M/520 - Mycotoxins in food

This document describes a method for the determination of the sum total of six ergot alkaloids (ergocornine, ergometrine, ergocristine, ergotamine, ergosine and ergocryptine) and their  inine epimer pairs by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) after clean-up by dispersive solid phase extraction (SPE).
The method has been validated for cereals and cereal-based food products.
The method has been validated in the range 13,2 µg/kg to 168 µg/kg for the sum of the twelve ergot alkaloids, in rye flour, rye bread and cereal products (breakfast cereal, infant breakfast cereal, and crispbread) that contained rye as an ingredient, as well as seeded wholemeal flour and a barley and rye flour mixture.
Method performance was satisfactory in the range 24,1 µg/kg to 168 µg/kg, however at lower concentrations RSDR values were greater than 44 %, and HorRat values exceeded 2,0, indicating the method may not be fully suitable at concentrations below 24 µg/kg for sum of ergot alkaloids, although it is suitable for screening at these concentrations. Method performance may be improved by inclusion of an isotopically labelled internal standard, but this was not available at the time of the method validation study.

This document describes a procedure for the determination of aflatoxins B1, B2, G1 and G2 and total aflatoxins (sum of B1, B2, G1 and G2) in spices for which EU maximum levels are established, other than paprika, by high performance liquid chromatography (HPLC) with post-column derivatization (PCD) and fluorescence detection (FLD) after immunoaffinity column clean-up.
The method is applicable to the spices capsicum, pepper, nutmeg, ginger, turmeric and mixtures thereof.
The method has been validated for aflatoxins B1, B2, G1 and G2 and total aflatoxins in a range of test samples that comprised: ginger, pepper, nutmeg, chilli, turmeric as individual spices and mixed pepper+chilli+nutmeg (90+5+5, m+m+m), mixed spice+ginger (6+4, m+m) mixed spice, mixed turmeric+ginger (2+8, m+m).
The validation was carried out over the following concentration ranges: aflatoxin B1 = 1 µg/kg to 16 µg/kg and total aflatoxins = 2,46 µg/kg to 36,1 µg/kg.

This document describes a procedure for the determination of ochratoxin A (OTA) in chilli, paprika, black and white pepper, nutmeg, spice mix, liquorice (root and extracts), cocoa and cocoa products by high performance liquid chromatography (HPLC) with immunoaffinity column clean-up and fluorescence detection.
This method has been validated in interlaboratory studies via the analysis of both naturally contaminated and spiked samples ranging from 1,0 μg/kg to 84,9 μg/kg for spices (paprika and chili [5], black and white pepper, nutmeg and spice mix [6]), ranging from 7,7 μg/kg to 96,8 μg/kg for liquorice [7] and ranging from 2,1 μg/kg to 26,3 μg/kg for cocoa and cocoa products [6].
For further information on the validation see clause 9 and Annex B.

This document describes a procedure for the determination of phomopsins in lupin seeds and lupin-derived products based on liquid chromatography with tandem mass spectrometry (LC-MS/MS). Several phomopsins exist, i.e. phomopsin A, B, C and D, but the method only deals with the quantitative measurement of phomopsin A due to lack of commercially available analytical reference standards for the other phomopsins.
The method has been validated for phomopsin A in naturally contaminated lupin seeds, lupin flour and crisp bread at levels ranging from approximately 5 µg/kg to 60 µg/kg.

This document describes a procedure for the determination of ochratoxin A (OTA) in pork products specifically ham, pork based products (canned chopped pork) and pork liver using high performance liquid chromatography with fluorescence detection (HPLC-FLD).
The method has been validated for ochratoxin A with naturally contaminated ham, pork based products (canned chopped pork) and pork liver containing 0,5 μg/kg to 11 μg/kg [4, 5, 6].
Laboratory experiences have shown that this method is also applicable to pâté and kidney [4].

