en Nutrition Nutrition پژوهشي Research <p style="margin: 0px; text-align: justify; line-height: 200%;"><strong>Background and Objectives: </strong><strong>:</strong>&nbsp;Vitamin D₃&nbsp;measurement in food and fortified food matrixes is usually interrupted by several variables. This study aimed to elaborate a specific vitamin D₃&nbsp;quantification method for fortified <em>Taftoon</em> bread considering different influential variables in each step of measurement.<br> <strong>Materials and Methods: </strong>Dough was fortified with (4&mu;g/100g) and the standard protocol No.13579 was followed with some modification in the extraction and determination steps.<br> <strong>Results:</strong> Addition of ethanol before adding potassium hydroxide and&nbsp;heating up the samples in tight&nbsp;containers in saponification step ceased clumping the extracts and helps it&rsquo;s homogeneity. In extraction, distilled water addition following&nbsp;extraction with diethyl ether and Petroleum ether,&nbsp;adding ethanol at each step of washing and using SPE column result in&nbsp;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&nbsp;was recovered from fortified <em>Taftoon</em> dough with 82-100% recovery and recovery of spiked vitamin D₂&nbsp;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^-1respectively, which equaled 5&mu;g/Kg and 10&mu;g/Kg. True retention for the added commercial vitamin D₃in <em>Taftoon</em> bread was obtained as 83.4% which increased to 92% after six months storage in freezer.<br> <strong>Conclusions: </strong>Results of this study can be used to specify a protocol for vitamin D₃&nbsp;measurement in fortified bread samples.&nbsp;Modified experiment&nbsp;which is projected in&nbsp;this study&nbsp;would specify&nbsp;the general protocol of vitamin D₃&nbsp;measurement that was previously proposed in the national standard of Iran for developing&nbsp;a new and practical national standard&nbsp;document.</p> Vitamin D3, Fortified bread, Extraction, High-performance liquid chromatography​ 47 54 http://nfsr.sbmu.ac.ir/browse.php?a_code=A-10-584-1&slc_lang=en&sid=1 Mehrnaz Tabibian 10031947532846002605 10031947532846002605 Yes Department of Food Science and Technology, Faculty of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran Malihe Sadeghi 10031947532846002606 10031947532846002606 No Department of Food Sciences and Technology, School of Nutrition and Food Sciences, Isfahan University of Medical Sciences, Isfahan, Iran Javad Mohtadinia 10031947532846002607 10031947532846002607 No Department of Food Science and Technology, Faculty of Nutrition and Food Sciences, Tabriz University of Medical Sciences, Tabriz, Iran Mina Babashahi 10031947532846002608 10031947532846002608 No Students’ Research Committee, Faculty of Nutrition Sciences& Food Technology, Shahid Beheshti University if medical Sciences, Tehran, Iran Maryam Mirlohi 10031947532846002609 10031947532846002609 No Department of Food Sciences and Technology, School of Nutrition and Food Sciences, Isfahan University of Medical Sciences, Isfahan, Iran