1、 STD-BSI BS EN 32823-2-ENGL 2000 3b24bb 0853230 210 m BRITISH STANDARD Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Part 2: Measurement of p-carotene The European Standard EN 12823-22000 has the status of British Standard ICs 67.050 3s EN 12823-2:2000 I NO COPY
2、ING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYBIGFIT LAW - STD-BSI BS EN 12823-2-ENGL 2000 II 1b24bb9 0851231 157 = BS EN 12823-2:2000 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of
3、the Standards Committee and comes into effect on 15 May ZOO0 O BSI 05-2000 ISBN O 680 34206 9 National foreword This British Standard is the official English language version of EN 12823-2:2000. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food anaysis - Horiz
4、ontal methods, which has the responsibility to: - aid enquirers to understand the text; - present to the responsible European committee any enquiries on the - monitor related international and European developments and promulgate interpretation, or proposals for change, and keep the UK interests inf
5、ormed them in the UK. A list of organizations represented on this committee can be obtained on request to its secrem. Cross-references The British Standarcis which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the sect
6、ion entitled “International Standards Correspondence Index”, or by using the “Find facity of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Co
7、mpliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 13 and a back cover. The BSI copyright notice displayed in this document indicates when the documen
8、t was last issued. Amendments issued since publication Amd. No. 1 Date 1 Comments I l STD-ES1 ES EN 32823-2-ENGL 2000 I 3b24bb7 0853232 O73 I EUROPEAN STANDARD EN 12823-2 NORME EUROPEENNE EUROPISCHE NORM February 2000 ICs 67.040 English version Foodstuffs - Determination of vitamin A by high perform
9、ance liquid chromatography - Part 2: Measurement of B-carotene Produits alimentaires - Dosage de la vitamine A par chromatographie liquide haute performance - Partie 2: Dosage du bcarotne Lebensmittel - Bestimmung von Vitamin A mit Hochleistungs-Flssigchromatographie - Teil 2: Bestimmung von p-Carot
10、in This European Standard was approved by CEN on 2 January 2000. CEN members are bound to comply with the CENICENELEC Internal Regulations which stipulate the conditions for giving this Europeen Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical r
11、eferences concerning sich national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three oficial versions (English, French, German). A version in any other language made by traislation under the responsibility of a CEN member
12、 into its own language and notifed to the Central Secretariat has the same status as tie oficial 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,
13、 Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Central Secretariat: rue de Stassatt, 36 B-1050 Brussels Q 2000 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN n
14、ational Members. Ref. No. EN 12823-2:2000 E Page 2 EN 12823-2:2000 Contents Page 1 2 3 4 5 6 7 8 9 10 Foreword 2 Introduction 2 Scope . 3 Principle .3 Reagents Apparatus .5 Sampling . .5 Procedure . .5 Calculation . 7 Precision . .7 Test report 8 Annex A (informative) Example of a HPLC chromatogram
15、9 Annex B (informative) Precision data . 1 O Annex C (informative) Alternative HPLC-systems . .12 Normative references . 3 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 DI
16、N. 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, and conflicting national standards shall be withdrawn at the latest by August 2000. According to the CENEENELEC Internal Regulations
17、, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United
18、Kingdom. This European Standard, Foodstuffs - Determination of vitamin A by high performance liquid chromatography, consists of two parts: Part 1 : Measurement of all-trans-retinol and 13-cis-retinol; Part 2: Measurement of 0-carotene. This European Standard provides the base for the analytical meth
19、ods. 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 European Standard deals with the measurement of total-carotene in foodstuffs, reference is made to the literature for the calculation an
