PMID- 23101768 OWN - NLM STAT- MEDLINE DCOM- 20131112 LR - 20161125 IS - 1520-6882 (Electronic) IS - 0003-2700 (Linking) VI - 84 IP - 23 DP - 2012 Dec 4 TI - Metabolism of growth hormone releasing peptides. PG - 10252-9 LID - 10.1021/ac302034w [doi] AB - 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. FAU - Thomas, Andreas AU - Thomas A AD - Center for Preventive Doping Research/Institute of Biochemistry, German Sport University, Cologne, Germany. thomas@biochem.dshs-koeln.de FAU - Delahaut, Philippe AU - Delahaut P FAU - Krug, Oliver AU - Krug O FAU - Schanzer, Wilhelm AU - Schanzer W FAU - Thevis, Mario AU - Thevis M LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20121113 PL - United States TA - Anal Chem JT - Analytical chemistry JID - 0370536 RN - 0 (Oligopeptides) RN - 0 (Peptide Fragments) RN - 12629-01-5 (Human Growth Hormone) RN - AR09D82C7G (Deuterium) RN - E6S6E1F19M (growth hormone-releasing peptide-2) SB - IM MH - Animals MH - Chromatography, High Pressure Liquid/*methods MH - Deuterium MH - Doping in Sports/prevention & control MH - Female MH - Human Growth Hormone/*urine MH - Humans MH - Male MH - Oligopeptides/administration & dosage/*metabolism MH - Peptide Fragments/*analysis MH - Rats MH - Rats, Wistar MH - Solid Phase Extraction MH - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods EDAT- 2012/10/30 06:00 MHDA- 2013/11/13 06:00 CRDT- 2012/10/30 06:00 PHST- 2012/10/30 06:00 [entrez] PHST- 2012/10/30 06:00 [pubmed] PHST- 2013/11/13 06:00 [medline] AID - 10.1021/ac302034w [doi] PST - ppublish SO - Anal Chem. 2012 Dec 4;84(23):10252-9. doi: 10.1021/ac302034w. Epub 2012 Nov 13.