A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry.
Abstract: The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the original four LC/MS methods. The new method has better detection limits than the superseded methods. In addition, it covers new target analytes that could not be adequately detected by the four LC/MS methods. The new method involves solid-phase extraction (SPE) of two aliquots of equine urine using two Abs Elut Nexus cartridges. One aliquot of the urine sample is treated with β-glucuronidase before subjecting to SPE. A second aliquot of the same urine sample is processed directly using another SPE cartridge, so that drugs that are prone to decomposition during enzyme hydrolysis can be preserved. The combined eluate is analysed by UPLC/MS/MS using alternating positive and negative electrospray ionisation in the selected-reaction-monitoring mode. Exceptional chromatographic separation is achieved using an UPLC system equipped with a UPLC(®) BEH C18 column (10 cm L×2.1 mm ID with 1.7 μm particles). With this newly developed UPLC/MS/MS method, the simultaneous detection of 140 drugs at ppb to sub-ppb levels in equine urine can be achieved in less than 13 min inclusive of post-run equilibration. Matrix interference for the selected transitions at the expected retention times is minimised by the excellent UPLC chromatographic separation. The method has been validated for recovery and precision, and is being used regularly in the authors' laboratory as an important component of the array of screening methods for doping control analyses of equine urine samples.
Copyright © 2011 Elsevier B.V. All rights reserved.
Publication Date: 2011-04-23 PubMed ID: 21641418DOI: 10.1016/j.aca.2011.04.030Google Scholar: Lookup
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- Journal Article
- Analytical Methods
- Beta-Glucuronidase
- Corticosteroids
- Diagnostic Technique
- Doping
- Drug
- Equine Diseases
- Equine Health
- High-performance Liquid Chromatography (HPLC)
- Horse Management
- Laboratory Methods
- Pharmaceuticals
- Urine Analysis
- Veterinary Care
- Veterinary Medicine
- Veterinary Procedure
- Veterinary Research
- Veterinary Science
Summary
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The research article centers on a newly developed method using ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) for more efficient and effective screening of a multitude of drugs in equine urine. With this new method, the simultaneous detection of 140 drugs can be achieved in less than 13 minutes, greatly reducing labor and instrumental workload.
Original Screening Methods and their Limitations
- The authors’ laboratory formerly used four different liquid chromatography/mass spectrometric (LC/MS) methods to detect a wide range of drugs in equine urine, including anti-diabetic drugs, sedatives, steroids, diuretics, and more.
- Despite their range, these methods were labor-intensive and time-consuming, and they did not cover some target analytes, limiting their effectiveness.
Development and Mechanism of the New UPLC/MS/MS Method
- In response to these limitations, the authors developed a new method utilizing UPLC/MS/MS that consolidates all target analytes detected by the original four LC/MS methods, resulting in improved detection limits and coverage.
- The new method uses a solid-phase extraction (SPE) of two aliquots (portions) of equine urine with two Abs Elut Nexus cartridges.
- One aliquot of the urine sample undergoes treatment with β-glucuronidase prior to SPE. This enzyme breaks down glucuronic acid in the urine, facilitating drug detection.
- The second aliquot is processed directly, allowing preservation of drugs that may decompose during enzyme hydrolysis.
- The combined eluate, which is the dissolved drug portion, is analyzed by UPLC/MS/MS using positive and negative electrospray ionisation in the selected-reaction-monitoring mode, an optimized detection scheme.
- These steps altogether allow simultaneous detection of 140 drugs at ppb (parts per billion) to sub-ppb levels in equine urine in less than 13 minutes.
Benefits and Results of the New Method
- The new method drastically reduces matrix interference, or background noise, during the detection process due to the superior chromatographic separation achieved with the UPLC system, ensuring more accurate measurements.
- Moreover, the method has been validated for recovery (how well the extraction process works) and precision, making it a reliable resource for drug detection.
- Given its efficiency and effectiveness, the authors’ lab now uses this method as an important part of their array of screening techniques for doping control analysis in equine urine samples.
Cite This Article
APA
Wong CH, Tang FP, Wan TS.
(2011).
A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry.
Anal Chim Acta, 697(1-2), 48-60.
https://doi.org/10.1016/j.aca.2011.04.030 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, N.T., Hong Kong, China. colton.hf.wong@hkjc.org.hk
MeSH Terms
- Animals
- Cattle
- Chromatography, High Pressure Liquid / economics
- Chromatography, High Pressure Liquid / methods
- Doping in Sports
- Glucuronidase / metabolism
- Horses / urine
- Hydrolysis
- Patella / enzymology
- Peptide Hydrolases / metabolism
- Pharmaceutical Preparations / chemistry
- Pharmaceutical Preparations / isolation & purification
- Pharmaceutical Preparations / metabolism
- Pharmaceutical Preparations / urine
- Solid Phase Extraction
- Tandem Mass Spectrometry / economics
- Tandem Mass Spectrometry / methods
- Time Factors
- Urinalysis / economics
- Urinalysis / methods
Citations
This article has been cited 1 times.- Rodríguez-Palazón MC, Arroyo-Manzanares N, Viñas P, López-García I, Hernández-Córdoba M, Campillo N. Dispersive magnetic solid-phase extraction for capsaicinoid compounds in human serum using LC-HRMS: targeted and non-targeted approaches. Anal Bioanal Chem 2023 May;415(11):2133-2145.
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