~ ~ STD BSI BS EN 32823 2 ENGL 2000 3b24bbî 0853230 210 m BRITISH STANDARD Foodstuffs Determination of vitamin A by high performance liquid chromatography Part 2 Measurement of p carotene The European[.]
~ ~ S T D - B S I BS EN 32823-2-ENGL 2000 b b b ỵ 0853230 210 m 3s EN BRITISH STANDARD 12823-2:2000 ```,-`-`,,`,,`,`,,` - 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 I NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYBIGFIT LAW Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale -~ ~ ~ ~~~ S T D - B S I BS EN 12823-2-ENGL 2000 II b b b 0851231 157 = BS EN 12823-2:2000 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 anaìysis - Horizontal methods, which has the responsibility to: - aid enquirers to understand the text; - present to the responsible European committee any enquiries on the interpretation,or proposals for change, and keep the UK interests informed - monitor related international and European developments and promulgate 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 section entitled “International Standards Correspondence Index”, or by using the “Findfaciïty of the BSI StandardsElectronic Catalogue A British Standard does not purport to include all the necessary provisions of a contract Users of British Standardsare responsible for their correct application Compliance 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 to 13 and a back cover The BSI copyright notice displayed in this document indicates when the document was last issued This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 May ZOO0 O BSI 05-2000 Amendments issued since publication Amd No Date Comments I l ISBN O 680 34206 ```,-`-`,,`,,`,`,,` - Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale ~ S T D - E S ES EN 32823-2-ENGL 2000 I 3b24bb7 0853232 O73 I EN 12823-2 EUROPEANSTANDARD NORME EUROPEENNE EUROPÄISCHE NORM February 2000 ICs 67.040 ```,-`-`,,`,,`,`,,` - English version Foodstuffs - Determination of vitamin A by high performance liquid chromatography - Part 2: Measurement of B-carotene Produits alimentaires - Dosage de la vitamine A par chromatographie liquide haute performance Partie 2: Dosage du bcarotène Lebensmittel - Bestimmung von Vitamin A mit Hochleistungs-Flüssigchromatographie- Teil 2: Bestimmung von p-Carotin - This European Standard was approved by CEN on 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 references 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 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, Spain, Sweden, Switzerland and United Kingdom EUROPEAN COMMITEE FOR STANDARDIZATION C O M I T É EUROPÉEN D E NORMALISATION E U R O P Ä I S C H E S KOMITEE F Ü R N O R M U N G Central Secretariat: rue de Stassatt, 36 Q 2000 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale B-1050 Brussels Ref No EN 12823-2:2000 E Page EN 12823-2:2000 Contents Page Foreword Introduction Scope Normative references 3 Principle Reagents Apparatus Sampling .5 Procedure Calculation Precision .7 10 Test report Annex A (informative) Example of a HPLC chromatogram Annex B (informative) Precision data Annex C (informative) Alternative HPLC-systems 1O 12 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 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, 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 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 Introduction As this draft European Standard deals with the measurement of total-@-carotenein foodstuffs, reference is made to the literature for the calculation and 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 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale ```,-`-`,,`,,`,`,,` - 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 to the standard procedure ~ ~ S T D - B S I BS E N 12823-2-ENGL 0 b b b 0851234 ï b b Page EN 12823-2:2000 Scope This European Standard specifies a method for the determination of total$-carotene in foodstuffs by high performance liquid chromatography (HPLC) 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 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 3696 Water for analytical laboratory use - Specification and test methods (IS0 3696: 1987) EN IS0 5555 Animal and vegetable fats and oils Sampling (IS0 55551991) EN 12823-12000 Foodstuffs - Determination of vitamin A by high performance liquid chromatography Pati 1: Measurement of all-trans-retinol and 13-cis-retinol - - 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 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 itself Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade according to EN I S 3696 4.1 Methanol 4.2 Ethanol abs., volume fraction cp (CzH50H)= 100 % 4.3 Ethanol, cp (C2H50H)= 96 % 4.4 Sodium sulfate, anhydrous 4.5 KOH solutions for saponification, in suitable concentrations, e.g p (KOH) = 50 g l l 0 