1、BRITISH STANDARD PD CEN/TR 15298:2006 Foodstuffs Sample comminution for mycotoxins analysis Comparison between dry milling and slurry mixing ICS 67.050 PD CEN/TR 15298:2006 This Published Document was published under the authority of the Standards Policy and Strategy Committee on 31 October 2006 BSI
2、 2006 ISBN 0 580 49336 9 National foreword This Published Document was published by BSI. It is the UK implementation of CEN/TR 15298:2006. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal methods. A list of organizations represented on AW/
3、-/3 can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Amendments issued since publication Amd. No. Date CommentsTECHNICALREPORT RAPPORTTECHNIQUE TECHNISCHERBERICHT C
4、EN/TR15298 May2006 ICS67.050 EnglishVersion FoodstuffsSamplecomminutionformycotoxinsanalysis Comparisonbetweendrymillingandslurrymixing ProduitsalimentairesPrparationdchantillonsgros volumepourlanalysedesmycotoxinesComparaison entrebroyagesecetbroyageparvoiehumide LebensmittelProbenvorbereitungfrdie
5、 MycotoxinanalytikVergleichzwischenTrockenvermahlung undAufschlmmung ThisTechnicalReportwasapprovedbyCENon30November2005.IthasbeendrawnupbytheTechnicalCommitteeCEN/TC275. CENmembersarethenationalstandardsbodiesofAustria,Belgium,Cyprus,CzechRepublic,Denmark,Estonia,Finland,France, Germany,Greece,Hung
6、ary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal, Romania, Slovakia,Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG ManagementCentre:ruedeStassart,36B1050Bruss
7、els 2006CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.CEN/TR15298:2006:E2 Contents Page Foreword3 Introduction .4 1 Scope 5 2 Test methods5 3 Results and discussion.6 4 Acknowledgements 17 Bibliography. 18 CEN/TR 15298:20063 Foreword This Technical
8、Report (CEN/TR 15298:2006) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal method”, the secretariat of which is held by DIN. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall
9、not be held responsible for identifying any or all such patent rights. CEN/TR 15298:20064 Introduction Since 1999-01-01, EC directives for aflatoxins entered into force, which consisted of sampling plans resulting in sample weights of up to 30 kg. This raised questions on how these relatively big sa
10、mples could fulfil the requirement to “finely grind and mix thoroughly each laboratory sample using a process that has been demonstrated to achieve complete homogenisation” 1. Since the analytical sample is taken out of this big sample, the critical step is to take a representative increment out of
11、it. As such this topic has been subject of several studies in the past. Dickens and Satterwhite 2 developed a mill that could handle up to 25 kg peanut samples. They presented results of tests on 5 kg samples from which they withdrew 50 g sub-samples, but gave no data on larger samples. Velasco and
12、Morris 3 considered use of a water slurry to obtain finer particles and a more uniform particle distribution. Another advantage of slurry preparation is the avoidance of clogging of samples that have high oil content. They presented experiments on different matrices with sample weights up to 4,5 kg,
13、 whereas they mentioned that slurry preparation is limited only by the capacity of the equipment. Whitaker et al. 4 considered a compromise. They prepared a slurry from a sample, which was first comminuted by another milling process. Due to the regulations of the USDA they limited themselves to an a
14、mount of only 1 100 g. Nevertheless this restriction in their method was developed into the alternative best foods method used for aflatoxin in peanuts 5. Dorner and Cole 6 started all over again from the beginning: the 218 kg sample of raw, shelled peanuts for analysis in official USDA approved lab
15、oratories. They compared variability by grinding with four different mills, but only with sub-sample sizes up to 4 kg. So the question how the result would be on 21,8 kg samples remained unanswered. Their statistical data, especially CV values, on the 2 kg and 4 kg sub-samples were less favourable t
16、han the ones that can be achieved by applying the slurry method. Scholten and Spanjer 7 published data on slurry preparation for samples up to 10 kg, whereas the laboratory of Wiertz, Eggert and Jrissen had similar experiences, even when applying samples up to 30 kg. Data of the latter are compiled
17、in this report. Worldwide however, sub-sampling mills are in favour because they are easy to apply and fast in comminuting samples into analytical portions. Calori-Domingues et al. 8 demonstrated this with a poster presentation at the X thInternational IUPAC symposium on mycotoxins and phycotoxins i
18、n May 2000. They tested variability for aflatoxin analysis in peanuts associated with sample preparation by dry milling with a RAS mill. Unfortunately however they only investigated samples up to 5 kg. So the labs of the Inspectorate for Health Protection, a delivery unit of the Dutch Food and non-f
19、ood Authority, and of Wiertz, Eggert and Jrissen, a member of the Eurofins Scientific group, decided to perform new experiments with following goals: 1. what CV values are achieved when milling 10 kg samples, and 2. are correct aflatoxin values measured while doing so? The choice of matrices has bee
20、n discussed at a CEN/TC 275/WG 5 (Comit Europen de Normalisation, Technical Committee 275, Working Group 5, Biotoxins) meeting, considering existing and upcoming legislation for different mycotoxins and food types. Combining both items lead to the conclusion that a lot of matrices, existing as dried
