1、June 2016 English price group 11No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 65.120!%VpR“2517747www.din.deDIN EN
2、 ISO 13904Animal feeding stuffs Determination of tryptophan content (ISO 13904:2016);English version EN ISO 13904:2016,English translation of DIN EN ISO 13904:2016-06Futtermittel Bestimmung des Tryptophangehalts (ISO 13904:2016);Englische Fassung EN ISO 13904:2016,Englische bersetzung von DIN EN ISO
3、 13904:2016-06Aliments des animaux Dosage du tryptophane (ISO 13904:2016);Version anglaise EN ISO 13904:2016,Traduction anglaise de DIN EN ISO 13904:2016-06SupersedesDIN EN ISO 13904:2005-08www.beuth.deDocument comprises 20 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-languag
4、e original shall be considered authoritative.06.16DIN EN ISO 13904:2016-06 2 A comma is used as the decimal marker. National foreword This document (ISO 13904:2016) has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcommittee SC 10 “Animal Feeding Stu
5、ffs” (Secretariat: ISIRI, Iran). Based on the results of parallel voting under the leadership of ISO/TC 34/SC 10, Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands) decided to adopt the International Standard as a European Standard
6、. The responsible German body involved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-03-03 AA Futtermittel (Spiegelausschuss zu CEN/TC 327). Amendments This standard differs
7、from DIN EN ISO 13904:2005-08 as follows: a) Subclause 3.17 “Calibration standard solutions of tryptophan and internal standard”, Clause 6 “Calculation of results”, and Subclauses 7.1 to 7.3 of Clause 7 “Precision” have been subdivided into “Feeding stuffs” and “Commercial pure substances and premix
8、tures containing more than 2 % of tryptophan”; b) a new informative Annex C “Sample preparation: Example for analyses in pure products and premixtures” has been added; c) a new informative Annex D “Results of an interlaboratory test for tryptophan in pure products and premixtures containing more tha
9、n 2 % of tryptophan” has been added; d) the standard has been editorially revised. Previous editions DIN EN ISO 13904: 2005-08 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 13904 March 2016 ICS 65.120 Supersedes EN ISO 13904:2005English Version Animal feeding stuffs Determination of trypt
10、ophan content (ISO 13904:2016) Aliments des animaux Dosage du tryptophane (ISO 13904:2016) Futtermittel Bestimmung des Tryptophangehalts (ISO 13904:2016) This European Standard was approved by CEN on 30 January 2016. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which sti
11、pulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European
12、 Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are th
13、e national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romani
14、a, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by an
15、y means reserved worldwide for CEN national Members. Ref. No. EN ISO 13904:2016 EEN ISO 13904:2016 (E) 2 Contents Page European foreword . 3 Foreword 4 1 Scope 5 2 Principle . 5 3 Reagents and materials . 5 4 Apparatus . 7 5 Procedure. 8 5.1 Preparation of samples . 8 5.1.1 Feeding stuffs 8 5.1.2 Co
16、mmercial pure substances and premixtures containing more than 2 % of tryptophan . 8 5.2 Determination of free tryptophan (extract) 8 5.2.1 Feeding stuffs 8 5.2.2 Commercial pure substances and premixtures containing more than 2 % of tryptophan . 8 5.3 Determination of total tryptophan (hydrolysates)
17、 . 9 5.4 HPLC determination . 9 6 Calculation of results 10 6.1 Feeding stuffs . 10 6.2 Commercial pure products and premixtures containing more than 2 % of tryptophan . 10 6.2.1 Control of the calibration 10 6.2.2 Calculation 10 7 Precision 11 7.1 Interlaboratory test . 11 7.1.1 Feeding stuffs . 11
18、 7.1.2 Commercial pure substances and premixtures containing more than 2 % of tryptophan 11 7.2 Repeatability 11 7.2.1 Feeding stuffs . 11 7.2.2 Commercial pure substances and premixtures containing more than 2 % of tryptophan 11 7.3 Reproducibility . 11 7.3.1 Feeding stuffs . 11 7.3.2 Pure products
19、 and premixtures containing more than 2 % of tryptophan 12 8 Test report 12 Annex A (informative) Results of an interlaboratory test 13 Annex B (informative) Observations on the method . 15 Annex C (informative) Sample preparation: Example for analyses in pure products and premixtures 16 Annex D (in
20、formative) Results of an interlaboratory test for tryptophan in pure products and premixtures containing more than 2 % of tryptophan 17 Bibliography . 18 DIN EN ISO 13904:2016-06 EN ISO 13904:2016 (E) 3 European foreword This document (EN ISO 13904:2016) has been prepared by Technical Committee ISO/
21、TC 34 “Food products” in collaboration with Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” the secretariat of which is held by NEN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endor
22、sement, at the latest by September 2016, and conflicting national standards shall be withdrawn at the latest by September 2016. 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 i
23、dentifying any or all such patent rights. This document supersedes EN ISO 13904:2005. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech R
24、epublic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. E
25、ndorsement notice The text of ISO 13904:2016 has been approved by CEN as EN ISO 13904:2016 without any modification. DIN EN ISO 13904:2016-06 ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of prepa
26、ring International Standards is normally carried out through ISO technical committees. Each member body interested 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 l
27、iaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC D
28、irectives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the possibility that
29、 some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent dec
