DIN EN 12822-2014 Foodstuffs - Determination of vitamin E by high performance liquid chromatography - Measurement of α- β- γ- and δ-tocopherols German version EN 12822 2014《食品 高效液相.pdf

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1、August 2014Translation by DIN-Sprachendienst.English price group 12No part of this translation 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

2、67.050!%:;)“2232406www.din.deDDIN EN 12822SupersedesDIN EN 12822:2000-07www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Foodstuffs Determination of vitamin E by high performance liquid chromatography Measurement of -, -, - and -tocopherols; English transl

3、ation of DIN EN 12822:2014-08 Lebensmittel Bestimmung von Vitamin E mit HochleistungsFlssigchromatographie Bestimmung von -, , und Tocopherol Englische Fassung EN 12822:2014, Englische bersetzung von DIN EN 12822:201408 Produits alimentaires Dtermination de la teneur en vitamine E par chromatographi

4、e liquide hauteperformance Dosage de -, , at tocophrols; Version anglaise EN 12822:2014, Traduction anglaise de DIN EN 12822:201408 Document comprises 22 pages07.14 DIN EN 12822:2014-08 2 A comma is used as the decimal marker. National foreword This document (EN 12822:2014) has been prepared by Tech

5、nical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und land-wirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057

6、-01-13 AA Vitamine und Carotinoide. The DIN Standards corresponding to the International Standards referred to in this document are as follows, whereby EN ISO Standards are only listed below if these have not been published as DIN EN ISO Standards with the same number: EN ISO 3696 DIN ISO 3696 ISO 5

7、725 (all parts) DIN ISO 5725 (all parts) Amendments This standard differs from DIN EN 12822:2000-07 as follows: a) the scope of the standard has been extended; b) the Bibliography has been updated; c) the standard has been editorially revised. Previous editions DIN EN 12822: 2000-07 National Annex N

8、A (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725 (all parts), Accuracy (trueness and precision) of measurement methods and results EN 12822 June 2014 ICS 67.050 Supersedes EN 12822:2000English Version Foodstuffs - Determinatio

9、n of vitamin E by high performance liquid chromatography - Measurement of -, -, - and -tocopherol Produits alimentaires - Dtermination de la teneur en vitamine E par chromatographie liquide haute performance - Dosage des -, -, - et -tocophrolsLebensmittel - Bestimmung von Vitamin E mit Hochleistungs

10、-Flssigchromatographie - Bestimmung von -, -, - und -Tocopherol This European Standard was approved by CEN on 17 April 2014. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard wi

11、thout any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any othe

12、r language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic,

13、Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. CEN-CENELEC Ma

14、nagement Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12822:2014 E EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGEUROPEAN STANDAR

15、DNORME EUROPENNEEUROPISCHE NORM2 Contents PageForeword 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Principle 5 4 Reagents .5 5 Apparatus .8 6 Procedure .9 7 Calculation . 11 8 Precision 11 9 Test report . 12 Annex A (informative) Examples of HPLC chromatograms . 14 Annex B (informative) P

16、recision data . 16 Annex C (informative) Alternative HPLC systems 18 Bibliography . 19 DIN EN 12822:2014-08EN 12822:2014 (E) 3 Foreword This document (EN 12822:2014) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. Thi

17、s European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by December 2014 and conflicting national standards shall be withdrawn at the latest by December 2014. Attention is drawn to the possibility that some of

18、the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 12822:2000. Annexes A, B and C are informative. According to the CEN-CENELEC Internal Regulations, the nation

19、al standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithu

20、ania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. WARNING The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety

21、 problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. DIN EN 12822:2014-08EN 12822:2014 (E)4 Introduction This European Standard provides th

22、e 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. As the method in this European Standard deals with the measurement of the mass fraction of -, -, - and -tocopherol in food, reference

