Probing for corticotropin-releasing hormone (CRH) in human blood for doping control purposes using immunoaffinity purification and LC-HRMS/MS

Corticotropin-releasing hormone (CRH), a peptide hormone whose secretion leads to adrenal cortisol release, is classified as prohibited substance by the World Anti-Doping Agency (WADA). In order to comprehensively enforce anti-doping regulations, a detection method for CRH in blood (serum and plasma) is required. In this study, two different immunoaffinity purification strategies were optimized and employed for sample preparation, followed by nano-ultra high performance liquid chromatography (UHPLC) coupled to high resolution/high accuracy tandem mass spectrometry (HRMS/MS). For that purpose, a CRH primary polyclonal antibody was immobilized to either IgG-covered paramagnetic particles or a monolithic protein A/G surface. The first approach using magnetic beads was fully validated in both human plasma and serum, while the Mass Spectrometric Immunoassay (MSIA (TM)) procedure was cross-validated for selected parameters. The resulting assays' LLODs were estimated at 200 pg mL(-1) (magnetic beads) and 500 pg mL(-1) (MSIA (TM)). In addition to human CRH, also animal analogs such as ovine and bovine CRH were found to be traceable using the established approach. For all target analytes comprising of 41 amino acids (approximately 4.7 kDa), high-resolution/high-accuracy product ion mass spectra were generated, and diagnostic y-and b-ions were identified. Proof-of-concept data were obtained by the analysis of plasma samples collected in the course of CRH stimulation blood tests. Specimens were collected from three patients 15 and 30 min after intravenous application of a 100 mu g single dose of human CRH, yielding plasma concentrations between 7.6 and 18.9 ng mL(-1).