A novel method for the determination of isoxazoline derivatives in plasma by ultra-high performance liquid chromatography–tandem mass spectrometry: validation and applicability to screening tests
Paulina Markowska-Buńka
Profil Orcid, Jerzy J. Jaroszewski
Profil Orcid, et Hubert Ziółkowski
Profil Orcid 
Publié en ligne: 24 sept. 2025
Reçu: 07 mars 2025
Accepté: 17 sept. 2025
DOI: https://doi.org/10.2478/jvetres-2025-0050
Mots clés
© 2025 Paulina Markowska-Buńka et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
Introduction
Isoxazoline derivatives such as fluralaner, sarolaner, lotilaner, and afoxolaner are a new class of insecticide and acaricide compounds. These compounds are characterised by rapid action and high efficacy over a period of up to several weeks. A method for identifying all four isoxazoline derivatives in animal and human plasma has not been proposed to date. Therefore, the aim of this study was to develop and validate a novel analytical method for the determination of fluralaner, sarolaner, lotilaner, and afoxolaner in laying hen plasma and to revalidate the proposed method in human, canine and feline plasma.
Material and Methods
The plasma concentrations of isoxazoline derivatives were determined by ultra-high performance liquid chromatography–tandem mass spectrometry. Analytical samples were prepared by precipitating proteins with a mixture of 96% acetonitrile and 4% ammonium hydroxide (v/v). Chromatographic separation was achieved on a chromatographic column (1.8 μm 2.1 × 100 mm) using 0.1% formic acid in water and 0.1% formic acid in acetonitrile with gradient elution.
Results
The results indicate that the described method is replicable, linear (r2 = 0.99), precise (1.66% to 14.97%), accurate (1.19% to 11.67%), selective and sensitive (limit of quantitation = 1 ng/mL). Depending on the studied compound and animal species, total recovery reached 85–99%, and the matrix effect did not exceed 15% in any of the analyses.
Conclusion
The proposed method is simple, effective, inexpensive and rapid because it requires only a single-step sample preparation protocol.