Nasal GHRPs yield distinct urinary metabolite profiles, enabling extended anti-doping detection windows up to 47 hours

Growth Hormone Releasing Peptides (GHRPs) are synthetic substances that stimulate the body's natural secretion of growth hormone. Because of their performance-enhancing potential, they are included on the World Anti-Doping Agency (WADA) Prohibited List. Developing effective anti-doping controls requires a deep understanding of how these compounds are processed and eliminated by the body. Standard methods often look for the parent drug, but these can be cleared quickly. Investigating the metabolic byproducts (metabolites) of GHRPs can significantly extend the window of detection, making it harder for athletes to evade testing. This study specifically explores the urinary metabolites following nasal administration, a less common but relevant route of administration that needs characterization for comprehensive screening protocols.

Each of five GHRPs (GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin) was administered nasally to a single volunteer (N=1 per compound). Urine samples were collected from each volunteer for 2 days following administration. The collected samples underwent solid-phase extraction using weak cation exchange cartridges. The processed samples were then analyzed using a highly sensitive technique: nano-liquid chromatography - high resolution mass spectrometry (nano-LC-HRMS). This method allowed for the identification and tracking of the parent peptides and their various metabolites over time. The most significant metabolites were then integrated into a routine ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) procedure for validated, routine screening.

The study revealed distinct metabolic fates for each GHRP. GHRP-1 was extensively metabolized; the parent compound was not detected at all, but six different metabolites were identified. Its metabolite GHRP-1 (2-4) free acid was detectable for up to 27 hours. GHRP-2 and two of its metabolites, GHRP-2 free acid and GHRP-2 (1-3) free acid, were found in urine for up to 47 hours, offering the longest detection window. In contrast, GHRP-6 was mostly excreted unchanged and was detectable for 23 hours, while its metabolites were only present for 12 hours. Hexarelin and Ipamorelin were metabolized intensively.

Key metabolites like Hexarelin (1-3) free acid and Ipamorelin (1-4) free acid were still detectable in urine even after the parent compounds had been completely eliminated. The results confirm that detection windows are highly variable and depend on the specific peptide, individual metabolism, and administration route.