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Mycotoxins: occurrence, legislation and analysis

Prof. Dr. Biljana Škrbić

University of Novi Sad, Faculty of Technology, Centre of Excellence in Food Safety and Emerging Risks (CEFSER)

E-mail: [email protected]://www.tf.uns.ac.rs/CEFSERweb/CEFSERindex.html

5th CEFSER Training CourseAnalysis of chemical contaminants in food and the environmentFaculty of Technology, University of Novi Sad,Novi Sad, Republic of Serbia7-11 May 2012

Mycotoxins are secondary metabolites produced by a wide variety of fungal species that cause nutritional losses and represent a significant hazard to the food

chain.In fact, mycotoxins have been ranked as the most important chronic dietary risk factor, higher than

synthetic contaminants, plant toxins, food additives or pesticide residues.

Centre of Excellence in Food Safety and Emerging Risks - (CEFSER)

Several environmental factors such as temperature, humidity and soil or storage conditions influence the

occurrence of these toxic substances on agricultural commodities.

Prolonged periods of high humidity and warm temperatures (25-30oC) are the most favorable conditions

for mold growth, while several other factors, such as mechanical injury, insect damage, chemical treatment,

rapidity of drying, leakage in storage and hot spots can also affect the mold growth.


Approximately 300 to 400 substances are

recognized as mycotoxins, comprising a broad

variety of chemical structures, but the most

important groups of mycotoxins that occur

quite often in food are:


• aflatoxins (AFs): AFB1, AFG1, AFB2, AFG2, AFM1 produced by Aspergillus species,

• ochratoxin A (OTA) produced by both Aspergillus and Penicillium species,

• patulinproduced by both Aspergillus and Penicillium species,


• trichothecenes

- type A: HT-2 and T-2 toxin, and

- type B: deoxynivalenol (DON)

zearalenone (ZON), and

• fumonisins B1 and B2 (FBs: FB1 and FB2) produced

by Fusarium species.


Examples of high mycotoxin occurrence in cereals in Europe

• Hungaria:1998: DON was found in 88%(n=99) wheat samples, with thehighest concentration of 4300 μg/kg,being 3.4 times higher than the EClimit (Fazekas et al., 2000)

•Croatia:2004: maximum level of DON incereal grains was 2.7 times higherthan the EC limit (Sokolović andŠimpraga, 2006)

Northern Italy:

aflatoxin contamination exceeding the EU limit in corn(Giorni et al. 2007)

Romania:

1997: in all analyzed wheat samples (n=25) DON waspresented 100%, while 32% of samples had the concentrationabove the EC limit with the maximum concentration of 5600μg/kg , being about 4.5 times higher than the limit (Curtui et al.1998)

2002-2004: 30% of the examined corn samples had of AFs,20% of them above the EU limit (Tabuc et al. 2009)


Germany:1999 frequency of DON occurrence in wheat floursamples (n=60) was very high, with almost 100%, withthe maximum concentration of 1379 μg/kg, being about 2times higher than the EC limit (Schollenberger et al.2002).

Czech Republic:1999 in all analyzed wheat samples (n=48) DON waspresented 100%, with the maximum concentration of2265.2 μg/kg, being about 2 times higher than the EC limit(Hajslova et al. 2007).


When present in food in sufficiently high amounts, these fungal metabolites can have toxic effects that

range from acute to chronic symptoms.

The chemical and biological properties of the mycotoxins, as well

as their toxic effects are extremly variable. Some mycotoxins were

shown to be mutagenic, teratogenic, or/and carcinogenic.


The International Agency for Research in Cancer (IARC) has classified aflatoxins (AFs) as

carcinogenic to humans, while ochratoxin A (OTA) and fumonisins B (FBs) were classified as possibly

carcinogenic.

Trichothecenes (TRs) and zearalenone (ZON) were classified as noncarcinogenic but cause other

adverse effects.


