Investigation of the presence of endogenous prednisolone in equine urine by high-performance liquid chromatography mass spectrometry and high-resolution mass spectrometry.

This research article delves into the investigation of detected prednisolone in racehorse urine samples. Using advanced lab techniques, scientists explore whether the prednisolone detected could be endogenous (originating within the horse), or if there could be other explanations, such as fermentation of another hormone, cortisol.

Objective and Methods

• The researchers initiated this study after the detection of prednisolone in urine samples from racehorses in Italy. They aimed to double-check the accuracy of the tests that led to these findings, and to explore whether the presence of this steroid might be endogenous.
• The experimental approach involved using mass spectrometry (MS), a technique that measures the mass-to-charge ratio of ions to identify and quantify molecules in samples. Several methods were used which include multiple reaction monitoring (MRM) with a triple quadrupole and full scan MS with linear ion trap (a kind of mass spectrometer).
• High-resolution mass spectrometry (HRMS) was also used as an additional confirmation method on ten urine samples.

Results

• The results revealed challenges in identifying prednisolone, likely due to the presence of other compounds in the urine (matrix) with the same molecular weight.
• Despite this, the characteristic transitions for prednisolone were identified in both second (MS2) and third (MS3) stages of mass spectrometry.
• The concentrations of prednisolone detected using the triple quadrupole and the linear ion trap were not statistically different, suggesting the reliability of both methods.
• The exact mass of the formate adduct (a compound formed from prednisolone during the testing process) was identified using high-resolution mass spectrometry.

Conclusions

• The high frequency of samples that tested positive for prednisolone (78.5%), the low concentration levels of the steroid, and the narrow range of the 95% confidence limits could all suggest that its presence is endogenous. Essentially, this means that the prednisolone may actually be produced within the horse.
• The researchers ultimately conclude that this endogenous hypothesis is the most probable, but they also note that further studies are still needed for confirmation.
• The possibility of a microbiological origin, such as the fermentation of cortisol after the sample collection, couldn’t be ignored altogether.