This document specifies a procedure for the determination of ochratoxin A (OTA) in chilli, paprika, black and white pepper, nutmeg, spice mix, liquorice (root and extracts), cocoa and cocoa products by high performance liquid chromatography (HPLC) with immunoaffinity column clean-up and fluorescence detection (FLD).
This method has been validated in interlaboratory studies via the analysis of both naturally contaminated and spiked samples ranging from 1,0 μg/kg to 84,9 μg/kg for spices (paprika and chili [5], black and white pepper, nutmeg and spice mix [6]), ranging from 7,7 μg/kg to 96,8 μg/kg for liquorice and liquorice products [7] and ranging from 2,1 μg/kg to 26,3 μg/kg for cocoa and cocoa products [6].
For further information on the validation, see Clause 10 and Annex B.

This document describes a procedure for the determination of ochratoxin A (OTA) in pork products specifically ham, pork-based products (canned chopped pork) and pork liver using high performance liquid chromatography with fluorescence detection (HPLC-FLD).
The method has been validated for ochratoxin A in naturally contaminated ham, pork based products (canned chopped pork) and pork liver containing 0,5 μg/kg to 11 μg/kg [4], [5], [6].
Laboratory experiences have shown that this method is also applicable to pâté and kidney [4].

This document specifies a procedure for the determination of phomopsin A in lupin seeds and lupin-derived products based on liquid chromatography with tandem mass spectrometry (LC-MS/MS). Several phomopsins exist, i.e. phomopsin A, B, C and D, but the method only deals with the quantitative measurement of phomopsin A due to lack of commercially available analytical reference standards for the other phomopsins.
The method has been validated for phomopsin A in naturally contaminated lupin seeds, lupin flour and crisp bread at levels ranging from approximately 5 µg/kg to 60 µg/kg

This document describes a screening method for the determination of aflatoxin B1, deoxynivalenol, fumonisin B1 and B2, ochratoxin A, HT-2 and T-2 toxins, and zearalenone in foodstuffs by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS).
The aim of the screening method is to test compliance of foodstuff with regulatory limits or to determine whether a certain pre-defined level (the screening target concentration, STC) is exceeded or not. The result of the screening is either "negative" or "suspect". "Negative" (screen negative) means that the targeted mycotoxins are not detected or potentially present but below the STC. "Suspect" (screen positive) means that the established cut-off level is exceeded and the sample can contain one or more mycotoxins at a level higher than the STC.
For full identification and accurate quantification a second confirmatory quantitative analysis method is required which is outside the scope of this document.
The method is suitable for various types of foodstuff and has been validated for representative matrices from four commodity groups:
-   high starch and/or protein content and low water and fat content: wheat, cereal mixture, wheat flour and cornflakes;
-   high oil content: peanuts;
-   high sugar low water content: figs;
-   high water content: grape juice.
During validation, cut-off levels were established for the following screening target concentrations:
-   aflatoxin B1: 2 µg/kg to 5 µg/kg;
-   deoxynivalenol: 250 µg/kg to 865 µg/kg;
-   fumonisin B1: 200 µg/kg to 790 µg/kg;
-   fumonisin B2: 110 µg/kg to 230 µg/kg;
-   ochratoxin A: 4 µg/kg to 9 µg/kg;
-   T-2 toxin: 25 µg/kg;
-   HT-2 toxin: 25 µg/kg to 50 µg/kg;
-   zearalenone: 30 µg/kg to 100 µg/kg.

