High-throughput doping control analysis of 28 amphetamine-type stimulants in equine plasma using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The research presents a method using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) to identify 28 different kinds of amphetamine-type stimulants (ATSs) in horse blood samples. The focus of this research is to improve doping control for racehorses.

Introduction to the Study

This study is centered on the development and implementation of a method to detect various amphetamine-type stimulants in equine plasma in order to control doping in race horses. The technology employed is hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), which allows for the simultaneous detection of several stimulants within a sample.

Methodology

• The researchers leveraged a liquid-liquid extraction (LLE) at pH 9.5 using methyl tert-butyl ether, to recover the stimulants from the horse’s plasma.
• The extracted substances were then detected using a Thermo Finnigan triple quadrupole mass spectrometer operating in positive-ion mode electrospray ionization.
• All of the stimulants were successfully separated and eluted within 7 minutes.
• HILIC chromatography helped in achieving baseline separation for isomeric and isobaric compounds.

Results and Analysis

• Extraction efficiency was found to be more than 80%, indicating the methodology is highly efficient at retrieving the target compounds from equine plasma.
• The recorded limit of detection (LOD) ranged from 10-50 pg/mL and the lower limit of quantification (LLOQ) was in the 50-100 pg/mL range, showcasing the method’s high sensitivity and precision.
• Quadratic regression was applied for quantification, with a dynamic range of quantification from 50-10000 pg/mL.
• The limit of confirmation (LOC) was between 20-100 pg/mL, cementing the protocol’s ability to confirm the presence of stimulants.

Further Observations

• The team identified that some stimulants were not stable in horse plasma at room temperature and 4 degrees Celsius.
• But all of the stimulants that were part of the study were found to be stable at -20°C and -80°C for the duration of the 6-month study period.