A GC-MS method for the determination of isoxsuprine in biological fluids of the horse utilizing electron impact ionization.

The study involves the development and validation of a faster and more sensitive GC-MS method, based on electron impact ionization, to detect and quantify the drug isoxsuprine in horse plasma and urine. Isoxsuprine is a drug used to treat certain lower-limb diseases in horses and requires monitoring due to its classification as a class 4 compound by the Association of Racing Commissioners, International (ARCI).

Method and Procedure

• The researchers used gas chromatography-mass spectrometry (GC-MS), a method effective for identifying molecules in a tested sample by breaking it into individual components through separating the gases and identifying the constituents in the mass spectrometer.
• The new protocol they developed uses electron impact ionization, a widely used ionization method in mass spectrometry. This method removes an electron from the molecular ion, resulting in a positive ion with an unpaired electron – a radical cation.
• The study implements robotic solid-phase extraction, a common sample preparation method that isolates the analytes (the substances to be measured). Here, it was used to extract isoxsuprine from the biological samples.
• Different products of derivatization – chemical reactions used to transform the analyte into a product that can be more easily detected – were used in the study, specifically bis-trimethylsilyl (TMS) isoxsuprine and tris-TMS ritodrine. The latter was used as an internal standard, meaning it was present in all samples, standards, and blanks, aiding in the quantification of isoxsuprine.

Results and Conclusion

• The process generated intense quantifier ions m/z 178 for isoxsuprine and m/z 236 for ritodrine, as products of C-C cleavage (a chemical reaction breaking carbon-carbon bonds).
• Concentrations of isoxsuprine in the urine and plasma were calculated from a calibrator curve created alongside unknowns.
• The validation data showed that the limit for detecting isoxsuprine in plasma was 2 ng/mL, and the limit for quantifying it was less than 5 ng/mL.
• The average coefficient of determination for the calibrator curves was 0.9925 in plasma and 0.9904 in urine, indicating a high degree of correlation between predicted and observed values.
• The recovery efficiencies (the measure of a method’s ability to extract and measure an analyte from a sample) at concentrations of 50, 200, and 300 ng/mL were 76%, 73%, and 76% in plasma and 92%, 89% and 91% in urine respectively. This suggests that the method is efficient and reliable at different isoxsuprine concentrations.