BS EN 15792-2009 动物饲料.动物饲料中玉米烯酮的测定.高效液相色谱法结合荧光检测和免疫亲和柱净化.pdf

《BS EN 15792-2009 动物饲料.动物饲料中玉米烯酮的测定.高效液相色谱法结合荧光检测和免疫亲和柱净化.pdf》由会员分享，可在线阅读，更多相关《BS EN 15792-2009 动物饲料.动物饲料中玉米烯酮的测定.高效液相色谱法结合荧光检测和免疫亲和柱净化.pdf（18页珍藏版）》请在三一文库上搜索。

1、 W - - - - - - - 77 - - - - 7 7 - - 7 - -$- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - W W D - w - - - - EN 15792:2009 (E) 2 Contents Page Foreword . 3 1 Scope 4 2 Normative references 4 3 Principle 4 4 Reagents . 4 5 Apparatus . 7 6 Procedures . 8 7 HPLC determination 9

2、 8 Calculations . 10 9 Precision . 11 10 Test report 11 Annex A (informative) Precision data 13 Bibliography 15 EN 15792:2009 (E) 3 Foreword This document (EN 15792:2009) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretariat of which is held by NEN. This Europe

3、an Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by March 2010, and conflicting national standards shall be withdrawn at the latest by March 2010. Attention is drawn to the possibility that some of the elements

4、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 by the European Commission and the European Free Trade Association. According to the CEN/

5、CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Lu

6、xembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. EN 15792:2009 (E) 4 1 Scope This Standard is applicable to the determination of zearalenone in animal feed at concentrations from 30 g/kg to 3 000 g/kg. 2 Normative

7、 references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies: EN ISO 3696, Water for analytical lab

8、oratory use - Specification and test methods (ISO 3696:1987) 3 Principle Zearalenone is extracted from the commodity using organic solvent. The solvent extract is then diluted with phosphate buffered saline to give an aqueous extract which is applied to an immunoaffinity column containing antibodies

9、 specific for zearalenone. The analyte is isolated, purified and concentrated on the column and removed from the antibodies with elution solvent. Zearalenone is quantitatively determined by high performance liquid chromatography (HPLC) with fluorescence detection. 4 Reagents During the analysis, unl

10、ess otherwise stated, use only reagents of recognised analytical grade and only distilled water or water of grade 1 as defined in EN ISO 3696. Solvents shall be of quality for HPLC analysis. 4.1 Acetonitrile WARNING Acetonitrile is hazardous and handling shall be carried out inside a fume cupboard.

11、Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. 4.2 Methanol, technical grade WARNING Methanol is hazardous and handling shall be carried out inside a fume cupboard. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. Samples shall be blended using an expl

12、osion proof blender. 4.3 Methanol, HPLC grade WARNING Methanol is hazardous and handling shall be carried out inside a fume cupboard. Appropriate safety equipment (lab coat, goggles, gloves) shall be worn. Samples shall be blended using an explosion proof blender. 4.4 Sodium chloride 4.5 Disodium hy

13、drogen orthophosphate 4.6 Potassium dihydrogen phosphate EN 15792:2009 (E) 5 4.7 Potassium chloride 4.8 Hydrochloric acid (32%) WARNING Hydrochloric acid is hazardous and handling shall be carried out with the necessary precaution inside a fume cupboard. Appropriate safety equipment (lab coat, goggl

14、es, gloves) shall be worn. 4.9 Phosphate buffered saline (PBS) Dissolve 8 g sodium chloride (4.4), 1,2 g disodium hydrogen orthophosphate (4.5), 0,2 g potassium dihydrogen phosphate (4.6) and 0,2 g potassium chloride (4.7) in 1 l of distilled water. Adjust the pH to 7,4 with hydrochloric acid (4.8).

