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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
25、ml of dichloromethane, tetrahydrofuran or toluene (4.7), placing the volumetric flask for approximately 30 s in an ultrasonic bath (5.6). Dilute this solution with n-hexane up to a volume of 100 ml. Dilute 10,0 ml of this solution with n-hexane up to 100 ml. 1 ml of this standard solution contains a
26、pproximately 3 ?g ?-carotene in n-hexane/dichloromethane (98+2) (V/V), n-hexane/tetrahydrofuran (98+2) (V/V); or n-hexane/toluene (98+2) (V/V). Store the stock solution protected from light at less than 4?C. 4.12.2 Concentration and purity test Measure the absorbance of the ?-carotene stock solution
27、 (4.12.1) at the maximum wavelength of about 453 nm using a spectrometer (5.1). Calculate the mass concentration, ? , in micrograms per millilitre, using equation (1): 2592 104 453? ? A ? (1) where: A453is the absorption value of the stock solution at the maximum wavelength of about 453 mm; 2592is t
28、he 1% 1cm E value of ?-carotene in n-hexane. It may change considerably with the composition of the solvent 8. The ratio of A455/A340 should be greater than 15, and the ratio A455/A483 should be in range 1,14 to 1,18 for pure, all-trans-?-carotene 8. 4.12.3 Standard solution of ?-carotene Pipette 20
29、 ml of the ?-carotene stock solution (4.12.1) into a round-bottomed flask and remove the solvent under reduced pressure (5.2) at not more than 50? C. Dissolve the residue in 20 ml of a solvent compatible to the reversed phase HPLC. The standard solution shall be stored protected from light and at a
30、temperature below 4 C and is usually stable for up to 1 week. 1) This information is given for the convenience of users of this standard method and does not constitute an endorsement by CEN of the supplier named. Equivalent products may be used if they can be shown to lead to the same results. -,-,-
31、 Page 5 EN 12823-2:2000 4.12.4 Standard solutions of ? ?-carotene and lycopene2 For qualitative purposes, pre-dissolve approximately 0,3 mg of ?-carotene (4.11.3) or lycopene (4.11.4) in approximately 10 ml of tetrahydrofuran (4.7) and dilute to a volume of 100 ml with ethanol (4.3) or another solve
32、nt compatible to the HPLC-system. The standard solution shall be stored protected from light and at a temperature below 4 C and is usually stable for up to 1 week. 5 Apparatus Usual laboratory apparatus and, in particular, the following: 5.1 UV-VIS Spectrometer, capable of measuring absorbance at de
33、fined wavelengths, with appropriate quartz cells, e. g. of 1 cm path length. 5.2 Rotary evaporator, with water bath and vacuum unit. NOTE: The use of nitrogen is recommended for releasing the vacuum. 5.3 HPLC system, consisting of a pump, a sample injecting device, a UV-VIS detector and an evaluatio
34、n system such as an integrator or recorder. 5.4 HPLC column Analytical reversed phase column, e.g. C18 reversed phase, particle size 5 ?m, diameter 4,0 mm to 4,6 mm, length 250 mm. Column types and particle sizes other than specified in this European standard may be used. Chromatographic conditions
35、may have to be adapted for such materials to guarantee equivalent results. The performance criterion for suitable analytical columns is the resolution factor for all-trans-?-carotene and all- trans-?-carotene which should be greater than 1. Suitable RP column packing materials are e.g. Vydac 201TP54
36、3), Vydac 218TP54 3), Eurospher?100-C183), Ultraspher ODS3), Spherisorb ODS23), Zorbax ODS3) and LiChrospher RP 183).The columns may also be used in series. It is also advisable to use a guard column to increase longevity of the analytical column. 5.5 Filter device Large and small scale filter devic
37、es to filter HPLC mobile phases and sample solutions respectively, e.g. of 0,45 m pore size is appropriate. NOTE: Filtering of the mobile phase as well as of the sample test solution through a membrane filter prior to use or injection usually increases longevity of the columns. 5.6 Ultrasonic bath 5
38、.7 Phase separation filter (optional) 6 Sampling Sampling shall be in accordance with EN ISO 5555, if appropriate. 2) The standard solutions of ?-carotene and lycopene are not necessary for the quantification of the ?-carotene in the sample extract but help to identify clearly the different compound
39、s. 3) Vydac 201TP54, Vydac 218TP54, Eurospher? 100-C18, Ultrasphere ODS, Spherisorb ODS2, Zorbax ODS and LiChrospher RP18 are available examples of suitable products available commercially. This information is given for the convenience of users of this standard method and does not constitute an endo
40、rsement by CEN of these products. -,-,- Page 6 EN 12823-2:2000 7 Procedure 7.1 Preparation of the test sample Homogenize the test sample. Grind coarse material with an appropriate mill and mix again. Measures such as pre- cooling have to be taken to avoid exposing the sample to high temperatures for
41、 long periods of time. ?-carotene is sensitive to UV radiation and light. 7.2 Preparation of the sample test solution 7.2.1 Saponification Saponify 2 g to 10 g of the test sample by refluxing preferably under nitrogen using suitable amounts of ethanol (4.3) or methanol (4.1), water, an antioxidant (
42、4.6) and one of the potassium hydroxide solutions (4.5.1). Add the antioxidants to the sample prior to the addition of the potassium hydroxide. Sodium sulfide (4.6) may also be added to obviate the oxidative catalytic effects of trace metals. Examples of suitable ratios of reagents are given in tabl
43、e 1: Table 1 Sample mass AlcoholAntioxidantPotassium hydroxide 2 g to 5 g50 ml methanol0,25 g AA5 ml of a 50 g/100 ml solution 5 g to 10 g100 ml ethanol1,0 g AA + 0,04 g Na2S20 ml of a 60 g/100 ml solution 10 g150 ml ethanol1,0 g AA50 ml of a 60g/100 ml solution Typical saponification times range fr
44、om 15 min to 40 min at temperatures of 80 C to 100 C (reflux). If after saponification and cooling, fat or oil is present on the surface of the saponification mixture, additional ethanolic potassium hydroxide has to be added and saponification time extended. NOTE: It has been shown that low fat samp
45、les such as fruits or vegetables can be extracted directly with a suitable solvent with a corresponding method e.g. as described in 9 to 11. However it is advisable to check that the chromatographic separation fulfils the above defined criteria (problem of interference). 7.2.2 Extraction In order to
46、 avoid emulsions, an amount of water has to be added to the saponified sample solution so that the ratio of alcohol to water in the resulting solution is 1:1. Extract the ?-carotene from the saponified sample solution by means of a suitable solvent or solvent mixture (4.7). Repeat the extraction pro
47、cedure 3 to 4 times with volumes ranging from 50 ml to 150 ml. Wash the combined extracts to neutral with water (typically 2 to 4 times 50 ml to 150 ml). 7.2.3 Evaporation Evaporate the extract using a rotary evaporator (5.2) under partial vacuum and at a temperature not exceeding 50 C. Remove trace
48、s of water by drying with sodium sulfate or by azeotropic distillation with abs. ethanol (4.2) or toluene (4.6). Other equivalent techniques such as phase separation filter paper (5.7) to eliminate traces of water may be used provided they have been proven not to affect the result. 7.2.4 Dilution Re
49、-dissolve the residue in the same solvent mixture in which the standard solutions (4.12.3 and 4.12.4) has been prepared, preferentially the mobile phase or another HPLC - compatible solvent in such a way to obtain a concentration of up to 5 ?g/ml of ?-carotene. This is the sample test solution. 7.3 Identification Identify ? -carotene by