1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16006:2011Animal feeding stuffs Determination of the Sumof Fumonisin B1 160 g NaCl (4.5); 72 g Na2HPO4*12 H2O (4.6). Adjust to pH 7,4 with 10 mol/l HCl and make up to 2 000
2、 ml. 4.16 PBS Ready to use Dilute 100 ml of PBS concentrate (4.15) to 1 000 ml with water (4.1), or PBS tablets, Phosphate buffered saline tablets one tablet dissolved in 200 ml of water (4.1) yields 0,01 mol/l phosphate buffer, 0,002 7 mol/l potassium chloride and 0,137 mol/l sodium chloride, pH 7,
3、4, at 25C (e.g. Sigma P4417). 4.17 Diluent Mix 50 parts per volume methanol (4.2) with 50 parts per volume water (4.1). 4.18 Extraction solvent Mix 50 parts per volume methanol (4.2) with 50 parts per volume of PBS (4.16). BS EN 16006:2011EN 16006:2011 (E) 6 4.19 Reaction buffer 4.19.1 Post-column d
4、erivatisation (0,006 mol/l OPA, 0,006 mol/l NAC, 0,384 mol/l sodium carbonate, 0,216 mol/l boric acid and 0,108 mol/l potassium sulphate): Dissolve 40,7 g sodium carbonate (4.8), 13,4 g boric acid (4.9) and 18,8 g potassium sulphate (4.10) per 1,0 l of water (4.1); stir for 10 min; add 800 mg of OPA
5、 (4.12) per 1,0 l of the above solution; add 1 g of NAC (4.11) per 1,0 l of the above solution; stir for 10 min; sonicate for 15 min; stir for 10 min; sonicate again for 15 min and filter the solution through a 0,45 m nylon filter (5.17). Proper dissolution of the OPA is very important! The reaction
6、 buffer should not be changed within a sequence of HPLC runs. Prepare fresh for every sequence of HPLC runs. 4.19.2 Pre-column derivatisation (0,05 mol/l OPA, 0,12 mol/l BME, 0,08 mol/L disodium tetraborate, 16,7% methanol): Dissolve 40 mg OPA (4.12) in 1,0 ml methanol (4.2); mix until completely di
7、ssolved; add 5,0 ml of a 0,1 mol/l solution of disodium tetraborate decahydrate (3,8 g / 100 ml; 4.7); mix thoroughly; add 50 l of BME (4.13), and mix thoroughly. Alternatively: Phthaldialdehyde Reagent. 4.20 FB1or BS EN 16006:2011EN 16006:2011 (E) 7 Separate certified solutions of Fumonisin FB1and
8、FB2 in appropriate solvents that will be mixed such that a stock solution containing ca. 50 g/ml of each is obtained. Calculate exact concentrations from certificates and dilutions. NOTE 1 The above solutions (4.20) may also be prepared gravimetrically by accurately weighing the dry substance and th
9、e solvent used to dissolve it. Accurately measuring the volume of the solvent is also allowed. NOTE 2 The above solutions may be stored for up to six months at below -18C in the dark. 4.21 FB1 add 200 ml of extraction solvent (4.18), cap, and shake vigorously by hand, so that the material disperses
10、evenly; put on a shaker (5.4) for 120 min; choose speed such that the material is mixed well without collecting in the top of the flask; allow the extracted sample to settle after shaking; of the supernatant take 5,0 ml and dilute with PBS (4.16) to a total volume of 50,0 ml and mix; prepare a filte
11、r funnel (5.10) with a glass micro fibre filter (5.9), and BS EN 16006:2011EN 16006:2011 (E) 11 filter the diluted supernatant of the extracted sample into a new flask (5.5). The diluted filtered extract may be stored at 4C to 10C overnight. In case of a highly contaminated material above 10 000 g/k
12、g (see Clause 8), take 10,0 ml of the stored filtered diluted extract and dilute again with PBS (4.16) to a total volume of 50,0 ml and mix. 6.3 Clean up Take one IAC (immunoaffinity column; 4.23) per extract; attach a reservoir (5.12), do not empty storage solution from column; to the reservoir add
13、 25,0 ml of the filtered diluted extract (6.2); open the column outlet; allow everything to pass slowly through the column (flow rate should be one drop per second to two drops per second); after the extract has passed completely, wash the IAC with 10 ml of PBS (4.16); pass air through the IAC (e.g.
