1、BSI Standards PublicationBS EN 12823-1:2014Foodstuffs Determination of vitamin A by high performance liquid chromatographyPart 1: Measurement of all-E-retinol and 13-Z-retinolBS EN 12823-1:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 12823-1:2014. It sup
2、ersedes BS EN 12823-1:2000 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods.A list of organizations represented on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
3、 include all 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 77938 1ICS 67.050Compliance with a British Standard cannot confer immunity from legal obligations.This
4、British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 May 2014.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS EN 12823-1:2014EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12823-1 May 2014 ICS 67.050 Supersedes EN
5、12823-1:2000English Version Foodstuffs - Determination of vitamin A by high performance 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 to
6、ut-E-rtinol et du 13-Z-rtinol Lebensmittel - Bestimmung von Vitamin A mit Hochleistungs-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
7、 which stipulate the conditions for giving 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-CENELEC Management Centre or to any CEN member. Thi
8、s European Standard exists in three official versions (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-CENELEC Management Centre has the same status as the official versions. CEN memb
9、ers are the national standards bodies of Austria, Belgium, Bulgaria, 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, Portug
10、al, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, 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 for
11、m and by any means reserved worldwide for CEN national Members. Ref. No. EN 12823-1:2014 EBS EN 12823-1:2014EN 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
12、 Annex A (informative) Examples of HPLC chromatograms . 13 Annex B (informative) Precision data . 14 Annex C (informative) Alternative HPLC systems 15 Bibliography . 16 BS EN 12823-1:2014EN 12823-1:2014 (E) 3 Foreword This document (EN 12823-1:2014) has been prepared by Technical Committee CEN/TC 27
13、5 “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 by publication of an identical text or by endorsement, at the latest by November 2014, and conflicting national standards shall be withdraw
14、n at the latest by November 2014. Attention is drawn 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 Europ
15、ean Standard consists of two parts: Part 1: Measurement 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
16、to the standard procedure. According to the CEN-CENELEC 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 Republi
17、c 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 the United Kingdom. BS EN 12823-1:2014EN 12823-1:2014 (E) 4 1 Scope This Europea
18、n Standard specifies a method for the determination 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
19、with 13-Z-retinol levels ranging from 30 g/100 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
20、 method can be used for the determination of -carotene, 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 p
21、revent isomerisation and oxidation of -carotene. 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 editio
22、n of the referenced document (including any amendments) 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 det
23、ermination is carried out by high performance liquid 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 Reagen
24、ts 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, volume fraction, (C2H5OH) = 100 %. 4.3 Ethanol, (C2H5OH) = 96 %. 4.4 Sodium sulfate, anhydrous. 4.5 KOH soluti
25、on for saponification, in suitable mass concentrations, 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 sulfid
26、e (Na2S), butylated hydroxytoluene (BHT), pyrogallol 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. BS EN 12823-1:2014EN 1
27、2823-1:2014 (E) 5 4.8 HPLC phases Examples 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,
28、5 + 91,5) in 12 min. 4.9 Standard substances 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
29、vitamin A esters are used (e.g. retinyl palmitate 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 g
30、iven to the information on the vitamin A content 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) = 52
31、4,9 g/mol. 4.9.3.2 Retinyl acetate, vitamin 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 appr
32、oximately 50 mg of all-E-retinol (4.9.2) to 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
33、 retinyl palmitate (4.9.3.1), or 50 mg of retinyl 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 ma
34、sses and volumes may be used according to chromatographic 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. BS EN 12823-1:2014EN 12
35、823-1:2014 (E) 6 4.10.2 13-Z-retinol stock 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 contai
36、ns approximately 10 g/ml to 20 g/ml and 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
37、with the mobile phase. Pipette 5 ml of 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
38、 retinyl acetate stock solutions (4.10.1) 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 carr
39、y out a concentration test as described 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 s
40、tandard solution of all-E-retinol in ethanol 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 usi
41、ng Formula (1): P10MA3E-allE-all=EallEall (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)
42、 of all-E-retinol; all-Eis the molar extinction 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 o
43、f 328 nm; M13-Zis the molar mass (286,5 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 correctio
44、n factor for purity of all-E-retinol or 13-Z-retinol assessed by HPLC and calculated using Formula (3): BS EN 12823-1:2014EN 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 a
45、reas or heights for all-E-retinol or 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
46、 between 325 nm and 326 nm using a 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 p
47、ath. 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 vitamin A alcohol, the standard substance is suitable for use 6, 7. For retinyl palmitate (4.9.3.1), determine the ratio of E/E326at wavele
48、ngths of 300 nm, 350 nm and 370 nm with 2-propanol (or other suitable solvents) in the reference path. If the ratio does not exceed 0,593 (300 nm), 0,537 (350 nm) and 0,142 (370 nm), the standard substance is suitable for use 6, 7, 8. 5 Apparatus Usual laboratory apparatus and, in particular, the fo
49、llowing: 5.1 UV-VIS spectrometer, capable of measuring absorbance at defined wavelengths, with appropriate quartz cells, e.g. of 1 cm path length. 5.2 Rotary evaporator, with water bath and vacuum unit. The use of nitrogen is recommended for releasing of the vacuum. 5.3 HPLC system, consisting of a pump, sample injection device, a UV-VIS detector or a fluorescence detector and data integrator/processing device. 5.4 HPLC columns Suitable analytical normal phase columns are appropriate such as LiChrospherSi