This document describes a procedure for the determination of nivalenol (NIV), deoxynivalenol (DON) and its acetyl derivatives (3-acetyl-DON and 15-acetyl-DON), HT-2 and T-2 toxins (HT-2, T-2) and zearalenone (ZEA) in cereals and cereal products by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid phase extraction (SPE).
The method has been validated with both contaminated and spiked samples of wheat, wheat flour, and wheat crackers.
Validation levels for NIV ranged from 27,7μg/kg to 377,8 μg/kg.
Validation levels for DON ranged from 233,9μg/kg to 2420,0 μg/kg.
Validation levels for 3-acetyl-DON ranged from 18,5μg/kg to 136,5 μg/kg.
Validation levels for 15-acetyl-DON ranged from 11,4μg/kg to 141,8 μg/kg.
Validation levels for HT-2 ranged from 6,6 μg/kg to 133,8 μg/kg.
Validation levels for T-2 ranged from 2,1 μg/kg to 37,6 μg/kg.
Validation levels for ZEA ranged from 31,6μg/kg to 229,7 μg/kg
Laboratory experiences have shown that this method is also applicable to barley and oat flour, and rye based crackers [5], however, this has not been validated in a collaborative study.

This document describes a screening method for the determination of aflatoxin B1, deoxynivalenol, fumonisin B1 and B2, ochratoxin A, HT-2 and T-2 toxins, and zearalenone in foodstuffs by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS).
The aim of the screening method is to test compliance of foodstuff with regulatory limits or to determine whether a certain pre-defined level (the screening target concentration, STC) is exceeded or not. The result of the screening is either "negative" or "suspect". "Negative" (screen negative) means that the targeted mycotoxins are not detected or potentially present but below the STC. "Suspect" (screen positive) means that the established cut-off level is exceeded and the sample can contain one or more mycotoxins at a level higher than the STC.
For full identification and accurate quantification a second confirmatory quantitative analysis method is required which is outside the scope of this document.
The method is suitable for various types of foodstuff and has been validated for representative matrices from four commodity groups:
-   high starch and/or protein content and low water and fat content: wheat, cereal mixture, wheat flour and cornflakes;
-   high oil content: peanuts;
-   high sugar low water content: figs;
-   high water content: grape juice.
During validation, cut-off levels were established for the following screening target concentrations:
-   aflatoxin B1: 2 µg/kg to 5 µg/kg;
-   deoxynivalenol: 250 µg/kg to 865 µg/kg;
-   fumonisin B1: 200 µg/kg to 790 µg/kg;
-   fumonisin B2: 110 µg/kg to 230 µg/kg;
-   ochratoxin A: 4 µg/kg to 9 µg/kg;
-   T-2 toxin: 25 µg/kg;
-   HT-2 toxin: 25 µg/kg to 50 µg/kg;
-   zearalenone: 30 µg/kg to 100 µg/kg.

This document specifies a procedure for the determination of nivalenol (NIV), deoxynivalenol (DON) and its acetyl derivatives (3-acetyl-DON and 15-acetyl-DON), HT-2 and T-2 toxins (HT-2 and T-2) and zearalenone (ZEN) in cereals and cereal products by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after clean-up by solid phase extraction (SPE).
The method has been validated with samples of wheat, wheat flour, and wheat crackers. The wheat and the wheat flour was prepared from a mixture of wheat and fungi infected wheat kernels. The wheat crackers were baked from wheat flour and water spiked with the target mycotoxins.
Validation levels for NIV ranged from 27,7 μg/kg to 378 μg/kg.
Validation levels for DON ranged from 234 μg/kg to 2420 μg/kg.
Validation levels for 3-acetyl-DON ranged from 18,5 μg/kg to 137 μg/kg.
Validation levels for 15-acetyl-DON ranged from 11,4 μg/kg to 142 μg/kg.
Validation levels for HT-2 ranged from 6,6 μg/kg to 134 μg/kg.
Validation levels for T-2 ranged from 2,1 μg/kg to 37,6 μg/kg.
Validation levels for ZEN ranged from 31,6 μg/kg to 230 μg/kg.
Laboratory experiences have shown that this method is also applicable to barley and oat flour, and rye based crackers [5], however, this has not been validated in a collaborative study.

