EN-12823-2-2000.pdf
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1、EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12823-2 February 2000 ICS 67.040 English version Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Part 2: Measurement of ?-carotene Produits alimentaires - Dosage de la vitamine A par chromatographie liquide haut
2、e performance - Partie 2: Dosage du ?-carotne Lebensmittel - Bestimmung von Vitamin A mit Hochleistungs-Flssigchromatographie - Teil 2: Bestimmung von ?-Carotin This European Standard was approved by CEN on 2 January 2000. CEN members are bound to comply with the CEN/CENELEC Internal Regulations whi
3、ch 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 Central Secretariat or to any CEN member. This European Sta
4、ndard 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 Central Secretariat has the same status as the official versions. CEN members are the national sta
5、ndards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE F
6、R NORMUNG Central Secretariat: rue de Stassart, 36 B-1050 Brussels 2000 CENAll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12823-2:2000 E -,-,- Page 2 EN 12823-2:2000 Contents Page Foreword .2 Introduction2 1Scope 3 2Normative referenc
7、es3 3Principle.3 4Reagents .3 5Apparatus 5 6Sampling5 7Procedure6 8Calculation.7 9Precision7 10Test report.8 Annex A (informative) Example of a HPLC chromatogramme.9 Annex B (informative) Precision Data.10 Annex C (informative) Alternative HPLC-Systems.12 Bibliography.13 Foreword This European Stand
8、ard 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 by publication of an identical text or by endorsement, at the latest by August 2000,
9、and conflicting national standards shall be withdrawn at the latest by August 2000. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, Fr
10、ance, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. This European Standard “Foodstuffs - Determination of vitamin A by high performance liquid chromatography“ consists of two parts: Part 1: Measurement of all-t
11、rans-retinol and 13-cis-retinol. Part 2: Measurement of ?-carotene. This European Standard provide 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. Introduction As this draft Europ
12、ean Standard deals with the measurement of total-?-carotene in foodstuffs, reference is made to the literature for the calculation and expression of?-carotene as vitamin A equivalents 1, 2. Vitamin A activity can be calculated from the ?-carotene data assuming appropriate factors. -,-,- Page 3 EN 12
13、823-2:2000 1 Scope This European Standard specifies a method for the determination of total-?-carotene in foodstuffs by high performance liquid chromatography (HPLC). 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These n
14、ormative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated r
15、eferences the latest edition of the publication referred to applies. EN ISO 3696 Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) EN ISO 5555 Animal and vegetable fats and oils Sampling (ISO 5555:1991) EN 12823-1 : 2000Foodstuffs - Determination of vitamin A by hi
16、gh performance liquid chromatography - Part 1: Measurement of all-trans-retinol and 13-cis-retinol 3 Principle Determination of the sum of ?-carotene isomers in an appropriate sample solution by HPLC and spectrometric detection in the visible range. The extract obtained after saponification as descr
17、ibed in EN 12823 -1 may be used for quantification. Identification on the basis of the retention times, and determination by the external standard method using peak areas or peak heights, see 3 to 7. Internal standard methods may also be used if the corresponding recovery tests have proven the same
18、behaviour of the internal standard during the analysis as the analyte itself. 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 4.2Ethanol abs., volume fraction ? (C2H5OH) = 1
19、00% 4.3Ethanol, ? (C2H5OH) = 96% 4.4 Sodium sulfate, anhydrous 4.5 KOH solutions for saponification, in suitable 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 an ethanol/water mixture (9+1)(V+V) 4.6Antioxidants, such as ascorbic acid (A
20、A), sodium ascorbate, sodium sulfide (Na2S), butylated hydroxytoluene (BHT), pyrogallol or hydroquinone. 4.7Solvents and extraction solvents such as acetonitrile, diethyl ether (peroxide-free), di-isopropylether, light petroleum (boiling range of 40 C to 60 C), n-hexane, dichloromethane, tetrahydrof
21、uran, toluene or appropriate mixtures thereof. 4.8Methanolic ammonium acetate solution, e.g. c (CH3CO2NH4) = 0,05 mol/l 4.9Triethylamine 4.10 HPLC mobile phase, for example acetonitrile (4.7) + methanolic ammonium acetate solution (4.8) + dichloromethane (4.7) (75+20+5) (volume parts) containing 0,1
22、 percent by mass of butylated hydroxy toluene (4.6) and 0,05 percent by mass of triethylamine (4.9). For mobile phases of alternative HPLC-systems see Annex C. -,-,- Page 4 EN 12823-2:2000 4.11 Standard substances 4.11.1 General ?-Carotene and ?-carotene can be obtained from various suppliers, e.g.
23、Sigma1). The purity of the standards can vary between 90 % and 100 %. It is therefore necessary to determine the concentration of the calibration solution spectrometrically (see concentration and purity test (4.12.2). 4.11.2 ?-Carotene, M (C40H56) = 536,85 g/mol, with a known mass content of at leas
24、t 95 %. 4.11.3 ? ?-Carotene, M (C40H56) = 536,85 g/mol, for qualitative purposes. 4.11.4 Lycopene, M (C40H56) = 536,85 g/mol, for qualitative purposes. 4.12 Stock and standard solutions 4.12.1 ?-Carotene stock solution Dissolve approximately 3 mg, of the ?-carotene standard substance (4.11.2) in 20
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