Ultrapure water Volasertib cancer (resistance > 18m��) was obtained from Milli-Q system (Millipore, France). 2.2. Standard SolutionsStock standard solutions (1mg/mL) were prepared by weighing 25.0mg of each reference standard and dissolving in 25mL methanol. These solutions were kept in the temperature below ?18��C for 12 months. Mixed working standard solution (20��g/mL MAD and 100��g/mL of remaining ionophores) was stored in 6�C10��C up to three months.2.3. Sample TreatmentFor the method development and validation, animal feed samples without ionophore coccidiostats were used. The feed was ground with rotor mill ZM200 (Retsch, Germany) and sieved through 0.5mm sieve. The sample (5g �� 0.01g) was weighted into the polypropylene centrifuge tube.

The appropriate amount of mixed standard solution was added to the spiked samples, and the sample was let to stand for at least one hour. Methanol was added to the sample so that the total solvent volume was 25mL (the volume of methanol added with the standard solution was subtracted), and the sample was shaken for 30 minutes at 200 cycles/min (MaxQ 2000 Orbital Shaker, Thermo Scientific, USA). The sample was then centrifuged (3500rpm, 10min), and 0.5mL of supernatant was transferred to a glass tube. Internal standard (monensin methyl ester, 10��L of 10��g/mL solution) and 0.5mL DMSO were added, and the sample was evaporated under the stream of nitrogen (45��C). The extract in DMSO was transferred into a vial and analysed with liquid chromatography.2.4.

Instrumental AnalysisThe instrumental analysis of coccidiostats was performed using Varian Prostar HPLC system equipped with quaternary pump, autosampler, column oven, postcolumn derivatisation module, and two detectors��fluorescence and UV-Vis, controlled by Galaxie Workstation software. Chromatographic separation of compounds was performed on Kinetex C18 column (150 �� 4.6mm, 2.6��m, Phenomenex, USA) connected with precolumn (4 �� 3mm, SecurityGuard, Phenomenex, USA). The isocratic elution was applied, with mobile phase consisting of 88% methanol and 12% 0.02M KH2PO4, adjusted to pH 7.0 Anacetrapib at 0.7mL/min flow rate. Column oven temperature was controlled at 32��C. After HPLC separation, eluate was transferred through fluorescence detector (excitation and emission wavelength 310 and 420nm, resp.) for the lasalocid detection. Next, the eluate was passed through the reaction cell (1.4mL) of the postcolumn derivatisation reactor (Pinnacle PCX, Pickering, USA). The derivatisation was performed with 6% vanillin solution in methanol (0.35mL/min) in the presence of 4% sulfuric acid in methanol (0.35mL/min), and the coil was maintained at 110��C. The derivatives of ionophores were then detected at �� 520nm. The injection volume was 50��L.2.5. Validation2.5.1.