Background:
The present study was focused on the development of HPLC method for purity
testing of sofosbuvir by the Design of Experiments and determination of the activation energy of hydrolytic
degradation reactions of sofosbuvir using HPLC based on the kinetics of sofosbuvir degradation.
Methods:
Following four factors for the Design of Experiments were selected, stationary phase, an
organic modifier of the mobile phase, column temperature and pH of the mobile phase. These factors
were examined in two or three level experimental design using Modde 11.0 (Umetrics) software. The
chromatographic parameters like resolution, USP tailing and discrimination factor were calculated and
analysed by partial least squares. The chromatography was performed based on Design of Experiments
results with the mobile phase containing ammonium phosphate buffer pH 2.5 and methanol as an organic
modifier. Separation was achieved using gradient elution on XBridge BEH C8 at 50 °C and a
flow rate of 0.8 mL/min. UV detection was performed at 220 nm. The activation energy of hydrolytic
degradation reactions of sofosbuvir was evaluated using two different calculation methods. The first
method is based on the slope of dependence of natural logarithm of the rate constant on inverted thermodynamic
temperature and the second approach is the isoconversional method.
Results and Conclusion:
Calculated activation energies were 77.9 ± 1.1 kJ/mol for the first method
and 79.5 ± 3.2 kJ/mol for the isoconversional method. The results can be considered to be identical,
therefore both calculation methods are suitable for the determination of the activation energy of degradation
reactions.
