In Vitro Plasma Protein Binding Determination of Lefamulin by Equilibrium Dialysis and High‐Performance Liquid Chromatography Coupled With Evaporative Light Scattering Detector

A credible method utilizing equilibrium dialysis and high-performance liquid chromatography with an evaporative light scattering detector (HPLC-ELSD) was developed to determine the plasma protein binding of lefamulin in swine. The method demonstrated excellent linearity, with a logarithmic calibration curve in the range of 0.1 to 2 μg/mL (r > 0.999). Extraction recoveries from plasma samples ranged from 81.0% to 90.8%, with coefficients of variation less than 7.21%. The limits of detection and quantitation were established at 0.05 and 0.1 μg/mL, respectively. The mean plasma protein binding rates of lefamulin were found to be 70.2 ± 8.0%, 68.4 ± 4.9%, and 68.4 ± 3.6%, respectively, with coefficients of variation below 11.4%. This method was satisfactorily applied to the determination of plasma protein binding rate of lefamulin with high precision, accuracy, and sensitivity.