Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry.
Abstract: A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment (17)LKKTETQ(23) is the active site within the protein thymosin β(4) responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography-mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses.
Copyright © 2012 Elsevier B.V. All rights reserved.
Publication Date: 2012-09-23 PubMed ID: 23084823DOI: 10.1016/j.chroma.2012.09.043Google Scholar: Lookup
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Summary
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The study described in this abstract details the development of a new method for detecting the synthetic peptide TB-500, a performance-enhancing drug used in horse racing, in equine urine and plasma samples.
Background of the Study
- The main focus of the reported research is on TB-500, a synthetic version of the peptide LKKTETQ found in Thymosin β(4), a naturally occurring protein responsible for various cell activities such as actin binding, cell migration, and wound healing.
- TB-500 (preparation containing the synthetic version of the peptide) is believed to enhance various biological effects such as endothelial cell differentiation, dermal tissue angiogenesis, keratinocyte migration, collagen deposition, and inflammation reduction. These characteristics make it potentially desirable as a doping agent in equine sports.
Objective of the Study
- The primary goal of this research was to develop a definitive method of identifying the use and misuse of TB-500 in equine sports by detecting an artificial acetylation of the peptide in horse urine and plasma.
Methodology and Results
- The researchers carried out in vitro studies to identify the potential metabolites of N-acetylated LKKTETQ. They then used solid-phase extraction and ion-exchange cartridges to isolate the parent peptide and its metabolites from equine urine or plasma.
- These isolated substances were then analyzed through liquid chromatography-mass spectrometry (LC/MS) by comparing their LC retention times and the relative abundance of major product ions to confirm the presence of N-acetylated LKKTETQ.
- The developed method was able to detect and confirm the presence of the peptide in concentrations as low as 0.02 ng/mL in plasma and 0.01 ng/mL in urine.
- The method was further tested and confirmed the presence of N-acetylated LKKTETQ and its metabolites in samples collected from horses after a single dose of TB-500 administration.
Significance of the Study
- This research presents the first successful identification of TB-500 and its metabolites in post-administration samples from horses, marking an advancement in efforts to curb doping in equine sports.
- The developed method will allow for a more accurate and definitive identification of the use of this performance-enhancing drug in equine sports.
Cite This Article
APA
Ho EN, Kwok WH, Lau MY, Wong AS, Wan TS, Lam KK, Schiff PJ, Stewart BD.
(2012).
Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry.
J Chromatogr A, 1265, 57-69.
https://doi.org/10.1016/j.chroma.2012.09.043 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China. emmie.nm.ho@hkjc.org.hk
MeSH Terms
- Animals
- Chromatography, Liquid / methods
- Horses
- Limit of Detection
- Mass Spectrometry / methods
- Reproducibility of Results
- Thymosin / analysis
- Thymosin / blood
Citations
This article has been cited 5 times.- Chang W, Yan S, Yan X, Wang Z, Gu B, Liu Y, Zhang Y, Yang S. The sensitive detection of low molecular mass peptide drugs in dried blood spots by solid-phase extraction and LC-HRMS. Anal Bioanal Chem 2024 Nov;416(26):5655-5669.
- Salehpour N, Bayatloo MR, Nojavan S. Magnetic solid-phase extraction of high molecular weight peptides using stearic acid-functionalized magnetic hydroxyapatite nanocomposite: determination of some hypothalamic agents in biological samples. Anal Bioanal Chem 2021 Dec;413(30):7609-7623.
- Maar K, Hetenyi R, Maar S, Faskerti G, Hanna D, Lippai B, Takatsy A, Bock-Marquette I. Utilizing Developmentally Essential Secreted Peptides Such as Thymosin Beta-4 to Remind the Adult Organs of Their Embryonic State-New Directions in Anti-Aging Regenerative Therapies. Cells 2021 May 28;10(6).
- Chi LH, Chang WM, Chang YC, Chan YC, Tai CC, Leung KW, Chen CL, Wu AT, Lai TC, Li YJ, Hsiao M. Global Proteomics-based Identification and Validation of Thymosin Beta-4 X-Linked as a Prognostic Marker for Head and Neck Squamous Cell Carcinoma. Sci Rep 2017 Aug 22;7(1):9031.
- 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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