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 hydroxytoluene (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 O C ) , n-hexane, dichloromethane, tetrahydrofuran, toluene or appropriate mixtures thereof 4.8 Methanolic 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 0,05% by mass of triethylamine (4.9) For mobile phases of alternative HPLC-systems, see annex C OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS ```,-`-`,,`,,`,`,,` - Not for Resale Page EN 12823-212000 4.11 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 % and 1O0 % 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) = 536,85g/mol, for qualitative purposes 4.1 I.4 Lycopene, M (C40ii56) = 536,85g/mol, for qualitative purposes 4.12 Stock and standard solutions ```,-`-`,,`,,`,`,,` - 4.12.1 /3-Carotene stock solution Dissolve approximately mg, of the P-carotene standard substance (4.1I 2)in 20 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 1O0 ml Dilute 10,Oml of this solution with n-hexane up to O0 ml ml of this standard solution contains approximately pg 0-carotene in n-hexaneldichloromethane (98+2)(VIV), n-hexaneltetrahydrofuran (98+2)(VIV);or n-hexaneltoluene (98+2)(VIV) Store the stock solution protected from light at less than 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 453 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& value of 0-carotene in n-hexane It may change considerably with the composition of the solvent 181 The ratio of A455/A340 should be greater than 15, and the ratio&j5/A483 should be in range ,I4to I,I8 for pure, all-trans-0-carotene [8] 4.12.3 Standard solution of &carotene Pipette 20 ml of the 0-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 OC 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 temperature below "C and is usually stable for up to week 4.12.4 Standard solutions of acarotene and lycopene*' For qualitative purposes, pre-dissolve approximately 0,3 mg of acarotene (4.1 I.3) or lycopene (4.1 1.4)in approximately 1O ml of tetrahydrofuran (4.7) and dilute to a volume of O0 ml with ethanol (4.3)or another solvent compatible to the HPLC-system Tht standard solution shall be stored protected from light and at a temperature below "C and is usually stable for up to week ') This information is given for the convenience of users of this standard method and does not constitute an endorsement by CEN ofthe supplier named Equivalent products may be used if they can be shown to lead to the same results The standard solutions of &carotene and lycopene are not necessary for the quantification of thepcarotene in the sample extract but help to identify clearly the different compounds OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale S T D - B S I BS EN 12823-2-ENGL Lb2LibbS 085123b 2000 Page EN 12823-2:2000 Apparatus Usual laboratory apparatus and, in particular, the following: 5.1 UV-VIS Spectrometer, capable of measuring absorbance at defined wavelengths, with appropriate quartz cells, e.g of 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 evaluation system such as an integrator or recorder 5.4 HPLC column cl8 reversed phase, particle size l m , diameter 4,O mm to 4,6 mm, length Column types and particle sizes other than specified in this European standard may be used Chromatographic conditions 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-a-carotene and all-trans-0-carotene which should be greater than I ```,-`-`,,`,,`,`,,` - Analytical reversed phase column, e.g 250 mm Suitable RP column packing materials are e.g V y d a a 201TP543),Vyda& 218TP543),EurospherB100-Cla3), UltraspherQS ODs3),SpherisoMl ODSZ3),ZorbaxQ ODs3)and LiChrosphetfB RP 183).Thecolumns 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 devices to filter HPLC mobile phases and sample solutions respectively, e.g of 0,45 pm 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 5.7 Phase separation filter (optional) Sampling Sampling shall be in accordance with EN I S 5555, if appropriate 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 long periods of time.B-carotene is sensitive to UV radiation and light 7.2 Preparation of the sample test solution 7.2.1 Saponification Saponify 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 (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 b y d a d 201TP54, Vyda& 218TP54, Eurosphea lOO-C, , Ultrasphere@ODs, SpherisoW ODS2,Zorbax@ODS and LiChrosphetfB RP18 are available examples of suitable products available commercially This informationis given for the convenience of usersof this standard method and does not constitute an endorsement by CEN of these products OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale ~~ S T D * B S I BS EN 32823-2-ENGL 2000 D L b b b 0853237 b75 D Page EN 12823-2:2000 Sample mass g to g Alcohol Antioxidant 50 ml methanol 0,25 g AA ml of a 50 g/l O0 ml solution