EN 12821:2009

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

Lebensmittel - Bestimmung von Vitamin D mit Hochleistungs-Flüssigchromatographie - Bestimmung von Cholecalciferol (D3) oder Ergocalciferol (D2)

Produits alimentaires - Dosage de la vitamine D par chromatographie liquide haute performance - Dosage du cholécalciférol (D3) et de l' ergocalciférol (D2)

La présente Norme européenne spécifie une méthode de dosage de la vitamine D3 (cholécalciférol) ou D2
(ergocalciférol) présente dans les produits alimentaires, par chromatographie liquide à haute performance
(CLHP).
La vitamine D3 se trouve principalement dans les produits alimentaires d’origine animale tandis que la
vitamine D2 est surtout présente dans les champignons sauvages. Les vitamines D3 et D2 peuvent être
présentes dans des produits alimentaires supplémentés. La présente Norme européenne n’est pas applicable
aux échantillons contenant à la fois de la vitamine D3 et de la vitamine D2.
Mise à part l’activité de la vitamine D des formes parentes, la vitamine D3 et la vitamine D2, les métabolites
correspondants, à savoir le 25-hydroxy vitamine D et le 1,25-dihydroxy vitamine D, contribuent également à
l’activité de la vitamine D. La présente Norme européenne traite uniquement du dosage de la vitamine D3 ou
de la vitamine D2.
La présente Norme européenne fournit la base des méthodes d’analyse. Elle est destinée à servir de cadre
sur lequel l’analyste peut s’appuyer pour définir son travail d’analyse conformément au mode opératoire
normalisé.
La présente méthode a été validée par des essais interlaboratoires portant sur des échantillons supplémentés
et non supplémentés comme la margarine, le lait, la poudre de lait, la formulation infantile liquide pour bébé,
la formulation infantile lactée, l’huile de cuisson et l’huile de poisson à des concentrations allant de
0,4 μg/100 g à 14 μg/100 g. Des informations supplémentaires sur les données de validation sont données à
l’Annexe D.

Živila - Določevanje vitamina D s tekočinsko kromatografijo visoke ločljivosti - Merjenje holekalciferola (D3) in ergokalciferola (D2)

General Information

• Status: Published
• Publication Date: 31-Mar-2009
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 9 - Vitamins and Carotenoids
• Current Stage: 9060 - Closure of 2 Year Review Enquiry - Review Enquiry
• Start Date: 02-Sep-2024
• Completion Date: 02-Sep-2024
• Ref Project: SIST EN 12821:2009 - Foodstuffs - Determination of vitamin D by high performance liquid chromatography - Measurement of cholecalciferol (D3) or ergocalciferol (D2)

Relations

• Replaces: EN 12821:2000 - Foodstuffs - Determination of vitamin D by high performance liquid chromatography - Measurement of cholecalciferol (D3) and ergocalciferol (D2)
• Effective Date: 08-Apr-2009

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von Vitamin D mit Hochleistungs-Flüssigchromatographie - Bestimmung von Cholecalciferol (D3) oder Ergocalciferol (D2)Produits alimentaires - Dosage de la vitamine D par chromatographie liquide haute performance - Dosage du cholécalciférol (D3) et de l' ergocalciférol (D2)Foodstuffs - Determination of vitamin D by high performance liquid chromatography - Measurement of cholecalciferol (D3) or ergocalciferol (D2)67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 12821:2009SIST EN 12821:2009en,fr,de01-julij-2009SIST EN 12821:2009SLOVENSKI
STANDARDSIST EN 12821:20001DGRPHãþD

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 12821April 2009ICS 67.050Supersedes EN 12821:2000
English VersionFoodstuffs - Determination of vitamin D by high performanceliquid chromatography - Measurement of cholecalciferol (D3) orergocalciferol (D2)Produits alimentaires - Dosage de la vitamine D parchromatographie liquide haute performance - Dosage ducholécalciférol (D3) et de l' ergocalciférol (D2)Lebensmittel - Bestimmung von Vitamin D mitHochleistungs-Flüssigchromatographie - Bestimmung vonCholecalciferol (D3) oder Ergocalciferol (D2)This European Standard was approved by CEN on 21 February 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 12821:2009: ESIST EN 12821:2009

Examples of suitable HPLC systems . 14 Annex B (informative)
Examples of suitable extraction and saponification conditions . 15 Annex C (normative)
Examples of suitable semi-preparative and analytical HPLC chromatograms . 16 Annex D (informative)
Precision data . 18 Annex E (informative)
Additional cleanup step for the determination of vitamin D with use of preparative TLC, column chromatography and or SPE . 20 Bibliography . 24
4.9.2 Examples of solvent and solvent mixtures for reverse-phase analytical HPLC Examples of appropriate solvent and solvent mixtures (given as volume fractions) for reverse-phase analytical HPLC include:  methanol;  methanol and water (95 + 5) or (93 + 7);  acetonitrile and methanol (80 + 20), (90 + 10) or (70 + 30);  acetonitrile, chloroform and methanol (93 + 4 + 3).
= 18 843
m2/mol, calculated from the % 1cm 1E value, see [9]); b is the optical path length of the quartz cell in centimetres. 4.12.2 Vitamin D3 standard solution Pipette 1 ml of the vitamin D3 stock solution (4.11.2) into a one mark 100 ml volumetric flask and dilute to the mark with ethanol (4.4). This solution contains approximately 10 µg/ml of vitamin D3. Prepare this solution on the day of use. NOTE The mass concentration of the standard solution can be adjusted if necessary to suit the analytical requirements. Measure the absorption of the vitamin D3 standard solution in a 1 cm quartz cell at a wavelength of 265 nm using ethanol (4.4) in the reference path. Calculate the mass concentration of vitamin D3, D3, in microgram per millilitre of the standard solution using Equation (2): bMA×××=ερ1000D3265D3 (2) where: A265 is the absorption of the vitamin D3 standard solution at 265 nm; MD3 is the molar mass of vitamin D3 (MD3 = 384,6 g/mol); 0 is the molar absorption coefficient of vitamin D3 (here: 0
= 18 461
m2/mol, calculated from the % 1cm 1E value, see [9]); b is the optical path length of the quartz cell in centimetres. 4.13 Internal standard solutions 4.13.1 Vitamin D2 internal standard solution Pipette 10 ml of the vitamin D2 standard solution (4.12.1) into a one mark 100 ml volumetric flask and dilute to the mark with ethanol (4.4). Prepare this solution on the day of use. 4.13.2 Vitamin D3 internal standard solution Pipette 10 ml of the vitamin D3 standard solution (4.12.2) into a one mark 100 ml volumetric flask and dilute to the mark with ethanol (4.4
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