LC-MS/MS method for confirmation of recombinant human erythropoietin and darbepoetin alpha in equine plasma.
Abstract: Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS. Two specific proteotypic peptides, 46VNFYAWK52 and 144VYSNFLR150 from rhEPO and DPO were employed for confirmation of the analytes based on chromatographic retention times and major product ions. The limit of confirmation of this method was 0.2 ng/mL, and the limit of detection was 0.1 ng/mL for rhEPO and DPO in equine plasma. This method was successful in confirming the presence of rhEPO and DPO in plasma samples collected from research horses to which rhEPO or DPO was administered and from racehorses following competition and in noncompetition samples in North America. To our knowledge, this is the first LC-MS method with adequate sensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples. This method provides a powerful enforcement tool that was lacking in the fight against the abuse of rhEPO and DPO in the horse racing industry.
Publication Date: 2007-05-15 PubMed ID: 17500535DOI: 10.1021/ac070135oGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article entails the development of a novel method using LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) to confirm the presence of recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) in equine plasma. These drugs, commonly used to treat anemia, are at times abused in equine sports for performance enhancement. The new method has proved to be efficient, sensitive, and specific in detecting these prohibited substances in equine sports.
LC-MS/MS Detection Method
- The paper describes a method utilizing LC-MS/MS to detect and confirm rhEPO and DPO in horse plasma. LC-MS/MS is a technique that combines the physical separation capabilities of liquid chromatography (LC) with the mass analysis capabilities of mass spectrometry (MS). It’s a powerful tool in identifying the components of a sample.
- This method includes separation and enrichment of the target substances (rhEPO and DPO) using an immunoaffinity process with anti-rhEPO antibodies attached to magnetic beads. This step increases the concentration of analytes relative to other substances to increase detection sensitivity.
- In the next step, the samples are digested with trypsin – an enzyme that cuts proteins into smaller peptides. The resulting fragments are then analyzed using LC-MS/MS.
Confirmation of Analytes
- For confirming the substances, two specific “proteotypic” peptides (small pieces of the protein) were used. These peptides, 46VNFYAWK52 and 144VYSNFLR150, are specific to rhEPO and DPO.
- The method focuses on comparing the chromatographic retention times and the major product ions for each peptide, which would ensure the presence and identity of the drugs.
Performance of the Method
- The method has been effective in confirming the presence of rhEPO and DPO in equine plasma with high sensitivity. The limit of detection was as low as 0.1 ng/mL, and the limit of confirmation was 0.2 ng/mL, making it a highly sensitive detection method.
- The new methodology was successfully applied in real settings, with plasma samples from research horses and racehorses in competition and non-competition samples.
Impact on the Horse Racing Industry
- The mention of the method’s usability in the horse racing industry points to its potential in curbing the abuse of performance-enhancing drugs in the sport. By providing a reliable tool for detecting these substances, it assists in ensuring fair competition and animal welfare.
- The authors note that this method is the first of its kind for providing unambiguous confirmation of rhEPO and DPO in equine plasma, marking a notable contribution to drug testing methodologies in equine sports.
Cite This Article
APA
Guan F, Uboh CE, Soma LR, Birks E, Chen J, Mitchell J, You Y, Rudy J, Xu F, Li X, Mbuy G.
(2007).
LC-MS/MS method for confirmation of recombinant human erythropoietin and darbepoetin alpha in equine plasma.
Anal Chem, 79(12), 4627-4635.
https://doi.org/10.1021/ac070135o Publication
Researcher Affiliations
- University of Pennsylvania School of Veterinary Medicine, New Bolton Center Campus, 382 West Street Road, Kennett Square, Pennsylvania 19348, USA.
MeSH Terms
- Animals
- Antibodies / immunology
- Chromatography, Liquid / methods
- Darbepoetin alfa
- Doping in Sports
- Erythropoietin / analogs & derivatives
- Erythropoietin / blood
- Horse Diseases / blood
- Horses
- Humans
- Mass Spectrometry / methods
- Recombinant Proteins
- Reproducibility of Results
- Sensitivity and Specificity
- Substance Abuse Detection / methods
Citations
This article has been cited 8 times.- Dahlgren AR, Knych HK, Arthur RM, Durbin-Johnson BP, Finno CJ. Transcriptomic Markers of Recombinant Human Erythropoietin Micro-Dosing in Thoroughbred Horses.. Genes (Basel) 2021 Nov 24;12(12).
- Sun XY, Ma RT, Chen J, Shi YP. Magnetic boronate modified molecularly imprinted polymers on magnetite microspheres modified with porous TiO(2) (Fe(3)O(4)@pTiO(2)@MIP) with enhanced adsorption capacity for glycoproteins and with wide operational pH range.. Mikrochim Acta 2018 Nov 29;185(12):565.
- Jin S, Liu L, Zhou P. Amorphous titania modified with boric acid for selective capture of glycoproteins.. Mikrochim Acta 2018 May 22;185(6):308.
- Liu X, Zeng L, Zhao Z, Xie Y, Wang S, Zhang J, He Y, Zou Z, Zhang J, Tao A. Construction, Expression, and Characterization of rSEA-EGF and In Vitro Evaluation of its Antitumor Activity Against Nasopharyngeal Cancer.. Technol Cancer Res Treat 2018 Jan 1;17:1533033818762910.
- Liu L, Xing Y, Zhang H, Liu R, Liu H, Xia N. Amplified voltammetric detection of glycoproteins using 4-mercaptophenylboronic acid/biotin-modified multifunctional gold nanoparticles as labels.. Int J Nanomedicine 2014;9:2619-26.
- Llewellyn AC, Zhao J, Song F, Parvathareddy J, Xu Q, Napier BA, Laroui H, Merlin D, Bina JE, Cotter PA, Miller MA, Raetz CR, Weiss DS. NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis.. Mol Microbiol 2012 Nov;86(3):611-27.
- Lee B, Lopez-Ferrer D, Kim BC, Na HB, Park YI, Weitz KK, Warner MG, Hyeon T, Lee SW, Smith RD, Kim J. Rapid and efficient protein digestion using trypsin-coated magnetic nanoparticles under pressure cycles.. Proteomics 2011 Jan;11(2):309-18.
- López-Ferrer D, Petritis K, Lourette NM, Clowers B, Hixson KK, Heibeck T, Prior DC, Pasa-Tolić L, Camp DG 2nd, Belov ME, Smith RD. On-line digestion system for protein characterization and proteome analysis.. Anal Chem 2008 Dec 1;80(23):8930-6.
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