1、DD CEN/TS15754:2008ICS 65.120,NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWDRAFT FOR DEVELOPMENTAnimal feeding stuffs Determination ofsugar content Highperformance exchangechromatographicmethod (HPAEC-PAD)This Draft for Developmentwas published underthe authority of theStand
2、ards Policy andStrategy Committee on 30November 2008 BSI 2008ISBN 978 0 580 60796 7Amendments/corrigenda issued since publicationDate CommentsDD CEN/TS 15754:2008National forewordThis Draft for Development is the UK implementation of CEN/TS15754:2008.This publication is not to be regarded as a Briti
3、sh Standard.It is being issued in the Draft for Development series of publications andis of a provisional nature. It should be applied on this provisional basis,so that information and experience of its practical application can beobtained.Comments arising from the use of this Draft for Development
4、arerequested so that UK experience can be reported to the internationalorganization responsible for its conversion to an international standard.A review of this publication will be initiated not later than 3 years afterits publication by the international organization so that a decision can betaken
5、on its status. Notification of the start of the review period will bemade in an announcement in the appropriate issue of Update Standards.According to the replies received by the end of the review period,the responsible BSI Committee will decide whether to support theconversion into an international
6、 Standard, to extend the life of theTechnical Specification or to withdraw it. Comments should be sent tothe Secretary of the responsible BSI Technical Committee at BritishStandards House, 389 Chiswick High Road, London W4 4AL.The UK participation in its preparation was entrusted to TechnicalCommitt
7、ee AW/10, Animal feeding stuffs.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 are responsible for its correct application.Compliance with a British Standar
8、d cannot confer immunityfrom legal obligations.DD CEN/TS 15754:2008TECHNICAL SPECIFICATIONSPCIFICATION TECHNIQUETECHNISCHE SPEZIFIKATIONCEN/TS 15754September 2008ICS 65.120English VersionAnimal feeding stuffs - Determination of sugar content - Highperformance exchange chromatographic method (HPAEC-P
9、AD)Aliments des animaux - Dtermination de la teneur ensucre - Chromatographie dchange danions hauteperformance couple la dtection par ampromtriepulse (HPAEC-PAD)Futtermittel - Bestimmung des Zuckergehalts -Hochleistungs-Anionenaustausch-Chromatographieverfahren (HPAEC-PAD)This Technical Specificatio
10、n (CEN/TS) was approved by CEN on 17 May 2008 for provisional application.The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit theircomments, particularly on the question whether the CEN/TS can be converted into a E
11、uropean Standard.CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS availablepromptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)until the fi
12、nal decision about the possible conversion of the CEN/TS into an EN is reached.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malt
13、a, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploita
14、tion in any form and by any means reservedworldwide for CEN national Members.Ref. No. CEN/TS 15754:2008: EDD CEN/TS 15754:2008CEN/TS 15754:2008 (E) 2 Contents Page Foreword3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle5 5 Reagents.5 6 Apparatus .7 7 Sampl
15、ing.8 8 Preparation of the test sample .8 9 Procedure .8 10 Calculation and expression of the results 11 11 Precision.12 12 Test report 12 Annex A (informative) Results of an interlaboratory test for each individual sugar13 Bibliography 20 DD CEN/TS 15754:2008CEN/TS 15754:2008 (E) 3 Foreword This do
16、cument (CEN/TS 15754:2008) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs - Methods of sampling and analysis“, the secretariat of which is held by NEN. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CE
17、N and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association, and supports essential requirements of EU Directive(s). According to the CEN/
18、CENELEC Internal Regulations, the national standards organizations of the following countries are bound to announce this CEN Technical Specification: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lith
19、uania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. DD CEN/TS 15754:2008CEN/TS 15754:2008 (E) 4 Introduction This Technical Specification describes an application of the High Performance Anion Exchange Chromatog
