1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16160:2012Animal feeding stuffs Determination of Hydrocyanicacid by HPLCBS EN 16160:2012 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of
2、EN 16160:2012.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contra
3、ct. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 66995 8ICS 65.120Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority
4、of theStandards Policy and Strategy Committee on 31 March 2012.Amendments issued since publicationDate Text affectedBS EN 16160:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16160 March 2012 ICS 65.120 English Version Animal feeding stuffs - Determination of Hydrocyanic acid by HPLC Alime
5、nts pour animaux - Dosage 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 Europe
6、an 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 Standard exists in three official versions
7、(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 the national standards bodies of Austria, Belg
8、ium, Bulgaria, Croatia, Cyprus, 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. EUROP
9、EAN COMMITTEE FOR STANDARDIZATION 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: EBS EN 16160:2012
10、EN 16160:2012 (E) 2 Contents Page Foreword 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 . 13An
11、nex B (informative) Results of the collaborative study without recovery correction . 14Annex C (informative) Alternative procedure for enzymatic breakdown (8.3.3) and steam distillation (8.3.4). 15Bibliography . 16BS EN 16160:2012EN 16160:2012 (E) 3 Foreword This document (EN 16160:2012) has been pr
12、epared by Technical Committee CEN/TC 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
13、standards shall be withdrawn at the latest 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 p
14、repared under a mandate given to CEN 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, Croat
15、ia, Cyprus, 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. BS EN 16160:2012EN 16160:
16、2012 (E) 4 1 Scope This European 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 m
17、ethod is used outside this range 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
18、 application. For dated references, 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 f
19、eed as cyanoglycosides. Cyanoglycosides 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 hydro
20、xide solution by steam distillation. 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 disti
21、lled or demineralised water or water 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
22、.1.21, minimum 2 units/mg (e.g. 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 endo
23、rsement by CEN of this product. 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. BS EN 16160:2012EN 16160:2012 (E) 5 4.4 Potas
24、sium dihydrogen phosphate, 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
25、 H2O 4.12 NDA, 2,3 Naphthalene 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
26、 and does not constitute an 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: Tran
27、sfer 200 ml 0,1 mol/l H3PO4solution 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
28、 flask of 100 ml containing 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 s
29、olution to a volumetric flask 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
30、 mol/l sodium hydroxide solution (4.16) (use a pH meter (5.12). Transfer to a BS EN 16160:2012EN 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 neare
31、st 0,1 mg, 100 mg KCN (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
32、stored in the refrigerator. 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
33、 calibration standard 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
34、,40 Standard 3 5,00 0,20 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 -glucosida
35、se (4.1) to obtain a final 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 -glucos
36、idase can be tested by 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, concentrat
37、ion c(amygdalin) 0,019 mol/l Dissolve 85,0 mg amygdalin (4.3) in 10 ml water. For every series of analysis, a fresh solution is prepared. BS EN 16160:2012EN 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
38、ml water. Adjust 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
39、 c(PO43-) 0,05 mol/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 Phos
40、phate buffer for 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 f
41、lask of 500 ml. Fill 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 w
42、ith phosphate buffer 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 soluti
43、on (4.24) and dissolve 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 HP
44、LC mobile phase (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
45、 and screw cap glass bottles. 5.2 Volume pipettes, 5 ml and 10 ml BS EN 16160:2012EN 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
46、system for HPLC 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
47、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 detector; analytical reversed phase HPLC column: C18 RP-column preferably 125 mm 4 mm, 5 m; precolumn: C18 RP-column preferably 7,5 mm
48、 4 mm, 5 m. 5.9 Analytical balance, able to measure with an accuracy of 0,1 mg 5.10 Balance, able to measure with an accuracy of 0,01 g 5.11 Mill, capacity to grind to a particle size of 1 mm 5.12 pH meter. 6 Sampling It is important that the laboratory receives a sample that is truly representative
49、 and has not been damaged or changed during transport and storage. Sampling is not part of the method specified in this European Standard. A recommended sampling method is given in EN ISO 6497 1. It is recommended that samples be stored frozen to prevent changes in hydrocyanic acid levels due to endogenous enzyme activity. 7 Preparation of test sample Prepare the test sample in accordance with prEN ISO 6498. Grind the laboratory sample (at least 500 g) so it passes c