23、 is made to the literature for the calculation and expression of the vitamin E content in terms of biological activities. For further information see 1, 2, 3 and 4. The differentiation of RRR-tocopherol and all racemic tocopherols is not possible with this method. DIN EN 12822:2014-08EN 12822:2014 (

24、E) 5 1 Scope 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 -, -, - and -tocopherol. This method has been validated in two interlaboratory

25、studies. The first study was for the analysis of -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 -, -, - and -tocopherol in milk powder and of -, and -tocopherol in oat powder ranging from 0,057 mg/100 g (-tocopherol) to

26、 10,2 mg/100 g (-tocopherol). NOTE The vitamin E activity can be calculated from the tocopherol content assuming appropriate factors as given in 1, 2, 3 and 4. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for i

27、ts application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696) 3 Principle -, -, - and -tocoph

28、erol are determined in a sample solution by HPLC separation and subsequent photometric (UV-range) or preferably fluorometric detection. In most cases a saponification of the test material followed by an extraction is necessary. Identification is carried out on the basis of retention times and quanti

29、tative determination by the external standard method using peak areas of peak heights. Internal standard methods can also be used if the corresponding recovery tests have proven the same behaviour of the internal standard during the analysis as the analyte itself, for more information see 4 to 14. N

30、OTE Using normal phase columns, the separation of tocopherols and tocotrienols is also feasible. 4 Reagents 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. 4.1 Methanol. 4.2 Ethanol absolute, volum

31、e fraction (C2H5OH) = 100 %. 4.3 Ethanol, (C2H5OH) = 96 %. 4.4 Sodium sulfate, anhydrous. 4.5 KOH solution, for saponification, in suitable mass concentrations, for example (KOH) = 50 g/100 ml or (KOH) = 60 g/100 ml or alcoholic solutions, for example 28 g of KOH in 100 ml of a mixture of 9 parts pe

32、r volume of ethanol and 1 part per volume of water. 4.6 Antioxidants, such as ascorbic acid (AA), sodium ascorbate, pyrogallol, sodium sulfide (Na2S), hydroquinone or butylated hydroxytoluene (BHT). DIN EN 12822:2014-08EN 12822:2014 (E)6 4.7 Solvents and extraction solvents, such as diethyl ether (p

33、eroxide free), dicholormethane, light petroleum (boiling range of 40 C to 60 C), n-hexane, ethylacetate or appropriate mixtures thereof. 4.8 HPLC mobile phase, appropriate mixtures expressed as volume fractions of for example 3 % 1,4-dioxane or 0,5 % 2-propanol, 3 % tert-butyl methyl ether in n-hexa

34、ne or n-heptane for normal phase chromatography (NP) or 1 % to 10 % water in methanol for reversed phase chromatography (RP). For alternative HPLC systems, see Annex C. 4.9 Standard substances 4.9.1 General -, - and -tocopherol can be obtained from Calbiochem1)-tocopherol can be obtained from variou

35、s suppliers. The purity of the tocopherol standards can vary between 90 % and 100 %. It is therefore necessary to determine the concentration of the calibration solution by UV spectrometry (for purity tests, see 4.10.5). 4.9.2 -tocopherol, M(C29H50O2) = 430,7 g/mol, with a known mass fraction of at

36、least 95 %. -tocopherol acetate, M(C31H52O3) = 472,7 g/mol, may also be used as standard after saponification. 4.9.3 -tocopherol, M(C28H48O2) = 416,7 g/mol, with a known mass fraction of at least 90 %. 4.9.4 -tocopherol, M(C28H48O2) = 416,7 g/mol, with a known mass fraction of at least 90 %. 4.9.5 -

37、tocopherol, M(C27H46O2) = 402,6 g/mol, with a known mass fraction of at least 90 %. 4.10 Stock solutions 4.10.1 -tocopherol stock solution Weigh, to the nearest milligram, an amount of the -tocopherol standard substance (4.9.2), e.g. approximately 10 mg, and dissolve it in a defined volume, e.g. 100

