Determination of LongR(3)-IGF-I, R(3)-IGF-I, Des1-3 IGF-I and their metabolites in human plasma samples by means of LC-MS

According to the regulations of the World Anti-Doping Agency (WADA), growth promoting peptides such as the insulin-like growth factor-I (IGF-I) and its synthetic analogues belong to the class of prohibited compounds. While several assays to quantify endogenous IGF-I have been established, the potential misuse of synthetic analogues such as LongR(3)-IGF-I, R(3)-IGF-I and Des1-3-IGF-I remains a challenge and superior pharmacokinetic properties have been described for these analogues. Within the present study, it was demonstrated that the target peptides can be successfully detected in plasma samples by means of magnetic beads-based immunoaffinity purification and subsequent nanoscale liquid chromatographic separation with high resolution mass spectrometric detection. Noteworthy, the usage of a specific antibody for LongR(3)-IGF-I enables the determination in low ng/mL levels despite the presence of an enormous excess of endogenous human IGF-I. In addition, different metabolism studies (in-vitro and in-vivo) were performed using sophisticated strategies such as incubation with skin tissue microsomes, degradation in biological fluids (for all analogues), and administration to rats (for LongR(3)-IGF-I). Herewith, several C-and N-terminally truncated metabolites were identified and their relevancy was additionally confirmed by in-vivo experiments with rodents. Especially for LongR(3)-IGF-I, a metabolite ((Des1-11)-LongR(3)-IGF-I) was identified that prolonged the detectability in-vivo by a factor of approximately 2. The method was validated for qualitative interpretation considering the parameters specificity, identification capability, recovery (26-60%), limit of detection (0.5ng/mL), imprecision (<25%), linearity, stability, and matrix effects. A stable isotope labelled ((15)N)-IGF-I was used as internal standard to control all sample preparation steps.