The development of a method to quantify encapsulated and free prednisolone phosphate in liposomal formulations.

This paper presents the development of a new method for the simple and reliable quantification of the free drug amount in liposomal preparations of prednisolone phosphate (PP). In this method the free drug is distinguished from the encapsulated drug by means of hydrolysis of the free PP into prednisolone (P) by alkaline phosphatase (AP). During method development reaction progress curves were recorded to determine the required AP concentration and the corresponding incubation time to achieve hydrolysis of all free PP. Reaction progress curves also showed that small changes in the amount of weighted AP and the incubation periods used do not cause a change in outcome. Further, several organic solvents were tested as precipitation solvent and the use of tetrahydrofuran (THF) yielded clean chromatograms, rapid AP deactivation and complete liposome rupture avoiding under- and overestimations of the encapsulated and free drug concentrations. Method accuracy was evaluated during a cross-validation involving dialysis. Intra- and interday precision were evaluated by determining the standard deviation (SD) and relative standard deviation (RSD) after applying the new method on one day (n=4) and on different days (n=3). The accuracy of the developed method is comparable to the accuracy determined by dialysis, while clearly the method using AP is more precise. In conclusion, comprehensive method development yielded an accurate and precise method, which can replace traditional methods like dialysis and solid phase extraction (SPE). With little effort the method can be upgraded and become part of the liposome certification prior to human use. The overall principle behind the method offers possibilities for many drug carrier systems.