Summary - vitamin D

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EN 12821 Foodstuffs - Determination of vitamin D by high performance liquid chromatography - Measurement of cholecalciferol (D3) or ergocalciferol (D2):


This European Standard specifies a method for the determination of vitamin D3 (cholecalciferol) or vitamin D2 (ergocalciferol) in foodstuffs by high performance liquid chromatography (HPLC).

Vitamin D3 is primary in foodstuffs of animal origin, while vitamin D2 is primary in wild mushrooms. Both vitamin D3 and vitamin D2 can be present in fortified foodstuffs. This European Standard is not applicable for samples with a content of vitamin D3 and vitamin D2.

Apart from the vitamin D activity from the parent forms, vitamin D3 and vitamin D2, the corresponding metabolites 25-hydroxy vitamin D and 1,25-dihydroxy vitamin D also contribute to the vitamin D activity. This European Standard does only include measurement of vitamin D3 or vitamin D2.

This European Standard provides the base for the analytical methods. It is intended to serve as a frame in which the analyst can define his own analytical work in accordance to the standard procedure.

This method has been validated in inter-laboratory tests on fortified and non-fortified samples such as margarine, milk, milk powder, liquid infant formula, infant formula, cooking oil, and fish oil at levels from 0,4 µg/100 g to 14 µg/100 g.


Vitamin D3 and vitamin D2 are saponified in the foodstuffs using alcoholic potassium hydroxide solution and extracted by an appropriate solvent. The determination of vitamin D3 or vitamin D2 in an appropriate sample extract solution is carried out by semi-preparative normal phase HPLC followed by reverse phase analytical HPLC.

If vitamin D3 is to be determined, then vitamin D2 is used as an internal standard. If vitamin D2 is to be determined, then vitamin D3 is used as an internal standard.

Vitamin D is detected by ultraviolet (UV) spectrometry and peaks are identified on the basis of retention times and additionally by UV spectral profile if diode-array detection is used. The determination is carried out by internal standard procedure using peak areas or peak heights.

Precision data


CRM 122

CRM 421
Milk powder

Repeatability standard deviation s r, µg/100 g



Repeatability coefficient of variation, %



Repeatability limit r = [2,8 x s r], µg/100 g



Reproducibility standard deviation s R, µg/100 g



Reproducibility coefficient of variation, %



Reproducibility limit r = [2,8 x s R], µg/100 g



Results obtained in 1994.

Data derived from EN 12821.