Advancements in the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to dried blood spots for the characterization of metabolic and hormonal derangement in endocrine diseases

The present study focused on the development and application of LC-MS/MS methodologies for hormonal and metabolite profiling. The project pursued four objectives: optimization of LC-MS/MS protocols for the quantification of extended exogenous and endogenous steroid panels in dried blood spots (DBS); analysis of DBS samples for a broad steroid panel using an internal calibration combined with multi-targeting strategy at the Biomedical and Metabolomics Analysis (BMA) group at the University of Geneva; development of a pre-analytical and analytical procedure for the highly sensitive LC-MS/MS quantification of estrogens; validation of the application in DBS of a broad targeted metabolomics method, and application to cohorts of patients with altered cortisol secretion. At the University of Bologna, two broad LC-MS/MS panels overall allowing the measurement of 34 among steroid hormones and drugs were optimized, and a dedicated DBS extraction protocol was refined to improve analytical recovery and matrix cleanliness. At the University of Geneva, 54 samples from a circadian steroid profiling study in 6 healthy subjects were analyzed using an internal calibration and multitargeted approach, which ensured absolute quantification of 8 key steroids. This work confirmed the feasibility of DBS sampling combined with LC-MS/MS for accurate characterization of diurnal steroidome rhythm. A second research line addressed one of the major analytical challenges of LC-MS/MS: the low ionization efficiency of estrogens. A dedicated sample purification and derivatization procedure was optimized, leading to a substantial improvement in detection sensitivity and enabling reliable quantification. Finally, a targeted metabolomics approach focusing on 187 molecules was applied to investigate metabolic pathway alterations associated with cortisol rhythm disorders. Since data demonstrated the general strong correlation of metabolite levels between DBS and serum matrices, the method was used to profile metabolites in patients with different adrenal disorders, revealing distinct metabolic patterns linked to the degree and timing of cortisol dysregulation.