A confirmatory, highly selective multi-residue method based on liquid chromatography coupled to hybrid high resolution mass spectrometry (LC-Q-Orbitrap) was developed and validated for the determination of 12 regulated coccidiostats in eggs and muscle. Particularly, ionophore antibiotics (lasalocid, maduramicin, monensin, narasin, salinomycin and semduramicin) and synthetic coccidiostats (diclazuril, halofuginone, nicarbazin as 4,4′-dinitrocarbanilide fraction, robenidine and toltrazuril as toltrazuril-sulphone) were included in the method. The sample preparation consisted in the extraction of the analytes from the matrix with acetonitrile, followed by a clean-up step with Oasis® PRiME HLB SPE and a defatting procedure with n-hexane. Validation was successfully performed according to Commission Implementing Regulation (EU) 2021/808, starting from 1 µg kg−1. The procedure was verified through the analysis of a certified reference material (CRM) and the occurrence of the residues was assessed in the context of the Italian National Residue Control Plan (NRCP).

The measure of hair cortisol concentration (HCC) is becoming an emerging approach to monitor mid-/long-term stress in animals, so it is more and more important to develop accurate and reliable methods. In the light of this, the aim of the present study was to compare mane HCCs of 47 horses with different managements, by means of an immunoassay (ELISA) and liquid chromatography coupled to hybrid high-resolution mass spectrometry (LC-HRMS/MS). After the washing step, the ground hair was extracted with methanol. The extract was evaporated and redissolved in two different aqueous solutions, depending on the detection technique. The methods were validated according to EMA guideline for bioanalytical method validation, in the range 2–50 pg mg−1 (ELISA) and 1–100 pg mg−1 (LC-HRMS/MS). Satisfactory quantitative performances were obtained for both of the approaches, but this latter demonstrated better precision. The detected concentrations in real samples were encompassing the range 1.3–8.8 pg mg−1 and 2.0–17.9 pg mg−1 by means of LC-HRMS/MS and ELISA, respectively. Overall, HCCs measured with ELISA technique were 1.6 times higher. The overestimation of immunoassay results might be caused by cross-reactivity phenomena of laboratory reagents and other structurally similar hormones present in the mane. Graphical abstract