Background and Objectives: : Vitamin D₃ measurement in food and fortified food matrixes is usually interrupted by several variables. This study aimed to elaborate a specific vitamin D₃ quantification method for fortified Taftoon bread considering different influential variables in each step of measurement.
Materials and Methods: Dough was fortified with (4μg/100g) and the standard protocol No.13579 was followed with some modification in the extraction and determination steps.
Results: Addition of ethanol before adding potassium hydroxide and heating up the samples in tight containers in saponification step ceased clumping the extracts and helps it’s homogeneity. In extraction, distilled water addition following extraction with diethyl ether and Petroleum ether, adding ethanol at each step of washing and using SPE column result in an appropriate experimental extract. In the chromatography step, mobile phase substitution from methanol to 5:95 water, methanol mixture provided the method with more differentiation power. Using the above condition, commercial vitamin D₃ was recovered from fortified Taftoon dough with 82-100% recovery and recovery of spiked vitamin D₂ from dough and bread were ranged as 94 and 86.5%, respectively, Limit Of Detection(LOD) and Limit Of Quantification(LOQ) of the method were 0.04 and 0.1mg L^-1 respectively, which equaled 5μg/Kg and 10μg/Kg. True retention for the added commercial vitamin D₃ in Taftoon bread was obtained as 83.4% which increased to 92% after six months storage in freezer.
Conclusions: Results of this study can be used to specify a protocol for vitamin D₃ measurement in fortified bread samples. Modified experiment which is projected in this study would specify the general protocol of vitamin D₃ measurement that was previously proposed in the national standard of Iran for developing a new and practical national standard document.