An Improved Reversed-Phase High-Performance Liquid Chromatography Method for the Analysis of Related Substances of Prednisolone in Active Ingredient

Abstract

Prednisolone, an important active pharmaceutical ingredient, is a synthetic glucocorticoid used for the preparation of various pharmaceutical products with anti-inflammatory and immunosuppressive properties. It is a challenge in high-performance liquid chromatography (HPLC) to separate the prednisolone peak and its structurally related substance (hydrocortisone), which only differs in a double bond at the C-1 position. Successful application of the HPLC method according to the European Pharmacopoeia monograph for related substances of prednisolone is very often limited to the chromatographic system available. This is due to the nonbaseline separation of the prednisolone and hydrocortisone peaks, which is strongly influenced by the instrument parameters and the chosen C18 column. First, an adjusted European Pharmacopoeia method for related substances of prednisolone was developed within the allowable adjustments. Next, an improved stability-indicating reversed-phase HPLC method for related substances of prednisolone was developed and validated for use in quality control laboratories for routine analysis. The optimized separation was performed on a Phenomenex Gemini C18 column (150 mm × 4.6 mm, 3 μm) using a gradient mobile-phase system consisting of acetonitrile/tetrahydrofuran/water (15:10:75 v/v/v), acetonitrile/water (80:20 v/v), and ultraviolet detection at 254 nm. A baseline separation was achieved, and stability indicating capability was demonstrated by a forced degradation study. A full validation procedure was performed in accordance with International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines.

Figure 1

Chemical structures of prednisolone and its related substances (impurities).

Figure 2

Chromatogram of prednisolone FSS obtained using the Venusil AQ C18 column (150 mm × 4.6 mm, 3 μm) with the official Ph. Eur. method (Table 1).

Figure 3

Chromatogram of prednisolone FPI obtained using the Venusil AQ C18 column (150 mm × 4.6 mm, 3 μm) with the official Ph. Eur. method (Table 1).

Figure 4

(a–l) Separation of prednisolone and impurity A and B peaks obtained with the official Ph. Eur. method using different columns: (a) Venusil AQ C18, (b) Gemini C18, (c) Synergi Hydro-RP, (d) Luna C18(2), (e) Gemini NX-C18, (f) Luna Omega Polar C18, (g) Kinetex F5, (h) Kinetex Biphenyl, (i) Kinetex C18, (j) Kinetex Phenyl-Hexyl, (k) Kinetex XB-C18, and (l) Kinetex Polar C18 (Table 2). All of the chromatograms are on the same y-axis scale and have a 5 min time interval.

Figure 5

Separation of the prednisolone (t_R = 12.972 min), impurity A (t_R = 13.511 min), and impurity B (t_R = 11.963 min) peaks obtained with the adjusted official Ph. Eur. method using the Gemini C18 column (150 mm × 4.6 mm, 3 μm) given in Table 3. (The y-scale is the same as in Figure 4.)

Figure 6

(a, b) Chromatograms of (a) prednisolone FSS and (b) prednisolone FPI obtained using the Gemini C18 column (150 mm × 4.6 mm, 3 μm) with the isocratic method in Table 4.

Figure 7

(a, b) Chromatograms of (a) prednisolone FSS and (b) prednisolone FPI obtained using the Gemini C18 column (150 mm × 4.6 mm, 3 μm) with the improved method reported in Table 5.