Single-run analysis of vitamin D photoproducts in oyster mushroom (Pleurotus ostreatus) after UV-B treatment

• A method for analysing UV-B photoproducts from provitamins D2/4 was developed.
• In total 8 isomers of vitamin D2/4 were quantified within a single-run HPLC method.
• LC–MS analysis was performed to distinguish D2 isomers from those of the D4 series.
• Hot alkaline hydrolysis yielded the highest recovery of vitamins D and side-products.
• Stability of intermediates in methanol extracts was examined from 20 to 130 °C.

A single-run high performance liquid chromatography (HPLC) with diode array detector (DAD) based method was developed for the separation, identification and comprehensive quantification of degradation products of ergosterol formed in oyster mushroom (Pleurotus ostreatus) after UV-B exposure. After 60 min, 10 substances involved in the photoprocess were separated, identified by their characteristic DAD spectrum and distinguished by their molecular weight, in cases where spectra were identical: vitamin D2, previtamin D2, tachysterol2, lumisterol2 and ergosterol, and, in minor quantity, their structural analogues of the 22,23-dihydroergocalciferol (vitamin D4) series. Sample preparation protocol affected the total yields and the ratios of previtamin and vitamin D2/D4. Hot alkaline hydrolysis resulted in the best digestion of the mushroom matrix and accordingly gave the highest vitamin D yield (D2: 141.32 μg g−1 dry matter, DM; D4: 22.72 μg g−1 DM). Limit of detection for vitamin D2/4 was 0.02 μg g−1 dry matter (DM) and was estimated for previtamin D2/4 (0.06 μg g−1 DM), tachysterol2/4 (0.02 μg g−1 DM) and lumisterol2/4 (0.06 μg g−1 DM). Recovery of spiked vitamin D2 was 97 ± 0.7%. The study provides an analytical tool to assess the process of vitamin D generation after UV-B treatment for the production of oyster mushrooms with a balanced nutritional profile of vitamin D compounds.