HPLC/ESI-MS(n) method for non-amino bisphosphonates: application to the detection of tiludronate in equine plasma.
Abstract: Tiludronate is a non-nitrogen-containing biphosphonate drug approved in equine veterinary medicine for the treatment of navicular disease and bone sparvin in horse. Its hydrophilic properties and its strong affinity for the bone have made the control of its use quite difficult. After an initial step of method development in plasma and urine, due to a strong matrix effect and erratic detection in urine, the final method development was conducted in plasma. After addition of (3-trifluoromethylphenyl) thiomethylene biphosphonic acid as internal standard, automated sample preparation consisted of a filtration on a Nexus cartridge followed by a Solid Phase Extraction on an Oasis WAX cartridge with weak anion exchange properties. After methylation of the residue with trimethyl orthoacetate (TMOA), analysis was conducted by HPLC/ESI-MS(n) on a LTQ mass spectrometer. The method has been validated with a LOD and LOQ of respectively 1 and 2.5ng/mL. Using a weighting factor of 1/concentration(2), a linear model was suitable in the range of 2.5 up to 500ng/mL. Precision and accuracy data determined at two concentrations were satisfactory (i.e. less than 15%). Carryover would have been a problem but this has finally been fixed using the additional steps of washing during robotised SPE extraction and analysis on both the autosampler and the analytical column. The method was successfully employed for the first time to the quantification of tiludronate in plasma samples collected from horses treated with Tildren™ (Intravenous administration at the dose of 0.1mg/kg/day for 10 days).
Copyright © 2014 Elsevier B.V. All rights reserved.
Publication Date: 2014-03-26 PubMed ID: 24732148DOI: 10.1016/j.jchromb.2014.03.018Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
- Analytical Methods
- Clinical Study
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Disease Treatment
- Drug
- Equine Diseases
- Equine Health
- High-performance Liquid Chromatography (HPLC)
- Horses
- Intravenous Administration
- Laboratory Methods
- Metabolites
- Pharmaceuticals
- Pharmacokinetics
- Plasma
- Veterinary Care
- Veterinary Medicine
- Veterinary Research
Summary
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The research paper focuses on the development and validation of a method for detecting the presence of a drug called tiludronate in horse plasma. Tiludronate is used in veterinary medicine to treat bone disorders in horses but its detection in plasma has been challenging due to various factors, necessitating the development of a reliable method.
Method Development
- The research first identified the challenges in detecting tiludronate – its hydrophilic properties and strong affinity to bone.
- They initially attempted method development on plasma and urine samples but shifted purely to plasma due to strong matrix effect and erratic detection in urine.
- An internal standard – (3-trifluoromethylphenyl) thiomethylene biphosphonic acid was added for the automated sample preparation.
- The sample preparation process involved filtration using a Nexus cartridge and Solid Phase Extraction employing an Oasis WAX cartridge with weak anion exchange properties.
- After the preparation, the residue was methylated with trimethyl orthoacetate (TMOA).
Analysis and Validation
- After sample preparation, the analysis was conducted using HPLC/ESI-MS(n) on a LTQ mass spectrometer.
- The method was validated with a limit of detection (LOD) and limit of quantification (LOQ) of 1 and 2.5 ng/mL respectively.
- The precision and accuracy of the method were tested at two concentrations and showed satisfactory results (less than 15% error).
- A major hurdle, carryover, was addressed using additional washing steps during robotised SPE extraction, and analysis on both the autosampler and the analytical column.
Application
- The developed method was used successfully to quantify tiludronate in plasma samples from horses undergoing treatment with Tildren™ (Intravenous administration at the dose of 0.1mg/kg/day for 10 days).
Cite This Article
APA
Popot MA, Garcia P, Hubert C, Bolopion A, Bailly-Chouriberry L, Bonnaire Y, Thibaud D, Guyonnet J.
(2014).
HPLC/ESI-MS(n) method for non-amino bisphosphonates: application to the detection of tiludronate in equine plasma.
J Chromatogr B Analyt Technol Biomed Life Sci, 958, 108-116.
https://doi.org/10.1016/j.jchromb.2014.03.018 Publication
Researcher Affiliations
- LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France. Electronic address: ma.popot@lchfrance.fr.
- LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
- LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
- LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
- LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
- LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
- CEVA Santé Animale, La Ballastière, BP 126, 33501 Libourne Cedex, France.
- CEVA Santé Animale, La Ballastière, BP 126, 33501 Libourne Cedex, France.
MeSH Terms
- Animals
- Bone Density Conservation Agents / blood
- Chromatography, High Pressure Liquid / methods
- Diphosphonates / blood
- Female
- Horses / blood
- Limit of Detection
- Solid Phase Extraction
- Spectrometry, Mass, Electrospray Ionization / methods
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 3 times.- Knych HK, Janes J, Kennedy L, McKemie DS, Arthur RM, Samol MA, Uzal FA, Scollay M. Detection and residence time of bisphosphonates in bone of horses. J Vet Diagn Invest 2022 Jan;34(1):23-27.
- Tou K, Cawley A, Noble G, Loy J, Bishop D, Keledjian J, Sornalingam K, Richards S, Fu S. Lipid and Corticosteroid Biomarkers Under the Influence of Bisphosphonates. Drug Test Anal 2025 Jul;17(7):1107-1117.
- Ayodele BA, Pagel CN, Mackie EJ, Armour F, Yamada S, Zahra P, Courtman N, Whitton RC, Hitchens PL. Differences in bone turnover markers and injury risks between local and international horses: A Victorian Spring Racing Carnival study. Equine Vet J 2025 Mar;57(2):333-346.
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