Generally, they are stable chemical

compounds and can neither be

completely removed from the food supply

nor destroyed during processing and

heat treatment.


Therefore, to protect consumers and

to control the mycotoxin occurrence

in products intended for human or

animal consumption,

regulatory authorities have set limits for

maximum residue levels of several toxins.


The major factors affecting maximum

levels are the toxicity of the respective

mycotoxin, its occurrence in food

products and the intake of the concerned

food products by the population.


The latest Official Bulletin of the Republic of

Serbia, No 28/11 (26 April 2011) established

the maximum level for mycotoxins (AFs,

OTA, DON, ZON, PAT, FB1 and FB2) in

different foodstuffs in line with the relevant

EU regulations.


Ochratoxin A Maximum levels (μg/kg)

Unprocessed cereals 5.0

2.2.9 and 2.2.10 All products derived from unprocessed cereals, including processed cereal products and cereals intended for direct human consumption with the exception of foodstuffs listed in

3.0

Dried vine fruit (currants, raisins and sultanas) 10.0

Roasted coffee beans and ground roasted coffee, excluding soluble coffee 5.0

Soluble coffee (instant coffee) 10.0

Wine (including sparkling wine, excluding liqueur wine and wine with an alcoholic strenghtof not less than 15% vol) and fruit wine 2.0

Automatised wine, aromatised wine-based drinks and aromatised wine-product cocktails 2.0

Grape juice, concentrated grape juice as reconstituted, grape nectar, grape must and concentrated grape must as reconstituted, intended for direct human consumption 2.0

Processed cereal-based foods and baby foods for infants and young children 0.50

Dietary foods for special medical purposes intended specifically for infants 0.50

Green coffee, dried fruit other than dried vine fruit, beer, cocoa and cocoa products, liqueur wines, meat products, spices and liquorice –

COMMISSION REGULATION (EC) No 1881/2006

of 19 December 2006

setting maximum levels for certain contaminants in foodstuffs

Patulin Maximum levels (μg/kg)

Fruit juices, concentrated fruit juices as reconstituted and fruit nectars 50

Spirit drinks, cider and other fermented drinks derived from apples or containing apple juice 50

Solid apple products, including apple compote, apple puree intended for direct consumption with the exception of food-stuffs 25

Apple juice and solid apple products, including apple compote and apple puree, for infants and young children and labelled and solid as such 10.0

Baby foods other than processed cereal-based foods for infants and young children 10.0

T2 and HT-2 toxin Sum of T-2 and HT-2 toxin

Unprocessed cereals and cereal products -

It should be noted that regulations regarding

HT-2 and T-2 toxins in food have not yet been

established due to scarce data on the occurrence

of these toxins; nevertheless, they are in the

preparation.


However, according to some literature data (Erikson and

Alexander, 1998), limit for T2 in food and feed is proposed to be

100 µg/kg.

Canada also recomends the maximum of

100 µg/kg of HT-2 in cattle and poultry

feed, Israel has the 100 µg/kg as a

tolerance limit for T-2, as well as Russia

uses the same values as a limit for cereals,

wheat flour and bran.

* Eriksen GS, Alexander J (eds.), 1998. Fusarium toxins in cereals – a risk assessment. Nordic Council of Ministers; TemaNord 1998: 502, pp. 7-27 and 45-58; Copenhagen.

