HPLC/ESI-MS(n) method for non-amino bisphosphonates: application to the detection of tiludronate in equine plasma.

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).