1、March 2008DEUTSCHE NORM English price group 13No part of this standard 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 67.050!$LV Miis the mola
2、r mass of each aflatoxin, in grams per mol; DIN EN 14123:2008-03 EN 14123:2007 (E) 6 iis the molar absorption coefficient of each aflatoxin in toluene and acetonitrile (4.18), in square metres per mol; b is the optical path length of the cell, in centimetres. Miand iof aflatoxins B1, B2, G1and G2are
3、 given in Table 1. Table 1 Molar mass and molar absorption coefficient of aflatoxins B1, B2, G1and G2(In mixture of toluene and acetonitrile (4.18) Aflatoxin Mig/mol im2/mol B1312 1930 B2314 2040 G1328 1660 G2330 1790 4.21 Mixed aflatoxins stock solution Prepare a mixed aflatoxins stock solution con
4、taining 1000 ng/ml of aflatoxin B1and G1, 200 ng/ml of aflatoxin B2and G2in the toluene and acetonitrile mixture (4.18) by appropriate dilution of aflatoxins (B1, B2, G1and G2) stock solutions (4.20). NOTE A commercial total aflatoxins standard solution which is ready to use in a vial containing 100
5、0 ng/ml of total aflatoxin may be used as an alternative. 4.22 Diluted mixed aflatoxins stock solution Prepare a diluted mixed aflatoxins stock solution containing 100 ng/ml of aflatoxin B1and G1, 20 ng/ml of aflatoxin B2and G2in the toluene and acetonitrile mixture (4.18) by pipetting exactly 1,0 m
6、l of the mixed aflatoxins stock solution (4.21) into a 10 ml calibrated volumetric flask (5.10), fill to the mark with the toluene and acetonitrile mixture (4.18) and mix well. Wrap the flask tightly in aluminium foil and store it at less than 4 C or in a freezer. Before use, do not open the flask u
7、ntil the contents have reached room temperature to avoid incorporation of water by condensation. 4.23 Mixed aflatoxins calibration solutions Use the diluted mixed aflatoxins stock solution containing 100 ng/ml of aflatoxin B1and G1, 20 ng/ml of aflatoxin B2and G2(see 4.22) for pipetting the volumes
8、as given in Table 2 into a set of 10 ml volumetric flasks (5.10). Evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. To each flask, add 4 ml of methanol, let aflatoxins dissolve, dilute to 10 ml with water, and shake well. Methanol and water a
9、re subject to volume contraction when mixed, so adjust the volume again to the given volume. DIN EN 14123:2008-03 EN 14123:2007 (E) 7 Table 2 Preparation of mixed aflatoxins calibration solutions Calibration solution Mass concentration of calibration solution ng/ml Taken from diluted stock solution
10、(4.22) l B1B2G1G21 40 0,400 0,080 0,400 0,080 2 120 1,200 0,240 1,200 0,240 3 200 2,000 0,400 2,000 0,400 4 280 2,800 0,560 2,800 0,560 5 360 3,600 0,720 3,600 0,720 4.24 Spiking solution Prepare a spiking solution by pipetting 2 ml of the mixed aflatoxins stock solution (containing 1000 ng/ml of af
11、latoxin B1and G1, 200 ng/ml of aflatoxin B2and G2, see 4.21) into a 10 ml calibrated volumetric flask. Evaporate the toluene/acetonitrile solution just to dryness under a stream of nitrogen at room temperature. Dilute to the mark with methanol and shake well. The concentration of this spiking soluti
12、on is 200 ng/ml of aflatoxin B1and G1, and 40 ng/ml of aflatoxin B2and G2. Wrap the flask tightly in aluminium foil and store it at less than 4 C. Before use, do not open the flask until the contents have reached room temperature to avoid incorporation of water by condensation. 5 Apparatus 5.1 Gener
13、al All glassware coming into contact with aqueous solutions of aflatoxins shall be washed with acid solution before use. Many laboratory washing machines do this as part of the washing program. Otherwise soak such laboratory glassware in sulfuric acid (2 mol/l) for several hours (e.g. 15 h overnight
14、), then rinse well (e.g. three times) with water to remove all traces of acid. Check the absence of acid with pH paper. This treatment is necessary, because the use of non-acid washed glassware may cause losses of aflatoxins. In practice, the treatment is necessary for round bottomed flasks, volumet
15、ric flasks, measuring cylinders, vials or tubes used for calibration solutions and final extracts (particularly autosampler vials), and Pasteur pipettes, if these are used to transfer calibration solutions or extracts. 5.2 Usual laboratory apparatus and, in particular, the following 5.3 Laboratory m
16、ill, or explosion proof high speed blender1) , necessary for the production and extraction of pastes from hazelnuts, peanuts, pistachios and figs, with suitable blender jar 5.4 Adjustable vertical or horizontal shaker, needed for the analysis of paprika powder 5.5 Paper filter, e.g. 24 cm diameter,
17、prefolded 5.6 Conical flask, with screw top or glass stopper 1) Contact your National Standardization Institute for appropriate high speed blenders. DIN EN 14123:2008-03 EN 14123:2007 (E) 8 5.7 Glass microfiber filter paper, retention size 1,6 m or smaller 5.8 Reservoir, 75 ml with luer tip connecto
