1、May 2012 Translation by DIN-Sprachendienst.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
2、.120!$|=“1892626www.din.deDDIN EN 16160Animal feeding stuffs Determination of Hydrocyanic acid by HPLCEnglish translation of DIN EN 16160:2012-05Futtermittel Bestimmung von Blausure mittels HPLCEnglische bersetzung von DIN EN 16160:2012-05Aliments pour animaux Dosage de lacide cyanhydrique par CLHPT
3、raduction anglaise de DIN EN 16160:2012-05www.beuth.deDocument comprises 18 pagesIn case of doubt, the German-language original shall be considered authoritative.05.12 DIN EN 16160:2012-05 2 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committ
4、ee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committe
5、e NA 057-03-03 AA Futtermittel. The DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2:
6、 Basic method for the determination of repeatability and reproducibility of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16160 March 2012 ICS 65.120 English Version Animal feeding stuffs - Determination of Hydrocyanic acid by HPLC Aliments pour animaux - Dosage
7、de lacide cyanhydrique par CLHP Futtermittel - Bestimmung von Blausure mittels HPLC This European Standard was approved by CEN on 14 January 2012. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of
8、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 Standard exists in three official versions (English, French, German).
9、 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 the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cy
10、prus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARD
11、IZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16160:2012: EEN 16160:2012 (E) 2 Contents Page Foreword
12、 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Sampling .87 Preparation of test sample 88 Procedure .99 Calculation of results . 1110 Precision 1211 Test report . 12Annex A (informative) Results of the collaborative study . 13Annex B (informative) Results of the collabo
13、rative study without recovery correction . 14Annex C (informative) Alternative procedure for enzymatic breakdown (8.3.3) and steam distillation (8.3.4). 15Bibliography . 16DIN EN 16160:2012-05 EN 16160:2012 (E) 3 Foreword This document (EN 16160:2012) has been prepared by Technical Committee CEN/TC
14、327 “Animal feeding stuffs”, 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 September 2012, and conflicting national standards shall be withdrawn at the l
15、atest by September 2012. 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 all such patent rights. This document has been prepared under a mandate given to CEN
16、by the European Commission and the European Free Trade Association. 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 Republic, Denmark,
17、Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. DIN EN 16160:2012-05 EN 16160:2012 (E) 4 1 Scope This European
18、 Standard is applicable to the quantitative analysis of (bound and free) hydrocyanic acid (HCN) in feed materials of plant origin and compound feed by High Performance Liquid Chromatography (HPLC). The method is validated from 10 mg HCN/kg to 350 mg HCN/kg. When the method is used outside this range
19、 it should be validated at least within the laboratory. A limit of quantification of 2 mg HCN/kg should normally be obtained. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated referenc
20、es, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. prEN ISO 6498, Animal feeding stuffs Guidelines for sample preparation (ISO/DIS 6498) 3 Principle Hydrocyanic acid occurs in feed as cyanoglycosides. Cyanogly
21、cosides are extracted from feed with an acid solution. After incubation with acid, the pH is adjusted to a value between 5,9 and 6,0 and cyanoglycosides are treated by -glucosidase at 38 C to release hydrocyanic acid. Hydrocyanic acid is collected in a potassium hydroxide solution by steam distillat
22、ion. Subsequently, cyanide is derivatized with taurine and 2,3 napthylene dicarboxy aldehyde (NDA) to form a fluorescent complex. The cyanide complex is analyzed by HPLC with fluorescence detection. 4 Reagents Use only reagents of recognized analytical grade and distilled or demineralised water or w
23、ater of equivalent quality, unless otherwise specified. WARNING Use all solvents and solutions in a fume hood. Wear safety glasses, protective clothing, and avoid skin contact. Take special care of the waste containing HCN or CN-. 4.1 -glucosidase from almonds, EC 3.2.1.21, minimum 2 units/mg (e.g.
24、Sigma G-0395)1)4.2 Potassium cyanide, KCN 4.3 Amygdalin (e.g. Sigma A-6005)2)1)Sigma G-0395 is an example of a suitable product available commercially. This information is given for the convenience of the users of this European Standard and does not constitute an endorsement by CEN of this product.