Deoxynivalenol (17) μg/kg

Unprocessed cereals (18) (19) other than durum wheat, oats and maize 1 250Unprocessed durum wheat and oats (18) (19) 1 750Unprocessed maize (18), with the exception of unprocessed maize intended to be processed by wet milling (*) 1 750 (20)

Cereals intended for direct human consumption, cereal flour, bran and germ as end product marketed for human consumption, with the exception of foodstuffs listed in 2.4.7, 2.4.8 and 2.4.9

750

Pasta (dry) (22) 750Bread (including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals 500

Processed cereal-based foods and baby foods for infants and young children (3) (7) 200Milling fractions of maize with particle size > 500 micron falling within CN code 1103 13 or 1103 20 40 and other maize products with particle size > 500 micron not used for direct human consumption falling within CN code 1904 10 10

750 (20)

Milling fractions of maize with particle size ≤ 500 micron falling within CN code 1102 20 and other maize milling products with particle size ≤ 500 micron not used for direct human consumption falling within CN code 1904 10 10

1 250 (20)


of 28 Septembar 2007

amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards Fusariumtoxins in maize and maize products

Zearalenone (17) μg/kg

Unprocessed cereals (18) (19) other than maize 100Unprocessed maize (18) with the exception of unprocessed maize intended to be processed by wet milling (*) 350 (20)

Cereals intended for direct human consumption, cereal flour, bran and germ as end product marketed for direct human consumption, with the exception of foodstuffs listed in 2.5.6, 2.5.7, 2.5.8, 2.5.9 and 2.5.10

75

Refined maize oil 400 (20)Bread (including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals, excluding maize-snacks and maize-based breakfast cereals 50

Maize intended for direct human consumption, maize based snacks and maize-based breakfast cereals 100 (20)

Processed cereal-based foods (excluding processed maize-based foods) and baby foods for infants and young children (3) (7) 20

Processed maize-based foods for infants and young children (3) (7) 20 (20)Milling fractions of maize with particle size > 500 micron falling within CN code 1103 13 or 1103 20 40 and other maize milling products with particle size > 500 micron not used for direct human consumption falling within CN code 1904 10 10

200 (20)


300 (20)

Fumonisins Sum of B1 and B2

Unprocessed maize (18), with the exception of unprocessed maize intended to be processed by wet milling (*) 4 000 (23)

Maize intended for direct human consumption, maize-based foods for direct human consumption, with the exception of foodstuffs listed in 2.6.3 and 2.6.4 1 000 (23)

Maize-based breakfast cereals and maize-based snacks 8 00 (23)Processed maize-based foods and baby foods for infants and young children (3) (7) 200 (23)

Milling fractions of maize with particle size > 500 micron falling within CN code 1103 13 or 1103 20 40 and other maize milling products with particle size > 500 micron not used for direct human consumption falling within CN code 1904 10 10

1 400 (23)


2 000 (23)

(*) The exemption applied only for maize for which it is evident e.g. through labelling, destination, that is intended for use in a wet milling process only (starch production).’

Foodstuffs Maximum levels (μg/kg)

Aflatoxins B1Sum of B1, B2,

G1 and G2M1

Groundnuts (peanuts) and other oilseeds, to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs,with the exception of:– groundnuts (peanuts) and other oilseeds for crushing for refined vegetable oil production

8.0 15.0 –

Almonds, pistachios and apricot kernels to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs

12.0 15.0 –

Hazelnuts and Brasil nuts, to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs 8.0 15.0

Tree nuts, other than the tree nuts listed in 2.1.2 and 2.1.3, to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs

5.0 10.0 –

Groundnuts (peanuts) and other oilseeds and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs,With the exception of:– crude vegetables oils destined for refining– refined vegetable oils

2.0 4.0 –


of 26 February 2010

amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins



G1 and G2M1

Almonds, pistachios and apricot kernels, intended for direct human consumption or use as an ingredient in foodstuffs 8.0 10.0 –

Hazelnuts and Brasil nuts, intended for direct human consumption or use as an ingredient in foodstuffs 5.0 10.0

Tree nuts, other than the tree nuts listed in 2.1.6 and 2.1.7, and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs

2.0 4.0 –

Dried fruit to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs 5.0 10.0 –

Dried fruit and processed products thereof, intended for direct human consumption or use as an ingredient in foodstuffs 2.0 4.0 –

All cereals and all products derived from cereals, including processed cereal products, 2.0 4.0 –

