All organic solvents used were of an analytical grade. The tiamulin fumarate standard and internal standard (IS) of sulfaphenazole were purchased from LGC Standards (Teddington, Middlesex, UK). Acetonitrile was from J.T. Baker (Deventer, the Netherlands). Heptafluorobutyric acid and formic acid were from Sigma-Aldrich (St. Louis, MO, USA). Hydrophilic polyvinylidene fluoride (PVDF) membrane syringe filters, 0.22 μm, were provided by Restek (College, PA, USA). Nanosep centrifugal filters with 0.2 µm of Bio-Inert were from Pall Corporation (New York, NY, USA).
A tiamulin and sulfaphenazole stock standard solution at a concentration of 1000 μg/mL in methanol was kept stable for 6 months by storing at −18 °C in the dark. Working solutions were made in ultrapure water from a diluted stock solution and stored at 4–8 °C for 1 month.
A tiamulin and sulfaphenazole stock standard solution at a concentration of 1000 μg/mL in methanol was kept stable for 6 months by storing at −18 °C in the dark. Working solutions were made in ultrapure water from a diluted stock solution and stored at 4–8 °C for 1 month.