An LC-MS/MS Methodological Framework for Steroid Hormone Measurement from Human Serum

Abstract

AbstractEstrogens and androgens are important regulators of sexual development and physiological processes in men and women, acting on numerous organs throughout the body. Moreover, they can contribute to a variety of pathologies, including osteoporosis, cancer, and cardiovascular and neurologic diseases. Analysis of estrogens and androgens in biological samples has been commonly performed using immunoassays for many years. However, these assays are suboptimal, as there is cross-reactivity with similar analytes, and they have moderate specificity and sensitivity. Thus, there is a clinical need to develop highly sensitive and specific methods for the accurate measurement of estrogen and androgen concentrations. Herein, we describe the development of three liquid chromatography coupled tandem mass spectrometry-based methods that incorporate the use of a Triple Quadrupole Mass Spectrometer for quantitative measurement of endogenous concentrations of various steroid hormones in human serum samples: (1) the simultaneous measurement of testosterone, androstenedione, and cortisol, (2) dehydroepiandrosterone (DHEA), and (3) 17β-estradiol (E2). The use of derivatizing reagents, Girard’s reagent P and dansyl chloride, allowed for significant gains in sensitivity in the analysis of DHEA and E2, respectively, relative to the underivatized analyte. These procedures proved efficient and adequately sensitive for steroid hormone analysis in extracted patient sera samples from older men and postmenopausal women, providing reliable data down to low nanogram/ml and sub-nanogram/ml levels. Moreover, utilizing the combination of highly specific mass transitions associated with these analytes and their respective internal deuterated standards provided a high degree of specificity to the identity of these hormones.