1、BRITISH STANDARDBS EN ISO 13903:2005Animal feeding stuffs Determination of amino acids contentThe European Standard EN ISO 13903:2005 has the status of a British StandardICS 65.120g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36
2、g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 13903:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 24 October 2005 BSI 24 October 2005ISBN 0 580 46218 8National forewordThis British Standard is
3、 the official English language version of EN ISO 13903:2005. It is identical with ISO 13903:2005.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 ob
4、tained on request to its secretary.Cross-referencesThe British Standards which implement 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 E
5、lectronic Catalogue or of British Standards Online.This publication does not purport 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 t
6、o understand the text; present to the responsible international/European committee any 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 pagesThis document compris
7、es a front cover, an inside front cover, the EN ISO title page, the EN ISO foreword page, the ISO title page, pages ii to iv, pages 2 to 17 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Dat
8、e CommentsEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 13903May 2005ICS 65.120English versionAnimal feeding stuffs - Determination of amino acids content(ISO 13903:2005)Aliments des animaux - Dtermination de la teneur enacides amins (ISO 13903:2005)Futtermittel - Bestimmung des Aminosuregeh
9、alts (ISO13903:2005)This European Standard was approved by CEN on 19 April 2005.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bib
10、liographical references concerning such nationalstandards 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 by translationunder the responsibility of
11、a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvi
12、a, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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 2005 CEN A
13、ll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 13903:2005: EEN ISO 13903:2005 Foreword This document (EN ISO 13903:2005) has been prepared by Technical Committee ISO/TC 34 “Agricultural food products“ in collaboration with Technical
14、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 endorsement, at the latest by November 2005, and conflic
15、ting national standards shall be withdrawn at the latest by November 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, Cyprus, Czech Republic, Denmark, Estonia, Fin
16、land, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of ISO 13903:2005 has been approved by CEN as EN ISO 13903:2005
17、 without any modifications. Reference numberISO 13903:2005(E)INTERNATIONAL STANDARDISO13903First edition2005-05-15Animal feeding stuffs Determination of amino acids content Aliments des animaux Dtermination de la teneur en acides amins EN ISO 13903:2005iiiiiContents PageForeword iv1 Scope 12 Princip
18、le . 12.1 Free amino acids. 12.2 Total amino acids 23 Reagents and materials 24 Apparatus. 45 Procedure. 45.1 Preparation of test sample. 45.2 Determination of free amino acids in feeding stuffs and premixtures 45.3 Determination of total amino acids . 55.4 Chromatography . 66 Calculation of results
19、 77 Precision 87.1 Interlaboratory tests . 87.2 Repeatability 87.3 Reproducibility 88 Use of reference materials . 89 Observations on the method . 8Annex A (informative) Results of interlaboratory tests 10Annex B (informative) Examples of chromatograms 15Bibliography . 17EN ISO 13903:2005ivForeword
20、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 interested in a subject for which a technical
21、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 Commission (IEC) on all matters of elect
22、rotechnical 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 technical committees are circulated to the