21、, whole or ground raw material are to be considered. Also differences in sample weight, i.e. between nuts and spices, exist. Suggestions for representative commodities were: cereals, since for this staple food directives exist on as well as aflatoxins, as ochratoxin A and as DON; raisins, because th
22、ese are included in directives for aflatoxins and ochratoxin A; paprika powder as an example of a ground commodity. In practice however it turned out that the availability of naturally contaminated lots that could be used for these experiments was the limiting factor. The presented results show what
23、 exactly has been examined. After these experiments the detailed work of Schatzki and Toyofuku 9, who measured particle size distributions on pistachio slurries, became available. This lead to a joint presentation at the 2 ndWorld Mycotoxin Forum, February 2003, in The Netherlands 10. This report is
24、 a combined outline of both investigations. CEN/TR 15298:20065 1 Scope A comparison was made between dry milling and slurry mixing as comminution step preceding mycotoxins analysis. Such in respect to EC legislation that consists of sample schemes up to 30 kg. Cacao, green coffee, almonds and pistac
25、hio samples of 10 kg were milled by a RAS mill and all three sub-samples were completely analysed for aflatoxin B 1 or Ochratoxin A. The differences in analytical results are explained by measurements of particle size distributions of both milling types. The obtained data are compared with literatur
26、e data on coefficients of variation (CV) for various milling procedures. For dry milling CV values were generally not below 20 % for aflatoxin B 1 levels up to 38 g/kg in peanuts, whereas slurry mixing could achieve CV values below 5 % at aflatoxin B 1levels down to 4 g/kg in pistachios. Measurement
27、s also showed possible difference in mycotoxin content of a sample between both milling types. This could lead to false positive or negative results when rejecting or accepting a lot, as this is based on the sample result. It was concluded that slurries contain smaller particles than dry milled samp
28、les and thus generate the lowest possible CV values which in turn leads to better sample homogenisation. 2 Test methods 2.1 Apparatus 2.1.1 Slurry mixer, Slurry mixer - Silverson type EX mixer 1 ) ; 2.1.2 RAS mill, Romer Analytical Sampling mill 1)Other laboratory equipment and slurry preparation pr
29、ocedures as described before (see 7 and 9). The RAS mill was applied according to the manual (Release 2, January 1998) of the supplier. Before the dry milling process the pistachio samples were frozen overnight at minus 20 C. 2.2 Reagents and materials Aflatoxin measurements were performed as descri
30、bed in EN 14123. Ochratoxin measurements were carried out in cacao and in green coffee beans as described in EN 14132, including quality control. The only difference is that fluorescence detection for ochratoxin A is carried out as published by Zimmerli and Dick 11. 2.3 Procedure For each commodity,
31、 experiments were carried out by the following procedure: 1. sampling according to the EC directive, resulting in 10 kg sample; 2. milling the 10 kg sample by a Romer mill with a split ratio of 10 %; 3. taking a dry sample out of the 10 % part as usual for Romer mill users (sub-sample A); 4. slurry
32、mixing of the remaining part of the 10 % part of the sample (sub-sample B); 5. slurry preparation of the 90 % part by Silverson mixing (sub-sample C); 6. analysing the three sub-samples A, B and C by HPLC methods. 1Silverson type EX mixer is the trade name of a product supplied by Silverson Machines
33、 Ltd., Waterside, Chesham, Bucks, England. Romer Analytical Sampling (RAS) mill is the trade name of a product supplied by Coring-System Diagnostic GmbH, Robert-Bunsen-Stra e 4, D-64579 Gernsheim, Germany. This information is given for the convenience of the users of this Technical Report and does n
34、ot constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. CEN/TR 15298:20066 Doing so the complete mycotoxins content in the sample can be reconstructed afterwards by calculation. 3 Results and discussion The results o
35、f all experimental values are given in the second, third and fourth column of Table 1. They consist of measurements of ochratoxin A in cacao and green coffee beans and of aflatoxins, of which only aflatoxin B 1is useful for this purpose, in almonds, pistachios and a sample of mixed spices. All other
36、 columns in Table 1 are filled with figures that are calculated from these data. From the weight of each sub-sample and its mycotoxins content, it is possible to calculate what the mycotoxins content would have been in the total sample if it had been measured in one sample as a whole. This calculate
37、d value is presented in the column “sample value” in the first row, such as to facilitate several comparisons that will be made in the clause results and discussion. In the last three columns the mathematical mean of the A, B and C sub-sample results, the standard deviation and the coefficient of va
38、riation of these three measurements are given, which will be discussed later as well. Considering the results have to be done from the starting point of the experiments: milling the 10 kg sample by a Romer mill, which creates a division of the original sample in two sub-samples of different weight.
