Simultaneous Quantification of Steroid Hormones Using hrLC-MS in Endocrine Tissues of Male Rats and Human Samples

Abstract

Steroid hormones play a vital role in the regulation of cellular processes, and dysregulation of these metabolites can provoke or aggravate pathological issues, such as autoimmune diseases and cancer. Regulation of steroid hormones involves different organs and biological compartments. Therefore, it is important to accurately determine their levels in tissues and biofluids to monitor changes after challenge or during disease. In this work, we have developed and optimized the extraction and quantification of 11 key members of the different steroid classes, including androgens, estrogens, progestogens and corticoids. The assay consists of a liquid/liquid extraction step and subsequent quantification by high-resolution liquid chromatography coupled time-of-flight mass spectrometry. The recoveries range between 74.2 to 126.9% and 54.9 to 110.7%, using a cell culture or urine as matrix, respectively. In general, the signal intensity loss due to matrix effect is no more than 30%. The method has been tested in relevant steroidogenic tissues in rat models and it has also been tested in human urine samples. Overall, this assay measures 11 analytes simultaneously in 6 min runtime and it has been applied in adrenal gland, testis, prostate, brain and serum from rats, and urine and extracellular vesicles from humans.

Keywords: androgens; hormone-dependent disease; liquid chromatography–mass spectrometry; metabolomics; steroid hormones; time-of-flight; urinary extracellular vesicles.

Figure 1

Schematic representation of the steroid hormone biosynthesis pathway in relevant organs and its regulation. CRH stimulates the release of ACTH from the pituitary gland. ACTH stimulates the production of cortisol (exerts negative feedback on CRH and ACTH) and DHEAS in adrenal glands. Pulses of GnRH from hypothalamic neurons stimulate pulses of LH as well as FSH. LH stimulates testosterone production in testis. Liver maintains pathway’s homeostasis and several processes may happen: sulf desulfation makes metabolites available to feed the pathway while processes indicated with a flat end arrow inactivate metabolites that are in circulation. Bold arrows indicate a higher activity of the specific reaction. In bold, the metabolites that are majorly produced in each specific organ are represented. ACTH: adrenocorticotropin; CRH: corticotropin-releasing hormone; FSH: follicle stimulating hormone; GnRH: gonadotropin-releasing hormone; LH: luteinizing hormone; CYP17A1: Steroid 17-alpha-monooxygenase; CYP19A1: aromatase; SULT: hydroxysteroid sulfotransferase; STS: steroid sulfatase; 3β-HSD: 3β-Hydroxysteroid dehydrogenase; 17β-HSD: 17β-Hydroxysteroid dehydrogenase; DHEA: dehydroepiandrosterone; DHEAS: DHEA sulfate.