This European Standard describes a method for the determination of the content of T-2 toxin and HT-2 toxin in cereals and cereal based products e.g. oats, intended for nutrition of infants and young children by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid phase extraction (SPE) [5].
The method has been validated for HT-2 toxin in oat flour at levels of 9,3 µg/kg and 28,1 µg/kg, oat flakes at levels of 16,5 µg/kg and 21,4 µg/kg, and breakfast cereals (containing oat flakes) at a level of 8,1 µg/kg and for T-2 toxin in oat flour at levels of 4,4 µg/kg and 8,3 µg/kg, oat flakes at levels of 4,9 µg/kg and 6,6 µg/kg and breakfast cereals (containing oat flakes) at a level of 3,5 µg/kg.
Laboratory experiences [6] have shown that the method is also applicable to highly swelling materials (dry cereal based porridges and modified starches), but these were not examined in the method validation study. Details are outlined in 6.3.
The method can also be applied to oat-by-products at higher levels of T-2- and HT-2 toxin. In this case, the dilution steps need to be considered [6].

This European Standard describes a procedure for the determination of the zearalenone content in edible vegetable oils specifically maize germ oil by either of the following techniques: High performance liquid chromatography with fluorescence detection (LC-FLD) or high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) after basic extraction of the diluted oil.
The method has been validated for zearalenone in naturally contaminated maize germ oil at levels of 61,2 µg/kg to 515 µg/kg [5].
Laboratory experiences [6] have shown that this method is also applicable to vegetable oils such as wheat germ oil (n = 4), sunflower oil (n = 5), pumpkin seed oil (n = 1), soybean oil (n = 5), hemp seed oil (n = 5), rape seed oil (n = 11), and mixed oils including maize germ oils (n = 3). However occasionally, samples can result in interferences in the FLD-chromatograms. In this case, the detection with MS/MS is recommended.

This European Standard describes a method for the determination of T-2 toxin and HT-2 toxin in cereals and cereal based products e.g. oats, intended for nutrition of infants and young children by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) after cleanup by solid phase extraction (SPE) [5].
The method has been validated for HT-2 toxin in oat flour at levels of 9,3 µg/kg and 28,1 µg/kg, oat flakes at levels of 16,5 µg/kg and 21,4 µg/kg, and breakfast cereals (containing oat flakes) at a level of 8,1 µg/kg and for T-2 toxin in oat flour at levels of 4,4 µg/kg and 8,3 µg/kg, oat flakes at levels of 4,9 µg/kg and 6,6 µg/kg and breakfast cereals (containing oat flakes) at a level of 3,5 µg/kg.
Laboratory experiences [6] have shown that the method is also applicable to highly swelling materials (dry cereal based porridges and modified starches), but these were not examined in the method validation study. Details are outlined in 6.3.
The method can also be applied to oat-by-products at higher levels of T-2- and HT-2 toxin. In this case, the dilution steps need to be considered [6].
The method can also be applied to cereals and cereal products for infants and young children based on e.g. wheat, barley, and rice. In this case, the method needs to be in-house-validated for each material. At the time of the interlaboratory study, planned range was 10 µg/kg to 100 µg/kg, and it is known from the pre-study that the method works well in the whole range, although final validation was only done in the range from 3,5 µg/kg to 28,1 µg/kg.

This European Standard describes a procedure for the determination of the zearalenone content in edible vegetable oils specifically maize germ oil by either of the following techniques: High performance liquid chromatography with fluorescence detection (LC-FLD) or high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) after basic extraction of the diluted oil.
The method has been validated for zearalenone in naturally contaminated maize germ oil at levels of 61,2 µg/kg to 515 µg/kg [5].
Laboratory experiences [6] have shown that this method is also applicable to other vegetable oils such as wheat germ oil (n = 4), sunflower oil (n = 5), pumpkin seed oil (n = 1), soybean oil (n = 5), hemp seed oil (n = 5), rape seed oil (n = 11), and mixed oils including maize germ oil (n = 3). However occasionally, samples can result in interferences in the FLD-chromatograms. In this case, the detection with MS/MS is recommended.

