Introduction
Fortification of milk, dairy products, infant and adult nutritionals with vitamin D is required in many countries to provide additional dietary vitamin D. The fortification of milk in the USA, for example, is done at 400 International Units (IU) per eight ounces of milk. The recently adopted AOAC method 2011.11 describes the procedure for the analysis of vitamin D in fortified infant formula. This method was used in the current work to measure vitamin D in the fortified whole milk and in an infant formula sample. Two forms of vitamin D are recognized: vitamin D2 or ergocalciferol and vitamin D3 or cholecalciferol. Although both forms may be present in the fortified food, vitamin D3 is more commonly used for fortification of dairy products. Vitamin D hydroxy-metabolites can also be present in food products at low levels. While quantitative analysis was performed for vitamins D2 and D3, the samples were scanned for the presence of 25-hydroxy metabolites.
Method
AOAC method 2011.11 was followed for analysis of vitamin D in both milk and infant formula. Vitamin D2 and D3 standards and their deuterated analogues were used. The UHPLC column was Titan C18, 10 cm x 2.1 mm packed with 1.9 μm particles. The HPLC method was adopted from the standard AOAC method, including mobile phases, gradient, and flow rates. Baseline separation between vitamin D3 and D2 peaks, and between all vitamins and matrix component peaks was obtained on the Titan C18 UHPLC column. Analysis of vitamin D was done using MS/MS with APCI detection. MS transitions used in this study are shown in Table 1.
The sample preparation for both milk and infant formula samples was done through saponification, extraction, evaporation, and reconstitution of extracts into LC-compatible solvents. The sample preparation steps following AOAC method 2011.11 are shown in Figure 1.
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