Detection, quantification, and identification of dermorphin in equine plasma and urine by LC-MS/MS for doping control.
Abstract: Dermorphin is a unique opioid peptide that is 30-40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC-MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20-10,000 pg/mL in plasma and 50-20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.
Publication Date: 2013-04-10 PubMed ID: 23571464DOI: 10.1007/s00216-013-6907-0Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article describes the first-ever LC-MS/MS method for detecting, quantifying, and identifying the opioid peptide, dermorphin, in horse plasma and urine samples to control doping in horse racing.
Methodology
- The method involves pre-treatment of horse plasma and urine samples with ethylenediamine tetra-acetic acid and urea.
- These pre-treated samples are then subjected to solid-phase extraction (SPE) on Oasis MCX cartridges.
- The eluates obtained are then dried under vacuum and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect dermorphin.
Assessments Carried Out
- The researchers evaluated the matrix effect, solid-phase extraction efficiency, and the stability of dermorphin in the samples.
- Other assessments carried out include intra-day and inter-day accuracy and precision of the method.
Findings
- The method can detect as low as 10 pg/mL dermorphin in plasma and 20 pg/mL in urine.
- The confirmation limits for dermorphin were 20 pg/mL for plasma and 50 pg/mL for urine.
- Dermorphin was found to be stable at normal ambient temperatures, while its diastereomer showed instability.
- With the use of isotopically labeled dermorphin as an internal standard, the method efficiently quantified between 20-10,000 pg/mL dermorphin in plasma and 50-20,000 pg/mL in urine.
- The accuracy of the method on any given day and over different days ranged from 91% – 100% for low, medium, and high concentration levels.
- The method showed variation coefficients of less than 12% on the same day and across different days.
Specificity and Application
- The method was able to differentiate dermorphin from its diastereomer, indicating high specificity.
- The LC-MS/MS method’s specificity for identifying dermorphin in horse plasma and urine was confirmed through BLAST search, targeted SEQUEST search, and MS/MS spectrum library search.
- Finally, the successful application of the method was demonstrated by analyzing samples collected from research horses post administration of dermorphin, as well as official post-race samples.
Cite This Article
APA
Guan F, Uboh CE, Soma LR, Robinson M, Maylin GA, Li X.
(2013).
Detection, quantification, and identification of dermorphin in equine plasma and urine by LC-MS/MS for doping control.
Anal Bioanal Chem, 405(14), 4707-4717.
https://doi.org/10.1007/s00216-013-6907-0 Publication
Researcher Affiliations
- Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA 19483, USA.
MeSH Terms
- Analgesics, Opioid / analysis
- Animals
- Chromatography, Liquid / methods
- Chromatography, Liquid / veterinary
- Doping in Sports / prevention & control
- Horses / blood
- Horses / urine
- Opioid Peptides / analysis
- Substance Abuse Detection / methods
- Substance Abuse Detection / veterinary
- Tandem Mass Spectrometry / methods
- Tandem Mass Spectrometry / veterinary
Citations
This article has been cited 2 times.- Sekulic D, Tahiraj E, Zvan M, Zenic N, Uljevic O, Lesnik B. Doping Attitudes and Covariates of Potential Doping Behaviour in High-Level Team-Sport Athletes; Gender Specific Analysis.. J Sports Sci Med 2016 Dec;15(4):606-615.
- Zvan M, Zenic N, Sekulic D, Cubela M, Lesnik B. Gender- and Sport-Specific Associations Between Religiousness and Doping Behavior in High-Level Team Sports.. J Relig Health 2017 Aug;56(4):1348-1360.
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