g to 10 g 1O0 ml ethanol 1,O g AA + 0,04 g N+S 20 ml of a 60 g/l O0 ml solution 10 150 ml ethanol 1,O g AA 50 ml of a 60g/l O0 ml solution Potassium hydroxide Typical saponification times range from 15 to 40 at temperatures of 80 "C to 1O0 "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 samples such as fruits or vegetables can be extracted directly with a suitable solvent with a corresponding method e.g as described in [9] to [ I I ] 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 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:l Extract the P-carotene from the saponified sample solution by means of a suitable solvent or solvent mixture (4.7) Repeat the extraction procedure to times with volumes ranging from 50 ml to 150 rnl Wash the combined extracts to neutral with water (typically to times 50 ml to 150 mi) 7.2.3 Evaporation Evaporate the extract using a rotary evaporator (5.2) under partial vacuum and at a temperature not exceeding 50 OC Remove traces 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-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 pglml of p-carotene This is the sample test solution 7.3 Identification Identify p-carotene by comparison of the retention time of the individual peaks in the chromatograms obtained with the sample test solution (see 7.2.4) and with the standard solution (4.12.3 and 4.12.4) Peak identification can also be performed by adding small amounts of the appropriate standard solution to the sample test solution NOTE: The separation and the quantification have proven to be satisfactory if the following chromatographic conditions are followed (see also Figure A.l) Stationary phase: Mobile phase: Flow rate: Injection volume: Detection: Spheri~orb@~)ODS2,5 pm, 100 mm x 4,6 mm cartridge combined with Vydacd) 201TP54, 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; ml/min; 50 pl; 450 nm $pherisorb@is a product supplied by Phase Separations Inc; V y d a d 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 the same results ```,-`-`,,`,,`,`,,` - Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS OBSI 05-2000 Not for Resale STD-BSI BS EN 12823-2-ENGL 0 l b Z L i b b 0851238 501 = Page 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 quantitative 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 volumes of the sample test and of the standard solutions (4.12.3 and 4.12.4) or compensate with a corresponding factor in the calculationof 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 determinations Perform at least two independent determinations 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 sample using equation (2): where: 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 of thep-carotene in the standard solution in micrograms per c millilitre; is the total volume of sample test solution (7.2.4) in millilitres; Vs injection volume of the standard solution, in microlitres; V,, As, is the peak area or peak height forp-carotene obtained with the standard solution (4.12.3), in units of area or height; is the sample mass in grams; rn is the injection volume of the sample test solution, in microlitres; V,, O00 is the conversion factor (micrograms to milligrams); 1O0 is the conversion factor for the content per 1O0 g As Precision Details of the inter-laboratory test of the precision of the method according to I S 5725 [12] are summarized in annex 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 within the shortest feasible time interval will exceed the repeatability limitr in not more than % of the cases The values for total-p-carotene are: margarine vitamin drink pudding powder mixed vegetables X =0,253 Y =2,248 Y =1,531 X= 8,05 I mg/l O0 g mg/l O0 g mgil00 g mg/l O0 g r= r= r= r= 0,032 0,19 0,24 2,O mg/l00 g mg/100g mgll00 g 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 % of the cases ```,-`-`,,`,,`,`,,` - OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale Page EN 12823-2:2000 The values for total-fi-carotene are: margarine vitamin drink pudding powder mixed vegetables X =0,253 x =2,248 x = I ,531 X =I 8,05 mg/l O0 g mg/l O0 g mg/l O0 g mg/l O0 g R = 0,069 R = 0,41 R = 0,40 R = 7,6 mg/l00 g mg/100 g mg/l00 g mg/l00 g 10 Testreport The test report shall contain at least the following 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 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale STDmBSI B S EN 12823-2-ENGL 2000 L b b b S 0851290 b T Page EN 12823-212000 Annex A (informative) Example of a HPLC chromatogram 15 al C al c O J= m 9m -a 11 -88 ? I t a) wi -m v 18,18 1.58 9.98 O L 1 12 13 14 15 16 18 19 21 2 23 2L 26 27rn11-129 b) - a) Absorption; b) Time; Stationary Phase: Spherisorb6') ODS2,5 rn, 100 mm x 4,6 mm cartridge combined with Vydacd' 201TP54, pm, 250 mm x 4,6 mm; Mobile phase: Acetonitrile + methanolic ammonium acetate solution + Dichlorornethane (75+20+5) (volume parts) containing 0,l % by mass of butylated hydroxytoluene and 0,05% by mass of triethylamine; Flow rate: mllmin; Injection volume: 50 pl; Detection: 450 nm - Example for an HPLC separation of a- and &carotene and lycopene in a mixed vegetable sample SpherisorbQ is a trade-name of a product supplied by Phase Separations Inc; Vydam is a trade-name of a product supplied by The Separations Group This information is given for the convenience of users of this standard and does not constitute an endorsemait by CEN of this product named Equivalent products may be used if they can be shown to lead to the same results OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale ```,-`-`,,`,,`,`,,` - Figure A.1 Page 10 EN 12823-212000 Annex B (informative) Precision data Statistical results and precision data given in Table B l for the determination of total+-carotene were established by m inter-laboratory-test according to IS0 5725-2:1994 [12] organized by W Schüep and J Schierle [ I 11 Table B.l Sample Margarine Analyte Total Total Total Total @-carotene B-carotene B-carotene 0-carotene Year of the inter-laboratory test Vitamin Drink 1995 1995 1995 13 13 13 13 1 1 III 12 Number of samples I Number of laboratories retained after elimination of outliers l2I l2 Number of outliers 1 55 59 51 56 0,253 2,248 1,531 18,05 Number of accepted results I Repeatability value, r (2,83 x sr), mg/l O0 g 0,032 Reproducibility standard deviation, S , mg/l O0 g 0,024 ```,-`-`,,`,,`,`,,` - 0,011 Repeatability standard deviation, s , mg/l O0 g 0,065 I Repeatability relative standard deviation, RSD, ~ Reproducibility relative standard deviation, RSDR Reproducibilityvalue, R (2,83 x sR), mg/l O0 g I Mixed Vegetables 1995 Number of laboratories Mean value mgll00 g Pudding Powder $7 5,6 %I 0,085 3,9 % ~~ 0,191 02-0, 0,15 0,14 %I 0,069 0,71 2,7 15 % 0,41 0,40 7,6 OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale ~ ~~ S T D - B S I B S EN 12823-2-ENGL 2000 I I L b b b ï 0851242 TI2 Page Il EN 12823-2:2000 The validation data given in Table 8.2 have been defined in a comparison study which was conducted under the European Commissions Standard, Measurement and Testing program organized by Institute of Food Research, Norwich, United Kingdom, in 1996 on the measurement of all-trans+-carotene in a mixed vegetable sample [lo] Table 8.2 Mixed vegetables Sample 1996 Year of the inter-laboratory test I Number of laboratories Number of samples Number of laboratories retained after elimination of outliers 14 I Number of outliers Number of data sets 14 Number of accepted results 60 I Mean value mg/l O0 g Repeatability standard deviation, s,, mgll O0 g 0,159 Repeatability relative standard deviation, RSDr 6,7% Repeatability value, r (2,83 x sr),mg/l O0 g 0,450 Reproducibility standard deviation, sR, mg/l O0 g 0,241 Reproducibility relative standard deviation, RSDR 10,2 % Reproducibility value, R (2,83 x sR),mgll00 g 0,682 NOTE: The data obtained in this international comparison study have been produced using different established methods being identical with in house routine assay procedures of the participating laboratories with the HPLC-systems described in annex C ```,-`-`,,`,,`,`,,` - OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale Page 12 EN 12823-212000 Annex C (informative) Alternative HPLC-systems The separation and quantification have been proven to be satisfactory if the following chromatographic conditions given in Table C.1 are applied [IO] See also [Il] Table C.16) Mobile Phase Stationary Phase Column Dimension (mm) Sphericorb@ODS2,5 pm plus 100 x 4,6 Vydam 201TP54,5 pm 250 x 4,6 NucleosilB prn 125 x 4,6 VydacB TP54 pm 250 x 4,6 CH$N+(MeOH + 0,05 rnolll NH4Ac)+ DCM (70+20+10)containing 0,05 Yo TEA Vydam 201TP54, prn 250 x 4-6 MeOH + THF (95+5) + 0.1 Yo BHT 1,O mllmin diode array MeOH+THF (99+1) + 50 Dom AA 1,5 ml/min 450 nm to min: 1.8 mllrnin to 20 min: 3 milmin diode array 1,5 rnl/rnin CH$N+(MeOH + 0,05moll1 NH4Ac) + DCM 450 nrn (75+20+5) containing 0,l % BHT and 0,05 YO TEA ~ diode array ```,-`-`,,`,,`,`,,` - ~~ ~ Flow ~ Eurosphea 100-C18, pm 250 x 4,O CH3CN + MeOH (85+15) Spheri-5-ODS, pm 220 x 4,6 Gradient with: CH3CN + MeOH (85+15) and CH3CN + DCM + MeOH (70+20+10) diode array CH3CN + MeOH + DCM + H20 (700+150+100+25) UVNIS HypersilB ODS, pm Lichrosphea RP18, pm I 250 x 4,O MeOH + THF (95+5) 2.0 mllmin UVNIS Abbreviations: MeOH: EtOH: AA: KOH: THF: CHBCN: DCM: NH~Ac: TEA: BHT: Methanol Ethanol Ascorbic acid or sodium ascorbate Potassium hydroxide Tetrahydrofuran Acetonitrile Dichioromethane Ammonium acetate Triethylamine Butylated hydroxy toluene OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale S T D - B S I BS E N 12823-2-ENGL 2000 II 3ib24bb7 0853244 805 Page 13 EN 12823-2:2000 Bibliography World Health Organisation, Expert