1、BS EN ISO11701:2009ICS 67.200.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDVegetable fats andoils Determinationof phospholipidscontent in lecithins byHPLC using a light-scattering detector(ISO 11701:2009)This British Standardwas published under theauthority
2、 of the StandardsPolicy and StrategyCommittee on 31 January2010 BSI 2010ISBN 978 0 580 65445 9Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 11701:2009National forewordThis British Standard is the UK implementation of EN ISO 11701:2009.The UK participation in its preparation wa
3、s entrusted to TechnicalCommittee AW/307, Oil seeds, animal and vegetable fats and oils andtheir by products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users
4、are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11701 December 2009 ICS 67.200.10 English Version Vegetable fats and oils - Determination of phospholipids content in l
5、ecithins by HPLC using a light-scattering detector (ISO 11701:2009) Corps gras dorigine vgtale - Dtermination de la teneur en phospholipides dans les lcithines par CLHP avec dtecteur diffusion de la lumire (ISO 11701:2009) Pfanzliche Fette und le - Bestimmung des Gehaltes an Phospholipiden in Lecith
6、inen durch HPLC mittels eines Lichtstreudetektors (ISO 11701:2009) This European Standard was approved by CEN on 18 November 2009. 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 stand
7、ard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other
8、language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, F
9、inland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHE
10、S KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11701:2009: EBS EN ISO 11701:2009EN ISO 11701:2009 (E) 3 Foreword This document (EN ISO 11701:2009)
11、has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products - Methods of sampling and analysis” the secretariat of which is held by AFNOR. This European Standard shall be g
12、iven the status of a national standard, either by publication of an identical text or by endorsement, at the latest by June 2010, and conflicting national standards shall be withdrawn at the latest by June 2010. Attention is drawn to the possibility that some of the elements of this document may be
13、the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Bel
14、gium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notic
15、e The text of ISO 11701:2009 has been approved by CEN as a EN ISO 11701:2009 without any modification. BS EN ISO 11701:2009ISO 11701:2009(E) ISO 2009 All rights reserved iiiContents Page Foreword iv 1 Scope1 2 Normative references1 3 Terms and definitions .1 4 Principle .1 5 Reagents 2 6 Apparatus.2
16、 7 Sampling 3 8 Preparation of test sample .3 9 Procedure.3 10 Calculation and expression of results 5 11 Precision of the method5 12 Test report6 Annex A (informative) HPLC chromatogram7 Annex B (informative) Results of an interlaboratory test .8 Bibliography11 BS EN ISO 11701:2009ISO 11701:2009(E)
17、 iv ISO 2009 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body inter
18、ested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical
19、 Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the te
20、chnical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IS
21、O shall not be held responsible for identifying any or all such patent rights. ISO 11701 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils. BS EN ISO 11701:2009INTERNATIONAL STANDARD ISO 11701:2009(E) ISO 2009 All rights reserved 1Ve
22、getable fats and oils Determination of phospholipids content in lecithins by HPLC using a light-scattering detector 1 Scope This International Standard specifies a method for the quantitative determination of phospholipids content by high performance liquid chromatography (HPLC) using a diol column
23、and a light-scattering detector. The method is applicable to crude, oil-containing lecithins, and to oil-free lecithins and lecithin fractions from vegetable fats and oils. The method is not applicable to animal and ruminant lecithins and enzymatically hydrolysed lecithins as the peak separation of
24、lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI) and lysophosphatidic acid (LPA) is insufficient. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated refe
25、rences, the latest edition of the referenced document (including any amendments) applies. ISO 661, Animal and vegetable fats and oils Preparation of test sample 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 content of an individual phosphol
26、ipid mass fraction of N-acyl-phosphatidylethanolamine (N-acyl-PE) or phosphatidylcholine (PC) or phosphatidylethanolamine (PE) or phosphatidylinositol (PI) or phosphatidic acid (PA) or lysophosphatidylcholine (LPC), determined in accordance with the method specified in this International Standard NO
27、TE The content is expressed in grams per 100 g, numerically equal to a percentage mass fraction. 4 Principle The individual phospholipids are separated by HPLC using a diol column and a light-scattering detector. For the purpose of quantification, a certified reference mixture is used. BS EN ISO 117
28、01:2009ISO 11701:2009(E) 2 ISO 2009 All rights reserved5 Reagents WARNING Comply with any local regulations which specify the handling of hazardous substances. Technical, organizational and personal safety measures shall be followed. During the analysis, unless otherwise stated, use only reagents of
29、 recognized analytical grade. 5.1 Water, HPLC grade. 5.2 n-Hexane, HPLC grade. 5.3 2-Propanol, HPLC grade. 5.4 Acetic acid, wmin, 99,8 % mass fraction. 5.5 Triethylamine. 5.6 Solvent mixture: A mixture of 80 ml n-hexane (5.2) and 20 ml 2-propanol (5.3) (volume fraction = 80 ml/100 ml for n-hexane an