Maize and rice to be subjected to sorting or other physical treatment before human consumption or use as an ingredient in foodstuffs

5.0 10.0 –

Raw milk, heat treated milk and milk for the manufacture of milk-based products – – 0.05


G1 and G2M1

Following species of spices:Capsicum spp. (dried fruits thereof, whole or ground, including chillies, chilli powder, cayenne and paprika)Piper spp. (fruits thereof, including white and black pepper)Myristica fragrans (nutmeg)Zingiber officinale (ginger)Curcuma longa (turmeric)Mixtures of spices containing one or more of the above mentioned spices

5.0 10.0 –

Processed cereal-based foods and baby foods for infants and young children 0.10 – –

Infant formulae and follow-on formulae, including infant milk and follow-on milk – – 0.02

5

Dietary foods for special medical purposes intended specifically for infants 0.10 – 0.02

5’


Ochratoxin A

Capsicum spp. (dried fruits thereof, whole or ground, including chillies, chilli powder, cayenne and paprika)

30 μg/kg as from 1.7.2010 until

30.6.2012Piper spp. (fruits thereof. Including white and black pepper)

Myristica fragrans (nutmeg)

15 μg/kg as from 1.7.2012

Zingiber officinale (ginger)

Curcuma longa (turmeric)

Mixtures of spices containing one or more of the above mentioned spices

Liquorice (Glycyrrhiza glabra, Glycyrrhiza inflate snd other species)

Liquorice root, ingredient for herbal infusion 20 μg/kg

Liquorice extract, for use in food in particular beverages and confectionary 80 μg/kg


of 5 February 2010amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A

Products

Maximum levels aflatoxins(μ/kg or ppb) Sampling

method

Reference analysismethodB1 B1+B2+G1+G2 M1

‘2.1.5. Baby foods and processed cereal-based foods for infants and young children

0.10 – – Directive 1998/53/EC

Directive 1998/53/EC

2.1.6 Infant formulae and follow-on formulae, including infant milk and follow-on-milk

– – 0.025 Directive 1998/53/EC


2.1.7 Dietary foods for special medical purposes intended spacifically for infants

0.10 – 0.025 Directive 1998/53/EC


COMMISSION REGULATION (EC) No 683/2004of 13 April 2004

amending Regulation (EC) No 466/2001 as regards aflatoxins and ochratoxin A in food for infants and young children

ProductMaximum levels

Ochratoxin A(μg/kg or ppb)

Samplingmethod

Referenceanalysis method

‘2.2.4 Baby foods and processed cereal-based foods for infants and young children 0.50 Directive

2002/26/ECDirective

2002/26/EC

2.2.5 Dietary foods for special medical purposes intended spacifically for infants 0.50 Directive

2002/26/ECDirective

2002/26/EC

COMMISSION REGULATION EC No 401/2006 defines the quality control parameters in

order to assure the reliability of methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs....

CriterionConcentration

rangeRecommended

ValueMaximum permitted

Value

Blanks All Negligible –

Recovery-AflatoxinM1

0.01-0.05 μg/kg 60 to 120 %

> 0.05 μg/kg 70 to 110 %

Recovery-AflatoxinB1, B2, G1, G2

< 1.0 μg/kg 50 to 120 %

1-10 μg/kg 70 to 110 %

> 10 μg/kg 80 to 110%

Precision RSDR All As derived from Horwitz Equation

2 x value derived from Horwitz

EquationPrecision RSDr may be calculated as 0.66 times Precision RSDR at the concentration of interest.

