A Sensitive Method for Quantitative Profiling of Estrogens, Progesterone, Androgens, and Corticosteroids in Human...

Determination of steroid hormones by mass spectrometry (MS) offers several advantages over immunoassays. Modern MS instruments have high selectivity and sensitivity and permit measurements of several compounds at picomolar concentrations in one sample volume, which is advantageous when conducting biobank studies.

We developed a LC-MS/MS method for the determination of 17β-estradiol, 17α-ethinylestradiol, estrone, estrone 3-sulfate, estriol, progesterone, testosterone, dehydroepiandrosterone sulfate, aldosterone, cortisone, and cortisol in 300 μL human serum. Samples were subjected to robotized protein precipitation with acetonitrile and liquid-liquid extraction with ethyl acetate/heptane, and the aqueous and organic phases were used to determine conjugated and unconjugated steroids, respectively. The steroids were separated from isobars and compounds with similar mass (M + 2) on a C18 reversed phase column. Ammonium hydroxide was infused post-column to enhance ionization, and ions were measured in multiple reaction monitoring negative and positive mode.

The lower limits of quantification for 17β-estradiol and estrone were 3.6 (0.98 pg/mL) and 2.1 pmol/L (0.57 pg/mL), respectively. For medium concentrations of steroid hormones, total CVs were in the range 2.3% to 10.5%, and accuracies were 97% to 109%. The method was validated in terms of linearity (r2 ≥ 0.9779) and assay recovery (83%-114%), and certified control sera were analyzed. Steroid profiles were obtained in 921 women with a mean age of 56 years (range 44-66 years) from the European Community Respiratory Health Survey.

We developed a selective and sensitive LC-MS/MS method for 11 steroids in human serum. It is suitable for biobank studies.