Confirmatory analysis of continuous erythropoietin receptor activator and erythropoietin analogues in equine plasma by LC-MS for doping control.
Abstract: Continuous erythropoietin receptor activator (CERA) is the third generation of recombinant human erythropoietin (rhEPO) medication that retains the effect of promoting red blood cell production but has longer duration of action in the body. CERA, rhEPO, and darbepoetin alpha (DPO) can be misused to enhance performance in both human and equine athletes. To deter such misuse, a very selective and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has now been developed for identification of CERA, rhEPO, and DPO in equine plasma. The method employs a new signature tryptic peptide, T8 ((54)MEVGQQAVEVWQGLALLSEAVLR(76), common to the three proteins), and improved immunoaffinity extraction. The analytes were extracted by anti-rhEPO antibodies from plasma samples that were pretreated with polyethylene glycol (PEG) 6000. The extracted analytes were digested by trypsin and analyzed by LC-MS/MS. The limit of identification was 0.5 ng/mL for CERA, 0.2 ng/mL for rhEPO, and 0.1 ng/mL for DPO in equine plasma; the limit of detection was 0.3 ng/mL for CERA, 0.1 ng/mL for rhEPO, and 0.05 ng/mL for DPO. Specificity of the method was assessed via BLAST and SEQUEST protein database searches, and the T8 is extremely specific at both peptide and product ion levels for the identification of CERA, rhEPO, and DPO. This method was successful in identifying CERA and DPO in plasma samples collected from research horses post the drug administrations. It provides a useful tool in the fight against blood doping with CERA, rhEPO, and DPO in racehorses. Additionally, the following two technical approaches adopted in this study may also be helpful in protein identifications and biomarker discoveries in a broad scope: precipitating plasma proteins with PEG 6000 to improve immunoaffinity extraction efficiency of the target proteins and making a large and more lipophilic peptide detectable at low concentrations by increasing its solubility in the sample solvent.
Publication Date: 2010-10-14 PubMed ID: 20945883DOI: 10.1021/ac102031wGoogle Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research focuses on the development of a highly selective and sensitive method for identifying doping substances – Continuous erythropoietin receptor activator (CERA), recombinant human erythropoietin (rhEPO), and darbepoetin alpha (DPO) in equine plasma, implementing liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Doping Substances and Testing Method
- CERA, rhEPO and DPO are substances that can be misused to boost performance in both human and equine athletes, by promoting red blood cell production.
- The testing method uses a new signature tryptic peptide, T8, which is common to all three substances and helps in their identification.
- This method uses improved immunoaffinity extraction, where the substances in concern were extracted by anti-rhEPO antibodies from plasma samples that were pretreated with polyethylene glycol (PEG) 6000.
- After extraction, the analytes were digested by trypsin and analyzed by LC-MS/MS.
Sensitivity and Specificity of the Method
- The limit of identification for the substances were 0.5 ng/mL for CERA, 0.2 ng/mL for rhEPO, and 0.1 ng/mL for DPO in equine plasma.
- The limit of detection was slightly lower at 0.3 ng/mL for CERA, 0.1 ng/mL for rhEPO, and 0.05 ng/mL for DPO.
- Specificity of the method was assessed via BLAST and SEQUEST protein database searches. It was found that the T8 peptide is extremely specific at both peptide and product ion levels for the identification of CERA, rhEPO, and DPO.
Efficacy of the Method
- The developed method was successful in identifying CERA and DPO in plasma samples collected from research horses after the drug administrations.
- This technique provides a valuable tool in the fight against doping with CERA, rhEPO, and DPO in racehorses.
- Additionally, the two technical approaches adopted in this study: precipitating plasma proteins with PEG 6000 to improve extraction efficiency and making a large and more lipophilic peptide detectable at low concentrations, can also be helpful in protein identifications and biomarker discoveries in a broader scope.
Cite This Article
APA
Guan F, Uboh CE, Soma LR, Maylin G, Jiang Z, Chen J.
(2010).
Confirmatory analysis of continuous erythropoietin receptor activator and erythropoietin analogues in equine plasma by LC-MS for doping control.
Anal Chem, 82(21), 9074-9081.
https://doi.org/10.1021/ac102031w Publication
Researcher Affiliations
- School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, 382 West Street Road, Kennett Square, Pennsylvania 19348, United States, Pennsylvania Equine Toxicology and Research Center, West Chester University, West Chester, Pennsylvania 19382, United States, and Equine Drug Testing and Research Program, College of Veterinary Medicine, Cornell University, 925 Warren Drive, Ithaca, New York 14850, United States.
MeSH Terms
- Amino Acid Sequence
- Animals
- Chemical Precipitation
- Chromatography, Liquid / methods
- Chromatography, Liquid / veterinary
- Darbepoetin alfa
- Doping in Sports
- Erythropoietin / analogs & derivatives
- Erythropoietin / analysis
- Erythropoietin / blood
- Horses / blood
- Humans
- Molecular Sequence Data
- Polyethylene Glycols / analysis
- Polyethylene Glycols / chemistry
- Recombinant Proteins / analysis
- Recombinant Proteins / blood
- Sensitivity and Specificity
- Substance Abuse Detection / methods
- Substance Abuse Detection / veterinary
- Tandem Mass Spectrometry / methods
- Tandem Mass Spectrometry / veterinary
Citations
This article has been cited 1 times.- Jamieson CA, Baillie SL, Johnson JP. Blood Transfusion in Equids-A Practical Approach and Review.. Animals (Basel) 2022 Aug 23;12(17).
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