1、August 2009DEUTSCHE NORM English price group 13No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.050!$Y(j“1540571www.
2、din.deDDIN EN 12821Foodstuffs Determination of vitamin D by high performance liquidchromatography Measurement of cholecalciferol (D3) or ergocalciferol(D2)English version of DIN EN 12821:2009-08Lebensmittel Bestimmung von Vitamin D mit Hochleistungs-Flssigchromatographie Bestimmung von Cholecalcifer
3、ol (D3) oder Ergocalciferol (D2)Englische Fassung DIN EN 12821:2009-08SupersedesDIN EN 12821:2000-07www.beuth.deDocument comprises pages26DIN EN 12821:2009-08 National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Ger
4、many). Preliminary work was done by Working Group WG 9 “Vitamins and Carotenoids”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-13 AA V
5、itamine und Carotinoide. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN IS0 3696 Amendments This standard differs from DIN EN 12821:2000-07 as follows: a) The title of the standard has been modified: “Measurement of cholecalci
6、ferol (D3) or ergocalciferol (D2)” instead of “Measurement of cholecalciferol (D3) and ergocalciferol (D2)”. b) The normative reference to EN ISO 5555 has been deleted. c) The validation data for the method based on external standard have been deleted. d) Specifications relating to the validated foo
7、dstuffs have been rendered more precise. e) Specifications relating to the reagents have been given in more detail. f) The figures have been updated. g) The text of this standard has been editorially revised. h) The new rules for drawing up European Standards have been taken into consideration. Prev
8、ious editions DIN EN 12821: 2000-07 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12821 April 2009 ICS 67.050 Supersedes EN 12821:2000 English Version Foodstuffs - D
9、etermination of vitamin D by high performance liquid chromatography - Measurement of cholecalciferol (D3) or ergocalciferol (D2) Produits alimentaires - Dosage de la vitamine D par chromatographie liquide haute performance - Dosage du cholcalcifrol (D3) de l ergocalcifrol (D2) ouLebensmittel - Besti
10、mmung von Vitamin D mit Hochleistungs-Flssigchromatographie - Bestimmung von Cholecalciferol (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 givi
11、ng this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official ver
12、sions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgiu
13、m, 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 S
14、TANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12821:2009: EEN 12821:2009 (E) 2 Contents Page F
15、oreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .8 6 Procedure .9 7 Calculation . 11 8 Precision 12 9 Test report . 13 Annex A (informative) Examples of suitable HPLC systems . 14 Annex B (informative) Examples of suitable extraction and saponification conditions
16、 . 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 Bibliogra
17、phy . 24 DIN EN 12821:2009-08 EN 12821:2009 (E) 3 Foreword This document (EN 12821:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either
18、by publication of an identical text or by endorsement, at the latest by October 2009, and conflicting national standards shall be withdrawn at the latest by October 2009. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or
19、CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 12821:2000. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belg
20、ium, 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 the United Kingdom. DIN EN 12821:2009-
21、08 EN 12821:2009 (E) 4 1 Scope 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 D3is primary in foodstuffs of animal origin, while vitamin D2is primary
22、in wild mushrooms. Both vitamin D3 and vitamin D2can be present in fortified foodstuffs. This European Standard is not applicable for samples with a content of vitamin D3and vitamin D2. Apart from the vitamin D activity from the parent forms, vitamin D3and vitamin D2, the corresponding metabolites 2
23、5-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 D3or vitamin D2. This European Standard provides the base for the analytical methods. It is intended to serve as a frame in which the analyst ca
24、n 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 g/100 g
25、to 14 g/100 g. Further information on the validation data is given in Annex D. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the ref
26、erenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987). 3 Principle Vitamin D3and vitamin D2are saponified in the foodstuffs using alcoholic potassium hydroxide solution and extracted by an appropriate so
27、lvent. The determination of vitamin D3or vitamin D2in an appropriate sample extract solution is carried out by semi-preparative normal phase HPLC followed by reverse-phase analytical HPLC. If vitamin D3is to be determined, then vitamin D2is used as an internal standard. If vitamin D2is to be determi
28、ned, then vitamin D3is 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 the internal standard pro
29、cedure using peak areas or peak heights, see 1 to 8. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade 1 according to EN ISO 3696. DIN EN 12821:2009-08 EN 12821:2009 (E) 5 4.2 Methanol 4.3 Ethanol, volume
