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

Abstract

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.

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.