This International Standard specifies a method for the determination of vitamin D in a test sample containing at least 10 μg of vitamin D per 100 g [equal to 400 International Units (IU) of vitamin D per 100 g] by using high-performance liquid chromatography (HPLC).

The results of the determination are only reliable if, in the case of determination of vitamin D₃, the test sample contains only vitamin D₃, and no vitamin D₂ (which will be added as an internal standard) and, in the case of determination of vitamin D₂, the test sample contains only vitamin D₂ and no vitamin D₃ (which will be added as an internal standard). This is to be verified by the procedure carried out without the addition of the internal standard (vitamin D₂).