Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry.
Abstract: Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples.
Copyright © 2010 Elsevier B.V. All rights reserved.
Publication Date: 2010-12-31 PubMed ID: 21256494DOI: 10.1016/j.chroma.2010.12.052Google Scholar: Lookup
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- Journal Article
- Analytical Methods
- Doping
- Equestrian Sports
- Equine Diseases
- Equine Health
- Equine Science
- Exercise Physiology
- High-performance Liquid Chromatography (HPLC)
- Horse Management
- Insulin
- Laboratory Methods
- Metabolic Health
- Metabolites
- Performance Horses
- Pharmaceuticals
- Urine Analysis
- Veterinary Care
- Veterinary Medicine
- Veterinary Research
Summary
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The study discusses the development of a new method to detect doping in horses using banned substances like insulin and its synthetic analogues, which can potentially enhance performance. This method successfully identifies the trace presence of these substances in horse urine samples.
Understanding the Context: Doping in Sports
- There are strict rules against doping violations in both human and equine sports due to the performance enhancement potential of various substances.
- Insulin and its synthetic analogues can boost muscular glycogen and prevent protein breakdown, impacting muscle size and performance.
- Human insulin and its counterpart substances from other species like bovines (cows) and porcines (pigs) are often available and could be misused for doping.
Objective of the Study
- The aim of this research was to establish an effective method for detecting the misuse of insulins in equine sports.
- Earlier, the same lab had established a detection method for bovine, porcine and human insulins, along with synthetic analogues likes Humalog (Lispro) and Novolog (Aspart) in equine plasma.
- This study expands upon that initial research by devising a technique to detect these substances and their metabolites in horse urine.
Research Method and Results
- The research utilized immunoaffinity purification to isolate the insulins and metabolites from the urine.
- The isolated substances were then analyzed with nano liquid chromatography-tandem mass spectrometry (LC/MS/MS).
- Insulins and their analogues were identified by comparing retention times and major product ions.
- The detection sensitivity for the five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin) was as low as 0.05ng/mL, indicating a high sensitivity of this method.
- The method was successfully applied to identify human insulin in urine samples collected from horses that had been administered a low dose of recombinant human insulin (Humulin R, Eli Lilly). This was within a 4-hour timeframe post administration, which is noteworthy for the rapid detection and short duration.
Concluding Remarks
- To the researchers’ knowledge, this is the first time exogenous insulin has been identified in post-administration horse urine samples.
- This implies that the developed method is potentially a breakthrough in enhancing fairness and integrity in equine sports through reliable detection of doping violations.
Cite This Article
APA
Ho EN, Wan TS, Wong AS, Lam KK, Stewart BD.
(2010).
Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry.
J Chromatogr A, 1218(8), 1139-1146.
https://doi.org/10.1016/j.chroma.2010.12.052 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China. emmie.nm.ho@hkjc.org.hk
MeSH Terms
- Amino Acid Sequence
- Animals
- Cattle
- Chromatography, Liquid / methods
- Doping in Sports
- Horses
- Humans
- Immunosorbent Techniques
- Insulin / analogs & derivatives
- Insulin / chemistry
- Insulin / urine
- Insulin Aspart
- Insulin Lispro
- Reproducibility of Results
- Sensitivity and Specificity
- Swine
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 5 times.- Zeng X, Wang H, Zeng Y, Yang Y, Zhang Z, Li L. Label-free Aptasensor for the Ultrasensitive Detection of Insulin Via a Synergistic Fluorescent Turn-on Strategy Based on G-quadruplex and AIEgens. J Fluoresc 2023 May;33(3):955-963.
- Leschke DH, Muir GS, Hodgson JK, Coyle M, Horn R, Bertin FR. Immunoreactive insulin stability in horses at risk of insulin dysregulation. J Vet Intern Med 2019 Nov;33(6):2746-2751.
- Amouzadeh Tabrizi M, Shamsipur M, Saber R, Sarkar S, Besharati M. An electrochemical aptamer-based assay for femtomolar determination of insulin using a screen printed electrode modified with mesoporous carbon and 1,3,6,8-pyrenetetrasulfonate. Mikrochim Acta 2017 Dec 18;185(1):59.
- Liang K, Wu H, Li Y. Immune-enrichment of insulin in bio-fluids on gold-nanoparticle decorated target plate and in situ detection by MALDI MS. Clin Proteomics 2017;14:5.
- Sistik P, Urinovska R, Handlosova K, Handlos P, Andelova K, Jurica J, Stejskal D. First Dimension Trap-and-Elute Combined with Second Dimension Reversed-Phase Liquid Chromatography Separation Using a Two-Dimensional-Liquid Chromatography-Tandem Mass Spectrometry System for Sensitive Quantification of Human Insulin and Six Insulin Analogs in Plasma: Improved Chromatographic Resolution and Stability Testing. J Sep Sci 2025 Feb;48(2):e70092.
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