Simultaneous determination of evobrutinib and its metabolite evobrutinib‐diol in dog plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography hyphenated with electrospray ionization tandem mass spectrometric method (LC–ESI–MS/MS) was developed and validated for simultaneous determination of evobrutinib and evobrutinib‐diol in dog plasma. The plasma sample was processed using acetonitrile and chromatographic separation was carried out on a Waters Acquity BEH C 18 column (50 × 2.1 mm, 1.7 μm). The mobile phase was composed of 0.1% formic acid and acetonitrile, with an optimized gradient elution at a flow rate of 0.4 mL/min. Detection was accomplished in selective reaction monitoring mode via electrospray ionization interface operated in positive ion mode. The precursor‐to‐product transitions for quantification were m/z 430.2 → 98.1 for evobrutinib, m/z 464.2 → 98.1 for evobrutinib‐diol and m/z 441.2 → 138.1 for ibrutinib (internal standard). The developed assay was linear over the tested concentration ranges with correlation coefficient >0.995. The LLOQ was 0.1 ng/mL for both analytes. The inter‐ and intra‐day precisions were <9.65% and the accuracy ranged from −3.94 to 6.37%. The extraction recovery was >85.41% and no significant matrix effect was observed. The developed assay was successfully applied to the pharmacokinetic study of evobrutinib and evobrutinib‐diol in dogs after oral administration of evobrutinib at a single dose of 5 mg/kg.