A high-throughput LC-MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography.
Abstract: The growth hormone releasing peptides (GHRPs) hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are all synthetic met-enkephalin analogues that include unnatural D-amino acids. They were designed specifically for their ability to stimulate growth hormone release and may serve as performance enhancing drugs. To regulate the use of these peptides within the horse racing industry and by human athletes, a method is presented for the extraction, derivatization, and detection of GHRPs from equine and human urine. This method takes advantage of a highly specific solid-phase extraction combined with a novel derivatization method to improve the chromatography of basic peptides. The method was validated with respect to linearity, repeatability, intermediate precision, specificity, limits of detection, limits of confirmation, ion suppression, and stability. As proof of principle, all eight GHRPs or their metabolites could be detected in urine collected from rats after intravenous administration.
Copyright © 2014 John Wiley & Sons, Ltd.
Publication Date: 2014-02-24 PubMed ID: 24574167DOI: 10.1002/dta.1624Google Scholar: Lookup
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- Journal Article
- Validation Study
Summary
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This research discusses a new method for detecting growth hormone releasing peptides (GHRPs) – common potential performance enhancing substances – in equine and human urine. It uses previously applied principles and advances them to improve the efficiency and reliability of drug testing.
Understanding Growth Hormone Releasing Peptides (GHRPs)
- GHRPs such as hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are synthetic met-enkephalin analogues with unnatural D-amino acids.
- These peptides are specially designed to stimulate the release of growth hormone, which makes them potentially useful as performance-enhancing drugs.
- The use of these substances is regulated in the horse racing industry as well as among human athletes.
New Method for Detecting GHRPs
- The article presents an innovative method for the extraction, derivatization, and detection of GHRPs from both equine and human urine.
- This method combines a highly specific solid-phase extraction with a unique derivatization to improve the chromatography of basic peptides, a process through which the chemical substances are separated.
- The method underwent validation regarding linearity, repeatability, intermediate precision, specificity, detection limits, confirmation limits, ion suppression, and stability.
Proof of Concept and Practical Application
- The proof of principle was demonstrated by successfully detecting all eight GHRPs, or their metabolites, in urine collected from rats after they were administered intravenously.
- Practical use of this method will be beneficial for enhancing the efficiency and reliability of drug testing in sports, and specifically in the horse racing industry, where these substances are often used illicitly to generate a performance edge.
Cite This Article
APA
Timms M, Hall N, Levina V, Vine J, Steel R.
(2014).
A high-throughput LC-MS/MS screen for GHRP in equine and human urine, featuring peptide derivatization for improved chromatography.
Drug Test Anal, 6(10), 985-995.
https://doi.org/10.1002/dta.1624 Publication
Researcher Affiliations
- Biological Research Unit, Racing Analytical Services Ltd, 400 Epsom Road, Flemington, Victoria, Australia, 3031.
MeSH Terms
- Administration, Intravenous
- Animals
- Chromatography, Liquid / methods
- Doping in Sports / prevention & control
- Growth Hormone-Releasing Hormone / analogs & derivatives
- Growth Hormone-Releasing Hormone / analysis
- Growth Hormone-Releasing Hormone / urine
- High-Throughput Screening Assays / methods
- Horses
- Humans
- Male
- Rats
- Rats, Sprague-Dawley
- Sensitivity and Specificity
- Solid Phase Extraction
- Substance Abuse Detection / methods
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 3 times.- Gómez-Guerrero NA, González-López NM, Zapata-Velásquez JD, Martínez-Ramírez JA, Rivera-Monroy ZJ, García-Castañeda JE. Synthetic Peptides in Doping Control: A Powerful Tool for an Analytical Challenge.. ACS Omega 2022 Nov 1;7(43):38193-38206.
- Lange T, Thomas A, Walpurgis K, Thevis M. Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS.. Anal Bioanal Chem 2020 Jun;412(15):3765-3777.
- Judák P, Grainger J, Goebel C, Van Eenoo P, Deventer K. DMSO Assisted Electrospray Ionization for the Detection of Small Peptide Hormones in Urine by Dilute-and-Shoot-Liquid-Chromatography-High Resolution Mass Spectrometry.. J Am Soc Mass Spectrom 2017 Aug;28(8):1657-1665.
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