CEN/TC 275/WG 9 - Vitamins and Carotenoids

This European Standard specifies a method for the determination of vitamin B6 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6 is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxine. The β-glycosylated forms are not taken into account. These can be determined with the method given in EN 14663 [1] by which the different vitamins of vitamin B6 (pyridoxal, pyridoxamine and pyridoxine) are separated and individually quantified. A third European Standard, EN 14166 [2], determines the total vitamin B6 by microbiological assay.

This European Standard specifies a method for the determination of vitamin B1 in food by high performance liquid chromatography (HPLC) with enzymatic treatment and pre- or post-column derivatization. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of whole meal flour, milk powder/spray dried milk, freeze-dried mixed vegetables and freeze-dried pig's liver ranging from 0,295 mg/100 g to 0,807 mg/100 g. The second study was for the analysis of samples of tube feeding solution, baby food with vegetables, powdered milk, meal with fruits, yeast, cereal, chocolate powder and food supplement ranging from 0,11 mg/100 g to 486 mg/100 g. Vitamin B1 is the mass fraction of total thiamin including its phosphorylated derivatives.
For further information on the validation, see Clause 8 and Annex B.

This European Standard specifies a method for the determination of vitamin E in foods by high performance liquid chromatography (HPLC). The determination of vitamin E content is carried out by measurement of α-, β , γ- and δ-tocopherol. This method has been validated in two interlaboratory studies. The first study was for the analysis of α-tocopherol in margarine and milk powder ranging from 9,89 mg/100 g to 24,09 mg/100 g. The second study was for the analysis of α-, β-, γ- and δ-tocopherol in milk powder and of α-, and β-tocopherol in oat powder ranging from 0,057 mg/100 g (β-tocopherol) to 10,2 mg/100 g (α-tocopherol).
NOTE   The vitamin E activity can be calculated from the tocopherol content assuming appropriate factors as given in [1], [2], [3] and [4].

This European Standard specifies a method for the determination of vitamin B2 in food by high performance liquid chromatography (HPLC) and fluorescence detection. This method has been validated in two interlaboratory studies. The first study was for the analysis of samples of milk powder and pig's liver ranging from 1,45 mg/100 g to 10,68 mg/100 g. The second study was for the analysis of samples of tube feeding solution, baby food, powdered milk, meal with fruits, yeast, cereal and chocolate powder ranging from 0,21 mg/100 g to 87,1 mg/100 g. Vitamin B2 is the mass fraction of total riboflavin including its phosphorylated derivatives.
For further information on the validation, see Clause 8 and Annex B.

This European Standard specifies a method for the determination of vitamin A in foodstuffs by high performance liquid chromatography (HPLC). This method has been validated in an interlaboratory study with samples of margarine and milk powder with all-E-retinol levels ranging from 653 µg/100 g to 729 µg/100 g and with 13-Z-retinol levels ranging from 30 µg/100 g to 39 µg/100 g. The determination of vitamin A content is carried out by the measurement of all-E-retinol, 13-Z-retinol and β-carotene. This part covers the measurement of all-E-retinol and 13-Z-retinol.
The extract obtained after saponification in this method can be used for the determination of β-carotene, as described in EN 12823 2:2000 Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Part 2: Measurements of β-carotene. In this case, the saponification temperature should preferably not exceed 80 °C in order to prevent isomerisation and oxidation of β-carotene.

This Technical Specification specifies a method for the determination of canthaxanthin and astaxanthin in fish flesh by high performance liquid chromatography (HPLC). The method can be applied at a range above 0,02 mg/kg. The method should not be applied to the determination of canthaxanthin in poultry tissues, egg yolks and shrimp tissues due to a possible interference of canthaxanthin with cryptoxanthin and xanthophyll esters sometimes present in these matrices.

This Technical Specification describes a method for the determination of the astaxanthin enantiomer ratio in fish flesh by high performance liquid chromatography (HPLC).

This European Standard specifies a method for the determination of total vitamin B6 in foodstuffs by microbiological assay (MBA). Vitamin B6 is determined as the mass fraction of pyridoxine, pyridoxal and pyridoxamine, including their phosphorylated or glycosylated derivatives. It is usually expressed as milligram vitamin B6 per 100 g of foodstuff. The method is applicable to samples that can be rendered homogeneous and do not contain high concentrations of antibiotics or other interfering substances. This method has been validated in an inter-laboratory test on fortified and non-fortified samples such as wholemeal flour, milk powder, mixed vegetables and pigs liver at levels from 0,5 mg/100 g to 1,9 mg/100 g. For further information on the validation data, see Annex B.

by high performance liquid chromatography (HPLC) by three different ways of hydrolysis, acid hydrolysis (A),
enzymatic hydrolysis (B) or acid/alkaline hydrolysis (C).
The method has been validated in interlaboratory tests on fortified and non-fortified samples such as breakfast
cereal powder, chocolate cereals, cooked ham, green peas, lyophilized green peas with ham, lyophilized soup,
nutritive orange juice, milk powder and wheat flour, at levels from 0,5 mg/100 g to 24 mg/100 g. For further
information on the validation data, see Annex B.
A and B give similar results for niacin. In options A and B niacin is calculated as the sum of nicotinamide and
nicotinic acid, and expressed as nicotinic acid [1]. Option C gives higher results than A and B for niacin with
non-supplemented cereals, but similar results for other products. In option C, niacin is calculated and
expressed as nicotinic acid after transformation of nicotinamide into nicotinic acid [2].
Option A is faster and cheaper than B and C.
Option B is used if an exact quantification of nicotinamide and nicotinic acid is needed. This cannot be done
with option A, because there is a slight transformation of nicotinamide into nicotinic acid during the acid
hydrolysis.
Option C quantifies total niacin. The alkaline hydrolysis is able to liberate other forms giving higher results for
niacin, which in some foods such as maize and cereals are not normally biologically available, see [3], [4] and
[5].
Information on a comparison between the three different ways of hydrolysis is given in Annex C.

