Sustainable Thin-Layer Chromatographic Method for the Determination of Ipriflavone and Vitamins in Bone Health Supplements

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Abstract
A simple, high-performance thin-layer chromatographic method was developed to separate and quantify ipriflavone, ascorbic acid, pyridoxine, and vitamin D3 in bulk and combined pharmaceutical formulations. Elution was achieved using a developing system consisting of toluene, dichloromethane, methanol, and glacial acetic acid (5 : 3 : 3 : 0.2, v/v) on an F254 silica gel stationary phase, followed by densitometric measurement of the separated bands at 254 nm. Validation of the proposed method was performed according to the ICH guideline Q2(R1). The method was validated in the concentration ranges of 2–12, 4–22, 0.4–2.2, and 0.15–1.35 µg/band for ascorbic acid, pyridoxine, ipriflavone, and vitamin D3, respectively. The method can be used for the selective analysis of the stated compounds in both bulk and combined pharmaceutical formulations. The developed method was eco-evaluated using two assessment tools, namely the Green Analytical Procedure Index and the Analytical GREEnness metric, confirming the method’ s eco-friendliness. The whiteness of the proposed method was evaluated and compared to that of a published high-performance liquid chromatography method for separating the same mixture. The thin-layer chromatography method showed a better whiteness profile and was deemed suitable for routine quality control analysis.

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