Florfenicol quantification in plasma samples

Florfenicol concentration in plasma samples was determined using high-performance liquid chromatography (HPLC) [41]. An Agilent HPLC system was employed for the separation and quantification of florfenicol. The optimal mobile phase was established using a mixture of acetonitrile and water in an 18:82 ratio, with a flow rate of 1 ml/min. Florfenicol concentration was detected by measuring the UV absorption at 224 nm. The mobile phase was filtered, degassed using 0.45-µm nylon filter under vacuum and was sonicated for 30 min. The flow rate was maintained at 1 ml/min and the injection volume used was 10 µl. Plasma samples were extracted using ethylene acetate (0.5 ml:1.25 ml). The collected tubes were centrifuged at 2000 g for 10 min. Afterwards, 1 ml of the organic layer was then aspirated and evaporated under nitrogen. The obtained residues were dissolved in 0.375 ml of solvent mixture that consisted of acetonitrile and water (1:2, v/v), vortexed and then centrifuged at 19,000 g for 20 min at 4 °C. The supernatant was collected, filtered through a 0.45-μm nylon filter, and finally transferred to auto-sampler vials. Florfenicol standards at various concentrations (0.05–5 µg/ml) were prepared. A correlation coefficient (r²) and accuracy of 0.99998 and 99.3 ± 1.36 were achieved, respectively. A calibration curve was constructed by plotting florfenicol peak areas against the known concentrations. The equation was determined through the least-squares method using linear regression. The pharmacokinetic parameters were analyzed using PK solver 2.0.