Lipid and Corticosteroid Biomarkers Under the Influence of Bisphosphonates

• Published in: Drug testing and analysis Vol. 17; no. 7; pp. 1107 - 1117
• Main Authors: Tou, Kathy, Cawley, Adam, Noble, Glenys, Loy, Jaymie, Bishop, David, Keledjian, John, Sornalingam, Kireesan, Richards, Stacey, Fu, Shanlin
• Format: Journal Article
• Language: English
• Published: England 01.07.2025
• Subjects: Adrenal Cortex Hormones - blood; Animals; anti‐doping; biomarkers; Biomarkers - blood; bisphosphonates; Bone Density Conservation Agents - administration & dosage; Bone Density Conservation Agents - blood; Bone Density Conservation Agents - pharmacokinetics; Chromatography, Liquid - methods; Diphosphonates - administration & dosage; Diphosphonates - blood; Diphosphonates - pharmacokinetics; Doping in Sports; Horses - blood; Imidazoles - administration & dosage; Imidazoles - blood; Imidazoles - pharmacokinetics; lipids; Lipids - blood; Male; plasma; Solid Phase Extraction - methods; Substance Abuse Detection - methods; Substance Abuse Detection - veterinary; Tandem Mass Spectrometry - methods; Tandem Mass Spectrometry - veterinary; Zoledronic Acid; lipids; bisphosphonates; plasma; biomarkers; anti‐doping
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.3811

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. The detection of bisphosphonates is important due to the potential integrity and welfare issues associated with use of these substances. Targeted detection of a nitrogenous and non‐nitrogenous bisphosphonate was detected for 8 h and 28 days post‐administration, respectively. With this, the need for complementary indirect detection using lipid and corticosteroid biomarkers was investigated. Putative biomarkers were identified, which are now subject to validation into routine analysis for improved detection capabilities of bisphosphonate misuse in equine plasma.