30、d = 20 ml/100 ml for 2-propanol) is used to dissolve the standards and sample. 5.7 Reference substance (external standard) ILPS-LE011), mixed soy phospholipid reference standard, is a certified reference mixture with defined contents of N-acyl-PE, PA, PE, PC, PI, and LPC. 5.8 Mobile phase for the HP
31、LC. 5.8.1 Eluent A. Mix 814,2 ml of n-hexane (5.2), 170,0 ml of 2-propanol (5.3), 15 ml of acetic acid (5.4), and 0,8 ml of triethylamine (5.5) (volume fraction = 81,42 ml/100 ml for n-hexane, = 17,00 ml/100 ml for 2-propanol, = 1,50 ml/100 ml for acetic acid, and = 0,08 ml/100 ml for triethylamine)
32、. In order to obtain a reproducible eluent composition, it is recommended that the solvents be weighed out taking into account their densities. For a batch size of 2,5 l: 1 341,4 g of n-hexane, 331,5 g of 2-propanol, 39,4 g of acetic acid, and 1,45 g (2,0 ml) of triethylamine. 5.8.2 Eluent B. Mix 84
33、4,2 ml of 2-propanol (5.3), 140 ml of water (5.1), 15,0 ml of acetic acid (5.4) and 0,8 ml of triethylamine (5.5) (volume fraction = 84,42 ml/100ml for 2-propanol, = 14,00 ml/100 ml for water, = 1,50 ml/100 ml for acetic acid and = 0,08 ml/100 ml for triethylamine). In order to obtain a reproducible
34、 eluent composition, it is recommended that the solvents be weighed out taking into account their densities. For a batch size of 2,5 l: 1 646,2 g of 2-propanol, 350,0 g of water, 39,4 g of acetic acid and 1,45 g (2,0 ml) of triethylamine. 6 Apparatus 6.1 Analytical balance, capable of being read to
35、the nearest 0,000 1 g. 6.2 HPLC basic equipment with gradient system and light-scattering detector. 6.3 HPLC column oven, adjustable to 55 C. 6.4 Degasser or similar equipment for degassing the eluent. 1) ILPS-LE01 is the trade name of a product supplied by the International Lecithin and Phospholipi
36、d Society. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to comparable results. BS EN ISO 11701:2009ISO 11701:2009(E) ISO 2009 All
37、rights reserved 36.5 HPLC column (250 mm 4,0 mm) with pre-column (20 mm 4,0 mm) packed with spherical microparticles (5 m) of diol-bounded silica, e.g. LiChrospher 100 diol (5 m)2). The age and history of the column, the packaging of the column filling material and the temperature may influence the
38、separation. 6.6 One-mark volumetric flasks, of capacities 50 ml, 100 ml and 2 500 ml, ISO 10421class A. 6.7 Microlitre syringe, of capacity 25 l, graduated in microlitres. 6.8 Filter for filtering the external standard and test sample solutions, e.g. Millex HV3). 6.9 Integration system. 7 Sampling A
39、 representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 55552. 8 Preparation of test sample See I
40、SO 661. The sample is heated to a maximum of 60 C to soften it (overheating shall be avoided) and then homogenized by vigorous stirring. 9 Procedure 9.1 Preparation of standard reference solutions and test portions 9.1.1 Standard reference solutions R1, R2, and R3Prepare three different standard ref
41、erence solutions. For this purpose, accurately weigh out in three different 100 ml one-mark volumetric flasks approximately 550 mg, 850 mg and 1 150 mg of the certified reference mixture (5.7), dissolve in the solvent mixture (5.6) and make up to the mark with the same solvent. Filter (6.8) the stan
42、dard reference solutions before injection into the HPLC. 9.1.2 Test sample solutions and test portions Weigh, to the nearest 0,001 g, 425 mg of the sample in the case of crude lecithin or 255 mg of the sample in the case of deoiled or fractionated lecithin into separate 50 ml one-mark volumetric fla
43、sks, dissolve in solvent mixture (5.6) and make up to the mark with the same solvent. Filter (6.8) the test sample solutions before drawing test portions S1aand S1bfrom them. 2) LiChrospher 100 diol is the trade name of a product supplied by Merck. This information is given for the convenience of us
44、ers of this International Standard and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results. 3) Example of a suitable product available commercially. This information is given for the convenience of users of
45、this International Standard and does not constitute an endorsement by ISO of this product. BS EN ISO 11701:2009ISO 11701:2009(E) 4 ISO 2009 All rights reserved9.2 HPLC analysis Adjust the working conditions in the equipment using the test samples and reference samples in order to get a separation ac
46、cording to the chromatogram in Figure A.1. Optimize the separation profile, depending on the type of column and gradient. The following conditions are recommended (see Table 1): Temperature of the oven: 55 C Sensitivity of the detector: 5 to 6 Temperature of the detector: 50 C Pressure of the detect
47、or: 0,20 MPa (2,0 bar) Flow: 1,0 ml/min Flow for column rinsing: 2,0 ml/min Table 1 Gradient programme for HPLC Time min Eluent A % Eluent B % Flow ml/min 0,0 95 5 1,0 5,0 80 20 1,0 8,5 60 40 1,0 15,0 0 100 1,0 17,5 0 100 1,0 17,6 95 5 1,0 21,0 95 5 1,0 22,0 95 5 2,0 27,0 95 5 2,0 29,0 95 5 1,0 9.3
48、Calibration Use 20 l injection volumes for calculation of the linear regression lines and for determination of the test sample. Plot peak area against concentration to obtain a calibration curve. NOTE Light-scattering detectors are not linear over the whole range (S-shaped calibration curves). The c
49、oncentrations of the standard reference solutions have been chosen to be in the linear range. The following analysis sequence is recommended for the quantitative determination of phospholipids: R1, R2, R3(one injection each), S1a, S1b(test portions drawn from the test sample, two injections each), R1, R2, R3(one injection each). 9.4 Determination Inject 20 l of the test sample solution into the HPLC and register the peak areas. Identify the peaks by comparing the retention times of the substa
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