1、August 2014 Translation by DIN-Sprachendienst.English price group 11No 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!%:;$“2232401www.din.deDDIN EN 12823-1Foodstuffs Determination of vitamin A by high performance liquidchromatography Part 1: Measurement of all-E-retinol and 13-Z-retinol;English version EN 12823-1:2014,English translation of DIN EN 12823-1:2014-08Lebensmittel Bestimmung von Vitamin A mit Hoc
3、hleistungs-Flssigchromatographie Teil 1: Bestimmung von all-E-Retinol und 13-Z-Retinol;Englische Fassung EN 12823-1:2014,Englische bersetzung von DIN EN 12823-1:2014-08Produits alimentaires Dtermination de la teneur en vitamine A par chromatographie liquide hauteperformance Partie 1: Dosage du tout-
4、E-rtinol et du 13-Z-rtinol;Version anglaise EN 12823-1:2014,Traduction anglaise de DIN EN 12823-1:2014-08SupersedesDIN EN 12823-1:2000-07www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 18 pages07.14 DIN EN 12823-1:2014-08 2 A comma is u
5、sed as the decimal marker. National foreword This document (EN 12823-1:2014) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtsc
6、haftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-01-13 AA Vitamine und Carotinoide. This document uses the designation “all-E-Retinol” instead of “all-trans-Retinol” and “13-Z-Retinol” instead of “13-cis-Retinol”. Amendments This standard differs
7、from DIN EN 12823-1:2000-07 as follows: a) the scope of the standard has been extended; b) the standard has been editorially revised. Previous editions DIN EN 12823-1: 2000-07 EN 12823-1May 2014 ICS 67.050 Supersedes EN 12823-1:2000English Version Foodstuffs - Determination of vitamin A by high perf
8、ormance liquid chromatography - Part 1: Measurement of all-E-retinol and 13-Z-retinol Produits alimentaires - Dtermination de la teneur en vitamine A par chromatographie liquide haute performance - Partie 1: Dosage du tout-E-rtinol et du 13-Z-rtinol Lebensmittel - Bestimmung von Vitamin A mit Hochle
9、istungs-Flssigchromatographie - Teil 1: Bestimmung von all-E-Retinol und 13-Z-Retinol This European Standard was approved by CEN on 24 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
10、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 (English, French, German).
11、 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, Bulgaria, Croatia, Cy
12、prus, 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, Turkey andUnited Ki
13、ngdom. 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 Members. Ref. No. EN 12823-1:2014 EEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR N
14、ORMUNGEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 12823-1:2014 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents .4 5 Apparatus .7 6 Procedure .8 7 Calculation . 10 8 Precision 11 9 Test report . 12 Annex A (informative) Examples of HPLC chromatograms .
15、 13 Annex B (informative) Precision data . 14 Annex C (informative) Alternative HPLC systems 15 Bibliography . 16 DIN EN 12823-1:2014-08EN 12823-1:2014 (E) 3 Foreword This document (EN 12823-1:2014) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secreta
16、riat 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 November 2014, and conflicting national standards shall be withdrawn at the latest by November 2014. Attention is draw
17、n to the 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 12823-1:2000. This European Standard consists of two parts: Part 1: Measure
18、ment of all-E-retinol and 13-Z-retinol; Part 2: Measurements of -carotene. 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. According to the CEN-CEN
19、ELEC Internal 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, I
20、celand, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. DIN EN 12823-1:2014-08EN 12823-1:2014 (E) 4 1 Scope This European Standard specifies a method for the determina
21、tion of vitamin A in foodstuffs by high performance liquid chromatography (HPLC). This method has been validated in an interlaboratory study with samples of margarine and milk powder with all-E-retinol levels ranging from 653 g/100 g to 729 g/100 g and with 13-Z-retinol levels ranging from 30 g/100
22、g to 39 g/100 g. The determination of vitamin A content is carried out by the measurement of all-E-retinol, 13-Z-retinol and -carotene. This part covers the measurement of all-E-retinol and 13-Z-retinol. The extract obtained after saponification in this method can be used for the determination of -c
23、arotene, as described in EN 12823-2:2000, Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Part 2: Measurements of -carotene. In this case, the saponification temperature should preferably not exceed 80 C in order to prevent isomerisation and oxidation of -carotene