18、r for immunoaffinity column (IAC) 5.9 Hand pump, 20 ml syringe with luer lock or rubber stopper for IAC 5.10 Volumetric glassware, flasks of e.g. 3 ml, 5 ml, 10 ml and 20 ml, with an accuracy of at least 0,5 % 5.11 HPLC system, consisting of 5.11.1 HPLC pump, suitable for flow rate at 1,0 ml/min 5.1
19、1.2 Injection system, capable for total loop injection. A 100 l loop is recommended. In the case that a different loop size than recommended is used it shall be guaranteed that the limit of detection (LOD) for the system is 0,2 ng/g (signal-to-noise-ratio = 3) and the limit of quantification (LOQ) i
20、s 0,5 ng/g (signal-to-noise-ratio = 6) for each aflatoxin (using the standard solutions). 5.11.3 RP-HPLC column, e.g. C18or ODS-2 (length of 25 cm, inner diameter of 4,6 mm and particle size of 5 m), which ensures a baseline resolution of the aflatoxin B1, B2, G1and G2peaks from all other peaks. The
21、 maximum overlapping of peaks shall be less than 10 %. It could be necessary to adjust the mobile phase for a sufficient baseline resolution. A suitable pre-column should be used. 5.11.4 Post-column derivatisation system, with PBPB (only to be used with mobile phase A (4.15) Consisting of an HPLC pu
22、lseless pump, zero-dead volume T-piece, reaction tubing min. 45 cm x 0,5 mm internal diameter PTFE. 5.11.5 System for derivatisation with electrochemically generated bromine, e.g. KOBRA cell2)(only to be used with mobile phase B (4.16). 5.11.6 Fluorescence detector, with a wavelength of = 360 nm exc
23、itation filter and a wavelength of = 420 nm cut-off emission filter, or equivalent (e.g. a detector with an adjustable monochromator). Recommended settings for adjustable detectors are 365 nm (excitation wavelength), 435 nm (emission wavelength) and a bandwidth of 18 nm. 5.12 Disposable filter unit,
24、 of pore size 0,45 m Prior to usage, verify that no aflatoxin losses occur during filtration (recovery testing). NOTE There is a possibility that various filter materials can retain aflatoxins. 5.13 Pipettes, 2 ml, 5 ml and 10 ml capacity, with an accuracy of at least 0,5 % 5.14 Analytical balance,
25、capable of weighing to 0,1 mg 5.15 Laboratory balance, capable of weighing to 0,01 g 2) KOBRA cell is the trade name of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the pro
26、duct named. Equivalent products may be used if they can be shown to lead to the same results. DIN EN 14123:2008-03 EN 14123:2007 (E) 9 5.16 Calibrated microliter syringe(s) or microliter pipette(s), 10 l to 1000 l 5.17 Vacuum manifold, optional 6 Procedures 6.1 Sample preparation Homogenize a suitab
27、le amount (e.g. 10 kg, see European legislation 3) of pistachios, peanuts, hazelnuts and figs appropriately to give a paste, e.g. using a high speed blender (5.3). Information on sample sizes and sampling is given in 3. 6.2 Conditioning of immunoaffinity columns Allow the immunoaffinity columns (4.1
28、4) to reach room temperature prior to conditioning. Connect the immunoaffinity column to the vacuum manifold (5.17) and attach the reservoir (5.8) to the immunoaffinity column. For conditioning transfer 10 ml of PBS (4.3) on the top of the column and let it pass at a speed of 2 ml/min to 3 ml/min th
29、rough the column (e.g. by gravity). Make sure that a small portion (0,5 ml) of the PBS remains on the column until the sample solution is applied. Different conditioning procedures shall be considered in accordance with the manufacturers instructions. 6.3 Extraction 6.3.1 General For the extraction
30、of hazelnut paste, fig paste, peanut butter and pistachio paste a high speed blender shall be used, since the fatty commodities (hazelnut paste, peanut butter and pistachio paste) need to form an emulsion to break the fatty layers and allow a sufficient extraction. In addition, fig paste needs to br
31、eak down in the solvent, which cannot be guaranteed if a shaker is used, due to its consistency. Paprika powder can be extracted by shaking (provided that the powder is ground sufficiently to a particle size up to 500 m) to process several samples simultaneously and reduces the risk of cross contami
32、nation. Before weighing the homogenized test portion for extraction, stir the sample container well if it is a paste to overcome segregation of the matrix particles in the container. 6.3.2 Hazelnuts Weigh, to the nearest 0,1 g, approximately 50 g of the homogenized test portion (6.1) into a blender
33、jar. Add 4 g of sodium chloride (4.4) and 100 ml of water (4.2). Blend for 1 min with a high speed blender (5.3) to produce a slurry. Add 150 ml of methanol (4.8) and blend again for 2 min with the high speed blender. Filter the extract using a paper filter (5.5) and collect the filtrate in a 100 ml
34、 conical flask. Transfer 5 ml of the clear filtrate (equivalent to 1 g of sample) into a glass beaker and add 15 ml of PBS (4.3) solution. Add the diluted sample extract to the reservoir connected to the conditioned immunoaffinity column (4.14) and proceed as described in 6.4. 6.3.3 Figs Weigh, to t
