0:16 this video is complementary to the
0:19 written method it concentrates on
0:20 critical aspects of the methods
0:22 important in achieving accurate and
0:25 reproducible results for further details
0:27 please refer to the printed description
0:28 of the method
0:30 the latter is based on the most
0:32 up-to-date techniques currently
0:34 available and was developed by the
0:38 German VDL UFA the method has not been
0:39 previously applied in this form to feedstuff
0:46 regarding the procedures described in
0:48 this video all normal laboratory safety
0:51 precautions must be taken additionally
0:54 special precautions must be made in
0:56 relation to the handling of mycotoxins
0:58 these include the wearing of gloves and
1:01 glasses the handling of spillages and
1:03 the disposal of contaminated waste these
1:05 precautions are also listed in the
1:27 a wide variety of food or feet stuff
1:30 such as cereals maize ground nuts
1:32 cottonseed and so on may be contaminated
1:34 by moles that produce a full range of
1:38 chemicals named mycotoxins among these
1:41 aflatoxins are toxic metabolites of
1:43 Aspergillus flavors and a specialist
1:46 parasitic as' and have been found in
1:49 many food products
1:52 moreover aflatoxins are important in
1:54 animal health because they may occur in
1:58 animal feed stuff when cattle are given
2:01 contaminated feed aflatoxins can find
2:04 their way into dairy foods some
2:06 mycotoxins present a potential hazard to
2:09 human or animal health even when present
2:12 at very low concentrations their levels
2:14 in food and animal feed stuff are
2:15 controlled by regulations in more than
2:26 validated methods for the analysis of
2:28 mycotoxins are necessary in order to
2:30 apply these controls and to set
2:33 standards for trading purposes therefore
2:36 the food analysis unit of the European
2:38 Commission's joint research centre has
2:40 undertaken the initiative to validate a
2:42 suitable analytical method for the
2:44 determination of aflatoxin b1 in
2:51 feedstuff naturally contaminated
2:53 feedstuff material with various
2:56 concentrations of aflatoxin b1 in low
2:59 ppb micro kilogram ranges are the basis
3:03 for the validation study the feedstuff
3:05 contains also commonly used ingredients
3:07 which might interfere with the detection
3:10 of aflatoxins the analysis of aflatoxin
3:13 b1 is based on the extraction from the
3:16 feedstuff followed by an immuno affinity
3:17 column cleanup step for sample
3:21 preparation and further analysis by
3:23 high-performance liquid chromatography
3:37 when the feedstuff sample consists of
3:39 compound material or grain an
3:41 appropriate milling has to be carried
3:44 out prior to extraction ground feedstuff
3:46 material may be extracted and analyzed
3:52 directly a ground feedstuff sample of
3:54 about 50 grams is weighed out in an
4:00 Erlenmeyer flask it is very important to
4:09 record the exact weight the feed staff
4:12 is then diluted in 250 milliliters of an
4:15 acetone water mixture shaken intensively
4:17 by hand for a few seconds to achieve a
4:19 homogenous suspension and then shaken
4:21 mechanically at moderate speed for 30 minutes
4:33 filtration is then carried out through
4:38 folded filter paper an aliquot of five
4:40 milliliters is taken from the filtrate
4:42 and diluted to 100 millilitres in a
4:45 volumetric flask by using phosphate
4:47 buffered saline or water according to
4:48 the specifications for the immuno
4:56 if the solution isn't clear it must be
4:58 refill turd through a glass fiber filter
5:01 prior to the immuno affinity clean up if
5:04 it's clear it's ready for being
5:06 transferred on to the immuno affinity
5:16 column no specific manufacturers columns
5:19 are recommended however minimum
5:21 performance criteria are specified for
5:23 this method if the columns are not
5:25 guaranteed to meet these criteria they
5:28 must be tested in the laboratory the
5:30 immuno affinity column contains
5:32 antibodies which are specific to the
5:36 target mycotoxin the antibodies are
5:38 bound to the packing material in the
5:42 column they are selectively bind the
5:44 target mycotoxin and remove it from the
5:46 extract when the sample is passed down
5:49 the column the right flow rate is
5:51 important in order to bond all a flow
5:56 toxin b1 to the antibody the column is
5:57 then washed to remove any other
6:00 potentially interfering substances which
6:06 may still be present finally the bond
6:08 between antibody and mycotoxin is broken
6:10 by Ellucian with the solvent and
6:13 aflatoxin b1 in a very pure form is collected
6:29 to collect pure aflatoxin b1 an aliquot
6:31 of exactly 50 milliliters of the diluted
6:33 sample extract is applied on the immuno
6:37 affinity column the sample is poured
6:38 into a syringe barrel attached to the
6:44 top of the column the solution may be
6:46 passed through the immuno affinity
6:56 column by gravity or a slight vacuum if
6:58 the vacuum is applied the column should
7:00 not be allowed to dry out the flow rate
7:02 should not exceed three to five
7:05 milliliters per minute so the total time
7:06 for loading onto the column will not be
7:13 less than 17 minutes after loading the
7:15 column is washed twice with 10
7:18 milliliters of water and then dried by
7:25 the aflatoxin b1 is eluted from the
7:28 column in two stages firstly North point
7:30 seven five milliliters of methanol is
7:32 applied to the column and allowed to
7:35 pass by gravity the Lu it is collected
7:36 in a calibrated five milliliters
7:39 volumetric flask after one minute
7:41 another portion of one millimeter of
7:44 methanol is added in this case the Eliot
7:46 is collected by applying pressure or
7:47 passing air through the column after
7:49 most of the solvent has passed through
7:52 this allows to collect most of the
7:53 applied solvent with the aflatoxin b1
7:57 from the immuno affinity column the
7:59 volumetric flask is made up to volume
8:01 with water and is ready for injection
8:05 into the HPLC system as methanol and
8:07 water contract while shaken adjust the
8:11 level after shaking in case the solution
8:13 is cloudy it should be passed through a
8:16 disposal filter unit if it's clear it's
8:30 200 microliters of the solution are
8:35 injected into the HPLC system the method
8:37 requires post column bromination of the
8:40 aflatoxin b1 prior to fluorescence
8:42 detection bromination can be carried out
8:46 either using Pb Pb reagent or using
8:49 electro chemically generated bromine the
8:51 experimental details for both approaches
8:53 are set out in the written method and
9:02 calibration curve should be established
9:04 by injecting solutions of the aflatoxin
9:08 b1 standards this curve which should be
9:10 linear is employed to determine the
9:17 concentration of aflatoxin b1 after HPLC
9:20 analysis the extracts from all feedstuff
9:22 samples should show a well resolved
9:24 aflatoxin b1 peak free from other
9:33 extracted components this is a typical
9:35 chromatogram from naturally contaminated
9:38 material showing a clearly discernible
9:46 fully automated systems such as the ASP
9:49 EC system can be used as well all
9:52 techniques can be applied provided the
9:54 analytical protocol is not modified in
9:57 terms of volumes of reagents or flow
11:26 covered and allowed to stand for 60
11:33 finally a 50 microliters of the stop
11:40 solution is added to each well the micro
11:41 plate is now ready for the measurement
11:52 of the color intensity the micro plate
11:54 is placed into a microplate reader a
11:57 photometric instrument designed to
11:59 measure the solutions the intensity of
12:01 the color of the solution each well is
12:05 measured automatically at 440 nanometer
12:10 or 405 nano meter for this application
12:12 the microplate reader uses air as reference
12:18 finally the printout of the microplate
12:19 reader and the template with the
12:21 information about the samples are used
12:32 to evaluate the results for further
12:34 details please refer to the printed
12:36 method description you