15、 NOTE Commercially available phosphate buffered saline tablets with equivalent properties may be used. 4.10 Extraction solvent, methanol/water = 75+25 parts by volume Mix 75 parts per volume methanol (4.2) with 25 parts per volume of water 4.11 Washing solvent, methanol/PBS = 15 + 85 parts by volume

16、 Mix 15 parts per volume methanol (4.3) with 85 parts per volume PBS (4.9). 4.12 Injection solvent for HPLC analysis, methanol/water = 50+50 parts by volume Mix 50 parts per volume methanol (4.3) with 50 parts per volume water. 4.13 HPLC mobile phase, methanol/water = 75+25 parts by volume Mix 75 pa

17、rts per volume methanol (4.3) and 25 parts per volume water. Mix well and degas. 4.14 Zearalenone, minimum purity of 98 % WARNING Zearalenone is an oestrogenic compound and shall be treated with extreme caution. Gloves and safety glasses shall be worn at all times and all standard and sample prepara

18、tion stages shall be carried out in a fume cupboard. 4.15 Zearalenone (ZON) stock solution 10 g Zearalenone per millilitre of Acetonitrile. May be prepared by the following: Add 4,0 ml of acetonitrile (4.1) to 5 mg of zearalenone (4.14) for a standard solution of 1,25 mg/ml. Dilute 800 l of the 1,25

19、 mg/ml standard solution to 5,0 ml with acetonitrile (4.1) for a standard solution of 200 g/ml. Dilute 250 l of the 200 g/ml standard solution to 5,0 ml of acetonitrile (4.1) to create the stock solution of 10 g/ml. To determine the exact concentration record the absorption curve of this 10 g/ml sto

20、ck solution with the spectrophotometer (5.26) in the range of 200 nm to 300 nm in a 1 cm quartz cell with acetonitrile (4.1) as reference. Determine the absorption of the second maximum at = 274 nm. Calculate the mass concentration of zearalenone, zon, in micrograms per millilitre using equation 1:

21、d MA zon 100 max (1) EN 15792:2009 (E) 6 where: Amax is the absorption determined at the second maximum of the absorption curve (here: at 274 nm) M is the molar mass of zearalenone (M = 318,4 g/mol); is the molar absorption coefficient of zearalenone in acetonitrile (4.1) (here: 1 262 m2/mol 1 m2/mo

22、l 1); d is the optical path length of the quartz cell in centimetres (here: 1 cm). Store standard solutions at below 18 C. 4.16 ZON spiking solution The calibrated stock solution, see (4.15). This solution is stable for 2 months if stored at below 18 C. 4.17 ZON working solution Transfer an aliquot

23、of the calibrated stock solution (4.15), equivalent to 10 g of ZON, into a volumetric flask (5.11). Add acetonitrile (4.1) to make the total volume up to 5 ml. This is a 2 g/ml working solution. This solution is stable for 2 months if stored at below 18 C. 4.18 ZON Calibration solutions for HPLC Pre

24、pare 5 HPLC calibration solutions in separate 10 ml volumetric flasks (5.11) by pipetting the volumes shown in Table 1 below. Make up each standard to volume (10 ml) with injection solvent for HPLC (4.12). Table 1 Preparation of calibration solutions Calibration solution Volume of ZON working soluti

25、on (4.17) l Mass concentration of calibration solution ng/ml 1 50 10 2 250 50 3 450 90 4 650 130 5 850 170 The procedures above for standard preparation can be performed either by the use of pipettes or calibrated glassware as available. 4.19 Immunoaffinity column The immunoaffinity (IA) column cont

26、ains antibodies raised against zearalenone. The column shall have a capacity of not less than 1 500 ng of zearalenone and a recovery of not less than 70% when 75 ng of zearalenone are applied in 10 ml of washing solvent (4.11). EN 15792:2009 (E) 7 5 Apparatus Usual laboratory equipment and in partic

27、ular the following: 5.1 Analytical balance, with d=0,001 g or better 5.2 Horizontal or vertical shaker 5.3 Homogeniser/ High Speed Blender 5.4 Vortex Mixer, or equivalent 5.5 pH meter 5.6 Mill (various screens) 5.7 Tumble mixer 5.8 Glass vials, various sizes 5.9 Graduated pipettes, with volumes of 5

28、 ml and 50 ml 5.10 Graduated cylinders with and without stoppers, with volumes of 5 ml and 250 ml 5.11 Volumetric flasks, with volumes of 3 ml, 5 ml and 10 ml 5.12 Beaker, 250 ml 5.13 Conical or screw cap flasks, with volumes of 100 ml and 250 ml to 500 ml 5.14 Glass funnels, of appropriate size 5.1

29、5 Folded filters, cellulose (ca. 30 m pore size) for the glass funnels (5.14) 5.16 Filter disks, binder-free glas microfibre ( 2 m pore size) of appropriate size for the solvent vacuum filtration system (5.22) 5.17 Pipettors or gas-tight glas syringes, with a volume of 100 l, 500 l and 1 000 l 5.18

