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Direct injection horse-urine analysis for the quantification and confirmation of threshold substances for doping control. IV. Determination of 3-methoxytyramine by hydrophilic interaction liquid chromatography/quadrupole time-of-flight mass spectrometry.
Abstract: Levodopa and dopamine have been abused as performance-altering substances in horse racing. Urinary 3-methoxytyramine is used as an indicator of dopaminergic manipulation resulting from dopamine or levodopa administration and is prohibited with a urinary threshold of 4 microg mL(-1) (free and conjugated). A simple liquid chromatographic (LC)/mass spectrometric (MS) (LCMS) method was developed and validated for the quantification and identification of 3-methoxytyramine in equine urine. Sample preparation involved enzymatic hydrolysis and protein precipitation. Hydrophilic interaction liquid chromatography (HILIC) was selected as a separation technique that allows effective retention of polar substances like 3-methoxytyramine and efficient separation from matrix compounds. Electrospray ionization (ESI) in positive mode with product ion scan mode was chosen for the detection of the analytes. Quantification of 3-methoxytyramine was performed with fragmentation at low collision energy, resulting in one product ion, while a second run at high collision energy was performed for confirmation (at least three abundant ions). Studies on matrix effects showed ion suppression depending on the horse urine used. To overcome the variability of the results originating from the matrix effects, isotopic labelled internal standard was used and linear regression calibration methodology was applied for the quantitative determination of the analyte. The tested linear range was 1-20 microg mL(-1). The relative standard deviations of intra- and inter- assay analysis of 3-methoxytyramine in horse urine were lower than 4.2% and 3.2%, respectively. Overall accuracy (relative percentage error) was less than 6.2%. The method was applied to case samples, demonstrating simplicity, accuracy and selectivity.
Copyright 2009 John Wiley & Sons, Ltd.
Publication Date: 2010-04-01 PubMed ID: 20355216DOI: 10.1002/dta.70Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
- Validation Study
- Analytical Methods
- Biochemistry
- Diagnostic Technique
- Dopamine
- Doping
- Equine Diseases
- Equine Health
- High-performance Liquid Chromatography (HPLC)
- Horse Management
- Horse Racing
- Laboratory Methods
- Performance Horses
- Pharmaceuticals
- Physiology
- Urine Analysis
- Veterinary Care
- Veterinary Medicine
- Veterinary Research
- Veterinary Science
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research article presents a new method of detecting substances that can be used for doping in horse-racing. The method relies on the quantification of 3-methoxytyramine, a substance that increases in the body after consumption of dopamine or levodopa, drugs often misused for performance enhancement in competitive equestrian sports.
Research Methodology
- The research sought to develop and validate a simple LCMS method to identify and quantify 3-methoxytyramine in horse urine.
- The process of sample preparation involved two steps: enzymatic hydrolysis and protein precipitation. These steps were crucial to isolate the 3-methoxytyramine in its purest form.
- The researchers used Hydrophilic Interaction Liquid Chromatography (HILIC) to effectively retain polar substances like 3-methoxytyramine and to separate it from other compounds in the matrix.
Results of the Study
- For the detection of the substance, Electrospray ionization (ESI) in positive mode with product ion scan mode was used. Fragmentation at low collision energy resulted in one product ion which they used to quantify 3-methoxytyramine.
- To confirm the presence of 3-methoxytyramine, they did a second run at high collision energy to get at least three abundant ions.
- Matrix effects were also studied; and they noted that ion suppression happened, depending on the horse urine used.
- To minimize the variability of results due to matrix effects, they used isotopic labelled internal standard and linear regression calibration.
- They tested the linearity within a range of 1-20 μg/mL and established that the relative standard deviations were being lower than 4.2% and 3.2% intra- and inter-assay respectively.
Conclusion of the Study
- The research validated the method’s overall accuracy to have a relative percentage error less than 6.2%.
- The method was also applied to actual case samples, wherein it demonstrated accuracy, selectivity, as well as simplicity in its application.
- This novel technique therefore offers a reliable tool for detecting doping in horse racing by quantifying 3-methoxytyramine levels in horse urine.
Cite This Article
APA
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.
(2010).
Direct injection horse-urine analysis for the quantification and confirmation of threshold substances for doping control. IV. Determination of 3-methoxytyramine by hydrophilic interaction liquid chromatography/quadrupole time-of-flight mass spectrometry.
Drug Test Anal, 1(8), 365-371.
https://doi.org/10.1002/dta.70 Publication
Researcher Affiliations
- Doping Control Laboratory of Athens, Olympic Athletic Centre of Athens Spiros Louis, 37 Kifissias Ave, 151 23 Maroussi, Greece.
MeSH Terms
- Animals
- Calibration
- Chromatography, Liquid / methods
- Dopamine / analogs & derivatives
- Dopamine / urine
- Female
- Horses
- Hydrophobic and Hydrophilic Interactions
- Male
- Spectrometry, Mass, Electrospray Ionization / methods
- Substance Abuse Detection / methods
- Urinalysis / methods
Citations
This article has been cited 1 times.- Fouché N, Gerber V, Gorgas D, Marolf V, Grouzmann E, van der Kolk JH, Navas de Solis C. Catecholamine Metabolism in a Shetland Pony with Suspected Pheochromocytoma and Pituitary Pars Intermedia Dysfunction. J Vet Intern Med 2016 Nov;30(6):1872-1878.
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