14、 using a properly fitted large syringe) in order to expel excess PBS; place a 5 ml volumetric flask (5.13) or a 5 ml graduated cylinder (5.6) underneath the IAC and add 5 x 500 l of methanol (4.2) to the IAC (add next aliquot only after previous has completely passed); collect all the eluate in the
15、volumetric flask (5.13) or graduated cylinder (5.6); add 2,0 ml of water (4.1) to the IAC after all of the methanol (4.2) has passed through the column; continue to collect the eluate in the same volumetric flask or graduated cylinder, and carefully pass air through the column in order to collect mo
16、st of the applied water (4.1). 6.4 Test solution For pre-column derivatisation: make up the content of the volumetric flask or graduated cylinder to the 5 ml mark with water (4.1); For post-column derivatisation: add 5 l of formic acid (4.14) and make up the content of the volumetric flask or gradua
17、ted cylinder to the 5 ml mark with water (4.1); Mix the content of the volumetric flask or graduated cylinder and transfer an aliquot to an autosampler vial (5.11). This test solution may be stored at 4C to 10C for up to two days. 6.5 Spiking procedure To determine recovery spike a fumonisin-free re
18、presentative compound animal feed material with FB12) aspire 40 l test solution (6.4); 3) aspire 20 l pre-column reaction buffer (4.19.2); 4) mix 20 times, and 5) inject all. The above can be done manually (adjusting the total volume while maintaining the relative volumes if necessary) if it is asce
19、rtained that the solution is injected within 3 minutes after mixing. It is also important that the time period between mixing and injecting is the same for all test and calibration solutions. b) Injection volume: 80 l; c) Column temperature: 40C; d) Flow: 1,0 ml/ min; e) Fluorescence detector: Excit
20、ation : 335 nm; Emission : 440 nm (it should be checked if these are local maxima for the fluorescence detector in use); f) Mobile phase: 1) A: 0,5% formic acid (4.14) in water (4.1); 2) B: 0,5% formic acid (4.14) in methanol (4.2). g) Gradient settings (HPLC dwell volume 0,8 ml) see Table 2. BS EN
21、16006:2011EN 16006:2011 (E) 13 Table 2 Gradient settings for the pre-column derivatisation Time (min) B (%) 0 69,514 79 14,01 10017,01 10017,02 69,5 20 69,5Instruments with different dwell volume will need adjustment of the gradient to achieve the same separation as shown in Annex A. The aim should
22、be an apparent capacity factor at elution for FB1of k 3. 7.1.3 Post-column derivatisation Instructions for self-assembled system (5.16.5): The flow path to the chromatographic column (5.16.3) is unchanged from normal operation. The outlet of the column is connected to one of the outside ports of a m
23、ixing Tee (see 5.16.5). The tubing from column to mixing Tee should be as short as possible. The other outside port of the mixing Tee is connected to the outlet of a pump (see 5.16.5) delivering the reagent flow. This connection should be made of a long piece of 0,005“ ID PEEK tubing (see 5.16.5) so
24、 that a sufficient back pressure is created for the reagent pump to work properly. It is of utmost importance that the reagent flow is delivered pulsation-free. A slight pulsation can be minimized be introducing a large damping volume between the pump and the back pressure creating PEEK tubing. Larg
25、e ID PEEK tubing can serve this purpose. The remaining centre port of the mixing Tee is connected through a reagent loop to the fluorescence detector. The length, and therefore the volume, of this reagent loop is a balance between retaining the resolution of the chromatographic column (short) and ac
26、hieving complete reaction (long). The internal diameter is of lesser importance. If chosen too small excessive back pressure will be created. Satisfying results were achieved with a 2,5 m length of 0,02“ ID PEEK tubing. Using the equipment outlined in 5.16, the following conditions have shown to pro
27、duce satisfying results: a) Injection volume: 50 l b) Column temperature: 45C c) Flow : 1,2 ml/ min (mobile phase); 0,45 ml/min (post-column reagent (4.19.1) d) Fluorescence detector: Excitation : 335 nm; Emission : 440 nm (it should be checked if these are local maxima for the fluorescence detector
28、 in use). e) Mobile phase: BS EN 16006:2011EN 16006:2011 (E) 14 1) A: 0,1% formic acid (4.14) in water (4.1); 2) B: 0,1% formic acid (4.14) in acetonitrile (4.3). Gradient settings: see Table 3. Table 3 Gradient settings for the post column-derivatisation Time (min) B (%) 0 3413 34 13,01 9516 9516,0
29、1 34 19 34This separation is isocratic but to avoid accumulation of matrix components a step-up to 95% B is included. The percentage of organic modifier should be adjusted such that the capacity factor for FB1 will be k 2. 7.2 Determination of fumonisins in test solutions Inject aliquots of the test
30、 solutions (6.4) into the chromatograph using the same conditions as used for the calibration solutions (4.22). 7.3 Batch (Sequence) composition Always start a batch of measurements with a reagent blank to check the system. Subsequently inject the calibration solutions. Before the injection of the f
31、irst test solution the reagent blank should be injected to prove that there is no carry-over of analytes. The test solutions should be run in duplicate and reruns of the calibration solutions should be interspersed in regular intervals. The frequency of these calibration reruns depend on the stabili