30、larations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information
31、about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs.This second edition cancels and replaces t
32、he first edition (ISO 13904:2005), which has been technically revised.4EN ISO 13904:2016 (E) DIN EN ISO 13904:2016-06 1 ScopeThis International Standard specifies a method for determination of the total and free tryptophan (Trp) content in feeding stuffs (e.g. complete and complementary feeds, suppl
33、ementary feeds, raw materials, ingredients, and concentrates) and determination of free tryptophan in commercial pure substances and premixtures containing more than 2 % of tryptophan.It does not distinguish between D- and L-forms.2 PrincipleFor the determination of the total tryptophan, the sample
34、is hydrolysed under alkaline conditions with saturated barium hydroxide solution and heated to 110 C for 20 h. After hydrolysis, an internal standard is added.For the determination of free tryptophan, the sample is extracted under mild acidic conditions in the presence of an internal standard. For c
35、ommercial pure substances and premixtures containing more than 2 % of tryptophan, it is possible to add the internal standard after the extraction.The tryptophan and the internal standard in the hydrolysate or in the extract are determined by reversed phase C18HPLC with fluorescence detection.3 Reag
36、ents and materialsUse only reagents of recognized analytical grade, unless otherwise specified.3.1 Double-distilled water, or water of equivalent purity (conductivity 98 %.3.19 1,1,1-Trichloro-2-methyl-2-propanol solution.Add 1 g of 1,1,1-trichloro-2-methyl-2-propanol to 100 ml of methanol (3.8).NOT
37、E 1,1,1-trichloro-2-methyl-2-propanol solution (3.19) could be considered critical for environmental reasons and may require special disposal.3.20 Mobile phase for HPLC.Dissolve 3,00 g of acetic acid in 900 ml of water (3.1) and add 50,0 ml of 1,1,1-trichloro-2-methyl-2-propanol solution (3.19). Adj
38、ust the pH to 5,00 using ethanolamine (3.18). Make up to 1 000 ml with water (3.1).Shelf life of the mobile phase (especially stability of the mixture of acetic acid and ethanolamine) has to be checked by the retention times.See also Annex B for an alternative mobile phase: The mixture of phosphate
39、buffer and methanol is cheaper and harmless; pH adjustment is not necessary. The mixture is very stable.4 ApparatusUsual laboratory apparatus and, in particular, the following.4.1 HPLC equipment with a spectrofluorimetric detector.4.2 Liquid chromatographic column, 125 mm 4 mm, with C18, 3 m packing
40、, or equivalent.4.3 pH-meter.4.4 Polypropylene flask, of capacity 125 ml, with wide neck and screw cap.4.5 Membrane filter consisting of cellulose acetate (0,45 m or 0,22 m pore size).7 EN ISO 13904:2016 (E) DIN EN ISO 13904:2016-06 4.6 Autoclave, capable of being maintained at (110 2) C, (140 10) k
41、Pa (1,4 0,1) bar.A pressure-tight covered dish that may be put into a drying oven adjustable to (110 2) C can be used.4.7 Mechanical shaker or magnetic stirrer.4.8 Vortex mixer.4.9 Glassware filters.4.10 Graduated Erlen meyers flasks: 200 ml, 250 ml, 600 ml.4.11 Volumetric: 100 ml, 500 ml, 1 000 ml
42、(all class A).5 Procedure5.1 Preparation of samples5.1.1 Feeding stuffsGrind the sample to pass through a 0,5 mm sieve. Samples high in moisture shall be either air-dried at a temperature not exceeding 50 C or freeze-dried prior to grinding. Samples with high fat content shall be extracted with ligh
43、t petroleum (3.9) prior to grinding.5.1.2 Commercial pure substances and premixtures containing more than 2 % of tryptophanGrind the sample to pass through to a 0,25 mm sieve and homogenize it well.5.2 Determination of free tryptophan (extract)5.2.1 Feeding stuffsWeigh, to the nearest 1 mg, an appro
44、priate amount (1 g to 5 g) of the prepared sample (5.1.1) into a conical flask. Add 100,0 ml of hydrochloric acid, (3.13) and 5,00 ml of concentrated internal standard solution (3.16). Shake or mix for 60 min using a mechanical shaker or a magnetic stirrer (4.7). Allow the sediment to settle and pip
45、ette 10,0 ml of the supernatant solution into a beaker. Add 5 ml of orthophosphoric acid (3.14). Adjust the pH to 3,0 using sodium hydroxide (3.10). Add sufficient methanol (3.8) to give a concentration of between 10 % and 30 % of methanol in the final volume. Transfer to a volumetric flask of appro
46、priate volume and dilute with water (3.1) to a volume necessary for the chromatography approximately, the same volume as the calibration standard solution (3.17.1).Filter a few millilitres of the solution through a 0,45 m or 0,22 m membrane filter (4.5) before injection on the HPLC column. Proceed t
47、o the chromatography step according to 5.4.Protect the standard solution and extracts against direct sunlight. If it is not possible to analyze the hydrolysates the same day, they may be stored at 5 C for a maximum of three days.5.2.2 Commercial pure substances and premixtures containing more than 2
48、 % of tryptophanWeigh to the nearest of 0,1 mg, 0,5 g to 5 g of well homogenized sample (5.1.2), depending on the expected concentration of tryptophan in the sample (for example, see Annex C) into a 1 000 ml volumetric flask.Fill up to volume with 0,1 mol/l hydrochloric acid (3.13).The mixture is st
49、irred during 30 min on a mechanical shaker or a magnetic stirrer (4.7). Allow the particles to settle.Transfer an aliquot of 2 ml of clear solution into a 100 ml volumetric flask. Add 2 ml of the concentrated internal standard (3.16).8EN ISO 13904:2016 (E) DIN EN ISO 13904:2016-06 Fill up to the mark with 0,1 mol/l hydrochloric acid (3.13).This diluted test solution should have a tryptophan concentration as close as possible as the tryptophan concentration in the calibration standard solution (3.17.2) and the internal standard concentration
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