EN 16160:2012

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Bestimmung von Blausäure mittels HPLCAliments des animaux - Dosage de l'acide cyanhydrique par CLHPAnimal feeding stuffs - Determination of Hydrocyanic acid by HPLC65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16160:2012SIST EN 16160:2012en,fr,de01-september-2012SIST EN 16160:2012SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16160
March 2012 ICS 65.120 English Version
Animal feeding stuffs - Determination of Hydrocyanic acid by HPLC
Aliments pour animaux - Dosage de l'acide cyanhydrique par CLHP
Futtermittel - Bestimmung von Blausäure 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 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). 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.
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Results of the collaborative study . 13Annex 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 . 16 SIST EN 16160:2012

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 ß-glucosidase at 38 °C to release hydrocyanic acid. Hydrocyanic acid is collected in a potassium hydroxide 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 distilled 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 3.2.1.21, 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 endorsement 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. SIST EN 16160:2012

Amount work solutionml Concentration µg CN-/ ml Standard 1 25,00 1,00 Standard 2 10,00 0,40 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 ß-glucosidase solution Weigh such an amount of ß-glucosidase (4.1) to obtain a final concentration of 200 IU per ml and dissolve in water. NOTE The amount of ß-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 ß-glucosidase 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, concentration 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. SIST EN 16160:2012

The solution remains stable for 3 months at room temperature. 4.23 Phosphate buffer for HPLC mobile phase, concentration 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 Phosphate 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 flask 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 with 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 solution (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 HPLC 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 and screw cap glass bottles. 5.2 Volume pipettes, 5 ml and 10 ml SIST EN 16160:2012
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