Endocrine Abstracts

Estrogens are important for the development of the adult female phenotype and are intracellular mediators of androgen effects in many tissues. Measuring several estrogenic compounds in blood or other body fluids may be helpful for the assessment of estrogen status. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) is a versatile technique, which allows for the measurement of conjugated and unconjugated steroids in a single sample volume without derivatization. We used 300 ml of human serum for the analysis. Sample processing was robotized (Hamilton STAR) and included protein precipitation with acetonitrile and liquid–liquid extraction with ethylacetate–heptane. The samples were analyzed on a Waters Acquity UPLC system connected to a Waters Xevo TQ-S tandem mass spectrometer. The compounds were separated on a C-18 column (50×2.1 mm, 1.7 mm particle size), which was developed by gradient elution over 11.5 min, using an aqueous solution of ammonium hydroxide and methanol as mobile phases. 17β-estradiol, 17α-estradiol, ethinylestradiol, estrone, estrone sulfate and DHEA-S were detected in negative ion MRM mode, while progesterone was detected in positive mode. Two product ions were monitored for each compound to check for interferences. Limits of quantification were in the lower picomolar range and total imprecision was <10%. The method was virtually free from ion suppression. In conclusion, we have developed a sensitive method for the determination of multiple estrogens (endogenous, synthetic, unconjugated, and conjugated) in a single run. The method has been used to analyze several hundred samples from men and fertile and postmenopausal women.