Determination of vitamin D3 and 25-hydroxyvitamin D3 in foodstuffs by HPLC UV-DAD and LC–MS/MS

In order to generate new data for vitamin D content for the Canadian Nutrient File, a method for the quantification of vitamin D3 and 25(OH)D3 in foodstuffs has been modified and improved. Vitamin D3 was quantified using reverse phase liquid chromatography (LC) with UV-diode array detector (UV-DAD), while 25(OH)D3 was measured by triple quadrupole mass spectrometry (APCI MS/MS). Quantification was by internal standards (IS) using vitamin D2 and 25(OH)D2. A Certified Reference Material (CRM-421 containing vitamin D3) and a control sample (internally generated reference material of ground pork containing both vitamin D3 and 25(OH)D3) were used as validation and quality control tools. Limit of detection for both compounds was 0.04 μg/100 g. Accuracy for vitamin D in the CRM-421 was 99% (0.142 mg/kg for a target of 0.143, n = 10). Recovery of vitamin D3 in ground pork was 97% (88% absolute recovery). For 25(OH)D3, a recovery of 94% (73% absolute recovery) was obtained. Using this method, data for vitamin D3 and 25(OH)D3 content in a variety of foods (pork, beef, eggs, poultry, fish, and dinners) have been generated.