25、2)Sigma A-6005 is an example of a suitable product available commercially. This information is given for the convenience of the users of this European Standard and does not constitute an endorsement by CEN of this product. DIN EN 16160:2012-05 EN 16160:2012 (E) 5 4.4 Potassium dihydrogen phosphate,
26、KH2PO44.5 di-Potassium hydrogen phosphate, K2HPO44.6 Sodium hydroxide, NaOH 4.7 Methanol, CH3OH, HPLC grade 4.8 Sodium acetate trihydrate, CH3COONa.3H2O 4.9 Acetic acid, CH3COOH 4.10 Orthophosphoric acid, H3PO4, 85 % (15 mol/l) 4.11 Sodium meta borate tetrahydrate, BNaO2.4 H2O 4.12 NDA, 2,3 Naphthal
27、ene dicarboxy aldehyde, C12H8O24.13 Taurine, C2H7NO3S 4.14 EDTA, Titriplex III - C10H14N2Na2O8.2H2O NOTE Merck art. No. 1.08418.0250 is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an
28、 endorsement by CEN of this product. 4.15 Orthophosphoric acid solutions, concentrations c(H3PO4) = 0,1 mol/l and 0,02 mol/l: 0,1 mol/l: Add 6,9 ml H3PO485 % (4.10) to a volumetric flask of 1 000 ml containing 500 ml water, fill to mark with water and mix; 0,02 mol/l: Transfer 200 ml 0,1 mol/l H3PO4
29、solution to a volumetric flask of 1 000 ml, fill to mark with water and mix. Prepare these solutions for every series. 4.16 Sodium hydroxide solutions, concentrations c(NaOH) = 1,0 mol/l, 0,1 mol/l and 0,01 mol/l: 1,0 mol/l: Add 4,0 g sodium hydroxide (4.6) to a volumetric flask of 100 ml containing
30、 50 ml water, dissolve, cool to room temperature and fill to mark with water and mix; 0,1 mol/l: Transfer 100 ml 1,0 mol/l sodium hydroxide solution to a volumetric flask of 1 000 ml, fill to mark with water and mix; 0,01 mol/l: Transfer 100 ml 0,1 mol/l sodium hydroxide solution to a volumetric fla
31、sk of 1 000 ml, fill to mark with water and mix. These solutions are stable for three months. 4.17 EDTA solution, concentration = 0,20 mol/l Weigh 37,2 g EDTA (4.14) in a 500 ml beaker, add 300 ml water, dissolve and then adjust the pH to 7,0 - 8,0 with a 1,0 mol/l and 0,1 mol/l sodium hydroxide sol
32、ution (4.16) (use a pH meter (5.12). Transfer to a DIN EN 16160:2012-05 EN 16160:2012 (E) 6 volumetric flask of 500 ml; fill to mark with water and mix. The solution remains stable for 1 month when stored at room temperature. 4.18 KCN standard stock solution Weigh, to the nearest 0,1 mg, 100 mg KCN
33、(4.2) in a 100 ml volumetric flask. Add 50 ml 0,01 mol/l sodium hydroxide solution (4.16) and dissolve the KCN, fill to mark with 0,01 mol/l sodium hydroxide (4.16) and mix. This is the stock solution of 400 g cyanide ions (CN-) per ml. The solution is stable for 3 months when stored in the refriger
34、ator. 4.19 KCN working standard solutions Add, using a mechanic pipette (5.4), 1,00 ml KCN standard stock solution (4.18) in a 100 ml volumetric flask and fill to the mark with 0,01 mol/l sodium hydroxide solution (4.16) and mix; this is the work solution of 4 g CN-/ml. Prepare calibration standard
35、solutions by following schedule (Table 1) in 100 ml volumetric flasks and using volume pipettes (5 ml and 10 ml) (5.2) and/or a mechanic pipette (5.4): Table 1 calibration standard solutions Amount work solutionml Concentration g CN-/ ml Standard 1 25,00 1,00 Standard 2 10,00 0,40 Standard 3 5,00 0,
36、20 Standard 4 2,50 0,10 Standard 5 1,25 0,05 Standard 6 0,00 0,00 Use 0,01 mol/l sodium hydroxide solution (4.16) to fill the volumetric flasks to the mark. For every series of analysis, fresh standards are prepared. 4.20 -glucosidase solution Weigh such an amount of -glucosidase (4.1) to obtain a f
