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 modulate the immune response to inflammation and affect whole-body metabolism. Despite numerous studies using traditional GC-MS and LC-MS methods, the accurate quantification of steroid levels in biological matrices continues to pose significant analytical challenges. High structural similarity between steroid isomers and their active and inactive forms in various abundance levels with variable ionizability can result in significant interferences and false positive or negative results. The present study aimed to develop a selective and sensitive high-throughput reversed phase ultra-high performance liquid chromatography-tandem mass spectrometry (RP-UHPLC-MS/MS) method for the simultaneous quantification of 40 endo- and exogenous steroids covering progestogens, corticosteroids, androgens, estrogens, and synthetic steroids in human, rat and mouse plasma. Overlapping of retention times and masses or fragmentation patterns of 31 of 40 steroids and difficult ionizability had to be overcome when developing the 20-minute long RP-UHPLC-MS/MS method. The sample preparation method of protein precipitation (PP) was developed to eliminate matrix effects. The optimized method was validated according to the EMA guideline on bioanalytical method validation, and the PP-UHPLC-MS/MS workflow has been applied so far to analyze 140 mouse plasma samples of mice exposed to different stress conditions (immune stress, acute stress response, food allergy). The study was supported by the GA UK project No. 348221, STARSS project (Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000465) co-funded by ERDF, the project of specific research SVV 260548 and CSF project No. 21-10845S.