CEN/TR 15298:2006
(Main)Foodstuffs - Sample comminution for mycotoxins analysis - Comparison between dry milling and slurry mixing
Foodstuffs - Sample comminution for mycotoxins analysis - Comparison between dry milling and slurry mixing
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 pistachio samples of 10 kg were milled by a RAS mill and all three sub-samples were completely analysed for aflatoxin B1 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 literature data on coefficients of variation (CV) for various milling procedures. For dry milling CV values were generally not below 20 % for aflatoxin B1 levels up to 38 µg/kg in peanuts, whereas slurry mixing could achieve CV values below 5 % at aflatoxin B1 levels down to 4 µg/kg in pistachios. Measurements 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 samples and thus generate the lowest possible CV values which in turn leads to better sample homogenisation.
Lebensmittel - Probenvorbereitung für die Mycotoxinanalytik - Vergleich zwischen Trockenvermahlung und Aufschlämmung
Die Trockenvermahlung und Aufschlämmung als Zerkleinerungsschritt vor der Mycotoxinanalyse wurden mit-einander
verglichen. Das bezieht sich auf die EG-Gesetzgebung, in der Stichprobenanweisungen für bis zu
30 kg enthalten sind. Proben von Kakao, Rohkaffee, Mandeln und Pistazien von 10 kg wurden mit einer RAS-Mühle
gemahlen und alle drei Teilproben wurden vollständig auf Aflatoxin B1 oder Ochratoxin A analysiert. Die
Unterschiede bei den Analysenergebnissen sind durch die Messungen der Korngrößenverteilungen von
beiden Vermahlungsarten begründet. Die erhaltenen Daten wurden auf der Grundlage der Variationskoeffi-zienten
(CV) für verschiedene Mahlverfahren mit Literaturangaben verglichen. Die CV-Werte für die Trocken-vermahlung
lagen im Allgemeinen nicht unter 20 % für Aflatoxin B1-Konzentrationen in Erdnüssen von bis zu
38 µg/kg, während bei Aufschlämmungen CV-Werte unter 5 % bei Aflatoxin B1-Konzentrationen in Pistazien
von bis zu 4 µg/kg erreicht werden können. Die Messungen zeigten auch den möglichen Unterschied
zwischen beiden Vermahlungsarten im Mycotoxingehalt einer Probe. Das könnte zu falsch positiven oder
negativen Ergebnissen führen, wenn eine Partie angesichts des Probenergebnisses zurückgewiesen oder
angenommen wird. Es wurde abschließend festgestellt, dass Aufschlämmungen kleinere Teilchen enthalten
als trocken vermahlene Proben und somit den kleinsten möglichen CV-Wert erzeugen, was wiederum zu
einer besseren Probenhomogenisierung führt.
Produits alimentaires - Préparation d'échantillons gros volume pour l'analyse des mycotoxines - Comparaison entre broyage à sec et broyage par voie humide
Le broyage à sec et le broyage par voie humide ont fait l'objet d'une comparaison réalisée en considérant
cette étape de broyage préalablement à l'analyse des mycotoxines. Cette comparaison s'inscrivait dans le
respect de la législation CE qui consiste à appliquer des programmes sur des échantillons jusqu'à 30 kg. Des
échantillons de cacao, de café vert, d'amandes et de pistaches de 10 kg ont été broyés au moyen d'un
broyeur RAS et les trois sous-échantillons ont fait l'objet d'une analyse complète pour déterminer la teneur en
aflatoxine B1 ou en ochratoxine A. Les distributions granulométriques des deux types de broyage expliquent
les différences au niveau des résultats des analyses. Les données obtenues sont comparées aux données
bibliographiques concernant les coefficients de variation (CV) pour diverses procédures de broyage. Dans le
cas du broyage à sec, les valeurs de coefficient de variation n'étaient en règle générale pas inférieures à 20 %
pour des niveaux d'aflatoxine B 1 jusqu'à 38 µg/kg dans les cacahuètes, tandis que le broyage par voie humide
pouvait donner des valeurs de coefficient de variation inférieures à 5 % pour des niveaux d'aflatoxine B 1
jusqu'à 4 µg/kg dans les pistaches. Les dosages ont également montré une différence éventuelle de teneur
en mycotoxines d'un échantillon, entre les deux types de broyage. Ceci pourrait entraîner des résultats faux
positifs ou négatifs lors du rejet ou de l'acceptation d'un lot, dans la mesure où ces procédés sont basés sur le
résultat fourni par l'échantillon. Il en a été conclu que les pâtes obtenues par voie humide contiennent des
particules plus petites que les échantillons broyés à sec, et génèrent ainsi les plus faibles valeurs de
coefficient de variation possible qui, à leur tour, donnent lieu à une meilleure homogénéisation des
échantillons.
