Bioanalysis of protein-unbound prednisolone in serum using equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry

Introduction: High-dose systemic prednisolone is the cornerstone treatment of many autoimmune- and inflammatory diseases. Since prednisolone shows non-linear protein binding at higher serum concentrations, quantification of the unbound prednisolone concentration is important to understand prednisolone pharmacokinetics. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify protein-unbound prednisolone in serum. Methods: Protein-unbound prednisolone was obtained using an equilibrium dialysis technique. Prednisolone was extracted from the dialysate using methyl tert-butyl ether. After evaporation to dryness, the organic phase residue was reconstituted and ready for injection onto the LC-MS/MS. Prednisolone was analysed by selected reaction monitoring with MS/MS operating in positive ion mode. Results and discussion: The equilibrium between bound and unbound prednisolone was stable after 24 h. The calibration model for prednisolone in serum ranged from 0.25 to 811 µg/L and had an average linearity of 0.998. The coefficient of variation (CV) for precision at the lower limit of quantification was ≤ 4.3 % and for the other quality control samples ≤ 7.8 %. Prednisolone protein binding showed no significant degradation after 30 months of storage at −80 °C and was not influenced by multiple cycles of freezing and thawing. The recovery for the tested matrix effects in serum ranged from 85 % to 115 % (CV 10.3 %) and throughout the validation, no carry-over was observed. Conclusion: An LC-MS/MS assay for prednisolone in serum was developed and validated, with a successful equilibrium dialysis technique to obtain protein-unbound prednisolone prior to quantification. This assay is considered suitable for pharmacokinetic studies.