20、raphy with Pulsed Amperometric Detection (HPAEC-PAD) for the determination of different individual sugars, both mono- and disaccharides, in dry animal feeding stuffs. HPAEC-PAD is a modern liquid chromatographic technique with high selectivity and sensitivity for especially carbohydrates. Due to thi
21、s high selectivity and sensitivity of HPAEC-PAD for carbohydrates just a minimum of sample clean-up is required in most carbohydrate applications in food and feed matrices. This technical specification is meant as an introduction of a new method for the determination of the content of the individual
22、 sugars present in animal feeding stuffs. The document should be used to get experienced with this new powerful technique and with the described method for the quantification of the individual sugars in animal feeding stuffs. Currently the HPAEC-PAD equipment is mainly used in research environments
23、and not wide spread in use in the feed and control laboratories. Therefore, the method is at this time not yet suitable for official control applications of the sugar content in feeding stuffs. It is however expected that within several years more and more laboratories in the field will have this in
24、strumentation at their disposal.DD CEN/TS 15754:2008CEN/TS 15754:2008 (E)51 Scope This Technical Specification describes the quantitative determination of specific sugars (glucose, fructose, galactose, sucrose, maltose, and lactose) in dry animal feeding stuffs at the g/kg level by a sophisticated h
25、igh performance anion exchange chromatography in combination with pulsed amperometric detection (HPAEC-PAD). 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 references,
26、 the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) ISO 6498:1998, Animal feeding stuffs Preparation of test samples 3 Terms and definitions For the purposes of this docum
27、ent, the following terms and definitions apply. 3.1 total sugar content sum of the content of the individual sugars, glucose, fructose, galactose, maltose, sucrose and lactose, expressed as g/kg in the sample as such 4 Principle The sugars present in the test potion of the animal feeding stuff sampl
28、e are extracted with water. The clean-up of the aqueous extract is done by either a C-18 SPE procedure, or by a deproteination with acetonitrile. After the clean-up, the sample solution is diluted and the sugars present are separated by high performance anion exchange chromatography and detected by
29、pulsed amperometric detection. Quantitation of the sugars present is done by comparison of the peak areas and/or heights with those of an external calibration graph prepared with standard solutions. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. DD CEN/TS 15
30、754:2008CEN/TS 15754:2008 (E) 6 5.1 Water, complying with EN ISO 3696, grade 3. 5.2 Acetonitrile 5.3 Dimethyl Sulfoxide (DMSO) 5.4 Sodium hydroxide solution, mass fraction, w(NaOH) = 50 % in water The reagent should contain the minimum amount of carbonate and mercury. Do not shake or stir the soluti
31、on before use. 5.5 Sodium acetate trihydrate (CH3COONa3H2O) 5.6 Eluent 1 (E1), aqueous solution of 500 mmol/l sodium hydroxide (NaOH) Add to a 1 000 ml volumetric flask about 800 ml degassed water (eluent 3, 5.8) followed by 40,0 g of sodium hydroxide 50 % (m/m) (5.4). Then dilute the aqueous soluti
32、on to the mark with degassed demineralised water (5.8). 5.7 Eluent 2 (E2), aqueous solution of 500 mmol/l sodium acetate Add to a 1 000 ml volumetric flask about 800 ml degassed demineralised water (eluent 3, 5.8) followed by 68,0 g sodium acetate trihydrate (CH3COONa3H2O) (5.5). Then dilute the aqu
33、eous solution to the mark with degassed demineralised water (5.8). 5.8 Eluent 3 (E3), degassed demineralized water Filter the demineralized water through a 0,2 membrane filter. Degas by sparging with helium for at least 30 min. 5.9 Sugar standard solutions 5.9.1 Preparation stock solution Prepare da
34、ily fresh aqueous solutions of glucose, fructose, galactose, maltose, sucrose, and lactose. Weigh approximately 40 mg of each sugar to the nearest 0,1 mg into separate 100 ml volumetric flasks (6.6) and dilute to the mark with water (stock standard solutions of 400 mg/l). NOTE 1 Mixed standard solut
35、ions can also be prepared once the retention time of the individual sugars is known under the prevailing chromatographic conditions. NOTE 2 The standard solutions can be further diluted to reach sugar concentrations similar to those found in the sample solutions. 5.9.2 Working standard solutions for
