Doping control analysis of 46 polar drugs in horse plasma and urine using a ‘dilute-and-shoot’ ultra high performance liquid chromatography-high resolution mass spectrometry approach.
Abstract: The high sensitivity of ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) allows the identification of many prohibited substances without pre-concentration, leading to the development of simple and fast 'dilute-and-shoot' methods for doping control for human and equine sports. While the detection of polar drugs in plasma and urine is difficult using liquid-liquid or solid-phase extraction as these substances are poorly extracted, the 'dilute-and-shoot' approach is plausible. This paper describes a 'dilute-and-shoot' UHPLC-HRMS screening method to detect 46 polar drugs in equine urine and plasma, including some angiotensin-converting enzyme (ACE) inhibitors, sympathomimetics, anti-epileptics, hemostatics, the new doping agent 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), as well as two threshold substances, namely dimethyl sulfoxide and theobromine. For plasma, the sample (200μL) was protein precipitated using trichloroacetic acid, and the resulting supernatant was diluted using Buffer A with an overall dilution factor of 3. For urine, the sample (20μL) was simply diluted 50-fold with Buffer A. The diluted plasma or urine sample was then analysed using a UHPLC-HRMS system in full-scan ESI mode. The assay was validated for qualitative identification purpose. This straightforward and reliable approach carried out in combination with other screening procedures has increased the efficiency of doping control analysis in the laboratory. Moreover, since the UHPLC-HRMS data were acquired in full-scan mode, the method could theoretically accommodate an unlimited number of existing and new doping agents, and would allow a retrospectively search for drugs that have not been targeted at the time of analysis.
Copyright © 2016 Elsevier B.V. All rights reserved.
Publication Date: 2016-05-03 PubMed ID: 27180888DOI: 10.1016/j.chroma.2016.05.002Google 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
- Validation Study
- Analytical Methods
- Diagnostic Technique
- Doping
- Drug
- Equestrian Sports
- Equine Diseases
- Equine Health
- Equine Science
- High-performance Liquid Chromatography (HPLC)
- Horse Management
- Horse Training
- Laboratory Methods
- Performance Horses
- Pharmaceuticals
- Plasma
- Urine Analysis
- Veterinary Care
- Veterinary Medicine
- Veterinary Research
Summary
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This research discusses using a fast and straightforward ‘dilute-and-shoot’ method with ultra high performance liquid chromatography-high resolution mass spectrometry for detecting prohibited substances in horse plasma and urine. This allows for easier detection of 46 polar drugs, including various types of doping agents.
Objective of the Research
- This research aims to present a new method for doping control in equine sports through the use of a ‘dilute-and-shoot’ UHPLC-HRMS screening approach.
- The authors of the study seek to improve the efficiency of doping control analysis and the detection of polar drugs that are challenging to detect with previously applied methods.
- The researchers illustrate this newly introduced screening method with its application on 46 polar drugs including some often utilized in doping.
Methodology
- The researchers use a ‘dilute-and-shoot’ UHPLC-HRMS method for the screening and detection of the targeted drugs.
- In the case of plasma, the sample was protein precipitated using trichloroacetic acid and then diluted using Buffer A. This led to an overall dilution factor of 3.
- For urine, the sample was directly diluted 50-fold with Buffer A.
- The diluted plasma or urine sample was then processed through the UHPLC-HRMS system for analysis in full-scan ESI mode.
Validation and Potential Applications
- The developed method was validated for qualitative identification purposes.
- This new approach is presented as a reliable and efficient method which could work in conjunction with other screening methods for doping control analysis.
- Since UHPLC-HRMS data was collected in full-scan mode, there’s potential for this method to theoretically detect an unlimited number of existing and new doping agents.
- This would enable a retrospectively search for drugs that hadn’t been targeted at the time of analysis, enhancing the capability of doping control.
Cite This Article
APA
Kwok WH, Choi TLS, Kwok KY, Chan GHM, Wong JKY, Wan TSM.
(2016).
Doping control analysis of 46 polar drugs in horse plasma and urine using a ‘dilute-and-shoot’ ultra high performance liquid chromatography-high resolution mass spectrometry approach.
J Chromatogr A, 1451, 41-49.
https://doi.org/10.1016/j.chroma.2016.05.002 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China. Electronic address: wh.kwok@hkjc.org.hk.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China.
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China. Electronic address: terence.sm.wan@hkjc.org.hk.
MeSH Terms
- Aminoimidazole Carboxamide / analogs & derivatives
- Aminoimidazole Carboxamide / blood
- Aminoimidazole Carboxamide / urine
- Animals
- Chromatography, High Pressure Liquid / methods
- Doping in Sports / prevention & control
- Horses / blood
- Horses / urine
- Mass Spectrometry / methods
- Pharmaceutical Preparations / blood
- Pharmaceutical Preparations / urine
- Ribonucleotides / blood
- Ribonucleotides / urine
- Substance Abuse Detection / methods
Citations
This article has been cited 4 times.- Mainero Rocca L, L'Episcopo N, Gordiani A, Vitali M, Staderini A. A 'Dilute and Shoot' Liquid Chromatography-Mass Spectrometry Method for Multiclass Drug Analysis in Pre-Cut Dried Blood Spots. Int J Environ Res Public Health 2021 Mar 16;18(6).
- Tozaki T, Kikuchi M, Kakoi H, Hirota KI, Mukai K, Aida H, Nakamura S, Nagata SI. Profiling of exercise-induced transcripts in the peripheral blood cells of Thoroughbred horses. J Equine Sci 2016;27(4):157-164.
- Sundström M, Kriikku P, Ojanperä I, Baessmann C, Pelander A. UHPLC-QTOFMS Urine Drug Screening With Dilute-and-Shoot Sample Preparation and Vacuum-Insulated Probe-Heated Electrospray Ionization. Drug Test Anal 2025 Aug;17(8):1208-1218.
- Wang Y, Li W, Deng X. Development and application of a dual isotopic labeling method for enhanced detection and quantification of stimulants in urine samples using high-resolution mass spectrometry. Anal Bioanal Chem 2024 Dec;416(29):7073-7084.
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