This European Standard specifies a method for the determination of the mass fraction of d-biotin by high performance liquid chromatography (HPLC). The method has been validated in an inter-laboratory test on fortified and non-fortifed samples such as cereal breakfast powder, infant milk powder, lyophilized green peas with ham, lyophilized chicken soup and on nutritive orange juice, at levels from 16 μg/100 g to 200 μg/100 g. For further information on the validation data, see Annex B. NOTE 1 d-biocytin can also be estimated by this method. But none of the samples used for the validation step contained d-biocytin. Nonetheless the recovery rate is more than 90 % for d-biotin and d-biocytin, see [2] and [3]. NOTE 2 The method underestimates the real biotin content when used for samples containing egg.

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. Further information on the validation data is given in Annex D.

This document specifies a method for the determination of vitamin B6 in foodstuffs by high performance liquid chromatography (HPLC).
Vitamin B6 is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives as well as the b-glycosylated forms that have been added to foodstuffs, calculated as pyridoxine.
This method has been successfully validated with semolina with milk (infant food), potato puree, vegetables with ham (convenient products) and a multi vitamin drink at levels from 0,034 mg/100 g to 1,21 mg/100 g.

This draft European Standard specifies a method for the determination of vitamin K1 in foodstuffs by high performance liquid chromatography (HPLC). The determination of Vitamin K1 content is carried out by measurement of reduced phylloquinone. The method has been validated for milk and milk products, however laboratory experiences exist which show that the method is also applicable to other type of foodstuffs.

This European Standard specifies a microbiological method for the determination of the total folate content of foodstuffs by turbidimetric detection of the growth of the microorganism Lactobacillus casei, subsp. rhamnosus (ATCC 7469).
The method allows for the determination of folates in foodstuffs, including naturally occurring folates and added folic acid (pteroylglutamic acid).

This European Standard specifies a method for the determination of total- beta-carotene in foodstuffs by high performance liquid chromatography (HPLC).

This European Standard specifies a method for the determination of vitamin B6 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B6 is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxine. The β-glycosylated forms are not taken into account. These can be determined with the method given in EN 14663 by which the different vitamers of vitamin B6 (pyridoxal, pyridoxamine and pyridoxine) are separated and individually quantified. A third European Standard (EN 14166 ) determines the total vitamin B6 by microbiological assay.

TC - Modifications to 4.2.15, footnote 1 and 6.2.2

TC - Modification to 4.2.14, footnote 1 and 6.3.2

This draft European Standard specifies a method for the determination of vitamin B2 in foodstuffs by high performance liquid chromatography (HPLC). The determination of vitamin B2 content is carried out by measurement of riboflavin.

This European Standard specifies a method for the determination of vitamin B1 in foodstuffs by high performance liquid chromatography (HPLC). Vitamin B1 is the mass fraction of total thiamin including its phosphorylated derivatives.

This European Standard specifies a method for the determination of Vitamin  E in foodstuffs by high performance liquid chromatography (HPLC). The determination of Vitamin E content is carried out by measurement of alpha-, beta-, Y- and delta-tocopherol. The vitamin E activity can be calculated from the tocopherol content assuming appropriate factors as given in the introduction.

This European Standard specifies a method for the determination of vitamin  A in foodstuffs by high performance liquid chromatography (HPLC). The determination of vitamin A content is carried out by the measurement of all- trans-retinol, 13-cis-retinol, and beta-carotene. This part covers the measurement of all-trans-retinol and 13-cis-retinol. The extract obtained after saponification in this method can be used for the determination of beta- carotene, as described in prEN 12823-2:1999 "Measurement of beta-carotene".

This European Standard specifies an HPLC-method for the determination of vitamin C in foodstuffs. Vitamin C is the sum of L(+) ascorbic acid and dehydro L(+) ascorbic acid.

This European Prestandard specifies a method for the determination of total vitamin B6 in foodstuffs by microbiological assay (MBA). Vitamin B6 is determined as the mass fraction of pyridoxine, pyridoxal and pyridoxamine, including their phosphorylated or glycosylated derivatives. It is usually expressed as milligram vitamin B6 per 100 g of foodstuff. The method is applicable to samples that can be rendered homogeneous and do not contain high concentrations of antibiotics or other interfering substances.

This European Standard specifies a method for the determination of vitamin  D in foodstuffs by high performance liquid chromatography (HPLC). In the majority of foodstuffs vitamin D is naturally present as cholecalciferol, vitamin D3, and this is the form of the vitamin determined. Vitamin D2, ergocalciferol, is sometimes present in fortified foodstuffs and can also be determined using the European Standard. Some foods will contain both vitamin D3 and D2. This method is not applicable to these samples.

This European Prestandard specifies a method for the determination of vitamin B6 in foodstuffs by HPLC.
Vitamin B6 is the mass fraction of the sum of pyridoxine, pyridoxal, pyridoxamine including their phosphorylated derivatives determined as pyridoxin. The b-glycosylated forms are not taken into account.