Quantification and confirmation of flunixin in equine plasma by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry.

This research presents a robust and reliable method for identifying and measuring the level of drug flunixin in horse plasma. The researchers successfully adapted a sophisticated mass spectrometry technique and used it to perform their investigation, highlighting its potential for use in horse racing regulatory enforcement.

Methodology

• The researchers primarily utilized a technique known as liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS/MS) in their study. This sophisticated scientific technique is used to identify and quantify unknown components in a sample. It is a powerful tool for the precise and specific measurement of chemical substances.
• Before subjecting samples to this analysis, they were first screened using an enzyme-linked immunosorbent assay (ELISA) to identify samples that could potentially contain the drug flunixin.
• The identified samples were then subjected to liquid-liquid extraction (LLE), a method used for the efficient extraction of drugs from biological fluids such as plasma. Prior to the extraction, the sample was diluted with 2 ml saturated phosphate buffer.

Analysis and Quantification

• After extraction, the sample undergoes reconstitution in a specific mixture of acetonitrile, water, and formic acid and is further analyzed with the mass spectrometer.
• The concentration of flunixin in the samples was determined by comparing the obtained results with the known data of an internal standard (IS), in this case, clonixin. This calibration method is commonly used to improve the reliability and accuracy of the analysis.
• The limits of detection (LOD) and quantification (LOQ) for flunixin were established. The LOD refers to the lowest amount of the drug that can be reliably detected, while the LOQ is the lowest amount that can be quantified with acceptable accuracy and precision. Additionally, the limit of confirmation (LOC) was specified, referring to the lowest levels where definitive confirmation can be established.

Results and Implications

• The measurement uncertainty, or margin of error, for the entire process was found to be 8.7%. This is a very acceptable value for this kind of complex analysis, indicating the method is adequately accurate and reliable.
• The method was demonstrated to be simple, sensitive, and fast, making it suitable for routine use in detecting and quantifying flunixin in equine plasma.
• Application of this method can assist racing authorities in enforcing regulations concerning drug use in racehorses, potentially leading to more efficient and reliable doping monitoring systems in the horse racing industry.