Endocrine Abstracts

Stress is increasingly pervasive in modern society and an unavoidable stimulus to the human organism. Stressors, whether of social or physical type, activate the hypothalamic-pituitary-adrenal (HPA) axis, resulting in the upregulation of glucocorticoid levels and, in some cases, its de novo biosynthesis. Aside from HPA axis regulation, corticosteroids also modulate the immune response to inflammation and affect the whole-body metabolism. Accurate quantification of endogenous steroids remains an analytical challenge. High structural similarity between steroid isomers and their active and inactive forms in various abundance levels can result in significant interferences and false positive results, especially when using immuno-assays. The present study aimed to develop a sensitive high-throughput reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry (RP-UHPLC-MS/MS) method for the simultaneous determination of 40 endo- and exogenous steroids. A set of C18 estrogens, C19 androgens, C21 progestogens and corticoids, and synthetic steroids in biologically active and inactive forms was selected based on cooperation with the Laboratory of Epithelial Physiology, Czech Academy of Science, to monitor the targeted panel of analytes in mouse plasma. Overlapping of retention times and masses or fragmentation patterns of 31 of 40 steroids had to be overcome when developing the 20minute long RP-UHPLC-MS/MS method. Sample preparation methods of supported liquid extraction (SLE) and protein precipitation (PP) were developed to eliminate matrix effects and achieve the highest degree of sample purification without the loss of target analytes. Both sample preparation methods have been validated according to the EMA guideline. Finally, the PP-RP-UHPLC-MS/MS method was selected to analyse mouse plasma samples due to lower obtained LLOQs.