1、BSI Standards PublicationBS EN 12822:2014Foodstuffs Determination of vitamin E by high performance liquid chromatography Measurement of null-, -, null- and null-tocopherolBS EN 12822:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 12822:2014. It supersedes
2、BS EN 12822:2000 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include a
3、ll the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2014.Published by BSI Standards Limited 2014ISBN 978 0 580 77942 8ICS 67.050Compliance with a British Standard cannot confer immunity from legal obligations.This British St
4、andard was published under the authority of the Standards Policy and Strategy Committee on 30 June 2014.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 12822:2014EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12822 June 2014 ICS 67.050 Supersedes EN 12822:2000 E
5、nglish Version Foodstuffs - Determination 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 -tocophrols Lebensmittel - B
6、estimmung von Vitamin E mit Hochleistungs-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 Stan
7、dard 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-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (Englis
8、h, 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-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bu
9、lgaria, Croatia, Cyprus, Czech Republic, 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,
10、Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management 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 Member
11、s. Ref. No. EN 12822:2014 E BS EN 12822:2014EN 12822:2014 (E) 2 Contents Page Foreword 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 chromatogr
12、ams . 14 Annex B (informative) Precision data . 16 Annex C (informative) Alternative HPLC systems 18 Bibliography . 19 BS EN 12822:2014EN 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
13、of which is held by DIN. This 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 t
14、he possibility that some of 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 Inte
15、rnal Regulations, the national 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, I
16、reland, Italy, Latvia, Lithuania, 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 purpo
17、rt to address all the safety 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. BS EN 12822:2014EN 12822:2014 (E) 4 Introduction This Euro
18、pean 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. As the method in this European Standard deals with the measurement of the mass fraction of -, -, - and -toco
19、pherol in food, reference 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. BS EN 1282
20、2:2014EN 12822:2014 (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
21、 two interlaboratory 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/1
22、00 g (-tocopherol) to 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 ar
23、e indispensable for its 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 Principl
24、e -, -, - and -tocopherol 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 reten
25、tion times and quantitative 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 infor
26、mation see 4 to 14. NOTE 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 Et
27、hanol absolute, volume 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
28、mixture of 9 parts per 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). BS EN 12822:2014EN 12822:2014 (E) 6 4.7 Solvents and extraction solvents, such a
29、s diethyl ether (peroxide 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 meth
30、yl ether in n-hexane 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 ob
31、tained from various 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 ma
32、ss fraction of at 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 l
33、east 90 %. 4.9.5 -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 define
34、d volume, e.g. 100 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.
35、 100 ml, of an appropriate 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
36、 appropriate solvent, 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 s
37、olvent, e.g. n-hexane 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. BS EN 1282
38、2:2014EN 12822:2014 (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
39、 and remove the 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.
40、Calculate the 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 methano
41、l in l x mol1x 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 29
42、2 nm 76 430,7 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 m
43、easured. The ratio 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 a
44、nd dilute to the 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 standa
45、rd solution shall 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 t
46、o the 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 each of the tocopherols. BS EN 12822:2014EN 12822:2014 (E) 8 The standard solution shall be stored protected from light and at a temper
47、ature below 4 C and should be checked as described in 4.10.5. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 UV spectrometer, capable of measuring absorbances at defined wavelengths, with appropriate cells, e.g. of 1 cm path length. 5.2 Rotary evaporator, with water ba
48、th and vacuum unit. The use of nitrogen is recommended for releasing the vacuum. 5.3 HPLC system HPLC system consisting of a pump, a sample injecting device, a fluorescence detector with an excitation wavelength set at 295 nm and an emission wavelength set at 330 nm and an evaluation system such as
49、an integrator. An UV detector may be used. The wavelength shall be set at 292 nm. In this case the standard and the sample solution should be more concentrated. In addition, the possibility of the detection of interfering compounds is increased. 5.4 HPLC column Analytical normal phase column, e.g. diameter of 4,0 mm to 4,6 mm, length of 100 mm to 250 mm, filled with silica, particle size 5 m. Other particle sizes or column
