Solid-phase extraction of small biologically active peptides on cartridges and microelution 96-well plates from human urine.

Currently liquid chromatography - mass spectrometry (LC-MS) analysis after solid-phase extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs), desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer provides good buffering capacity without affecting the peptides recoveries. SPE on microelution plates was performed on Waters Positive Pressure-96 Processor with subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample volume decreases the pre-concentration factor and increases the limits of detection of 5 out of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution extraction were comparable with cartridge SPE. The effectiveness of protocols was confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its metabolites. SPE after urine sample dilution with buffer can be used for faster sample preparation. The use of microelution plates decreases consumption of solvents and allows processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment remains the best option for confirmation. Copyright © 2015 John Wiley & Sons, Ltd.

Comparison of various in vitro model systems of the metabolism of synthetic doping peptides: Proteolytic enzymes, human blood serum, liver and kidney microsomes and liver S9 fraction.

Small peptides with a molecular weight of <2kDa represent a performance-enhancing substances. However, in vivo studies with human volunteers are limited because most of these peptides are not approved for human consumption. Thus, relevant in vitro models are a basic tool to study their metabolism for anti-doping purposes. To choose the best in vitro model the biotransformation of growth hormone releasing peptides (GHRPs), Desmopressin and TB-500 was investigated using various in vitro systems. High metabolic activity was observed during incubation of GHRPs and TB-500 with human kidney microsomes (HKM) and liver S9 fraction. Peptides degraded through cleavage of all bonds regardless protective modifications in primary structure. HKM and liver S9 fraction demonstrated enzymatic deamidation activity removing C-terminal amide group from all GHRPs. Fewer metabolites were produced during incubation with human serum. The metabolite pattern obtained with commercially available proteases was poor and included nonspecific hydrolyzed compounds. Thus, the maximum diversity of metabolites was achieved with HKM and liver S9 fraction which makes them the most efficient in vitro model systems for peptides biotransformation study. BIOLOGICAL SIGNIFICANCE: Currently, >60 peptide medicines are FDA approved and marketed in the United States as biopharmaceutical products. Approximately 140 peptide drugs are in clinical trials and about 500 therapeutic peptides in preclinical development. There is an emerging interest in small peptides with a molecular weight of <2kDa, which can be used as doping in modern sport due a wide spectrum of their physiological activity. Most of peptide doping products are not yet approved for human use and some of them undergo preclinical or clinical trials, which complicates the study of metabolism in vivo. The investigation of the metabolism with in vitro methods is an alternative that does not require a human participation and an approval by the Ethics Committee.

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

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.