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Drug testing and analysis2020; 12(7); 900-917; doi: 10.1002/dta.2799

A high-throughput and broad-spectrum screening method for analysing over 120 drugs in horse urine using liquid chromatography-high-resolution mass spectrometry.

Abstract: A high-throughput method has been developed for the doping control analysis of 124 drug targets, processing up to 154 horse urine samples in as short as 4.5 h, from the time the samples arrive at the laboratory to the reporting deadline of 30 min before the first race, including sample receipt and registration, preparation and instrument analysis and data vetting time. Sample preparation involves a brief enzyme hydrolysis step (30 min) to detect both free and glucuronide-conjugated drug targets. This is followed by extraction using solid-supported liquid extraction (SLE) and analysis using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). The entire set-up comprised of four sets of Biotage Extrahera automation systems for conducting SLE and five to six sets of Orbitrap for instrumental screening using LC-HRMS. Suspicious samples flagged were subject to confirmatory analyses using liquid chromatography-triple quadrupole mass spectrometry. The method comprises 124 drug targets from a spectrum of 41 drug classes covering acidic, basic and neutral drugs. More than 85% of the targets had limits of detection at or below 5 ng/mL in horse urine, with the lowest at 0.02 ng/mL. The method was validated for qualitative identification, including specificity, sensitivity, extraction recovery and precision. Method applicability was demonstrated by the successful detection of different drugs, namely (a) butorphanol, (b) dexamethasone, (c) diclofenac, (d) flunixin and (e) phenylbutazone, in post-race or out-of-competition urine samples collected from racehorses. This method was developed for pre-race urine testing in Hong Kong; however, it is also suitable for testing post-race or out-of-competition urine samples, especially when a quick total analysis time is desired.
© 2020 John Wiley & Sons, Ltd.
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Publication Date: 2020-05-18 PubMed ID: 32267632DOI: 10.1002/dta.2799Google Scholar: Lookup
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  • Journal Article

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.

This research presents a fast and all-inclusive method for detecting the use of over 120 drugs in horse urine, designed to maintain fair competition in horse racing.

Approach

The study presents a method to enable efficient and extensive screening of over 120 drugs used in doping in horse racing.

  • The method involves processing up to 154 horse urine samples in as short as 4.5 hours, from the time the samples enter the lab until 30 minutes before the start of the first race.
  • The process involves sample receipt and registration, preparation and instrument analysis, and data checking.

Procedure

The procedure involves several actions:

  • Initially, a brief enzyme hydrolysis step (30 min) is implemented to detect both free and glucuronide-conjugated drug targets.
  • Afterwards, extraction is executed using solid-supported liquid extraction (SLE).
  • Finally, analysis is carried out using liquid chromatography-high-resolution mass spectrometry (LC-HRMS).

Infrastructure

The research used a specific set-up that included:

  • Four sets of Biotage Extrahera automation systems for performing SLE.
  • Five to six sets of Orbitrap for instrumental screening using LC-HRMS.

Detection and Confirmation

Any suspicious samples were subjected to confirmatory analyses using liquid chromatography-triple quadrupole mass spectrometry.

Scope

The method targets 124 drugs from a spectrum of 41 drug classes, covering acidic, basic, and neutral drugs.

  • More than 85% of the targets had limits of detection at or below 5 ng/mL in horse urine, with the lowest at 0.02 ng/mL.
  • Successful detection of different drugs such as butorphanol, dexamethasone, diclofenac, flunixin, and phenylbutazone indicates the applicability of the method.

While the method was developed primarily for pre-race urine testing in Hong Kong, it is also adaptable for post-race or out-of-competition urine samples, especially when a quick total analysis time is needed.

Validation

This method was validated for qualitative identification, including specificity, sensitivity, extraction recovery, and precision. These factors ensure accurate and reliable results.

Cite This Article

APA
Wong JKY, Chan GHM, Choi TLS, Kwok KY, Lau MY, Leung GNW, Wan TSM, Ho ENM. (2020). A high-throughput and broad-spectrum screening method for analysing over 120 drugs in horse urine using liquid chromatography-high-resolution mass spectrometry. Drug Test Anal, 12(7), 900-917. https://doi.org/10.1002/dta.2799

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 12
Issue: 7
Pages: 900-917

Researcher Affiliations

Wong, Jenny K Y
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Chan, George H M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Choi, Timmy L S
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Kwok, Karen Y
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Lau, Ming Y
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Leung, Gary N W
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Wan, Terence S M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.
Ho, Emmie N M
  • Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N. T., Hong Kong, China.

MeSH Terms

  • Animals
  • Chromatography, Liquid / methods
  • Chromatography, Liquid / veterinary
  • Doping in Sports / prevention & control
  • High-Throughput Screening Assays / methods
  • High-Throughput Screening Assays / veterinary
  • Horses
  • Mass Spectrometry / methods
  • Mass Spectrometry / veterinary
  • Pharmaceutical Preparations / analysis
  • Pharmaceutical Preparations / chemistry
  • Pharmaceutical Preparations / urine
  • Substance Abuse Detection / methods
  • Substance Abuse Detection / veterinary
  • Time Factors

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This article includes 28 references
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Citations

This article has been cited 3 times.
  1. Nixon E, Mays TP, Routh PA, Yeatts JL, Fajt VR, Hairgrove T, Baynes RE. Plasma, urine and tissue concentrations of Flunixin and Meloxicam in Pigs. BMC Vet Res 2020 Sep 16;16(1):340.
    doi: 10.1186/s12917-020-02556-4pubmed: 32938437google scholar: lookup
  2. Zhou W, Pawliszyn J. Building a Bridge Between Ambient MS and LC-MS by Non-Exhaustive Microdesorption. Angew Chem Int Ed Engl 2025 Aug 18;64(34):e202504080.
    doi: 10.1002/anie.202504080pubmed: 40203020google scholar: lookup
  3. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Nagata SI. Construction of an individual identification panel for horses using insertion and deletion markers. J Equine Sci 2023 Sep;34(3):83-92.
    doi: 10.1294/jes.34.83pubmed: 37781568google scholar: lookup
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