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.
Highlights
Prednisolone (11β,17,21-trihydroxypregna-1,4-diene-3,20dione) is a synthetic glucocorticoid, a class of steroid hormones, which is produced by the adrenal gland and is known for its anti-inflammatory and immunosuppressive actions.[1,2] Glucocorticoids, the pregnane class containing C‐21 derivatives, are the most common therapeutic agents used in human and veterinary medicine.[3,4] Prednisolone was discovered and approved for medical use in 19555 and is listed in the World Health Organization’s List of Essential Medicines.[6]
The analytical methods for the quantification of prednisolone in pharmaceutical products and biological fluids are mainly reversed-phase high-performance liquid chromatography (RP-HPLC), liquid chromatography coupled with mass spectrometry (LC/MS), and even more hyphenated LC−MS/MS methods.[7−26] The methods used for routine analysis in quality control laboratories are either validated inhouse or incorporated within the regulatory procedures for a specific pharmaceutical final product
The obtained chromatogram for prednisolone FSS using this column was in accordance with the chromatogram that was delivered with the prednisolone FSS: a similar separation of peaks, comparable tR, and suitability test (SST) criteria were acceptable, i.e., passed
Summary
Prednisolone (11β,17,21-trihydroxypregna-1,4-diene-3,20dione) is a synthetic glucocorticoid, a class of steroid hormones, which is produced by the adrenal gland and is known for its anti-inflammatory and immunosuppressive actions.[1,2] Glucocorticoids, the pregnane class containing C‐21 derivatives, are the most common therapeutic agents used in human and veterinary medicine.[3,4] Prednisolone was discovered and approved for medical use in 19555 and is listed in the World Health Organization’s List of Essential Medicines.[6]. Manufacturers of the active ingredient must supply a certificate of analysis (CoA), which is issued by their quality control department Such analyses of related substances (impurities) of prednisolone in the active ingredient are performed according to official monograph methods (e.g., the European Pharmacopoeia monograph, hereinafter, Ph. Eur.) or other methods depending on the market/legislation or even customer requirements. Motivated by the above given facts, this work describes the analysis of the prednisolone active ingredient and the quantification of its related substances (in this work referred to as impurities) using the current Ph. Eur. The SST criteria did not pass (Figure 4d); tRa = 9.817 min; insufficient separation of impurity A and the prednisolone peak. The SST criteria did not pass (Figure 4f); tRa = 10.332 min; coelution of impurity A with the prednisolone peak
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