|Abstract:||An analysis of related substances is necessary in the development and production phase of the drug. In practice, in the implementation of Ph.Eur. HPLC method for the determination of related substances of prednisolone, we often encounter a problem with achieving a system suitability criteria. The problem is with the separation of the peaks prednisolone and its related substance hydrocortosone (impurity A), which are structurally very similar.
Ph.Eur. HPLC method for the determination of related substances of prednisolone was developed on the chromatography column Venusil AQ C18, 150 mm x 4.6 mm, 3 μm particles, which we tested as the starting point for our further optimization. In the continuation of the master's thesis we tried to optimize Ph.Eur. method on columns with the C18 stationary phase of the other manufacturers within the allowable adjustments to European pharmacopeia methods., and then tried to develop a new method.
All previously determined goals in the master's thesis have been achieved. In the master's paper is presented an optimized method (within the allowable adjustments to European pharmacopeia methods) on the column Gemini C18, 150 mm x 4.6 mm, 3 μm particles, with which we reached the criteria for the system suitability, the ratio peak/valley – Hp/Hv = 10,5. With the optimized method, we achieve a significantly improved result compared to the reference chromatogram obtained with the Ph.Eur. method on the column Venusil AQ C18, 150 mm x 4.6 mm, 3 μm particles, where the Hp/Hv ratio is 7,0. In the master's paper, a new developed method is introduced to achieve a very good separation of related substances and the separation of prednisolone and impurity A, which is almost on the base line (Hp/Hv = 21,0).|