30、Vacuum manifold or Automated SPE Vacuum System, capable of accommodating the immunoaffinity columns 5.19 Reservoirs, of appropriate volume with attachments to fit the immunoaffinity columns 5.20 Plastic syringes, 5 ml 5.21 Vacuum pump, capable of generating sufficient vacuum for the solvent vacuum f

31、iltration system (5.22) 5.22 Solvent vacuum filtration system, fitted with glass microfibre filter (5.16) EN 15792:2009 (E) 8 5.23 HPLC syringe filter unit, polyamide (nylon) with 0,45 m pore size 5.24 Ultrasonic bath 5.25 HPLC apparatus, comprising the following: 5.25.1 Injection system, manual or

32、autosampler, with loop suitable for 100 l to 300 l injections 5.25.2 Pump, isocratic, pulsation-free, capable of maintaining a volume flow rate of 0,5 ml/min to 1,5 ml/min 5.25.3 Analytical reversed phase HPLC column, Generally every RP-column is suitable that allows a sufficient separation of zeara

33、lenone from other interfering components. For example Phenomenex ODS3-Prodigy (150 mm x 4,6 mm i.d.), 5 m particle size, 250 pore size, or Spherisorb ODS2-Excel (250 mm x 4,6 mm i.d.), 5 m particle size, 250 pore size have been found to be suitable. 5.25.4 Pre-column (optional), appropriate for the

34、analytical column used 5.25.5 Fluorescence detector, fitted with a flow cell and suitable for measurements with excitation wavelength of 274 nm, and emission of 446 nm 5.25.6 Data system, integrator or PC workstation 5.26 UV spectrophotometer, for checking the concentration of the stock solution (4.

35、15) 6 Procedures 6.1 Sample preparation It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage. Samples should be taken and prepared in accordance with European legislation where applicable 2. Samples should

36、 be finely ground and thoroughly mixed using a mill (5.6) and a tumble mixer (5.7) or another process that has been demonstrated to give complete homogenisation before a test portion is removed for analysis. In all instances if the sample has been frozen allow it to thaw completely before sampling.

37、Stir the sample thoroughly before removing an analytical test portion. 6.2 Extraction Weigh 20,00 g (recorded to 2 decimal places) test portion into a screw cap flask of 250 ml to 500 ml (5.13). Add 150 ml extraction solvent (4.10). Mix briefly by hand to obtain a homogeneous suspension then either

38、shake for 1 h on a shaker (5.2), or sonicate for 15 min in an ultrasonic bath (5.24) and shake on a shaker (5.2) for another 15 min. Filtrate extract through folded filter paper (5.15) and collect the extract in a screw cap flask of 100 ml (5.13). Transfer exactly 30 ml (or 3 ml in case of results a

39、bove 500 g/kg, see section 8) of the filtered extract into a 250 ml graduated cylinder with stopper (5.10). Dilute the extract in the cylinder with PBS (4.9) to the 150 ml mark. Mix and filtrate approximately 20 ml of this diluted extract through a glass microfiber filter (5.16) into a glass beaker

40、by applying a slight vacuum (5.22). Do not apply too strong a vacuum in the beginning of the filtration process, as this can lead to turbid filtered extracts after filtration. Discard these 20 ml and filtrate another approximately 70 ml for analysis. Proceed immediately with the immunoaffinity colum

41、n cleanup procedure (6.3). EN 15792:2009 (E) 9 6.3 Immunoaffinity Clean-up Connect the immunoaffinity column (4.19) to the vacuum manifold (5.18) and attach a reservoir (5.19) to the top of the immunoaffinity column. Precondition the immunoaffinity column (4.19) with 20 ml of PBS (4.9) using a flow

42、rate of 3 ml/min to 5 ml/min. Pipette 50 ml of the filtered and diluted sample extract (6.2) into the reservoir. Let the extract pass through the column by gravity at a steady flow rate until all the extract has passed and the last solvent portion reaches the frit of the column. The flow rate should

43、 be 1 drop/s to 2 drops/s. After the extract has passed through the column wash the column with 5 ml of washing solvent (4.11) followed by 15 ml of water at a rate of 1 drop/s to 2 drops/s. Remove residual water from the column (4.19) by passing 3 ml of air or nitrogen through the column (1 s to 2 s). Discard all the e