Committee on Biological Standardisation, Techn Report No 147, 11, WHO, Geneva, 1958 and Techn Report 2 , lO, 51-52, WHO, Geneva, 1961 Int Union of Pure and Applied Chemistry (IUPAC), The Vitamin A Potency ofp-Carotene, Butterworths Scientific Publications, London 1959 Bushway, R.J.: Separation of Carotenoids in Fruits and Vegetables by High-Performance Liquid Chromatography, J Liq Chromatogr 8, 1985, 1527-47 Quackenbush, F.W.: Reversed Phase HPLC Separation of Cis- and Trans-Carotenoids and its Application in Food Materials, J Liq Chromatogr 10, 1987, 643-653 O Neil, C.A., Schwartz, S.J , Catignani, G.L.: Comparison of Liquid Chromatographic Methods for Determination of Cis-Trans Isomers offiCarotene, J Assoc Off Anal Chem 74, 1991, 36-42 Saleh, H.M., Tan, B.: Separation and Identification of Cisnrans Carotenoid Isomers, J Agric Food Chem.,39, 1991, 1438-1443 Lumley, i D.: in The Technology of Vitamins in Food, ed by P B Ottaway, Blacie Academic & Professional, Glasgow, 1993, 183-186 De Ritter, E and Purcell, A.E.: Carotenoids as Colorants and Vitamin A Precursors, ed by J Christopher Bauernfeind, Academic Press Inc., New York, 1981, 889 Hart, D.J.and Scott, K.J., 1995 Development and evaluation of an HPLC method for the analysis of carotenoids in foods, and the measurement of the carotenoid content of vegetables and fruits commonly consumed in the UK Food Chem 54, 101-1 11 Finglas, P.M., Scott, K.J., Wilthöft, C M., van der Berg, H 8, de Froidmont - Görtz, I., 1999 The certification of the mass fractions of vitamins in four reference materials: wholemeal flour (CRM 121), milk powder (CRM 421), lyophilized mixed vegetables (CRM 485) and lyophilized pig’s liver (CRM 487) EUR-Report DOC/BCR/O1/98 Commission of the European Union, Luxembourg Schüep, W and Schiede, J., 1997 Determination of p-Carotene in Commercial Foods: Interlaboratory Study Journal of AOAC International Vol 80 No 5, 1057-1064 ```,-`-`,,`,,`,`,,` - IS0 5725-2:1994 Accuracy (trueness and precision) of measurement methods and results - Part 2: Basic method for the determination of repeatability and reproducibility for a standard measurement method OBSI 05-2000 Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale BS EN 12823-2:2000 m, BSI - British Standards Institution BSI is the independent national body responsible for preparing British Standards It presents the UK view on standards in Europe and at the international level It is incorporated by Royal Charter Revisions British Standards are updated by amendment or revision Users of British Standards should make sure that they possess the latest amendments or editions It is the constant aim of BSI to improve the quaiity of our products and services We would be grateful if anyone fínding an ina~curacyor ambiguity while using this British Standard would inform the Secretary of the technical committee responsible, the identity of which can be found on the inside front cover Tel 020 8996 9OOO Fax: 020 8996 7400 BSI offers members an individual updating service called PLUS which ensures that subscribers automatically receive the latest editions of standards Buying standards Orders for all BSI, international and foreign standards publications should be addressed to Customer Services Tel 020 8996 9001 Fax: 020 8996 7001 In response to orders for international standards, it is BSI policy to supply the BSI implementation of those that have been published as British Standarch, unless otherwise requested Information on standards BSI provides a wide range of information on national, European and international standards through its Library and its Technical Help to Exporters Service Various BSI electsonic information services are also available which give details on all its products and services Contact the Information Centre Tel: 020 8996 7111 Fax: 020 8996 7048 Subscribing members of BSI are kept up to date with standards developments and receive Substantial dzscounts on the purchase price of standards For details of these and other benefits contact Membership Administrati'on Tel: 020 8996 7002 Fax: 020 8996 7001 Copyright This does not preclude the free use, in the course of implementing the standard, of necessary details such as symbols, and size, type or grade designations Lf these details are to be used for any other purpose than implementation then the prior written permission of BSI must be obtained üpermission is granted, the t e m may include royalty payments or a licensing agreement Details and advice can be obtained from the Copyright Manager Tel 020 8996 7070 BSI 389 Chiswick High Road London w4 4AL Copyright European Committee for Standardization Provided by IHS under license with CEN No reproduction or networking permitted without license from IHS Not for Resale ```,-`-`,,`,,`,`,,` - 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