The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance.

This research explored the use of in-vitro methods to study the metabolism of various drugs used in horses, which can aid drug testing in the horse racing industry. It demonstrated that the selected in-vitro procedures could recreate the metabolic pathways these drugs would undergo in the horse body.

Context and Objective

• This study was driven by a need in the horse racing industry for an efficient system to identify artificial enhancements (drug abuse) and ensure fair competition. The team sought to develop a method that effectively replicates how a horse’s body would metabolize certain drugs.
• Understanding the metabolic pathways of these drugs can provide crucial insights, mainly when urine is used as the sample source for drug testing.

Methodology

• The researchers studied eight relevant drugs for equine drug surveillance: acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate, and tripelennamine.
• The team employed equine liver and lung microsomes/S9 tissue fractions to replicate the in-vivo (inside a living organism) environment and observe the phase I metabolism of these drugs, which is the initial step of drug metabolism and often involves chemical modification of the drug.
• In-vitro samples were analyzed using two pieces of significant analytical equipment: a Thermo Orbitrap Discovery instrument (for high-resolution accurate mass LC-MS) and an Applied Biosystems Sciex 5500 Q Trap (for LC-MS/MS).

Results

• The in-vitro techniques were successful in producing at least the two most abundant phase I metabolites observed in-vivo for all eight drugs studied, affirming the technique’s effectiveness.
• In some cases, the in-vitro process also successfully created minor in-vivo metabolites and even some metabolites only seen in-vitro, suggesting it can offer more detailed insights into possible metabolic alterations.

Implications and Conclusion

• This research built a strong case for using in-vitro incubates (samples in which drugs are metabolized) as a reference in lieu of in-vivo post-administration samples, aligning with the 2009 International Laboratory Accreditation Cooperation-G7 (ILAC-G7) guidelines.
• The successful reproduction of the in-vivo metabolism of these drugs suggests that these in-vitro techniques can serve as useful tools in equine drug testing, reducing reliance on current in-vivo models.