39、When RAS milling is used in daily routine analysis this step is followed by taking an incremental sample out of the smallest sub-sample for further clean up and chemical analysis. This situation is comparable with the results for sub-sample A in this experiment with the crucial difference that data
40、as presented for sub-samples B and C are never measured in daily practice. In case of sample preparation by means of slurry, the whole sample is dealt with. A portion of the slurry is taken for further analysis. Regarding the methods in detail reveals that it will never be possible to do an experime
41、nt by applying both preparations towards one sample. Therefore the best estimate of a measurement of these samples, as if they were handled by preparing a slurry, can only be made by calculating the amount of mycotoxins from the individual A, B and C sub-sample values. This calculated value is prese
42、nted as “sample value” in Table 1. CEN/TR 15298:20067 Table 1 Results of sampling, milling and mixing experiments as described in 2.3 Ochratoxin A Sample Sub Sub Sub A,B,C Matrix a Value (g/kg) A (g/kg) B (g/kg) C (g/kg) Mean (g/kg) STD (g/kg) CV (%) Cacao 0,4 0,5 0,4 0,4 0,4 0,1 13,3 Cacao 0,6 0,4
43、0,5 0,6 0,5 0,1 20,0 Cacao 1,0 0,9 1,7 0,9 1,2 0,5 39,6 Cacao 1,1 0,8 0,4 1,2 0,8 0,4 50,0 Cacao 1,2 1,5 0,7 1,2 1,1 0,4 35,7 Cacao 1,2 2,6 1,5 1,2 1,8 0,7 41,7 Cacao 1,7 1,1 3 1,6 1,9 1,0 51,8 Cacao 1,7 1,5 1,5 1,7 1,6 0,1 7,4 Cacao 2,2 0,8 2,1 2,2 1,7 0,8 45,9 Cacao 3,5 5,2 1,5 3,7 3,5 1,9 53,7 Ca
44、cao 11,9 1,3 1,8 13 5,4 6,6 123,3 Green coffee 1,5 8,1 0,4 1,6 3,4 4,2 123,2 Green coffee 1,9 1,8 2,3 1,8 2,0 0,3 13,4 Green coffee 2,0 2,7 2,6 2,0 2,4 0,4 16,1 Green coffee 2,0 1,5 2,0 2,0 1,8 0,3 14,4 Aflatoxin B 1Sample Sub Sub Sub A,B,C Matrix Value (g/kg) A (g/kg) B (g/kg) C (g/kg) Mean (g/kg)
45、STD (g/kg) CV (%) Almonds 2,0 1,0 0,2 2,2 1,1 1,0 88,8 Almonds 2,4 1,0 4,2 2,2 2,5 1,6 65,5 Almonds 3,1 0 0 3,4 1,1 2,0 173,2 Almonds 4,1 0,5 6,7 3,8 3,7 3,1 84,6 Mixed spices 7,8 4,2 8,1 7,75 6,7 2,2 32,6 Pistachio in shell 33,8 88,2 38 33 53,1 30,5 57,5 Pistachio in shell 44,1 51,4 42,4 44,2 46,0
46、4,8 10,4 Pistachio kernels 114,1 250 108 114 157,3 80,3 51,0 Pistachio kernels 126,0 204 122 126 150,7 46,2 30,7 aMeasurements carried out according to the schedule as mentioned above, by Kastrup, WEJ Hamburg, Germany and Scholten, Inspectorate for Health Protection, Amsterdam, the Netherlands. For
47、the enforcement of a directive the analytical results are important at the point of accepting or rejecting a lot. Aflatoxin B 1is regulated in EC directives: 2 g/kg for nuts and 5 g/kg for spices. For ochratoxin A only values from a working document 12 can be used: 2 g/kg for cacao and 3 g/kg for co
48、ffee beans. The latter values are under discussion and are only used in this report to evaluate the presented measurements. With these figures, without adding measurement uncertainties, the differences between judgements of a lot based on dry milling (sub-sample A data) are compared with the data that would have been obtained after slurry preparation CEN/TR 15298:20068 of the sample as a whole (sample value data). Doing so for cacao 2 out of 11 lots would be rejected after a dry milling procedure and 3 out of these 11 lots after slurry preparation. Only in 1 of t