COMMISSION REGULATION (EC) No 401/2006of 23 February 2006

laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs

Performance criteria for aflatoxins

Level μg/kgOchratoxin A

RSDr % RSDR % Recovery %

< 1 ≤ 40 ≤ 60 50 to 120

1-10 ≤ 20 ≤ 30 70 to 110

Level μg/kgPatulin


< 20 ≤ 30 ≤ 40 50 to 120

20-50 ≤ 20 ≤ 30 70 to 105> 50 ≤ 15 ≤ 25 75 to 105

Performance criteria for ochratoxin A

Performance criteria for patulin

Level μg/kgDeoxynivalenol


> 100 - ≤ 500 ≤ 20 ≤ 40 60 to 110

> 500 ≤ 20 ≤ 40 70 to 120

Level μg/kgZearalenone


≤ 50 ≤ 40 ≤ 50 60 to 120

> 50 ≤ 25 ≤ 40 70 to 120

Level μg/kgFumonisin B1 and B2


≤ 500 ≤ 30 ≤ 60 60 to 120

> 500 ≤ 20 ≤ 30 70 to 110

Performance criteria for deoxynivalenol

Performance criteria for zearalenone

Performance criteria for fumonisin B1 and B2

Level μg/kgT-2 toxin


50-250 ≤ 40 ≤ 60 60 to 130

> 250 ≤ 30 ≤ 50 60 to 130

Level μg/kgHT-2 toxin


100-200 ≤ 40 ≤ 60 60 to 130

> 200 ≤ 30 ≤ 50 60 to 130

Performance criteria for T-2 and HT-2 toxin

In order to ensure compliance with the current legislation it is necessary to have reliable and

accurate mycotoxin analytical methods, which allow their unambiguous identification and

confirmation, as well as an accurate quantification at very low concentration levels.


Generally, they consist of preparatory step

based on extraction and clean-up

followed by the analysis.


The purpose of the preparatory step is to remove co-extracting compounds that can interfere

with the mycotoxin analysis;

these substances requires multiple extraction and clean-up steps before mycotoxins can be

quantified.


Thus, different strategies have been performed,

including solid phase extraction (SPE), liquid–liquid

extraction, supercritical fluid extraction, etc.


Also, multifunctional columns (MycoSep) and

selective or specific antibodies

(immunoaffinity columns: IAC) for isolation

and purification from the matrix

compounds have been extensively used.


Mycotoxins are usually extracted with

(combinations of) organic solvents (methanol,

acetonitrile or acetone) and water.


Modern analysis of mycotoxins relies mostly on high-performance liquid chromatography (HPLC) or

gas chromatography (GC) in combination with a variety of detectors:


fluorescence detection (FLD)

UV detection

flame ionization detection (FID)

electron-capture detection (ECD)

and more recently,

mass spectrometry (MS).


Different types of mycotoxins produced by a single or several fungal species may be present

simultaneously in a commodity, which is of importance since exposure to mixtures of mycotoxinsmay have unexpected synergistic or additive toxic

effects differing from those of an individual compound.


Thus, determination of all mycotoxins

subjected to the EU regulations in particular

matrix by routine analysis in one single

extract, and, if possible, in a single

analytical run is of great relevance.


However, several difficulties are found to reach this objective:

• Very different maximum levels are admitted for mycotoxins as a function of their toxicity and type of food (e.g. babyfood has more restricted regulation).

• The matrix food compositionis also highly variable, and, finally,

• Mycotoxins present a great diversity in their physicochemical properties.

Considering the wide range of polarities of the

analytes, it would be advantageous to keep

sample preparation to a minimum and to inject the crude extract.

While lower detection levels are achievable in more purified extracts using a clean-up step, excluding this

step provides a faster analysis.

Recently, several authors have validated the crude matrix extracts analysis in contrast to usual preparatory methods widely used by

many routine laboratories for analysis of single toxin or multiple toxins belonging to the same

group in one particular sample matrix.


Sulyok et al. firstly validated a method for the determination of 39 mycotoxins in wheat and maize

using a single extraction step followed by liquid chromatography coupled to triple qudrupole mass spectrometer equipped with electrospray ionization source (LC-ESI-MS/MS) without the need for any

clean-up.

Later, the authors extended this method to perform semi-quantitative analysis of 87

fungal metabolites.

