Liquid chromatography-mass spectrometry analysis of five bisphosphonates in equine urine and plasma.

The study presents a new method for detecting five types of bisphosphonates, which are drugs used to treat bone disorders, in horse urine and plasma. This method is useful for enforcing regulations that prohibit the use of these drugs in horse racing.

Background on Bisphosphonates Use in Horse Health

• Bisphosphonates are commonly used in both human and horse medical treatments to manage skeletal disorders.
• In horses, drug like tiludronic acid is used as a veterinary medicine to treat lameness related to degenerative joint diseases.
• However, the use of bisphosphonates is prohibited in horse racing because of potential misuse to provide competitive advantages.
• An effective method for detecting the presence of bisphosphonates in racehorses is therefore necessary for ensuring fair game and animal welfare.

The Challenge with Bisphosphonates Detection

• The detection of bisphosphonates presented particular challenges due to their hydrophilic properties which makes them difficult to isolate.
• Additionally, the complexity of equine biological samples further complicates the extraction process for drug testing.

New Method for Bisphosphonates Detection

• In this study, the researchers developed a method for simultaneous detection of five types of bisphosphonates — alendronic acid, clodronic acid, ibandronic acid, risedronic acid, and tiludronic acid — in horse urine and plasma samples.
• This method involves isolating bisphosphonates from samples through solid-phase extractions, followed by methylation with trimethylsilyldiazomethane.
• After isolation and methylation, the bisphosphonates are detected using liquid chromatography – tandem mass spectrometry analysis in the positive electrospray ionization mode.

Effectiveness and Applicability of the Method

• The method proved efficient in detecting as low as ppb levels of the five bisphosphonates in samples of equine plasma or urine.
• The turnaround time for the process is commendably low, and there was negligible matrix interference.
• The applicability of the method was successfully demonstrated in detecting tiludronic acid in an overseas equine urine sample and in a single-dose administration test in a horse.
• This is the first reported methodology capable of the effective screening and confirmation of both amino- and non-amino bisphosphonates in horse biological samples.