23、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. ISO shall not be held responsible for ident
24、ifying any or all such patent rights. ISO 13903 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal feeding stuffs.ISO 13903 is based on Commission Directive 98/64/EC of September 19981.EN ISO 13903:20051Animal feeding stuffs Determination of amino acids content
25、1 Scope This International Standard describes the determination of free (synthetic and natural) and total (peptide-bound and free) amino acids in feeding stuffs, using an amino acid analyser or HPLC equipment. It is applicable to the following amino acids: sum of cystine and cysteine; methionine; ly
26、sine; threonine; alanine; arginine; aspartic acid; glutamic acid; glycine; histidine; isoleucine; leucine; phenylalanine; proline; serine; tyrosine; valine. The method does not distinguish between the salts of amino acids, nor does it differentiate between D and Lforms of amino acids. It is not vali
27、d for the determination of tryptophan or hydroxy analogues of amino acids. Limits of quantification depend on the chromatographic equipment, but levels as low as: 0,3 g/kg total lysine; 0,25 g/kg total methionine; 0,35 g/kg total cystine plus cysteine; 0,2 g/kg total threonine; 0,035 g/kg free lysin
28、e; 0,035 g/kg free methionine; and 0,03 g/kg free threonine can typically be analysed. NOTE A lower limit of quantification or detection might be achievable but this is to be validated by the users. 2 Principle 2.1 Free amino acids The free amino acids are extracted with dilute hydrochloric acid. Co
29、-extracted nitrogenous macromolecules are precipitated with sulfosalicylic acid and removed by filtration. The filtered solution is adjusted to pH 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin with photometric detection at 570 nm. EN ISO
30、 13903:2005EN ISO 13903:200522.2 Total amino acids The procedure chosen depends on the amino acids under investigation. Cyst(e)ine and methionine shall be oxidized to cysteic acid and methionine sulphone, respectively, prior to hydrolysis. Tyrosine shall be determined in hydrolysates of unoxidized s
31、amples. All the other amino acids listed in Clause 1 may be determined in either the oxidized or unoxidized sample. Oxidation is performed at 0 C with a performic acid/phenol mixture. Excess oxidation reagent is decomposed with sodium disulfite. The oxidized or unoxidized sample is hydrolysed with h
32、ydrochloric acid (c 6 mol/l) for 23 h. The hydrolysate is adjusted to pH 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin, using photometric detection at 570 nm (440 nm for proline). 3 Reagents and materials Use only reagents of recognized
33、analytical grade, unless otherwise specified. 3.1 Water, double distilled water or water of equivalent quality shall be used (conductivity 10 PS). 3.2 Hydrogen peroxide, w 30 %. 3.3 Formic acid, w 98 % to 100 %. 3.4 Hydrochloric acid, density approximately 1,19 g/ml. 3.5 2,2-Thiodiethanol (thiodigly
34、col) 3.6 Light petroleum, boiling rate 40 C to 60 C 3.7 Norleucine, or any other compound suitable for use as internal standard. 3.8 Nitrogen gas ( 10 parts per million oxygen). 3.9 Amino acids.3.9.1 Standard substances listed under Clause 1. Use pure compounds containing no water of crystallization
35、. Dry under vacuum over P2O5or H2SO4for 1 week prior to use. 3.9.2 Cysteic acid.3.9.3 Methionine sulfone.3.10 Sodium hydroxide solution I, c 7,5 mol/l. Dissolve 300 g of NaOH (3.6) in water and make up to 1 l. 3.11 Sodium hydroxide solution II, c 1 mol/l. Dissolve 40 g of NaOH in water (3.1) and mak
36、e up to 1 l. 3.12 Formic acid-phenol solution.Mix 889 g of formic acid (3.3) with 111 g of water (3.1) and add 4,73 g of phenol. EN ISO 13903:200533.13 Hydrolysis mixture, c 6 mol/l HCl containing 1 g of phenol per litre. Add 1 g of phenol to 492 ml of HCl (3.4) and make up to 1 l with water (3.1).
