EN-12823-1-2000.pdf
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1、EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 12823-1 February 2000 ICS 67.040 English version Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Part 1: Measurement of all-trans-retinol and 13-cis-retinol Produits alimentaires - Dosage de la vitamine A par ch
2、romatographie liquide haute performance - Partie 1: Dosage du tout-trans-rtinol et du 13-cis-rtinol Lebensmittel - Bestimmung von Vitamin A mit Hochleistungs-Flssigchromatographie - Teil 1: Bestimmung von all-trans-Retinol und 13-cis-Retinol This European Standard was approved by CEN on 2 January 20
3、00. 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 without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained
4、on application to the Central Secretariat or to any CEN member. This 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 Central Secretari
5、at has the same status as the official versions. CEN members are the national standards 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 C
6、OMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR 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 12823-1:2000 E Page 2 EN 12823-
7、1:2000 Contents Foreword 2 1Scope 3 2Normative references 3 3Principle. 3 4Reagents . 3 5Apparatus 5 6Sampling 6 7Procedure 6 8Calculation. 8 9Precision 9 10Test report. 10 Annex A (informative) Examples of HPLC chromatograms 11 Annex B (informative) Precision Data. 12 Annex C (informative) Alternat
8、ive HPLC systems 13 Bibliography. 13 Foreword This European Standard 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
9、 identical text or by endorsement, at the latest by August 2000, 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
10、 Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, 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 ch
11、romatography“ consists of two parts: Part 1: Measurement of all-trans-retinol 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. -,-,- Page 4 EN 12823-1:2000 4.9 Stan
12、dard substances 4.9.1 General All-trans-retinol (all-trans vitamin A alcohol) and 13-cis-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 ar
13、e used (e.g. retinyl palmitate or acetate), check the concentration after saponification (see 7.3.1). NOTE: Particular attention should be given to the information on the vitamin A content of the standard substances supplied by different manufacturers. 4.9.2 All-trans-retinol, vitamin A alcohol, M (
14、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 A acetate, M (C22H32O2) = 328,5 g/mol, with a purity of at least 90%. 4.9.4 13-cis-retinol, M (C20H30O) = 286,5 g
15、/mol with a purity of at least 60% for qualitative purposes. 4.10 Stock and standard solutions 4.10.1 All-trans-retinol stock solution Weigh out approximately 50 mg of all-trans-retinol (4.9.2) to the nearest milligram into a 100 ml one-mark volumetric flask, dissolve in n-hexane or other suitable s
16、olvents (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 retinyl acetate (4.9.3.2) to the nearest milligram into a 100 ml one-mark volumetric flask, and treat as
17、for all-trans-retinol except the concentration should be determined (4.10.4) after saponification (7.3.1). Alternative masses and volumes may be used according to chromatographic separation and quantification. Store the stock solution protected from light at less than 4 ?C. 4.10.2 13-cis-retinol sto
18、ck solution Weigh out approximately 1 mg to 2 mg of 13-cis-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 an
19、d is used for identification purposes only. 4.10.3 All-trans-retinol standard solution Pipette 5 ml of the all-trans-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
20、 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 palmiate or retinyl acetate stock solution
21、s (4.10.1) may be used for the preparation of the standard solution. In that case, saponify the solutions using the conditions described in 7.3.1. After extraction and evaporation, redissolve the residue in n-hexane or other suitable solvent and proceed as for all-trans-retinol stock solution. Prote
22、ct the standard solution from light and store at a temperature of below 4 ?C. 4.10.4 Concentration and purity test Prepare a standard solution of all-trans-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
23、 nm with ethanol in the reference cell. Calculate the mass concentration, ?all-T , in microgram per millilitre, of all-trans-retinol using equation 1: -,-,- Page 5 EN 12823-1:2000 P A Tall Tall ? ? ? ? ? 1830 104 ?(1) Calculate the mass concentration, ?13cis , in microgram per millilitre, of 13-cis-
24、retinol using equation 2: P A cis cis ? ? ? 1686 104 13 13 ?(2) where: Aall-Tis the absorption value at the maximum at a wavelength of 325 nm to 326 nm; A13cisis the absorption value at the maximum at a wavelength of 328 nm; 1830 is the 1% 1cm Evalue for all-trans-retinol solved in ethanol. It may c
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