Development of an analytical protocol for the estimation of vitamin D2 in fortified toned milk

•Simple and rapid method for estimation of vitamin D2 in fortified toned milk.•Save chemicals and reagents in comparison to other methods.•Both crystalline and encapsulated vitamin D2 were estimated.•Vitamin D2 in fortified toned milk remained stable during storage and under exposure to light. A simple and rapid method has been developed for the isolation and determination of vitamin D2 in fortified milk samples by HPLC. The advantage of the proposed method is that sample preparation time is reduced and both crystalline and encapsulated forms of vitamin D2 can be estimated. The developed protocol involves a few extraction steps and requires less amounts of reagents. The HPLC separation of vitamin D2 was carried out on a reverse phase C18 column with photo diode array detector at 254nm. Some additional steps were required for extraction of microencapsulated vitamin D2 for breaking down coating material in comparison to crystalline vitamin D2. The recovery of vitamin D2 in fortified toned milk by the proposed method ranged from 96.46% to 99.05%. Non significant degradation was observed in vitamin D2 during seven days storage and also under light exposure (1485 lux) for 32h.