37、3.14 Extraction mixture, c 0,1 mol/l HCl containing 2 % thiodiglycol. Take 8,2 ml of HCl (3.4), dilute with approximately 900 ml of water (3.1). Add 20 ml of thiodiglycol (3.5) and make up to 1 l with water. Do not mix 3.4 and 3.5 directly. 3.15 5-Sulfosalicylic acid, E 6 %. Dissolve 60 g of 5-sulfo
38、salicylic acid dihydrate in water (3.1) and make up to 1 l with water. 3.16 Oxidation mixture (performic acid-phenol). Mix 0,5 ml of hydrogen peroxide (3.2) with 4,5 ml of formic acid-phenol solution (3.12) in a small beaker. Incubate at between 20 C and 30 C for 1 h in order to form performic acid,
39、 then cool in an ice-water bath (15 min) before adding to the sample. Avoid contact with skin and wear protective clothing.3.17 Citrate buffer, c 0,2 mol/l Na+, pH 2,20. Dissolve 19,61 g of sodium citrate dihydrate, 5 ml of thiodiglycol (3.5), 1 g of phenol and 16,50 ml of HCl (3.4) in approximately
40、 800 ml of water (3.1). Adjust the pH to 2,20. Make up to 1 l with water. 3.18 Elution buffers, prepared according to conditions for the analyser used (4.9). 3.19 Ninhydrin reagent, prepared according to conditions for the analyser used (4.9). 3.20 Standard solutions of amino acids.These solutions s
41、hall be stored below 5 C. 3.20.1 Stock standard solution of amino acids (3.9.1), c 2,5 Pmol/ml of each in hydrochloric acid. These may be obtained commercially. 3.20.2 Stock standard solution of cysteic acid and methionine sulfone, c 1,25 Pmol/ml. Dissolve 0,211 5 g of cysteic acid (3.9.2) and 0,226
42、 5 g of methionine sulphone (3.9.3) in citrate buffer (3.17) in a 1 l graduated flask and make up to mark with citrate buffer. Store below 5 C for not more than 12 months. This solution shall not be used if the stock standard solution (3.20.1) contains cysteic acid and methionine sulfone. 3.20.3 Sto
43、ck standard solution of the internal standard e.g. norleucine, c 20 Pmol/ml. Dissolve 0,656 0 g of norleucine (3.7) in citrate buffer (3.17) in a graduated flask and make up to 250 ml with citrate buffer. Store below 5 C for no more than 6 months. 3.20.4 Calibration solution of standard amino acids,
44、 for use with hydrolysates, c 0,1 Pmol/ml of cysteic acid and methionine sulfone and c 0,2 Pmol/ml of the other amino acids. Dissolve 2,2 g of sodium chloride in 100 ml beaker with 30 ml of citrate buffer (3.17). Add 4,00 ml of stock standard solution of amino acids (3.20.1), 4,00 ml of stock standa
45、rd solution of cysteic acid and methionine sulfone (3.20.2), and 0,50 ml of stock standard solution of internal standard (3.20.3) if used. Adjust the pH to2,20 with sodium hydroxide (3.11). Transfer quantitatively to a 50 ml graduated flask and make up to the mark with citrate buffer (3.17) and mix.
46、 Store below 5 C for not more than 3 months. See also 9.1. 43.20.5 Calibration solution of standard amino acids, for use with hydrolysates prepared according to 5.3.3.2 and for use with extracts (5.2). Prepare the calibration solution according to 3.20.4 but omitting sodium chloride. Store below 5 C
47、 for not more than 3 months. 4 Apparatus Usual laboratory apparatus and, in particular, the following. 4.1 Round bottomed flask, of capacity 100 ml or 250 ml, fitted with a reflux condenser. 4.2 Borosilicate glass bottle, of capacity 100 ml, with screw cap with rubber/teflon liner (e.g. Duran, Schot
48、t) for use in the oven. 4.3 Oven, with forced ventilation and a temperature regulator, with an accuracy better than r 2 C. 4.4 pH-meter, reading to three decimal places. 4.5 Membrane filter, 0,2 Pm. 4.6 Centrifuge.4.7 Rotary vacuum evaporator.4.8 Mechanical shaker or magnetic stirrer.4.9 Amino acid
49、analyser or HPLC equipment with ion exchange column, device for ninhydrin, post-column derivatization and photometric detector The column is filled with sulfonated polystyrene resins capable of separating the amino acids from each other and from other ninhydrin-positive materials. The flow in the buffer and ninhydrin lines is provided by pumps having a flow stability of r 0,5 % in the period covering both the standard calibration