35、he nearest 0,1 g, approximately 50 g of the homogenized test portion (6.1) into a 500 ml conical flask (5.6) or blender jar. Add 5 g of sodium chloride (4.4) and 300 ml of extraction solvent mixture (4.11). Blend for 3 min with a high speed blender (5.3). DIN EN 14123:2008-03 EN 14123:2007 (E) 10 Fi
36、lter the extract using a paper filter (5.5). Pipette 10,0 ml of the clear filtrate into a 100 ml glass beaker (or similar) and dilute with 60 ml of PBS (4.3). Add the diluted sample extract to the reservoir connected to the conditioned immunoaffinity column (4.14) and proceed as described in 6.4. Sl
37、urries or larger test portions may be used, provided that ratios (sample-to-extraction solvent as well as the extraction solvent composition for slurries) are maintained. 6.3.4 Peanuts Weigh, to the nearest 0,1 g, approximately 50 g of the homogenized test portion (6.1) into a 500 ml conical flask (
38、5.6) or blender jar. Add 5 g of sodium chloride (4.4), 200 ml of extraction solvent mixture (4.11) and 100 ml of n-hexane or cyclohexane (4.12). Blend for 3 min with a high speed blender (5.3). Filter the extract using a paper filter (5.5). In case of a solvent layer separation carry on with the low
39、er phase. Pipette 10,0 ml of the clear filtrate into a 100 ml glass beaker (or similar) and dilute with 60 ml of PBS (4.3). Add the diluted sample extract to the reservoir connected to the conditioned immunoaffinity column (4.14) and proceed as described in 6.4. Solvent layer separation should not o
40、ccur if filtration takes place immediately after blending since n-hexane/cyclohexane will be retained in the filter. A filter phase separator may be used if needed. Larger test portions may be used, provided that the sample-to-extraction solvent ratio is maintained. 6.3.5 Pistachios Weigh, to the ne
41、arest 0,1 g, approximately 50 g of the homogenized test portion (6.1) into a 500 ml conical flask (5.6) or blender jar. Add 5 g of sodium chloride (4.4), 200 ml of extraction solvent mixture (4.11) and 100 ml of n-hexane or cyclohexane (4.12). Blend for 3 min with a high speed blender (5.3). Filter
42、the extract using a paper filter (5.5). In case of a solvent layer separation carry on with the lower phase. Pipette 10,0 ml of the clear filtrate into a 100 ml glass beaker (or similar) and dilute with 60 ml of PBS (4.3). Add the diluted sample extract to the reservoir connected to the conditioned
43、immunoaffinity column (4.14) and proceed as described in 6.4. If significant precipitation occurs diluting with PBS, alternatively pipette 20 ml of the sample filtrate into a 250 ml glass beaker (or similar) and dilute with 140 ml of PBS (4.3) and then filter to a filter paper (5.7). In this case ad
44、d 70 ml of this filtered sample extract to the reservoir connected to the conditioned immunoaffinity column (4.14) and proceed as described in 6.4. Solvent layer separation should not occur if filtration takes place immediately after blending since n-hexane/cyclohexane will be retained in the filter
45、. A filter phase separator may be used if needed. Larger test portions may be used, provided that the sample-to-extraction solvent ratio is maintained. 6.3.6 Paprika powder Weigh, to the nearest 0,1 g, approximately 50 g of the homogenized test portion (6.1) into a 500 ml conical flask (5.6). Add 5
46、g of sodium chloride (4.4) and 300 ml of extraction solvent mixture (4.11). Shake vigorously by hand for the first 15 s to 30 s and then for 30 min with a shaker (5.4). For various types of shakers (e.g. horizontal platform shaker or vertical wrist shaker) the motion speed shall be adjusted to obtai
47、n maximum agitation of the extraction mixture. Filter the extract using a paper filter (5.5). Pipette 10,0 ml of the clear filtrate into a 100 ml glass beaker (or similar) and dilute with 60 ml of PBS (4.3). Add the diluted sample extract to the reservoir connected to the conditioned immunoaffinity
48、column (4.14) and proceed as described in 6.4. Larger test portions may be used, provided that the sample-to-extraction solvent ratio is maintained. DIN EN 14123:2008-03 EN 14123:2007 (E) 11 6.4 Immunoaffinity clean-up Pass the filtrate through the column at a flow rate of approximately 3 ml/min (ap
49、proximately one drop per second) or by gravity. Do not exceed a flow rate of 5 ml/min. Wash the column with approximately 20 ml of water (4.2) applied in little portions of approximately 10 ml at a flow rate of maximum 5 ml/min and dry by applying little vacuum for 5 s to 10 s or passing air through the immunoaffinity column by means of a syringe for 10 s. Elute the aflatoxins in a two step procedure. For hazelnut paste apply 0,50 ml of methanol (4.8) on the column and let it pass through by gravity. Collect the eluate in a calibrated volumetric flask of 3 m
