Phenylbutazone (PB) is a widely administered non-steroidal anti-inflammatory drug (NSAID) in the equine racing industry, primarily used to manage pain associated with performance-related injuries. Its use in racehorses is strictly regulated, with an established International Screening Limit (ISL) of 100 ng/mL in both plasma and urine matrices.
During a confirmatory analysis of PB using liquid chromatography-high resolution accurate mass-mass spectrometry (LC-HRAM MS), two chromatographic peaks were observed in both spiked control and case samples, whereas only a single peak appeared in the unextracted standard. A similar observation was noted for the deuterated internal standard, D9 PB. Analysis of the MS/MS spectra revealed that only one peak matched that of the unextracted standard and the reference library. The other peak exhibited identical fragment ions but with different ion ratios, suggesting the presence of a structural isomer.
PB, a pyrazolidine derivative, is known to undergo keto-enol tautomerism—a dynamic equilibrium between its keto and enol forms, involving proton transfer from the α-carbon adjacent to the ketone group to the carbonyl oxygen1,2. Although the keto form is thermodynamically favoured in most simple ketones, factors such as intramolecular hydrogen bonding, resonance, and conjugation may stabilize the enol form, making it the predominant tautomer in certain cases2. The presence of these tautomers can affect chromatographic behaviour and complicate mass spectral interpretation, causing challenges in accurate quantitation.
This study investigates the analytical implications posed by tautomerism of PB in some chromatographic techniques and its potential impact on determining whether routine case samples exceed the ISL or not. Additionally, it explores strategies to minimise these effects, ensuring reliable detection of PB in regulatory testing.