Solubility determination and characterisation of steroids

Abstract

Introduction: The pKa and logP of a compound affect its bioavailability and extent of action. The characterisation of properties of active pharmaceutical ingredients, such as solubility, aids in the development of analytical methods and formulations for finished dosage forms. To date, some of the physicochemical parameters of the steroids difluprednate and 6α,9α-difluoroprednisolone are only characterised using computational methods. Purpose: To experimentally determine the melting point and solubility of the selected steroids, difluprednate (DFPA) and 6α,9α-difluoroprednisolone (DFP). Method: The melting point of the selected steroids was investigated using a Griffin® melting point apparatus, and the result obtained was compared to the literature. An open-ended capillary was used to introduce the steroidal sample into the apparatus, and the temperature range over which the sample melted was recorded. The solubility of the steroids was assessed in different HPLC-grade solvents, namely water, methanol, and acetonitrile. These solvents were selected since they are the most commonly used in the analysis of active pharmaceutical ingredients. A set of stock solutions was prepared for each steroid in acetonitrile and methanol, respectively. The stock solutions were then sonicated using an IBX® ultrasonic bath for 30 minutes and left overnight in the fridge. A set of dilutions was prepared by adding HPLC-grade water to methanol or acetonitrile at ratios of 1:9, 3:7, 5:5, 7:3, and 9:1, respectively. UV spectroscopy (SPECTROstar Nano, BMG LABTECH) was used to determine the solubility of the steroids. The stock solutions of each steroid were used to determine the wavelength exhibiting the highest absorbance. The absorbance of the pure solvents was determined to eliminate interferences. A quartz cuvette was used for the analysis, and each sample was analysed in triplicate, with the average absorbance calculated. Spectra were plotted in Excel, and solubility was determined from the area under the peak. Results: The melting point of difluprednate was found to be 188–189 °C, and that of 6α,9α-difluoroprednisolone was found to be 210–220 °C. The wavelength that provided the highest absorbance was 242 nm. The maximum UV absorbance when analysing the diluted solutions of DFPA and DFP was 6.815 AU and 3.359 AU, respectively. These results indicate that the highest solubility of difluprednate was achieved when dissolved in a methanol–water mixture at a ratio of 1:9, with a solubility of 0.72 mg/mL. The highest solubility of 6α,9α-difluoroprednisolone was 1.92 mg/mL, observed when the steroid was dissolved in an acetonitrile–water mixture at a ratio of 1:9. Conclusion: The melting point of the analysed steroids was found to be in accordance with values reported in the literature. The experimental determination of the solubility of the analysed steroids will contribute towards improved analytical method development.