias 7 nmol/l). Comparison with other LC–MS/MS methods (UKNEQAS data) was good (y=1.02x+1.54, R²=0.98). No interference was observed from 11-deoxycortisol or 21-deoxycortisol. Mean (±S.D.) concentrations of 17-OHP in the follicular phase (n=40) were 0.75 nmol/l (0.38), giving a reference range of 0.3–1.50 nmol/l. Mean concentrations in the luteal phase (n=39) were 2.7 nmol/l (1.59) giving a reference range of 1.2–4.4 nmol/l.

Conclusions: A method has been developed for the measurement of and 17-OHP. Reference ranges have been established for 17-OHP in the follicular and luteal phases of the menstrual cycle.