36、 calibration Prepare dilutions of the sugar standard solutions as specified in Table 1. Dilute 0,25 ml of each obtained standard solution with 1,25 ml DMSO (5.3) in an HPLC vial. Close the vial and mix well. DD CEN/TS 15754:2008CEN/TS 15754:2008 (E) 7Table 1 Preparation aqueous DMSO sugar standard s
37、olutions Standard solution calibration graph Volume sugar standard stock solution ml Volume water ml Sugar concentration after dilution with DMSO g/mla Standard 1 0,00 10,00 0 Standard 2 0,10 9,90 0,67 Standard 3 0,30 9,70 2,00 Standard 4 0,70 9,30 4,67 Standard 5 1,40 8,60 9,33 Standard 6 2,00 8,00
38、 13,33 aBased upon a sugar content of exactly 400 mg/l in the aqueous stock solution. These aqueous DMSO sugar standard solutions will be used for preparing the calibration graphs. NOTE Aqueous DMSO is a good solvent for carbohydrates and the use of DMSO prevent microbial deterioration of the carboh
39、ydrates in the solutions. 6 Apparatus Usual laboratory equipment and, in particular the following: 6.1 Analytical balance, capable of weighting to an accuracy of 0,1 mg. 6.2 Glass centrifugation tubes, of a capacity of 50 ml. 6.3 Homogenizer16.4 Swing-out centrifuge, adjusted to a centrifugation for
40、ce of about 2 800 g. 6.5 Screw cap closed centrifugation tubes, with screw caps with a polytetrafluoroethylene (PTFE) inlay, of a capacity of about 10 ml. 6.6 One mark volumetric flasks, of a capacity of 100 ml and 1 000 ml. 6.7 Dispensors, resistant to organic solvents and adjusted to 2,5 ml and 20
41、 ml. 6.8 Disposable C-18 SPE cartridges, to be used according to the manufacturers recommendations. 1An IKA Ultra turrax with apprropriate probe is an example of a suitable commercially available homogenizer. DD CEN/TS 15754:2008CEN/TS 15754:2008 (E) 8 6.9 Metal free liquid chromatographic system2,
42、applicable for a ternair gradient elution profile. 6.10 Column oven, adjusted at 30 C. 6.11 High performance anion exchange column3, filled with pellicularpolystyrene-divinylbenzene resin and pre- column4(guard column), mounted in the column oven (6.10). 6.12 Pulsed amperometric detector5, with a go
43、ld electrode, situated together with the columns in the column oven at 30 C (6.10). Detector settings are as follows: 0 s 0,40 s potential working electrode + 0,1 V; 0,41 s 0,60 s potential working electrode + 0,7 V; 0,61 s 1,00 s potential working electrode 0,1 V; integration window 0,20 s 0,40 s.
44、6.13 Integrator chromatography data system 7 Sampling It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport and/or storage. Sampling is not a part of the method specified in this Technical Specification. A recommended
45、 sampling method is given in EN-ISO 6497 1. 8 Preparation of the test sample Prepare the test sample in accordance with ISO 6498. 9 Procedure 9.1 Extraction sugars Weigh approximately 600 mg of the test sample to the nearest 0,1 mg in a 100 ml glass centrifugation tube (6.2). Add with the dispensor
46、(6.7) 20,0 ml water and homogenize for at least 1 min with the homogenizer (6.3). Transfer quantitatively to a 100 ml volumetric flask, fill up with water to the mark and homogenize. Centrifuge an aliquot of the homogenized aqueous sample extract for 10 min at about 2 800 g with the swing out centri
47、fuge (6.4). 2 The Dionex ICS-3000 module is an example of a suitable commercially available metal free chromatographic system with a quaternair gradient elution pump. 3 The Dionex Carbopac PA1 analytical column (4 x 250 mm) is an example of a suitable commercially available high performance anion ex
48、change column. 4 The Dionex Carbopac PA1 Guard column (4 x 50 mm) is an example of a suitable commercially available guard column. 5 The Dionex model PAD-II is an example of a suitable commercially available pulsed amperometric detector. DD CEN/TS 15754:2008CEN/TS 15754:2008 (E) 99.2 Extract clean-u
49、p 9.2.1 Two extract clean-up methods For the extract clean-up, continue with either 9.2.2 or 9.2.3. After one of these clean-up procedures, continue with 9.3. 9.2.2 Extract clean-up, method 1, C-18 SPE procedure Dilute 2,0 ml of the clear solution of the centrifuged extract (9.1) with 2,5 ml water. Prepare and activate the C-18 SPE cartridge according to manufacturers instruction6. Elute about 5 ml of the clear solution of the centrifuged extract over the activated C-18 SPE cartridge. Discard the first 3 ml and collect 1 ml in a glass tu
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