《DD-CEN-TS-15754-2008.pdf》由会员分享，可在线阅读，更多相关《DD-CEN-TS-15754-2008.pdf（24页珍藏版）》请在三一文库上搜索。
1、DD CEN/TS 15754:2008 ICS 65.120, NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW DRAFT FOR DEVELOPMENT Animal feeding stuffs Determination of sugar content High performance exchange chromatographic method (HPAEC-PAD) Licensed Copy: London South Bank University, South Bank Univ
2、ersity, 31/01/2009 03:38, Uncontrolled Copy, (c) BSI This Draft for Development was published under the authority of the Standards Policy and Strategy Committee on 30 November 2008 BSI 2008 ISBN 978 0 580 60796 7 Amendments/corrigenda issued since publication DateComments DD CEN/TS 15754:2008 Nation
3、al foreword This Draft for Development is the UK implementation of CEN/TS 15754:2008. This publication is not to be regarded as a British Standard. It is being issued in the Draft for Development series of publications and is of a provisional nature. It should be applied on this provisional basis, s
4、o that information and experience of its practical application can be obtained. Comments arising from the use of this Draft for Development are requested so that UK experience can be reported to the international organization responsible for its conversion to an international standard. A review of t
5、his publication will be initiated not later than 3 years after its publication by the international organization so that a decision can be taken on its status. Notification of the start of the review period will be made in an announcement in the appropriate issue of Update Standards. According to th
6、e replies received by the end of the review period, the responsible BSI Committee will decide whether to support the conversion into an international Standard, to extend the life of the Technical Specification or to withdraw it. Comments should be sent to the Secretary of the responsible BSI Technic
7、al Committee at British Standards House, 389 Chiswick High Road, London W4 4AL. The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs. A list of organizations represented on this committee can be obtained on request to its secretary. This publicati
8、on does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard cannot confer immunity from legal obligations. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 03:38, Uncont
9、rolled Copy, (c) BSI DD CEN/TS 15754:2008 TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 15754 September 2008 ICS 65.120 English Version Animal feeding stuffs - Determination of sugar content - High performance exchange chromatographic method (HPAEC-PAD) Aliments des
10、animaux - Dtermination de la teneur en sucre - Chromatographie dchange danions haute performance couple la dtection par ampromtrie pulse (HPAEC-PAD) Futtermittel - Bestimmung des Zuckergehalts - Hochleistungs-Anionenaustausch- Chromatographieverfahren (HPAEC-PAD) This Technical Specification (CEN/TS
11、) 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 their comments, particularly on the question whether the CEN/TS can be converted into a European
12、 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 available promptly 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 final
13、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, Malta,
14、 Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2008 CENAll rights of exploi
15、tation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 15754:2008: E Licensed Copy: London South Bank University, South Bank University, 31/01/2009 03:38, Uncontrolled Copy, (c) BSI DD CEN/TS 15754:2008 CEN/TS 15754:2008 (E) 2 Contents Page Foreword3 Introdu
16、ction.4 1 Scope 5 2 Normative references5 3 Terms and definitions .5 4 Principle5 5 Reagents.5 6 Apparatus .7 7 Sampling.8 8 Preparation of the test sample.8 9 Procedure .8 10 Calculation and expression of the results 11 11 Precision.12 12 Test report12 Annex A (informative) Results of an interlabor
17、atory test for each individual sugar13 Bibliography20 Licensed Copy: London South Bank University, South Bank University, 31/01/2009 03:38, Uncontrolled Copy, (c) BSI DD CEN/TS 15754:2008 CEN/TS 15754:2008 (E) 3 Foreword This document (CEN/TS 15754:2008) has been prepared by Technical Committee CEN/
18、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. CEN and/or CENELEC shall not be held responsible for identifying any or al
19、l 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/CENELEC Internal Regulations, the national standards organizations of th
20、e 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, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania
21、, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 03:38, Uncontrolled Copy, (c) BSI DD CEN/TS 15754:2008 CEN/TS 15754:2008 (E) 4 Introduction This Technical Specification describes an application of the
22、 High Performance Anion Exchange Chromatography 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 sensitivit
23、y for especially carbohydrates. Due to this 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 dete
24、rmination of the content of the individual 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 equipmen
25、t is mainly used in research environments 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 l
26、aboratories in the field will have this instrumentation at their disposal. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 03:38, Uncontrolled Copy, (c) BSI DD CEN/TS 15754:2008 CEN/TS 15754:2008 (E) 5 1 Scope This Technical Specification describes the quantitative det
27、ermination of specific sugars (glucose, fructose, galactose, sucrose, maltose, and lactose) in dry animal feeding stuffs at the g/kg level by a sophisticated high performance anion exchange chromatography in combination with pulsed amperometric detection (HPAEC- PAD). 2 Normative references The foll
28、owing referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specif
29、ication 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 document, the following terms and definitions apply. 3.1 total sugar content sum of the content of the individual sugars, glucose, fructose, gala
30、ctose, 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 sample 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 acetonit
31、rile. After the clean-up, the sample solution is diluted and the sugars present are separated by high performance anion exchange chromatography and detected by pulsed amperometric detection. Quantitation of the sugars present is done by comparison of the peak areas and/or heights with those of an ex
32、ternal calibration graph prepared with standard solutions. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 03:38, Uncontrolled Copy, (c) BSI DD CEN/TS 15754:2008 CEN/TS 15754:2008
33、(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 solution before use. 5.5 Sodium
34、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 solution to the mark with degass
35、ed 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 aqueous solution to the mark
36、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 daily fresh aqueous solution
37、s 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 solutions can also be prepared
38、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 calibration Prepare dilut
39、ions 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. Licensed Copy: London South Bank University, South Bank University, 31/01/2009 03:38, Uncontrolled Copy, (c) BSI DD CE
40、N/TS 15754:2008 CEN/TS 15754:2008 (E) 7 Table 1 Preparation aqueous DMSO sugar standard solutions 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
41、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 13,33 a Based 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 s
42、olvent for carbohydrates and the use of DMSO prevent microbial deterioration of the carbohydrates 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 cap
43、acity of 50 ml. 6.3 Homogenizer1 6.4 Swing-out centrifuge, adjusted to a centrifugation force 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 m
44、l and 1 000 ml. 6.7 Dispensors, resistant to organic solvents and adjusted to 2,5 ml and 20 ml. 6.8 Disposable C-18 SPE cartridges, to be used according to the manufacturers recommendations. 1 An IKA Ultra turrax with apprropriate probe is an example of a suitable commercially available homogenizer.
45、 Licensed Copy: London South Bank University, South Bank University, 31/01/2009 03:38, Uncontrolled Copy, (c) BSI DD CEN/TS 15754:2008 CEN/TS 15754:2008 (E) 8 6.9 Metal free liquid chromatographic system2, applicable for a ternair gradient elution profile. 6.10 Column oven, adjusted at 30 C. 6.11 Hi
46、gh 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 gold electrode, situated together with the columns in the column oven at 30 C (6.10). Detector
47、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. 6.13 Integrator chromatography data system 7 Sampling It is important that the laboratory rec
48、eives 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 sampling method is given in EN-ISO 6497 1. 8 Preparation of the test sample Prepare the test
49、 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 (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 centrifuge (6.4). 2 The Dionex I