Several more simple multi-mycotoxinanalysis have been developed recently and they referred primarily to Fusarium toxins in

(crude extracts of) cereals and/or cereal-based products:


References MatrixNo. of

Mycotoxinsanalyzed

Sample preparation Separation/Detection

Herebian et al. J. Sep. Sci. 32

(2009) 939-948

wheat, maize

32 250 mg of sample + 950 µl ACN/H2O/HAc (79:20:1)

Vortex, Shaking, CentrifugationDilution 5:1 with the same extraction

solvent

HPLC-ESI-MS/MS

Spanjer et al. Food Addit.

Contam.25 (4) (2008)

472-489

peanut, pistacio, wheat, maize,

cornflake, raisin, fig

33 25 g of sample + 100 ml ACN/H2O (80:20),

25 g of sample + 70% MeOH/H2O - figs and raisisns

ShakingDilution 1:3 with H2O

HPLC-ESI-MS/MS

Sulyok et al.Rapid Commun. Mass Spectrom

20 (2006) 2649-2659

wheat, maize

39 0.5 g of sample + 2 ml ACN/H2O/HAc (79+20+1)

Shaking, CentrifugationDilution 1:1 with ACN/H2O/HAc

(20+79+1)

HPLC-ESI-MS/MS

Beltran et al. Rapid Commun. Mass Spectrom 23 (12) (2009)

1801-1809

maize, dry pasta,

multicereal baby food

11 2.5 g of sample + 10 ml ACN/H2O (80:20) + 0.1% HCOOH

Shaking, CentrifugationDilution 1:1 with HPLC grade water

UHPLC-ESI-MS/MS

Ferrer Amate et al. Anal Bioanal Chem397 (2010) 93-107

spices (e.g. paprika,

curry, black and white pepper,

chilli, curcuma, numteg, ginger)

4 1 g of sample +10 ml ACNShaking, Sonication, CentrifugationDilution 1:1 with high-purity water

HPLC-ESI-MS/MS

Frenich et al. Food Chem, 117 (2009)

705-712

maize, walnuts, biscuits,

breakfast cereals

12 5 g of sample +10 ml ACN/ H2O (80:20) (for biscuits- 20ml)

Vortex, Rotary agitator, Centrifugation

UHPLC-ESI-MS/MS

Elbert et al.AB Sciex Pte. Ltd.

(2010)

wheat, rye, barley, oat

10 10 g of sample + 40 ml ACN/H2O (84:16)Mixing, Filtrating

Dilution 1:10 with water and 5 mM NH4Ac

HPLC-ESI-MS/MS

Škrbić et al.Food Cont 22 (2011)

1261-1267

wheat 10 12.5 g of sample + 50 ml ACN/H2O (84:16)Mixing, Filtrating

Dilution 1:1 with water-methanol mixture (1:1, v/v)

HPLC-APCI-MS/MS

Škrbić et al.Food Control 25 (2012) 389-396

Wheat flour 11 5 g of sample + 20 ml ACN/H2O (84:16)Mixing, Filtrating

Dilution 1:3 with UHPLC mobile phase of the initial content

UHPLC-HESI-MS/MS

In these multimycotoxin analysis, ultra high pressure liquid chromatography (UHPLC)

coupled totandem mass (triple quadrupol) spectrometry (MS/MS or

QqQ)has been preferred analytical methods for selective

identification/quantification of target toxins.

Some of the first studies on the multitoxin

analysis in wheat, wheat flour, maize and spices

collected in Serbia have been conducted by the

CEFSER researchers and the presentation of

these results follows.


5th CEFSER Training CourseAnalysis of chemical contaminants in food and the environmentFaculty of Technology, University of Novi Sad,Novi Sad, Republic of Serbia7-11 May 2012

th CEFSER Training Course Analysis of chemical ... · carcinogenic to humans , while ochratoxinA...

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