OPTIMISATION AND VALIDATION OF AN HPLC-DAD METHOD FOR ANALYSIS OF ERGOSTEROL AND ERGOCALCIFEROL IN EDIBLE MUSHROOMS EXPOSED TO UV-B RADIATION

Abstract

Vitamin D is essential for maintaining bone health, calcium and phosphorus metabolism, and proper immune system function. Its deficiency is a global concern, and one potential solution for enhancement of vitamin D intake by consumption of biofortified mushrooms. The aim of this study was to optimise the analytical determination of ergosterol and ergocalciferol (vitamin D₂) using the HPLC-DAD method, evaluate the method based on selected validation parameters, and apply it to the analysis of shiitake mushrooms (Lentinula edodes). The experimental material was subjected to the influence of a selected stress factor (UV-B radiation) to potentially transform the precursor ergosterol into ergocalciferol. Method validation established detection limits and limits of quantification for ergosterol and ergocalciferol - LODs were 2.69 µg.mL^-1 and 1.84 µg.mL^-1, and LOQs were 8.06 µg.mL^-1 and 5.53 µg.mL^-1, respectively. Fruiting bodies of shiitake mushrooms were exposed to UV-B irradiation for different durations (0–60 min). The pre-treatment of the experimental material included drying, saponification, and subsequent liquid-liquid extraction of the analytes, followed by low-pressure evaporation and reconstruction. Using high-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis, the concentrations of ergosterol and ergocalciferol were determined. The results indicate a positive correlation between UV-B exposure duration and the formation of vitamin D₂. Longer exposure to UV-B radiation led to a higher ergocalciferol concentration, confirming the effectiveness of this method for mushroom biofortification. The findings highlight the importance of mushrooms as a natural source of vitamin D and confirm that UV-B exposure can enhance their nutritional value, making them a promising dietary component for addressing vitamin D deficiency.