Metabolism of Growth Hormone Releasing Peptides

Abstract

New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating Internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising four to seven amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, hexarelin, alexamorelin, and ipamorelin. With the exemption of GHRP-2, the entity of these peptides represents nonapproved pharmaceuticals; however, via Internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (po and iv administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation-exchange solid-phase extraction and analyzed by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution/high-accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyzer for the first metabolite screening and the speed of a quadrupole/time-of-flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high-accuracy product ion scan experiments. Two deuterium-labeled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimize the extraction and analysis procedure. Overall, 28 metabolites (at least three for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase-, or endopeptidase activity.

A high‐throughput LC‐MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography

“…However, the peptides GHRP-1 and GHRP-5 were metabolized differently in the rat administration study compared to the equine plasma metabolism experiment. All the other GHRP peptides behaved as predicted by the plasma modelling, although a small quantity of GHRP-6 M3 [(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2] [24] was detected in the GHRP-6 sample. In the GHRP-1 rat administration, GHRP-1 M1 [His-(D-2-Nal)-Ala-Trp-(D-Phe)-Lys-NH2] was present at significantly lower levels than the other peptides detected.…”

“…In [24] The degradation of alexamorelin and GHRP-1, and the formation of their primary metabolites during incubation in equine plasma, was quantified by SRM analysis and is shown in Figures 2B and 2C. The poor stability of alexamorelin and GHRP-1 can be simply explained by examining their amino acid sequence.…”

“…No acetylated GHRPs were observed in pre-administration urine samples (data not shown) while post-injection urine samples collected from 0-6 and 6--24 h all contained peaks for acetylated GHRPs or their metabolites (chromatogams from the 0-6 h samples are shown in Figure 3). This prompted a re-analysis of the sample and it was discovered that the GHRP-1 M3 peptide [(D-2-Nal)-Ala-Trp-(D-Phe)-Lys-NH2] [24] was the most abundant metabolite present ( Figure 3). However, the peptides GHRP-1 and GHRP-5 were metabolized differently in the rat administration study compared to the equine plasma metabolism experiment.…”

Detection of GHRP‐2 and GHRP‐6 in urine samples from athletes

“…[4] Use of the peptides is banned by the World Anti-doping Agency (WADA) and mass-spectrometry-based methods have been described to detect these peptides in urine at concentrations as low as 2-10 pg/mL. [10] However, the relevant metabolites in humans and the time course of metabolite excretion in urine are still unclear. Two studies have identified the major metabolite for GHRP-2 in humans which results from degradation at the C-terminus of the peptide to produce D -Alanine-D -βNaphthylalanine-Alanine-OH.…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…[98] Using des(1-3)-IGF-1 as internal standard, DBS were extracted into 0.1% formic acid, proteins were precipitated by means of acetonitrile, and the concentrated supernatant was analyzed by LC-MS/MS in MRM mode using a UHPLC set-up (2.1 x 50 mm, particle size 1.8 μm). [102] Urinary metabolites were identified by UHPLC-high resolution/high accuracy (tandem) mass spectrometry, and three metabolites per GHRP were characterized being potential target compounds for routine doping controls. In the absence of analytical proof, speculations as to the prevalence of IGF-1 as doping agent have arisen, particular with regard to elite sprinters, [99,100] and also deer antler velvet-derived nutritional supplements enriched with IGF-1 were recently reported.…”