Plasma, blood and liver tissue sample preparation methods for the separate quantification of liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone

Abstract

Besides the development of sample preparation methods for the determination of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in murine plasma and blood, this article also presents the first description of an accurate sample preparation method for the determination of such separate concentrations in the murine liver. The quantitative differentiation is based on the immediate hydrolysis of prednisolone phosphate (PP) into prednisolone (P) after its release from the liposomes in vivo: PP represents the encapsulated drug, while P represents the non-encapsulated drug. The use of 10 ml methanol/g tissue during homogenization of liver tissue ensures complete liposome rupture, prevention of the dephosphorylation of PP released during homogenization, sufficient clean supernatants, excellent extraction of P and sufficient extraction of PP and excellent accuracies and precision complying with the internal guidelines for pre-clinical studies (80–120% and maximal 20%, respectively). Similarly, the matching sample preparation methods for plasma and blood involve protein precipitation with four equivalents of methanol also ensuring accuracies and precision complying with the internal guidelines for pre-clinical studies. Application of these sample preparation methods is going to generate the first pharmacokinetic (PK) profile of a liposomal preparation, in which the encapsulated and non-encapsulated drug concentrations in a tissue are measured separately. Such separated concentration profiles can gain important insights into the PKs of liposomal PP and probably also with regard to liposomal formulations in general, like the quantification of the in vivo drug release from the liposomes.

• Bioanalysis
• liposomes
• phosphatase
• prodrug

Acknowledgements

The authors like to thank Foppe Bakker and Hans van Laarhoven from MSD Oss BV for the determination of the water content of murine liver tissue and plasma. The authors also like to thank Laura Hermens and Bianca Huisman for their assistance during the application of the General Laboratory Homogenizer. Coen Smits is acknowledged for his advice during the statistical analysis of the obtained results. We acknowledge MediTrans for the financial support.

Declaration of interest

This study has been carried out with financial support from the Commission of the European Communities, Priority 3 “Nanotechnologies and Nanosciences, Knowledge Based Multifunctional Materials, New Production Processes and Devices” of the Sixth Framework Programme for Research and Technological Development (Targeted Delivery of Nanomedicine: NMP4-CT-2006-026668). It does not necessarily reflect its views and in no way anticipates the Commission's future policy in this area.

Notes

¹ After finishing the method development for the preparation of tissues, methanol appeared to be the solvent of choice (see section “Selection of the homogenization and precipitation solvent”). Therefore, method development with regard to plasma sample preparation continued using methanol only.

² After development of the plasma sample preparation method it was evaluated whether the method could also be freely applied to whole blood samples.