Lipid and Corticosteroid Biomarkers Under the Influence of Bisphosphonates.
Abstract: Detecting the use of bisphosphonates (BPs) in equine athletes is of interest to regulators and laboratories due to the threat to welfare issues for the potential to provide analgesic effects and manipulating bone structure. The detection of BPs in biological matrices is challenging due to erratic biological elimination and inconsistent analytical recoveries. Therefore, complementary approaches are needed to provide evidence of their misuse in racehorses. BPs have two sub-classes: nitrogenous and non-nitrogenous. This study investigated plasma elimination following administration of one example from each sub-class, together with changes in endogenous eicosanoid and corticosteroids. Zoledronic acid (ZA) and tiludronic acid (TA) were administered by IV infusion to 8 thoroughbred horses with an 11-month washout period between each administration. Sample preparation for quantification of BPs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilised a two-step solid phase extraction (SPE) consisting of polymeric reversed-phase followed by weak anion exchange prior to derivatisation using trimethyl orthoacetate. Endogenous biomarkers were analysed after protein precipitation and SPE with polymeric reversed-phase prior to liquid chromatography-high resolution mass spectrometry (LC-HRMS) using data independent acquisition. The LC-MS/MS analysis showed ZA was undetectable after 8 h post-administration while TA was detected up to the final collection point of 28 days post-administration. The LC-HRMS analysis utilised targeted (i.e., prior inclusion list of compounds) approaches to monitor level changes of eicosanoid and corticosteroid biomarkers. Putative biomarkers were identified and now subject to validation for translation into routine sample analysis for improved retrospectivity to detecting BP misuse in equine plasma.
© 2024 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.
Publication Date: 2024-10-15 PubMed ID: 39407358PubMed Central: PMC12209688DOI: 10.1002/dta.3811Google Scholar: Lookup
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- Journal Article
Summary
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Overview
- This study investigated how two types of bisphosphonates (BPs)—zoledronic acid and tiludronic acid—are eliminated in horse plasma and examined changes in lipid and corticosteroid biomarkers to improve detection of BP misuse in racehorses.
Background and Purpose
- Bisphosphonates (BPs) are drugs that affect bone metabolism and can provide pain relief (analgesic effects), potentially giving racehorses an unfair advantage and raising welfare concerns.
- Detection of BPs in biological samples is difficult due to their irregular elimination from the body and variability in analytical methods.
- Because of this, researchers are looking for complementary biomarker approaches that indicate BP use indirectly, improving detection capabilities.
- BPs fall into two categories: nitrogenous (e.g., zoledronic acid) and non-nitrogenous (e.g., tiludronic acid), which may have different pharmacokinetics and biological effects.
Study Design
- Eight thoroughbred horses received both zoledronic acid (ZA) and tiludronic acid (TA) via intravenous infusion, with an 11-month washout period between treatments.
- Blood plasma samples were collected after administration to monitor drug levels and changes in endogenous lipid (eicosanoids) and corticosteroid biomarkers.
Analytical Methods
- Quantifying BPs:
- Used liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Sample preparation involved a two-step solid phase extraction (SPE) using polymeric reversed-phase and weak anion exchange columns.
- Derivatisation with trimethyl orthoacetate improved detection sensitivity.
- Measuring endogenous biomarkers:
- Sample preparation included protein precipitation and SPE with polymeric reversed-phase.
- Analysis performed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) using data independent acquisition.
- Targeted approaches were used with inclusion lists to monitor specific eicosanoids and corticosteroids known to change in response to drug administration.
Key Findings
- Pharmacokinetics:
- Zoledronic acid was rapidly eliminated and undetectable after 8 hours post-administration.
- Tiludronic acid remained detectable up to 28 days after administration, showing a longer presence in plasma.
- Biomarker changes:
- Identified potential lipid (eicosanoid) and corticosteroid biomarkers that changed in plasma following BP administration.
- These biomarkers were considered “putative,” meaning they require further validation to confirm reliability.
Implications and Future Directions
- This study provides evidence that combining direct BP detection with monitoring endogenous biomarkers could improve retrospective detection of BP use in racehorses.
- The identified biomarkers may enhance regulatory and laboratory ability to detect misuse, particularly when direct drug detection is challenging due to rapid elimination.
- Further validation of these lipid and corticosteroid biomarkers is necessary before integrating them into routine testing workflows.
- This approach presents a promising complementary strategy for safeguarding equine welfare and maintaining fair competition standards.
Cite This Article
APA
Tou K, Cawley A, Noble G, Loy J, Bishop D, Keledjian J, Sornalingam K, Richards S, Fu S.
(2024).
Lipid and Corticosteroid Biomarkers Under the Influence of Bisphosphonates.
Drug Test Anal, 17(7), 1107-1117.
https://doi.org/10.1002/dta.3811 Publication
Researcher Affiliations
- Centre for Forensic Science, University of Technology Sydney, Sydney, New South Wales, Australia.
- Racing Analytical Services Ltd, Flemington, Victoria, Australia.
- School of Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
- School of Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales, Australia.
- Hyphenated Mass Spectrometry Laboratory, University of Technology Sydney, Sydney, New South Wales, Australia.
- Australian Racing Forensic Laboratory, Racing NSW, Sydney, New South Wales, Australia.
- Australian Racing Forensic Laboratory, Racing NSW, Sydney, New South Wales, Australia.
- Australian Racing Forensic Laboratory, Racing NSW, Sydney, New South Wales, Australia.
- Centre for Forensic Science, University of Technology Sydney, Sydney, New South Wales, Australia.
MeSH Terms
- Animals
- Horses / blood
- Diphosphonates / blood
- Diphosphonates / administration & dosage
- Diphosphonates / pharmacokinetics
- Biomarkers / blood
- Tandem Mass Spectrometry / methods
- Tandem Mass Spectrometry / veterinary
- Doping in Sports
- Adrenal Cortex Hormones / blood
- Chromatography, Liquid / methods
- Substance Abuse Detection / methods
- Substance Abuse Detection / veterinary
- Zoledronic Acid
- Solid Phase Extraction / methods
- Bone Density Conservation Agents / blood
- Bone Density Conservation Agents / administration & dosage
- Bone Density Conservation Agents / pharmacokinetics
- Imidazoles / blood
- Imidazoles / administration & dosage
- Imidazoles / pharmacokinetics
- Lipids / blood
- Male
Conflict of Interest Statement
The Shimadzu LC‐QTOF 9030 was a demonstration instrument provided by Shimadzu Scientific Instruments (Australasia) to the ARFL for evaluation of biomarker profiling in equine anti‐doping.
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