37、inal concentration of 200 IU per ml and dissolve in water. NOTE The amount of -glucosidase weighed depends on the activity of the enzyme, given by the manufacturer of the enzyme. E.g. when the activity is 2 IU/mg enzyme, 100 mg enzyme/ml water is weighed. A new batch of -glucosidase can be tested by
38、 analyzing a sample with a known amount of hydrocyanic acid e.g. a reference sample. If the enzyme is active enough, the expected amount of hydrocyanic acid should be measured. For every series of analysis, a fresh solution is prepared. 4.21 Amygdalin spike solution, concentration c(amygdalin) 0,019
39、 mol/l Dissolve 85,0 mg amygdalin (4.3) in 10 ml water. For every series of analysis, a fresh solution is prepared. DIN EN 16160:2012-05 EN 16160:2012 (E) 7 4.22 Sodium acetate solution, concentration c(CH3COONa.3H2O) 0,75 mol/l Dissolve 100 g sodium acetate trihydrate (4.8) in 800 ml water. Adjust
40、pH to 7,9 with diluted (20x) acetic acid (4.9). Use a pH meter to control (5.12). Transfer to a volumetric flask of 1 000 ml. Fill to the mark with water and mix. The solution remains stable for 3 months at room temperature. 4.23 Phosphate buffer for HPLC mobile phase, concentration c(PO43-) 0,05 mo
41、l/l Dissolve 3,40 g potassium dihydrogen phosphate (4.4) and 4,35 g di-potassium hydrogen phosphate (4.5) in 100 ml water in a beaker. Transfer to a volumetric flask of 1 000 ml, fill to mark with water and mix. The solution remains stable for 3 months at room temperature. 4.24 Phosphate buffer for
42、NDA/taurine solution, concentration c(PO43-) 0,1 mol/l, concentration c(BNaO2) 0,025 mol/l Dissolve 3,40 g potassium dihydrogen phosphate (4.4), 4,35 g di-potassium hydrogen phosphate (4.5) and 3,45 g sodium meta borate tetrahydrate (4.11) in 100 ml water. Transfer to a volumetric flask of 500 ml. F
43、ill to the mark with water and mix. This solution remains stable for 3 months when stored in the refrigerator. 4.25 NDA solution; concentration c(NDA) 0,002 mol/l Weigh 36,8 mg NDA (4.12) in a 100 ml volumetric flask. Add 40 ml methanol (4.7) and dissolve the NDA. Fill to the mark with phosphate buf
44、fer for NDA/taurine solution (4.24) and mix. This solution remains stable 3 months when stored in the refrigerator. 4.26 Taurine solution, concentration c(taurine) 0,05 mol/l Weigh 0,626 g taurine (4.13) in a 100 ml volumetric flask. Add 40 ml phosphate buffer for NDA/taurine solution (4.24) and dis
45、solve taurine. Fill to the mark with phosphate buffer for NDA/taurine solution (4.24) and mix. This solution remains stable for 3 months when stored in the refrigerator. 4.27 HPLC mobile phase Weigh 675 g methanol (4.7) in a flask of 2 000 ml and add 1 000 ml phosphate buffer for HPLC mobile phase (
46、4.23), mix and cool to room temperature. Filter the mobile phase using a filtrate system (5.6). The solution remains stable for 3 months when stored at room temperature. 4.28 Liquid nitrogen. 5 Apparatus 5.1 Common laboratory glassware, such as graduated cylinders, volumetric flasks and screw cap gl
47、ass bottles. 5.2 Volume pipettes, 5 ml and 10 ml DIN EN 16160:2012-05 EN 16160:2012 (E) 8 5.3 One or two neck round bottom flask of 1 000 ml or a sample tube of 500 ml 5.4 Mechanic pipette, 100 l to 1 000 l, 1 000 l to 5 000 l and 5 000 l to 10 000 l 5.5 Oven, at 38 C 1 C 5.6 Filtration system for H
48、PLC mobile phase with a membrane filter 0,45 m 5.7 Steam distillation unit NOTE Landgraaf no. AS136721111 and Buchi K-350 are examples of suitable products available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. 5.8 HPLC system: Analytical pump; column oven; autosampler with a 10 l sample loop; optional: with cooled tray; HPLC Fluorescence d