38、 ml, of an appropriate solvent, e.g. n-hexane for a NP system or methanol for a RP system. 4.10.2 -tocopherol stock solution Weigh, to the nearest milligram, an amount of the -tocopherol standard substance (4.9.3), e.g. approximately 10 mg, and dissolve it in a defined volume, e.g. 100 ml, of an app

39、ropriate solvent, e.g. n-hexane for a NP system or methanol for a RP system. 4.10.3 -tocopherol stock solution Weigh, to the nearest milligram, an amount of the -tocopherol standard substance (4.9.4), e.g. approximately 10 mg, and dissolve it in a defined volume, e.g. 100 ml, of an appropriate solve

40、nt, e.g. n-hexane for a NP system or methanol for a RP system. 4.10.4 -tocopherol stock solution Weigh, to the nearest milligram, an amount of the -tocopherol standard substance (4.9.5), e.g. approximately 10 mg, and dissolve it in a defined volume, e.g. 100 ml, of an appropriate solvent, e.g. n-hex

41、ane for a NP system or methanol for a RP system. 1) This information is given for convenience of users of this European Standard and does not and does not constitute and endorsement by CEN. Equivalent products may be used if they can be shown to lead to the same results. DIN EN 12822:2014-08EN 12822

42、:2014 (E) 7 4.10.5 Concentration and purity tests Measure the absorbance of the stock solutions (4.10.1 to 4.10.4) at the appropriate wavelength using an UV spectrometer (5.1). If the solvent used is n-hexane, pipette 10 ml of the stock solution into an amber glass round bottomed flask and remove th

43、e solvent using a rotary evaporator (5.2) under reduced pressure at a temperature not higher than 50 C. After restoring atmospheric pressure with nitrogen, remove the flask and dissolve the residue in 10 ml of methanol by swirling. Take this solution for the spectrometric measurement. Calculate the

44、mass concentration of vitamin E, , of the respective of -, -, - and -tocopherol, in micrograms per millilitre by using Formula (1): 0001=MA(1) where A is the absorption value of each tocopherol in the respective stock solution in methanol; is the molar absorption coefficient in methanol in l x mol1x

45、 cm1at the specific wavelength as given in Table 1; M is the molar mass, in grams per mol, of each tocopherol as given in Table 1. Table 1 Examples for %11cmE values and calculated Substance Wavelength (in methanol) %11cnE Molar mass (in g mol1) (in l mol1 cm1) Reference -tocopherol 292 nm 76 430,7

46、3 273,3 12, 13, 15 -tocopherol 296 nm 89 416,7 3 708,6 12, 13, 15 -tocopherol 298 nm 91 416,7 3 782 12, 13, 15 -tocopherol 298 nm 87 402,6 3 502,6 12, 13, 15 In addition to the value for -tocopherol obtained at a wavelength of 292 nm, the absorbance at 255 nm (minimum) should also be measured. The r

47、atio at this wavelength should not exceed E255/E292= 0,18. Otherwise the substance has degraded (for more information see 15). 4.11 Standard solutions 4.11.1 -tocopherol standard solution Pipette 10 ml of the -tocopherol stock solution (4.10.1) into a one-mark 100 ml volumetric flask and dilute to t

48、he mark with the appropriate solvent (for NP e.g. n-hexane, for RP e.g. methanol). The standard solution should have a mass concentration of 1 g/ml to 10 g/ml of -tocopherol. If an UV-detector is used to monitor the chromatography, a more concentrated solution shall be used. The standard solution sh

49、all be stored protected from light and at a temperature below 4 C and should be checked as described in 4.10.5. 4.11.2 Standard solution of a mixture of -, -, - and -tocopherol Pipette e.g. 10 ml of each of the stock solutions (4.10) into a one-mark 100 ml volumetric flask and dilute to the mark with the appropriat