Sensitive hydrophilic interaction liquid chromatography/tandem mass spectrometry method for rapid detection, quantification and confirmation of cathinone-derived designer drugs for doping control in equine plasma.

The research involves the development of a method for detecting stimulant drugs, specifically cathinone derivatives, in racehorses using liquid chromatography and tandem mass spectrometry. This method provides a rapid, reliable, and sensitive measure for doping control in equine sports.

Objective and Background of the Research

• The aim of the study was to develop a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection, quantification, and confirmation of cathinone-derived designer drugs in racehorse plasma.
• Cathinone derivatives are new amphetamine-like stimulants; they are used for enhancing performance in sports including horse racing, and they can evade detection with currently available methods.
• The need to curb the misuse of these banned substances in horse racing led to this research.

Methodology

• The substances of interest (analytes) were recovered through liquid-liquid extraction using methyl tert-butyl ether. The separation of these substances was achieved on a hydrophilic interaction column using liquid chromatography.
• A QTRAP mass spectrometer was applied to perform the mass analysis. This was done in positive electrospray ionization (ESI) mode, coupling it with multiple reaction monitoring (MRM).
• Identification of the analytes was carried out by screening for a defined MRM transition. The process of quantification was conducted using an internal standard. Confirmation of the substances was done by establishing a match in terms of retention time and ion intensity ratios comparison.

Results and Conclusion

• The analytical method demonstrated linearity over the range of 0.2 to 50 ng/mL. The analysis performed on six different batches of blank plasma, and those spiked with each of the analytes showed good specificity.
• The recovery of these analytes from plasma at different concentrations was more than 70%, proving effective extraction techniques.
• The limits of detection, quantification, and confirmation ranged between 0.02-0.05, 0.2-1.0, and 0.2-10 ng/mL, respectively, showing good sensitivity of the method.
• The matrix effect on the analysis was insignificant, thereby eliminating false positives. The intra-day and inter-day precision were 1.94-12.08% and 2.58-13.32%, respectively, indicating good reproducibility.
• The developed method is currently used for screening eleven analytes in post-competition samples taken from racehorses in Pennsylvania, providing robust results for enforcing the ban on these performance-enhancing agents.
• Overall, this method was deemed sensitive, fast, effective, and reliably reproducible, making it an excellent tool for doping control in equine sports.