ImageVerifierCode 换一换
格式:PDF , 页数:26 ,大小:626.56KB ,
资源ID:665221      下载积分:10000 积分
快捷下载
登录下载
邮箱/手机:
温馨提示:
如需开发票,请勿充值!快捷下载时,用户名和密码都是您填写的邮箱或者手机号,方便查询和重复下载(系统自动生成)。
如填写123,账号就是123,密码也是123。
特别说明:
请自助下载,系统不会自动发送文件的哦; 如果您已付费,想二次下载,请登录后访问:我的下载记录
支付方式: 支付宝扫码支付 微信扫码支付   
注意:如需开发票,请勿充值!
验证码:   换一换

加入VIP,免费下载
 

温馨提示:由于个人手机设置不同,如果发现不能下载,请复制以下地址【http://www.mydoc123.com/d-665221.html】到电脑端继续下载(重复下载不扣费)。

已注册用户请登录:
账号:
密码:
验证码:   换一换
  忘记密码?
三方登录: 微信登录  

下载须知

1: 本站所有资源如无特殊说明,都需要本地电脑安装OFFICE2007和PDF阅读器。
2: 试题试卷类文档,如果标题没有明确说明有答案则都视为没有答案,请知晓。
3: 文件的所有权益归上传用户所有。
4. 未经权益所有人同意不得将文件中的内容挪作商业或盈利用途。
5. 本站仅提供交流平台,并不能对任何下载内容负责。
6. 下载文件中如有侵权或不适当内容,请与我们联系,我们立即纠正。
7. 本站不保证下载资源的准确性、安全性和完整性, 同时也不承担用户因使用这些下载资源对自己和他人造成任何形式的伤害或损失。

版权提示 | 免责声明

本文(DIN EN 12821-2009 Foodstuffs - Determination of vitamin D by high performance liquid chromatography - Measurement of cholecalciferol (D) or ergocalciferol (D) German version EN 128.pdf)为本站会员(ideacase155)主动上传,麦多课文库仅提供信息存储空间,仅对用户上传内容的表现方式做保护处理,对上载内容本身不做任何修改或编辑。 若此文所含内容侵犯了您的版权或隐私,请立即通知麦多课文库(发送邮件至master@mydoc123.com或直接QQ联系客服),我们立即给予删除!

DIN EN 12821-2009 Foodstuffs - Determination of vitamin D by high performance liquid chromatography - Measurement of cholecalciferol (D) or ergocalciferol (D) German version EN 128.pdf

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

copyright@ 2008-2019 麦多课文库(www.mydoc123.com)网站版权所有
备案/许可证编号:苏ICP备17064731号-1