Živila – Mletje vzorcev za analizo mikotoksinov – Primerjava med suhim mletjem in mešanjem ob prisotnosti tekočine
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-september-2006
äLYLOD±0OHWMHY]RUFHY]DDQDOL]RPLNRWRNVLQRY±3ULPHUMDYDPHGVXKLPPOHWMHPLQ
PHãDQMHPRESULVRWQRVWLWHNRþLQH
Foodstuffs - Sample comminution for mycotoxins analysis - Comparison between dry
milling and slurry mixing
Lebensmittel - Probenvorbereitung für die Mycotoxinanalytik - Vergleich zwischen
Trockenvermahlung und Aufschlämmung
Produits alimentaires - Préparation d'échantillons gros volume pour l'analyse des
mycotoxines - Comparaison entre broyage a sec et broyage par voie humide
Ta slovenski standard je istoveten z: CEN/TR 15298:2006
ICS:
67.050
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
TECHNICAL REPORT
CEN/TR 15298
RAPPORT TECHNIQUE
TECHNISCHER BERICHT
May 2006
ICS 67.050
English Version
Foodstuffs - Sample comminution for mycotoxins analysis -
Comparison between dry milling and slurry mixing
Produits alimentaires - Préparation d'échantillons gros Lebensmittel - Probenvorbereitung für die
volume pour l'analyse des mycotoxines - Comparaison Mycotoxinanalytik - Vergleich zwischen Trockenvermahlung
entre broyage à sec et broyage par voie humide und Aufschlämmung
This Technical Report was approved by CEN on 30 November 2005. It has been drawn up by the Technical Committee CEN/TC 275.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TR 15298:2006: E
worldwide for CEN national Members.
Contents Page
Foreword.3
Introduction.4
1 Scope .5
2 Test methods.5
3 Results and discussion.6
4 Acknowledgements . 17
Bibliography. 18
Foreword
This Technical 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 not be held responsible for identifying any or all such patent rights.
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 samples 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 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 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, 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
amount 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 laboratories. 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 than 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 Jörissen had similar experiences, even when applying samples up to 30 kg.
Data of the latter are compiled 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]
th
demonstrated this with a poster presentation at the X International IUPAC symposium on mycotoxins and
phycotoxins in 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-food Authority,
and of Wiertz, Eggert and Jörissen, 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 been discussed at a
CEN/TC 275/WG 5 (Comité Européen 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, 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 these 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 exactly has been examined. After these
experiments the detailed work of Schatzki and Toyofuku [9], who measured particle size distributions on
nd
pistachio slurries, became available. This lead to a joint presentation at the 2 World Mycotoxin Forum,
February 2003, in The Netherlands [10]. This report is a combined outline of both investigations.
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 pistachio samples of 10 kg were milled by a RAS mill and all three sub-samples were completely
analysed for aflatoxin B 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 literature data on
coefficients of variation (CV) for various milling procedures. For dry milling CV values were generally not
below 20 % for aflatoxin B levels up to 38 µg/kg in peanuts, whereas slurry mixing could achieve CV values
below 5 % at aflatoxin B levels down to 4 µg/kg in pistachios. Measurements 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 samples 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 ®
Other laboratory equipment and slurry preparation procedures 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 described 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, 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 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.
Silverson type EX mixer is the trade name of a product supplied by Silverson Machines 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-Straβe 4, D-64579 Gernsheim, Germany. This information is given for the convenience
of the users of this Technical Report and does not 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.
Doing so the complete mycotoxins content in the sample can be reconstructed afterwards by calculation.
3 Results and discussion
The results of 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 is useful for this purpose, in almonds, pistachios and a sample of mixed spices. All other 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 calculated 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 variation 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. 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 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 experiment 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 presented as “sample value” in Table 1.
Table 1 — Results of sampling, milling and mixing experiments as described in 2.3
Ochratoxin A Sample Sub Sub Sub A,B,C
Value A B C Mean STD CV
a
Matrix (µg/kg) (µg/kg) (µg/kg) (µg/kg) (µg/kg) (µg/kg) (%)
Cacao 0,4 0,5 0,4 0,4 0,4 0,1 13,3
Cacao 0,6 0,4 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
Cacao 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 Sample Sub Sub Sub A,B,C
Value A B C Mean STD CV
Matrix (µg/kg) (µg/k
...
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