20、d expression of p-carotene as vitamin A equivalents I, 2. Vitamin A activity can be calculated from the 0-carotene data assuming appropriate factors. OBSI 05-2000 STD-BSI BS EN 12823-2-ENGL 2000 1b24bb7 0851234 bb Page 3 EN 12823-2:2000 1 Scope This European Standard specifies a method for the deter
21、mination 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 normative references are cited at the appropriate places in the text and the
22、 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 references the latest edition of the publication referred to applies. EN IS0
23、 3696 EN IS0 5555 EN 12823-1 2000 Water for analytical laboratory use - Specification and test methods (IS0 3696: 1987). Animal and vegetable fats and oils - Sampling (IS0 55551 991). Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Pati 1: Measurement of all-trans
24、-retinol and 13-cis-retinol. 3 Principle Determination of the sum of fl-carotene isomers in an appropriate sample solution by HPLC and spectrometric detection in the visible range. The extract obtained after saponification as described in EN 12823 -1 may be used for quantification. Identification on
25、 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 behaviour of the internal standard during the analysis as the analyte
26、 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 IS0 3696. 4.1 Methanol 4.2 Ethanol abs., volume fraction cp (CzH50H) = 100 %. 4.3 Ethanol, cp (C2H50H) = 96 %. 4.4 Sodium sulfate, anhydrou
27、s. 4.5 KOH solutions for saponification, in suitable concentrations, e.g. p (KOH) = 50 gll00 ml or 60 gl100 ml, or alcoholic solutions, e.g. 28 g KOH in 100 ml of an ethanollwater mixture (9+1)(V+V). 4.6 Antioxidants, such as ascorbic acid (AA), sodium ascorbate, sodium sulfide (NqS), butylated hydr
28、oxytoluene (BHT), pyrogallol or hydroquinone. 4.7 Solvents and extraction solvents such as acetonitrile, diethyl ether (peroxide-free), di-isopropylether, light petroleum (boiling range of 40 “C to 60 OC), n-hexane, dichloromethane, tetrahydrofuran, toluene or appropriate mixtures thereof. 4.8 Metha
29、nolic ammonium acetate solution, e.g. c (CH3COzNH4) = 0,05 molll. 4.9 Triethylamine 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 % by mass of butylated hydroxy toluene (4.6) and 0,0
30、5 % by mass of triethylamine (4.9). For mobile phases of alternative HPLC-systems, see annex C. OBSI 05-2000 Page 4 EN 12823-212000 4.1 1 Standard substances 4.11.1 General 0-Carotene and a-carotene can be obtained from various suppliers, e.g. Sigma“. The purity of the standards can vary between 90
31、% and 1 O0 %. It is therefore necessary to determine the concentration of the calibration solution spectrometrically (see concentration and purity test 4.12.2). 4.1 I .2 /3-Carotene, M (C40H56) = 536,85 g/mol, with a known mass content of at least 95 %. 4.1 1.3 a-Carotene, M (C40H56) = 536,85 g/mol,
32、 for qualitative purposes. 4.1 I .4 Lycopene, M (C40ii56) = 536,85 g/mol, for qualitative purposes. 4.12 Stock and standard solutions 4.12.1 /3-Carotene stock solution Dissolve approximately 3 mg, of the P-carotene standard substance (4.1 I .2) in 20 ml of dichloromethane, tetrahydrofuran or toluene
33、 (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 1 O0 ml. Dilute 10,O ml of this solution with n-hexane up to 1 O0 ml. 1 ml of this standard solution contains approximately 3 pg 0-carotene in n-hexaneldichlo
34、romethane (98+2) (VIV), n-hexaneltetrahydrofuran (98+2) (VIV); or n-hexaneltoluene (98+2) (VIV). Store the stock solution protected from light at less than 4 OC. 4.12.2 Concentration and purity test Measure the absorbance of the 0-carotene stock solution (4.12.1) at the maximum wavelength of about 4
35、53 nm using a spectrometer (5.1). Calculate the mass concentration, p , in micrograms per millilitre, using equation (1): . I o4 = 2592 where: A453 is the absorption value of the stock solution at the maximum wavelength of about 453 mm; 2592 is the .Ei Injection volume: 50 pl; Detection: 450 nm. Sph
36、eriorb)ODS2,5 pm, 100 mm x 4,6 mm cartridge combined with Vydacd) 201TP54, 5 pm, 250 mm x 4,6 mm; Acetonitrile + methanolic ammonium acetate solution + Dichloromethane (75+20+5) (volume parts) containing 0,l % by mass of butylated hydroxytoluene and 0,05 % by mass of triethylamine; $pherisorb is a p
