Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP‐1, GHRP‐2, GHRP‐6, Hexarelin, and Ipamorelin

Abstract

Growth hormone releasing peptides (GHRPs) stimulate secretion of endogenous growth hormone and are listed on the World Anti-Doping Agency (WADA) Prohibited List. To develop an effective method for GHRPs anti-doping control we have investigated metabolites of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin in urine after nasal administration. Each compound was administrated to one volunteer. Samples were collected for 2 days after administration, processed by solid-phase extraction on weak cation exchange cartridges and analyzed by means of nano-liquid chromatography - high resolution mass spectrometry. Six metabolites of GHRP-1 were identified. GHRP-1 in the parent form was not detected. GHRP-1 (2-4) free acid was detected in urine up to 27 h. GHRP-2, GHRP-2 free acid and GHRP-2 (1-3) free acid were detected in urine up to 47 h after administration. GHRP-6 was mostly excreted unchanged and detected in urine 23 h after administration, its metabolites were detectable for 12 h only. Hexarelin and Ipamorelin metabolized intensively and were excreted as a set of parent compounds with metabolites. Hexarelin (1-3) free acid and Ipamorelin (1-4) free acid were detected in urine samples after complete withdrawal of parent substances. GHRPs and their most prominent metabolites were included into routine ultra-pressure liquid chromatography-tandem mass spectrometry procedure. The method was fully validated, calibration curves of targeted analytes were obtained and excretion curves of GHRPs and their metabolites were plotted. Our results confirm that the detection window after GHRPs administration depends on individual metabolism, drug preparation form and the way of administration.

Zebrafish (Danio rerio) water tank model for the investigation of drug metabolism: Progress, outlook, and challenges

“…49,50 Therefore, the evaluation of hexarelin by the ZWT model is a big challenge because of the temperature, the time of drug exposure and the possibility of glass interaction in the tank. An increase of hydroxybutyl metabolites in the first hours are observed followed by a decrease after 72 hours.…”

Simplifying and expanding the screening for peptides <2 kDa by direct urine injection, liquid chromatography, and ion mobility mass spectrometry

“…Unless simplicity and speed are requited, sub-ng/mL levels are crucial to detect due to fast clearance rates of the administered peptides, although concentrations after intranasal application of different GHRPs reach urinary C max levels in moderate ng/mL range. [27] The untargeted screening strategy with high resolution MS represents an effective tool for preventive doping control analysis due to the potential to evaluate the acquired data retrospectively. Thus, new performance enhancing compounds or new metabolites were acquired and identified with generic parameter settings.…”

“…In addition, several assays for the detection of small prohibited peptides were developed recently and adverse analytical findings followed accordingly [20]. These assays apply LC-MS after different sample preparation procedures [3,[13][14][15][21][22][23][24][25][26][27]. For the main GHRP-2 metabolite, also a direct injection of urine in a comprehensive screening assay was described formerly [28].…”

Detection of Growth Hormone Releasing Peptides in Serum by a Competitive Receptor Binding Assay

“…Anti-doping detection methods based in liquid chromatographymass spectrometry (LC-MS) have been developed in recent years using urine as main matrix [17][18][19][20][21]. These methods are focused especially on those GHSs with known structure and metabolism which could be used more frequently in sport abuse.…”

“…Therefore, this matrix has been used or GHSs (no: or GHSs) detection in athlete samples with anti-doping purposes [21]; although the use of another matrices, as serum, could be highly desirable. However, the GHRPs studies performed so far with serum samples have been carried out only in a few clinical trials with healthy males and children administered with GHRP-2 and GHRP-6 respectively [26,27].…”