32、ty of your chromatographic system. 7.4 Calibration Plot the signals (peak area or height) of all the measured calibration solutions against the corresponding concentrations for FB1and, separately, for FB2. Do not use means of the multiple injections. With linear regression estimate slope and interce
33、pt of each of the two calibration functions (FB1mSMPis the weight (g) of the test material used for extraction (20,0 g); V1is the total volume (ml) of the extraction solvent (200,0 ml); V2is the volume (ml) of the aliquot of the filtered raw extract used for dilution (5,0 ml); V3is the total volume
34、(ml) of the diluted filtered raw extract (50,0 ml); V4is the volume (ml) of the aliquot of the diluted filtered raw extract applied to the IAC (25,0 ml); V5is the total volume (ml) of the test solution (5,0 ml). If the weight of the test material and the volumes described herein before are kept the
35、above Equation (3) can be simplified to: 20=TSMPcw (g/kg) (4) Should the result of Equation (4) be larger than 10 000 g/kg or if it is known beforehand that the contamination level might exceed that value clean-up the respective diluted filtered extract (see 6.1) using an additional dilution (additi
36、onal dilution factor 50/10 = 5). The simplified equation will then be: 100520 =TTSMPccw (g/kg) (5) BS EN 16006:2011EN 16006:2011 (E) 16 Carry out the above calculations for FB1and FB2. The sum of both will then be calculated as follows: 2,1, FBSMPFBSMPSMPwww += (g/kg) (6) 9 Precision 9.1 Interlabora
37、tory study Details of an interlaboratory study on the precision of the method are shown in 3. The values derived from this interlaboratory study may not be applicable to concentration ranges and/or matrices other than those given. 9.2 Repeatability The absolute difference between two single test res
38、ults found on identical test materials by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r in not more than 5% of the cases. Table 4 Precision data repeatability x= 3 110 g/kg r = 229 g/kg x= 5 000 g/kg r = 282 g/kg x= 5 600 g/kg
39、r = 826 g/kg x= 8 200 g/kg r = 809 g/kg x= 16 000 g/kg r = 1 120 g/kg The relationship between r and xcan be sufficiently approximated by the following function: xr = 031.08.2 (7) meaning that the relative repeatability standard deviation is constant over the working range. 9.3 Reproducibility The a
40、bsolute difference between two single test results on identical test materials reported by two laboratories will exceed the reproducibility limit R in not more than 5% of the cases. BS EN 16006:2011EN 16006:2011 (E) 17 Table 5 Precision data reproducibility x= 3 110 g/kg R = 1 650 g/kg x= 5 000 g/kg
41、 R = 2 140 g/kg x= 5 600 g/kg R = 2 420 g/kg x= 8 200 g/kg R = 3 450 g/kg x= 16 000 g/kg R = 8 980 g/kg The relationship between R and xcan be sufficiently approximated by the following function: xR = 18.08.2(8) meaning that the relative reproducibility standard deviation is constant over the workin
42、g range. 10 Test report The test report shall contain the following data: a) information necessary for the identification of the sample (kind of sample, origin of sample, designation); b) a reference to this European Standard; c) the date and type of sampling procedure (if known); d) the date of rec
43、eipt; e) the date of test; f) the test results and the units in which they have been expressed; g) whether the repeatability has been verified; h) particular points observed in the course of the test; i) operations not specified in the method or regarded as optional, which might have affected the re
44、sults. BS EN 16006:2011EN 16006:2011 (E) 18 Annex A (informative) Precision data The following data were obtained in an interlaboratory study 3 according to AOAC Guidelines for collaborative study procedures to validate characteristics of a method of analysis 2. Table A.1 Precision data Sample Anima
45、l feed Animal feed Animal feed Animal feed Animal feed Year of inter-laboratory study Number of laboratories Number of outliers (laboratories) Number of non-compliant laboratories Number of accepted results 2008 16 0 4 12 2008 16 0 4 12 2008 16 0 4 12 2008 16 0 4 12 2008 16 0 4 12 Mean value x(g/kg)
46、 3 110 5 000 5 600 8 200 16 000 Repeatability standard deviation sr(g/kg) Repeatability relative standard deviation RSDr(%) 81,9 2,6 101 2,0 295 5,3 289 3,5 398 2,5 Repeatability limit r a(g/kg) 229 282 826 809 1 120 Reproducibility standard deviation sR(g/kg) Reproducibility relative standard devia
47、tion RSDR( %) 589 19 766 15 863 15 1 230 15 3 210 20 HORRATR1,4 1,2 1,3 1,3 1,9Reproducibility limit R b(g/kg) 1 650 2 140 2 420 3 450 8 980 Recovery ( %) n.a c 69 n.a.c 79 n.a.c a r = 2,8 sr b R = 2,8 sR c not applicable BS EN 16006:2011EN 16006:2011 (E) 19 Table A.2 Material composition Test Mater
48、ial Ingredient Parts Principal components Blank Rabbit feed 4 cereals, seeds, crop by-products, vegetables, minerals Horse feed 5 oat, barley flakes, flour pellets, corn flakes, pea flakes, fibres, oil, molasses Pig feed 5 peas, roasted soy, wheat, barley, tapioca, cabbage seeds, animal grease, corn
49、, salt Level B Blank 4 See “Blank“. Maize 1 Naturally contaminated Maize Level C Blank 2 See “Blank“. Maize 1 Naturally contaminated Maize Level SH Maize 1 Naturally contaminated Maize The following table lists the precision data computed after correcting the reported values of each laboratory with the apparent mean recovery of the respective laboratory. The table has been added purely for informative purposes. BS EN 16006:2011EN