24、. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any ame
25、ndments) applies. EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696) 3 Principle Retinol is saponified by using methanolic or ethanolic potassium hydroxide solution and extracted by an appropriate solvent. The determination is carried out by high performance l
26、iquid chromatography (HPLC) with either fluorometric (F) or ultraviolet (UV) detection. The substances are identified on the basis of the retention times and determined by the external standard procedure using peak areas or heights, see 1 to 4. 4 Reagents During the analysis, unless otherwise stated
27、, 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, volume fraction, (C2H5OH) = 100 %. 4.3 Ethanol, (C2H5OH) = 96 %. 4.4 Sodium sulfate, anhydrous. 4.5 KOH solution for saponification, in suitable mass concent
28、rations, e.g. (KOH) = 50 g/100 ml or 60 g/100 ml, or alcoholic solutions, e.g. 28 g KOH in 100 ml of a 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, sodium sulfide (Na2S), butylated hydroxytoluene (BHT), pyrog
29、allol or hydroquinone. 4.7 Solvents and extraction solvents, such as diethyl ether (peroxide-free), di-isopropylether, light petroleum (boiling range of 40 C to 60 C), n-hexane, butanol, iso-octane or appropriate mixtures thereof. DIN EN 12823-1:2014-08EN 12823-1:2014 (E) 5 4.8 HPLC phases Examples
30、of appropriate mixtures (expressed as volume parts) include: n-hexane + 2-propanol (98 + 2); iso-octane + 2-propanol (98,5 + 1,5); iso-octane+ iso-butanol (98 + 2); n-hexane + n-butanol (98 + 2); and gradient with 2-propanol + n-heptane, (0,5 + 99,5) to (8,5 + 91,5) in 12 min. 4.9 Standard substance
31、s 4.9.1 General All-E-retinol (all-E vitamin A alcohol) and 13-Z-retinol can be obtained in several forms, and from different suppliers. It is therefore necessary to determine the concentration of the calibration solution spectrometrically (see 4.10.4). If vitamin A esters are used (e.g. retinyl pal
32、mitate or acetate), check the concentration after saponification (see 6.3.1). Vitamin A and its derivatives are sensitive to oxygen and light. Standard substances should be stored in the dark under nitrogen or argon at 20 C. Particular attention should be given to the information on the vitamin A co
33、ntent of the standard substances supplied by different manufacturers. 4.9.2 All-E-retinol, vitamin A alcohol, M (C20H30O) = 286,5 g/mol, with a purity of at least 90 %. 4.9.3 Vitamin A esters. 4.9.3.1 Retinyl palmitate, vitamin A palmitate, M(C36H60O2) = 524,9 g/mol. 4.9.3.2 Retinyl acetate, vitamin
34、 A acetate, M(C22H32O2) = 328,5 g/mol, with a purity of at least 90 %. 4.9.4 13-Z-retinol, M(C20H30O) = 286,5 g/mol with a purity of at least 60 % for qualitative purposes. 4.10 Stock and standard solutions 4.10.1 All-E-retinol stock solution Weigh out approximately 50 mg of all-E-retinol (4.9.2) to
35、 the nearest milligram into a 100 ml one-mark volumetric flask, dissolve in n-hexane or other suitable solvents (4.7), and dilute the solution to the mark. The stock solution contains approximately 0,5 mg/ml. Alternatively, weigh out approximately 100 mg of retinyl palmitate (4.9.3.1), or 50 mg of r
36、etinyl acetate (4.9.3.2) to the nearest milligram into a 100 ml one-mark volumetric flask, and dilute the solution to the mark. The stock solution concentrations calculated as retinol are approximately 0,55 mg/ml and 0,44 mg/ml, respectively. Alternative masses and volumes may be used according to c
37、hromatographic separation and quantification. Store the stock solution protected from light at approximately 20 C. A maximum storage time should be defined based on stability tests carried out by the user under designated conditions. DIN EN 12823-1:2014-08EN 12823-1:2014 (E) 6 4.10.2 13-Z-retinol st
38、ock solution Weigh out approximately 1 mg to 2 mg of 13-Z-retinol (4.9.4) to the nearest 0,1 mg into a 100 ml one-mark volumetric flask, dissolve it in absolute ethanol (4.2), or other suitable solvents, and dilute the solution to the mark. This solution contains approximately 10 g/ml to 20 g/ml and
39、 is used for identification purposes only. 4.10.3 All-E-retinol standard solution Pipette 5 ml of the all-E-retinol stock solution (4.10.1) into a 100 ml one-mark volumetric flask and dilute to the mark with n-hexane (4.7) or other suitable solvents compatible with the mobile phase. Pipette 5 ml of
40、this solution into a 50 ml one-mark volumetric flask, and dilute to the mark with the same solvent. The standard solution contains approximately 2,5 g/ml. Then carry out a concentration and purity test as described in 4.10.4. Alternatively, retinyl palmitate or retinyl acetate stock solutions (4.10.