30、 fraction (C2H5OH) = 100 %. 4.4 Ethanol, (C2H5OH) = 96 %. 4.5 Sodium sulfate, anhydrous. 4.6 KOH solutions for saponification, in suitable concentrations, e.g. mass concentration (KOH) = 50 g/100 ml or (KOH) = 60 g/100 ml, or alcoholic solutions, e.g. 28 g of KOH in 100 ml of an ethanol and water mi
31、xture with a volume fraction of ethanol of 90 %. 4.7 Antioxidants, such as ascorbic acid (AA), sodium ascorbate, pyrogallol, sodium sulfide (Na2S) or butylated hydroxytoluene (BHT). 4.8 Solvents and extraction solvents, such as diethyl ether (peroxide-free), dichloromethane, light petroleum, n-hexan
32、e, ethylacetate or appropriate mixtures thereof. 4.9 HPLC Mobile phases 4.9.1 Examples of solvent mixtures for normal phase semi-preparative HPLC Examples of appropriate solvent mixtures (given as volume fractions) for normal phase semi-preparative HPLC include: n-hexane and 2-propanol (98 + 2), (99
33、 + 1) or (95 + 5); n-hexane and isoamyl alcohol (99 + 1); n-hexane, 2-propanol and tetrahydrofuran (98 + 1 + 1); iso-octane and iso-butanol (99 + 1); n-heptane and 2-propanol (97 + 3). 4.9.2 Examples of solvent and solvent mixtures for reverse-phase analytical HPLC Examples of appropriate solvent an
34、d 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). DIN EN 12821:2009-08 EN 12821:2009 (E) 6 4.10
35、Standard substances 4.10.1 Ergocalciferol standard substance (vitamin D2), M(C28H44O) = 396,7 g/mol Vitamin D2standard substance shall be of the highest purity obtainable (having a mass fraction of greater than 98 %) and shall be stored according to the suppliers instructions (in the absence of ligh
36、t, typically less than 4 C). 4.10.2 Cholecalciferol standard substance (vitamin D3), M(C27H44O) = 384,6 g/mol Vitamin D3standard substance shall be of the highest purity obtainable (having a mass fraction of greater than 98 %) and shall be stored according to the suppliers instructions (in the absen
37、ce of light, typically less than 4 C). 4.11 Stock solutions 4.11.1 Vitamin D2stock solution Weigh about 100 mg of vitamin D2(4.10.1) to the nearest milligram into a one mark 100 ml volumetric flask, dissolve in ethanol (4.4) and dilute to the mark with ethanol. This solution contains approximately 1
38、 mg/ml of vitamin D2. Store below 4 C and protect from light. Calculate the mass concentration of the stock solution and the mass fraction of the vitamin D2standard by the procedure described in 4.12.1. This solution is stable for 6 months at - 18C. 4.11.2 Vitamin D3stock solution Weigh about 100 mg
39、 of vitamin D3(4.10.2) to the nearest milligram into a one mark 100 ml volumetric flask, dissolve in ethanol (4.4) and dilute to the mark with ethanol. This solution contains approximately 1 mg/ml of vitamin D3. Store below 4 C and protect from light. Calculate the mass concentration of the stock so
40、lution and the mass fraction of the vitamin D3standard by the procedure described in 4.12.2. This solution is stable for 6 months at - 18 C. 4.12 Standard solutions 4.12.1 Vitamin D2standard solution Pipette 1 ml of the vitamin D2stock solution (4.11.1) into a one mark 100 ml volumetric flask and di
41、lute to the mark with ethanol (4.4). This solution contains approximately 10 g/ml of vitamin D2. 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 D2sta
42、ndard solution in a 1 cm quartz cell at a wavelength of 265 nm using ethanol in the reference path. Calculate the mass concentration of vitamin D2, D2, in microgram per millilitre of the standard solution using Equation (1): bMA=1000D2265D2(1) DIN EN 12821:2009-08 EN 12821:2009 (E) 7 where: A265is t
43、he absorption of the vitamin D2standard solution at 265 nm; MD2is the molar mass of vitamin D2(MD2= 396,7 g/mol); is the molar absorption coefficient of vitamin D2(here: = 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.
44、2 Vitamin D3standard solution Pipette 1 ml of the vitamin D3stock 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 g/ml of vitamin D3. Prepare this solution on the day of use. NOTE The mass concentration of t
45、he standard solution can be adjusted if necessary to suit the analytical requirements. Measure the absorption of the vitamin D3standard 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
46、 per millilitre of the standard solution using Equation (2): bMA=1000D3265D3(2) where: A265is the absorption of the vitamin D3standard solution at 265 nm; MD3is the molar mass of vitamin D3(MD3= 384,6 g/mol); is the molar absorption coefficient of vitamin D3(here: = 18 461 m2/mol, calculated from th
47、e % 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 D2internal standard solution Pipette 10 ml of the vitamin D2standard solution (4.12.1) into a one mark 100 ml volumetric flask and dilute to the mark with ethanol
48、 (4.4). Prepare this solution on the day of use. 4.13.2 Vitamin D3internal standard solution Pipette 10 ml of the vitamin D3standard solution (4.12.2) 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. NOTE If vitamin D3is to b
49、e determined, then vitamin D2is used as an internal standard. If vitamin D2is to be determined, then vitamin D3is used as an internal standard. DIN EN 12821:2009-08 EN 12821:2009 (E) 8 4.14 Vitamin D2and vitamin D3semi-preparative standard solution Pipette 5 ml of the vitamin D2standard solution (4.12.1) and 5 ml of the vitamin D3standard solution (4.12.2) into a rotary evaporator flask and carefully remove the solvent (at not more than 40 C). Re-dissolve the residue in 100 ml of the semi-preparative HPLC
