1. ETH Zurich, Animal Physiology, Institute of Agricultural Sciences, Switzerland
2. University of Zurich, Department of Chemistry, Switzerland
Abstract Text: Steroid hormones including glucocorticoids, mineralocorticoids, androgens, estrogens, and progestogens produced by the adrenal cortex, the gonads, the placenta are small lipophilic compounds with important biological functions including sexual differentiation and reproduction, growth, metabolism regulation, and immune system modulation. Metabolism of steroids includes oxidation and reduction reactions (phase I) and esterification reactions (phase II). During phase II metabolism, polar moieties, commonly a sulfate or glucuronide group, are added to the steroid skeleton. Analytics of steroids is often challenging due to isomeric structures and the low endogenous concentrations in body fluids. Mass spectrometry (MS)-based techniques have evolved as a gold standard for steroid analytics providing increased specificity compared to immunoassays. We aimed at developing a plasma/serum steroidome high-throughput MS based platform to study follicular and luteal dynamics across species including women. We extended our previously published ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) approach for progestogen and glucocorticoid profiling (Rehm et al. 2024) by adding androgens, estrogens and steroid conjugates. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) were employed to extract low-polarity (free steroids) and relatively more polar steroids (conjugated steroids), respectively. We utilized a semi-targeted approach to determine the steroidome in bovine and human plasma, employing a hybrid quadrupole-orbitrap mass spectrometer operating in full scan MS1/data dependent acquisition (DDA) MS2 mode to perform both targeted and untargeted analysis in a single sample injection. Specifically, thirteen progestogens, eleven glucocorticoids, seventeen androgens, nine estrogens and six steroid conjugates were of interest. Optimized ionization of the steroids was performed by polarity switching and the addition of different additives to the mobile phases of LC improving method sensitivity. While progestogens ionized best in positive mode using formic acid (FA) as an additive, resulting in limits of quantification (LOQs) between 50-500 pg/ml, steroid conjugates performed best in negative mode using FA as an additive with a LOQ at 50 pg/ml. Estrogens obtained improved sensitivity when using ammonium fluoride or ammonium hydroxide instead of FA as an additive. Among the 56 target steroids, 14 and 15 displayed changes across the female and bovine cycle, respectively. Of these, several bioactive steroid metabolites have not been described in detail in the context of follicular and luteal dynamics. Taken together, semi-targeted steroidomics enables to simultaneously monitor a large panel of steroid compounds and thereby allows to study steroid-related feedback mechanisms, elucidate biological functions of active steroid metabolites across species and identify biomarkers for endocrine physiological and pathological conditions such as reproductive health.
