Investigations into the In Vitro Metabolism of hGH and IGF-I Employing Stable-Isotope-Labelled Drugs and Monitoring Diagnostic Immonium Ions by High-Resolution/High-Accuracy Mass Spectrometry

Studying the metabolism of prohibited substances is an essential element in anti-doping research in order to facilitate and improve detectability. Whilst pharmacokinetic studies on healthy volunteers are valuable, they are often difficult, not least due to safety reasons and ethical constraints, especially concerning peptidic substances, which must be administered parenterally. Hence, there is a growing need for suitable in vitro models and sophisticated analytical strategies to investigate the metabolism of protein- and peptide-derived drugs. These include human growth hormone (hGH) and its main mediator insulin-like growth factor-I (IGF-I), both prohibited in professional sports for their anabolic and lipolytic effects, while challenging in their detection, as they occur naturally in the human body.Within this study, the in vitro metabolism of hGH and IGF-I was investigated using a stable-isotope-labelled reporter ion screening strategy (IRIS). A combination of liquid chromatography, high-resolution mass spectrometry, and characteristic immonium ions generated by internal dissociation of the stable-isotope-labelled peptidic metabolites enabled the detection of specific fragments. Several degradation products for hGH and IGF-I were identified within this study. These metabolites, potentially even indicative for subcutaneous administration of the drugs, could serve as promising targets for the detection of hGH and IGF-I misuse in future anti-doping applications.