41、1) may be used for the preparation of the standard solution. In that case, saponify an aliquot of the stock solution using the conditions described in 6.3.1. After extraction and evaporation, redissolve the residue in n-hexane or other suitable solvent and carry out a concentration test as described
42、 in 4.10.4. Protect the standard solution from light and store at a temperature of below 4 C. A maximum storage time should be defined based on stability tests carried out by the user under designated conditions. 4.10.4 Concentration and purity test Prepare a standard solution of all-E-retinol in et
43、hanol and measure the absorbance in a quartz cell having an optical path length of 1 cm at the maximum wavelengths of 325 nm to 326 nm with ethanol in the reference cell. Calculate the mass concentration, all-E, in microgram per millilitre, of all-E-retinol using Formula (1): P10MA3E-allE-all=EallEa
44、ll (1) Calculate the mass concentration,13-Z, in microgram per millilitre, of 13-Z-retinol using Formula (2): P10MAZ-13313Z-13Z-13=Z(2) where Aall-Eis the absorption value at the maximum at a wavelength of 325 nm to 326 nm; Mall-Eis the molar mass (286,5 g/mol) of all-E-retinol; all-Eis the molar ex
45、tinction coefficient (52 400) for all-E-retinol dissolved in ethanol, calculated from an 1%1cmE value of 1 830 5, and rounded to 3 significant digits. It may change significantly with other solvents; A13-Zis the absorption value at the maximum at a wavelength of 328 nm; M13-Zis the molar mass (286,5
46、 g/mol) of 13-Z-retinol; 13-Zis the molar extinction coefficient (48 300) for 13-Z-retinol dissolved in ethanol, calculated from an 1%1cmE value of 1 686 5, and rounded to 3 significant digits. It may change significantly with other solvents; P is the correction factor for purity of all-E-retinol or
47、 13-Z-retinol assessed by HPLC and calculated using Formula (3): DIN EN 12823-1:2014-08EN 12823-1:2014 (E) 7 totalBBP = (3) where B is the peak area or height for all-E-retinol or 13-Z-retinol obtained with the standard solution (4.10.3); Btotalis the sum of peak areas or heights for all-E-retinol o
48、r 13-Z-retinol obtained with the standard solution (4.10.3). When using newly purchased vitamin A standard substances, or ones that have been stored for a prolonged period, check whether the absorption maximum of the all-E-retinol standard solution (4.10.3) used is between 325 nm and 326 nm using a
49、suitable spectrometer. For further checks on the vitamin A standards, measure the absorbance of the standard solution in quartz cells (5.1) at wavelengths of 300 nm, 325 nm, 350 nm and 370 nm, with 2-propanol (or other suitable solvents, see 4.7) in the reference path. Determine the following ratio at each wavelength: 325EEfor all-E-retinol If the ratio does not exceed 0,602 (300 nm), 0,452 (350 nm) and 0,093 (370 nm) for vit
