Fluralaner, an innovative ectoparasiticide for use in cattle: UFLC–MS/MS method development and validation for its pharmacokinetic characterization

Abstract

Fluralaner (FLR) is an isoxazoline ectoparasiticide of increasing interest for use in cattle due to growing resistance to conventional antiparasitic compounds. Reliable analytical methods are required to support its pharmacokinetic characterisation and future residue and formulation studies. This study aimed to develop and validate an Ultra-Fast Liquid Chromatography-Tandem Mass Spectrometry (UFLC-MS/MS) method for determining FLR in bovine plasma and to investigate its pharmacokinetic behaviour following intravenous administration. Plasma samples were processed by solid-phase extraction and analysed using UFLC–MS/MS under optimised chromatographic and mass spectrometric conditions. The method was validated according to regulatory guidelines, showing linear response within the working range (10–200 ng/mL), limits of detection and quantification of 5 and 10 ng/mL, recovery rates of 95.4–114.5%, negligible matrix effects, and a coefficient of variation ≤ 10% for accuracy and precision. Stability under frozen storage was confirmed. Method applicability was demonstrated in a pharmacokinetic study conducted in four calves following intravenous administration of FLR (2.5 mg/kg). Plasma concentrations remained quantifiable up to 28 days post-administration, with detectable trace levels (LOD ≤ C ≤ LOQ) persisting until day 42. FLR showed high initial plasma concentrations (C₀: 1162.9 ng/mL), prolonged systemic exposure (AUC: 4400.5 ± 819.6 ng·day/mL), extensive tissue distribution (Vss: 3.7 ± 0.8 L/kg), and a long terminal elimination half-life (t₁/₂β: 6.1 ± 0.5 days). Overall, these results confirm the suitability of the validated UFLC–MS/MS method for pharmacokinetic applications and provide the first intravenous pharmacokinetic characterisation of FLR in cattle, establishing a baseline for future studies exploring its behaviour following administration by other therapeutic routes.