This document describes a method for the determination of the sum of six ergot alkaloids (ergocornine, ergometrine, ergocristine, ergotamine, ergosine and ergocryptine) and their -inine epimer pairs by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) after clean-up by dispersive solid phase extraction (dSPE).
The method has been validated in the range 13,2 µg/kg to 168 µg/kg for the sum of the twelve ergot alkaloids, in rye flour, rye bread and cereal products (breakfast cereal, infant breakfast cereal, and crispbread) that contained rye as an ingredient, as well as seeded wholemeal flour and a barley and rye flour mixture.
Method performance was satisfactory in the range 24,1 µg/kg to 168 µg/kg, however at lower concentrations RSDR values were greater than 44 %, and HorRat values exceeded 2,0, indicating the method may not be fully suitable at concentrations below 24 µg/kg for sum of ergot alkaloids, although it is suitable for screening at these concentrations.

This document describes a procedure for the determination of aflatoxins B1, B2, G1 and G2 and total aflatoxins (sum of B1, B2, G1 and G2) in spices for which EU maximum levels are established, other than paprika, by high performance liquid chromatography (HPLC) with post-column derivatization (PCD) and fluorescence detection (FLD) after immunoaffinity column (IAC) clean-up.
The method is applicable to the spices capsicum (excluding paprika), pepper, nutmeg, ginger, turmeric and mixtures thereof.
The method has been validated for aflatoxins B1, B2, G1 and G2 and total aflatoxins in a range of test samples that comprised: ginger, pepper, nutmeg, chilli, turmeric as individual spices and mixed pepper + chilli + nutmeg (90 + 5 + 5, m + m + m), mixed spice+ginger (6 + 4, m + m) mixed spice, mixed turmeric+ginger (2 + 8, m + m).
The validation was carried out over the following concentration ranges: aflatoxin B1 = 1 µg/kg to 16 µg/kg and total aflatoxins = 2,46 µg/kg to 36,1 µg/kg.

This European Standard specifies a method for the determination of five Alternaria toxins in wheat, tomato juice and sunflower seed samples by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method includes the analysis of Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME) in the range of 1 μg/kg to 100 μg/kg, and Tentoxin (TEN) in the range of 5 μg/kg to 500 μg/kg, and Tenuazonic acid (TEA) in the range of 10 μg/kg to 1000 μg/kg.

This European Standard specifies a method for the determination of five Alternaria toxins in wheat, tomato juice and sunflower seed samples by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method includes the analysis of Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME) in the range of 1 μg/kg to 100 μg/kg, and Tentoxin (TEN) in the range of 5 μg/kg to 500 μg/kg, and Tenuazonic acid (TEA) in the range of 10 μg/kg to 1000 μg/kg.

This European Standard describes a procedure for the determination of the citrinin content in food (cereals, red rice), herbs and food supplements by liquid chromatography tandem mass spectrometry (LC-MS/MS).
This method has been validated for red yeast rice in the range of 2,5 μg/kg to 3000 μg/kg and for wheat flour in the range of 2,5 μg/kg to 100 μg/kg.
Laboratory experiences have shown that this method is also applied to white rice, herbs such as a powder of ginkgo biloba leaves and the formulated food supplements.

This document describes a procedure for the determination of the citrinin content in food (cereals, red yeast rice (RYR)), herbs and food supplements by liquid chromatography tandem mass spectrometry (LC MS/MS).
This method has been validated for citrinin in red yeast rice and in the formulated food supplements in the range of 2,5 µg/kg to 3000 µg/kg and in wheat flour in the range of 2,5 µg/kg to 100 µg/kg.
Laboratory experiences have shown that this method is also applicable to white rice, herbs such as a powder of ginkgo biloba leaves and the formulated food supplements in the range of 2,5 µg/kg to 50 µg/kg.