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1、BRITISH STANDARD BS EN ISO 13904:2005 Animal feeding stuffs Determination of tryptophan content The European Standard EN ISO 13904:2005 has the status of a British Standard ICS 65.120 ? Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS EN I
2、SO 13904:2005 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 13 June 2005 BSI 13 June 2005 ISBN 0 580 46088 6 National foreword This British Standard is the official English language version of EN ISO 13904:2005. It is identical with ISO 139
3、04:2005. The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal Feeding Stuffs, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which impleme
4、nt international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport
5、to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee an
6、y enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN ISO title page, the EN ISO foreword
7、 page, the ISO title page, pages ii to iv, pages 1 to 9 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:
8、00 2006, Uncontrolled Copy, (c) BSI EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 13904 May 2005 ICS 65.120 English version Animal feeding stuffs - Determination of tryptophan content (ISO 13904:2005) Aliments des animaux - Dtermination de la teneur en tryptophane (ISO 13904:2005) Futterm
9、ittel - Bestimmung des Tryptophangehalts (ISO 13904:2005) This European Standard was approved by CEN on 4 April 2005. 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 standard without a
10、ny alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made b
11、y translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Gr
12、eece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Cent
13、re: rue de Stassart, 36 B-1050 Brussels 2005 CENAll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 13904:2005: E Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Foreword This
14、document (EN ISO 13904:2005) has been prepared by Technical Committee ISO/TC 34 “Agricultural 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
15、 the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November 2005, and conflicting national standards shall be withdrawn at the latest by November 2005. According to the CEN/CENELEC Internal Regulations, the national standards organizati
16、ons of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Sloveni
17、a, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of ISO 13904:2005 has been approved by CEN as EN ISO 13904:2005 without any modifications. EN ISO 13904:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Refere
18、nce number ISO 13904:2005(E) INTERNATIONAL STANDARD ISO 13904 First edition 2005-05-01 Animal feeding stuffs Determination of tryptophan content Aliments des animaux Dtermination de la teneur en tryptophane EN ISO 13904:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2
19、006, Uncontrolled Copy, (c) BSI ii EN ISO 13904:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2006, Uncontrolled Copy, (c) BSI iii Contents Page Forewordiv 1Scope1 2Principle.1 3Reagents and materials1 4Apparatus.2 5Procedure.3 5.1Preparation of samples 3 5.2Determin
20、ation of free tryptophan (extract)3 5.3Determination of total tryptophan (hydrolysate).3 5.4HPLC determination4 6Calculation of results4 7Precision5 7.1Interlaboratory test .5 7.2Repeatability5 7.3Reproducibility5 8Test report5 Annex A (informative) Results of an interlaboratory test.6 Annex B (info
21、rmative) Observations on the method.8 Bibliography .9 EN ISO 13904:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2006, Uncontrolled Copy, (c) BSI iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodi
22、es (ISO member bodies). The work of preparing 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
23、, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the IS
24、O/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the
25、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. ISO shall not be held responsible for identifying any or all such patent rights. ISO 13904 was prepared by Technical Committee ISO/TC 34, Food products,
26、 Subcommittee SC 10, Animal feeding stuffs. EN ISO 13904:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 1 Animal feeding stuffs Determination of tryptophan content 1 Scope This International Standard describes determination of the tota
27、l and free tryptophan content in feeding stuffs (e.g. complete and complementary feeds, supplementary feeds, raw materials, ingredients, premixtures and concentrates). It does not distinguish between D- and L-forms. 2 Principle For the determination of the total tryptophan, the sample is hydrolysed
28、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. The tryptophan an
29、d the internal standard in the hydrolysate or in the extract are determined by reversed phase C18 HPLC with fluorescence detection. 3 Reagents and materials Use only reagents of recognized analytical grade, unless otherwise specified. 3.1 Double-distilled water, or water of equivalent purity (conduc