37、roduct supplied by Phase Separations Inc; Vydad is a product supplied by The Separations Group. This infomidion is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product named. Equvalent products may be used if they can be shown to lead to t
38、he same results. OBSI 05-2000 STD-BSI BS EN 12823-2-ENGL 2000 lbZLibb7 0851238 501 = Page 7 EN 12823-2:2000 7.4 Determination Inject appropriate volumes (e.g. 20 pl) of the standard solution (4.12.3) as well as the sample test solution (see 7.2.4) into the HPLC-system (5.3). To carry out a quantitat
39、ive determination by the external standard method, integrate the peak areas or determine the peak heights obtained for sample test solutions, and compare the results with the corresponding values for the standard substance with similar retention time or construct a calibration curve. Inject equal vo
40、lumes of the sample test and of the standard solutions (4.12.3 and 4.12.4) or compensate with a corresponding factor in the calculation of the results. Check the linearity of the calibration function using a minimum of three dilution levels of the /?-carotene stock solution (4.12.1). 7.5 Number of d
41、eterminations Perform at least two independent determinations. 8 Calculation Base the calculation on a calibration graph, or use the corresponding programs of the integrator, or use the following simplified procedure. Calculate the mass concentration, p, of total-p-carotene in mg/l O0 g of the sampl
42、e using equation (2): where: As c Vs V, As, rn V, 1 O00 is the conversion factor (micrograms to milligrams); 1 O0 is the peak areas or peak heights forp-carotene-isomers obtained with the sample test solution (see 7.2.4), in units of area or height; is the purity corrected (see 4.12.2) concentration
43、 of thep-carotene in the standard solution in micrograms per millilitre; is the total volume of sample test solution (7.2.4) in millilitres; injection volume of the standard solution, in microlitres; is the peak area or peak height forp-carotene obtained with the standard solution (4.12.3), in units
44、 of area or height; is the sample mass in grams; is the injection volume of the sample test solution, in microlitres; is the conversion factor for the content per 1 O0 g. 9 Precision Details of the inter-laboratory test of the precision of the method according to IS0 5725 12 are summarized in annex
45、B. The value derived from the inter-laboratory test may not be applicable to analyte concentration ranges and matrices other than given in annex B. 9.1 Repeatability The absolute difference between two single test results found on identical test material by one operator using the same apparatus with
46、in the shortest feasible time interval will exceed the repeatability limitr in not more than 5 % of the cases. The values for total-p-carotene are: margarine X =0,253 mg/l O0 g r= 0,032 mg/l00 g vitamin drink Y =2,248 mg/l O0 g r= 0,19 mg/100g pudding powder Y =1,531 mgil00 g r= 0,24 mgll00 g mixed
47、vegetables X =I 8,05 mg/l O0 g r= 2,O mg/l00 g 9.2 Reproducibility The absolute difference between two single test results obtained on identical material reported by two laboratories will exceed the reproducibility limit R in not more than 5 % of the cases. OBSI 05-2000 Page 8 EN 12823-2:2000 The va
48、lues for total-fi-carotene are: X =0,253 mg/l O0 g R= 0,069 mg/l00 g mg/l O0 g R= 0,41 mg/100 g margarine vitamin drink pudding powder x =I ,531 mg/l O0 g R = 0,40 mg/l00 g mixed vegetables X =I 8,05 mg/l O0 g R = 7,6 mg/l00 g - x =2,248 - 10 Testreport The test report shall contain at least the fol
49、lowing data: all information necessary for the identification of the sample; a reference to this European Standard, or to the method used; the results and the units in which the results have been expressed; the date and type of sampling procedure (if known); the date of receipt; the date of test; any particular points observed in the course of the test; any operations not specified in the method or regarded as optional which might have affected the results. OBSI 05-2000 STDmBSI BS EN 12823-2-ENGL 2000 Lb29bbS 0851290 1bT Page 9 EN 12823-212000
