title: 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).
authors.affiliation: LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France. Electronic address: ma.popot@lchfrance.fr.
authors.forename: M A
authors.initials: MA
authors.lastname: Popot
authors.affiliation: LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
authors.forename: P
authors.initials: P
authors.lastname: Garcia
authors.affiliation: LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
authors.forename: C
authors.initials: C
authors.lastname: Hubert
authors.affiliation: LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
authors.forename: A
authors.initials: A
authors.lastname: Bolopion
authors.affiliation: LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
authors.forename: L
authors.initials: L
authors.lastname: Bailly-Chouriberry
authors.affiliation: LCH, Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France.
authors.forename: Y
authors.initials: Y
authors.lastname: Bonnaire
authors.affiliation: CEVA Santé Animale, La Ballastière, BP 126, 33501 Libourne Cedex, France.
authors.forename: D
authors.initials: D
authors.lastname: Thibaud
authors.affiliation: CEVA Santé Animale, La Ballastière, BP 126, 33501 Libourne Cedex, France.
authors.forename: J
authors.initials: J
authors.lastname: Guyonnet
mesh_terms: D000818:Animals; D050071:Bone Density Conservation Agents / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004164:Diphosphonates / Q000097:blood*; D005260:Female; D006736:Horses / Q000097:blood*; D057230:Limit of Detection; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D050071:Bone Density Conservation Agents; D004164:Diphosphonates; C058651:tiludronic acid
keywords: Horse doping control; LC/MS; Plasma; Quantification; SPE; TMOA methylation; Tiludronate