30、tivity 10 S/cm). 3.2 Standard substance: tryptophan (purity/content W 99 %) dried under vacuum over phosphorus pentoxide. 3.3 Internal standard substance: ? ? ? ?-methyltryptophan (purity/content W 99 %), dried under vacuum over phosphorus pentoxide. 3.4 Barium hydroxide octahydrate. Care should be
31、taken not to expose the Ba(OH)2?8H2O excessively to air in order to avoid formation of BaCO3, which could disturb the determination (see observation in B.3). 3.5 Sodium hydroxide. 3.6 Orthophosphoric acid, w = 85 %. 3.7 Concentrated hydrochloric acid,?20 = 1,19 g/ml. 3.8 Methanol, HPLC grade. 3.9 Li
32、ght petroleum, boiling range 40 ?C to 60 ?C. 3.10 Sodium hydroxide solution, c = 1 mol/l. Dissolve 40,0 g of NaOH (3.5) in water (3.1) and make up to 1 l with water (3.1). EN ISO 13904:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 2 3
33、.11 Hydrochloric acid, c = 6 mol/l. Take 492 ml of HCl (3.7) and make up to 1 l with water (3.1). 3.12 Hydrochloric acid, c = 1 mol/l. Take 82 ml of HCl (3.7) and make up to 1 l with water (3.1). 3.13 Hydrochloric acid, c = 0,1 mol/l. Take 8,2 ml of HCl (3.7) and make up to 1 l with water (3.1). 3.1
34、4 Orthophosphoric acid, c = 0,5 mol/l. Take 34 ml of orthophosphoric acid (3.6) and make up to 1 l with water (3.1). 3.15 Concentrated tryptophan solution (3.2), c = 0,000 5000 g/ml. In a 500 ml volumetric flask, dissolve 0,25 g of tryptophan (3.2) (weighed to the nearest 0,1 mg) in hydrochloric aci
35、d (3.13) and make up to the mark with hydrochloric acid (3.13). Store at 18 ?C for a maximum of four weeks. 3.16 Concentrated internal standard solution, c = 0,000 54 g/ml. In a 500 ml volumetric flask, dissolve 0,27 g of ?-methyltryptophan (3.3) (weighed to the nearest 0,1 mg) in hydrochloric acid
36、(3.13) and make up to the mark with hydrochloric acid (3.13). Store at 18 ?C for a maximum of four weeks. 3.17 Calibration standard solution of tryptophan and internal standard. Take 2,00 ml of the concentrated tryptophan solution (3.15) and 2,00 ml of concentrated internal standard solution (?-meth
37、yltryptophan) (3.16). Dilute with water (3.1) and methanol (3.8) to approximately the same volume and to approximately the same concentration of methanol (10 % to 30 %) as the finished hydrolysate. This solution shall be prepared freshly before use. Protect from direct sunlight during preparation. 3
38、.18 Ethanolamine ? 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). 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
39、(3.19). Adjust the pH to 5,00 using ethanolamine (3.18). Make up to 1 000 ml with water (3.1). 4 Apparatus Usual 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, or
40、 equivalent. 4.3 pH-meter. EN ISO 13904:2005 Licensed Copy: sheffieldun sheffieldun, na, Sun Nov 19 13:52:01 GMT+00:00 2006, Uncontrolled Copy, (c) BSI 3 4.4 Polypropylene flask, of capacity 125 ml, with wide neck and screw cap. 4.5 Membrane filter, 0,45 m. 4.6 Autoclave, capable of being maintained
41、 at (110 ? 2) ?C, (140 ? 10) kPa (1,4 ? 0,1) bar. A pressure-tight covered dish that may be put into a drying oven adjustable to (110 ? 2) ?C may be used. 4.7 Mechanical shaker or magnetic stirrer. 4.8 Vortex mixer. 5 Procedure 5.1 Preparation of samples Grind the sample to pass through a 0,5 mm sie
42、ve. 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 light petroleum (3.9) prior to grinding. 5.2 Determination of free tryptophan (extract) Weigh, to the nearest 1 mg, an
43、appropriate amount (1 g to 5 g) of the prepared sample (5.1) 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
44、pipette 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 ap
45、propriate 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). Filter a few millilitres of the solution through a 0,45 m membrane filter (4.5) before injection on the HPLC column. Proceed to the chr
46、omatography step according to 5.4. Protect the standard solution and extracts against direct sunlight. If it is not possible to analyse the extracts the same day, the extracts may be stored at 5 ?C for a maximum of three days. 5.3 Determination of total tryptophan (hydrolysate) Weigh, to the nearest
47、 0,2 mg, from 0,1 g to 1 g of the prepared sample (5.1) into the polypropylene flask (4.4). The weighed test portion should have a nitrogen content of about 10 mg. Add 8,4 g of barium hydroxide octahydrate (3.4) and 10 ml of water (3.1). Mix on a vortex mixer (4.8) or magnetic stirrer (4.7). Leave t
48、he Teflon-coated magnet in the mixture. Wash down the walls of the vessel with 4 ml of water (3.1). Put on the screw cap and close the flask loosely. Transfer to an autoclave (4.6) which contains boiling water, and steam for 30 min to 60 min. Close the autoclave and autoclave at (110 ? 2) ?C for 20
49、h. Before opening the autoclave, reduce the temperature to just under 100 ?C. In order to avoid crystallization of Ba(OH)28H2O, add to the warm mixture 30 ml of water (3.1) which is at room temperature. Shake or stir gently. Add 2,00 ml of concentrated internal standard solution (?-methyltryptophan) (3.16). Cool the vessel in a water/ice bath for 15 min. Then, add 5 ml of orthophosphoric acid (3.14). Keep the vessel in the cooling bath and neutralize with 6 mol/l HCl (3.11) whilst stirring