Abstract
Introduction
Famotidine and omeprazole are widely used for the treatment of acid-related disorders in racehorses, and their combined administration is expected to provide complementary therapeutic effects due to their distinct mechanisms of action. However, their markedly different physicochemical properties present substantial challenges for simultaneous quantification in biological matrices. This study aimed to develop and validate complementary analytical strategies for the simultaneous determination of famotidine and omeprazole in equine plasma using high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
Methods
Dual-platform analytical methods were developed, with sample preparation conditions tailored to the physicochemical properties of each compound. For HPLC analysis, the effects of sample preparation conditions on the chromatographic behavior and linearity of famotidine and omeprazole were evaluated. For UHPLC-MS/MS analysis, a pH-neutral sample preparation strategy was established to preserve the stability of acid-labile omeprazole. The developed methods were validated according to relevant analytical validation parameters and subsequently applied to equine plasma samples collected following drug administration for pharmacokinetic evaluation.
Results
In HPLC analysis, famotidine exhibited pH-dependent peak splitting associated with changes in its ionization state, which was resolved by acidifying the sample preparation solvent. Omeprazole showed limited solubility in aqueous environments, resulting in nonlinearity at higher concentrations; this was overcome by acidifying the plasma matrix, enabling quantification over 100-5,000 μg/mL. Although the HPLC method effectively characterized the analytical behavior of both compounds, its sensitivity was insufficient for trace-level pharmacokinetic analysis. Therefore, a UHPLC-MS/MS method was developed using pH-neutral sample preparation, enabling the simultaneous determination of omeprazole and its metabolite, omeprazole sulfone. The UHPLC-MS/MS method achieved an LLOQ of 50 ng/mL, representing an approximately 2,000-fold improvement in analytical sensitivity compared with HPLC. The developed methods demonstrated acceptable validation performance and were successfully applied to equine plasma samples, enabling the determination of concentration-time profiles and key pharmacokinetic parameters.
Discussion
The findings demonstrate that differences in the physicochemical properties and stability of famotidine and omeprazole require platform-specific sample preparation strategies for reliable simultaneous quantification. The complementary use of HPLC and UHPLC-MS/MS provided both characterization of compound-specific analytical behavior and sensitive quantification of trace drug concentrations in equine plasma. This analytical framework may support pharmacokinetic studies of famotidine and omeprazole in equine models.