Determination of growth hormone releasing peptides (GHRP) and their major metabolites in human urine for doping controls by means of liquid chromatography mass spectrometry |
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Authors: | Andreas Thomas Sebastian Höppner Hans Geyer Wilhelm Schänzer Michael Petrou Dorota Kwiatkowska Andrzej Pokrywka Mario Thevis |
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Institution: | 1.Center for Preventive Doping Research/Institute of Biochemistry,German Sport University Cologne,Cologne,Germany;2.Cyprus Anti-Doping Authority (CyADA) ?Lefkotheo? Indoor Hall,Nicosia,Cyprus;3.Department of Anti-Doping Research,Institute of Sport,Warsaw,Poland |
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Abstract: | A family of small peptides has reached the focus of doping controls representing a comparably new strategy for cheating sportsmen.
These growth hormone releasing peptides (GHRP) are orally active and induce an increased production of endogenous growth hormone
(GH). While the established test for exogenous GH fails, the misuse of these prohibited substances remains unrecognized. The
present study provides data for the efficient extraction of a variety of known drug candidates (GHRP-1, GHRP-2, GHRP-4, GHRP-5,
GHRP-6, alexamorelin, ipamorelin, and hexarelin) from human urine with subsequent mass spectrometric detection after liquid
chromatographic separation. The used method potentially enables the retrospective evaluation of the acquired data for unknown
metabolites by means of a non-targeted approach with high-resolution/high-accuracy full-scan mass spectrometry with additional
higher collision energy dissociation experiments. This is of great importance due to the currently unknown metabolism of most
of the targets and, thus, the method is focused on the intact peptidic drugs. Only the already characterised major metabolite
of GHRP-2 (d-Ala-d-2-naphthylAla-l-Ala, as well as its stable isotope-labelled analogue) was synthesised and implemented in the detection assay. Method validation
for qualitative purpose was performed with respect to specificity, precision (<20%), intermediate precision (<20%), recovery
(47–95%), limit of detection (0.2–1 ng/mL), linearity, ion suppression and stability. Two stable isotope-labelled internal
standards were used (deuterium-labelled GHRP-4 and GHRP-2 metabolite). The proof-of-principle was obtained by the analysis
of excretion study urine samples obtained from a single oral administration of 10 mg of GHRP-2. Here, the known metabolite
was detectable over 20 h after administration while the intact drug was not observed. |
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