Martien C Spanjer

Peter Rensen

Jos Scholten

Keywords: Baby food, Cereals and grain, Dried fruit, multi-mycotoxin, matrix effect, ion suppression, LC-MS/MS, validation, baby food, ergot alkaloids, silage

published or not. The documents may come

Introduction

Until now mycotoxins are mainly determined by single compound analytical methods based on immunoaffinity column cleanup. These methods were studied for aflatoxins [START_REF] Stroka | Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study[END_REF], ochratoxin A [START_REF] Entwisle | Liquid chromatographic method with immunoaffinity column cleanup for determination of ochratoxin A in barley: collaborative study[END_REF], fumonisins B 1 and B 2 [START_REF] Visconti | Determination of fumonisins B 1 and B 2 in corn and corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative study[END_REF], deoxynivalenol [START_REF] Cahill | Quantification of deoxynivalenol in wheat using an immunoaffinity column and liquid chromatography[END_REF] and zearalenone [START_REF] Schuhmacher | Immunoaffinity columns versus conventional cleanup: a method comparison study for the determination of zearalenone in corn[END_REF]. Since some years immunoaffinity columns are available for the simultaneous determination of aflatoxin & ochratoxin A [START_REF] Chan | Simultaneous determination of aflatoxins and ochratoxin A in food using a fully automated immunoaffinity column cleanup and liquid chromatography-fluorescence detection[END_REF] and aflatoxin, ochratoxin A & zearalenone [START_REF] Göbel | Simultaneous determination of aflatoxins, ochratoxin A and zearalenone in grains by new immunoaffinity column/liquid chromatography[END_REF]. Despite minor possibilities for cross-reactions, assays are generally considered to be so specific that confirmation is hardly necessary. Increasing quality demands changed these views in the nineties. Methods were developed in which mass spectrometry was applied for confirmation purposes, still based on individual mycotoxins. [START_REF] Hurst | The use of HPLC/thermospray MS for the confirmation of aflatoxins in peanuts[END_REF] applied thermospray mass spectrometry for the confirmation of aflatoxins in peanuts, [START_REF] Xiao | Synthesis and structural elucidation of analogs of Ochratoxin A[END_REF] used electron impact mass spectrometry to elucidate ochratoxin structures, and [START_REF] Holcomb | HPLC and FAB mass spectrometry analysis of fumonisins B 1 and B 2 produced by Fusarium moniliforme on food substrates[END_REF] choose fast atom bombardment mass spectrometry to confirm the presence of fumonisin B 1 in food. In our laboratory the need for confirmation has emerged when application of DON assays revealed peaks in the chromatogram, which could interfere with the DON peak. We [START_REF] Spanjer | Evaluation of effect of measures taken after a DON outbreak[END_REF] started to use mass spectrometry (MS) by applying the Thermo Finnigan LCQ, an ion trap detector, for confirmation of DON as described by [START_REF] Xiao | Synthesis and structural elucidation of analogs of Ochratoxin A[END_REF].

Aflatoxins and ochratoxin A are already subject to European Union (EU) legislation for some years (European Commission (EC), 2002 and[START_REF] Ec | Commission Regulation (EC) 683/2004 of April 13th 2004 amending Regulation (EC) 466/2001 as regards aflatoxins and ochratoxin A in foods for infants and young children[END_REF]. EU legislation on DON and zearalenone came into force [START_REF] Ec | Commission Regulation (EC) 856/2005 of 6 June 2005 amending Regulation (EC) 466/2001 as regards Fusarium toxins[END_REF]. The fumonisins B 1 +B 2 will follow as per October 1, 2007(EC, 2006). The European Commission is considering legislation for Fusarium toxins like T-2, HT-2 and nivalenol. Facing all this upcoming legislation there grew a need to analyse as many mycotoxins as possible in one liquid chromatography-mass spectrometry (LC-MS) run. This principle originates from the mycological field, where measurements are made preferably in crude fungal extracts to identify mould species [START_REF] Shu | Using direct electrospray mass spectrometry in taxonomy and secondary metabolite profiling of crude fungal extracts[END_REF]. These authors applied single quadrupole equipment to Penicillium isolates, in which they identified 13 mycotoxins, of which ochratoxin A, citrinin, penicillic acid and roquefortine C are the most important for food control. To perform quantification and identification at the same time, a triple quadrupole is needed. In that case one transition reaction is used for quantification and identification is obtained from a second transition. [START_REF] Kussak | Determination of aflatoxins in dust and urine by liquid chromatography/electrospray ionization tandem mass spectrometry[END_REF] applied this technique for the determination of aflatoxins in dust, [START_REF] Lukacs | Identification and determination of fumonisin FB 1 and FB 2 in corn and corn products by high-performance liquid chromatographyelectrospray-ionization tandem mass spectrometry (HPLC-ESI-MS-MS)[END_REF] identified fumonisins in corn, and [START_REF] Razzazi-Fazeli | Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography-mass spectrometry with negative ion atmospheric pressure chemical ionisation[END_REF] determined nivalenol and DON in wheat. However, these authors still applied a cleanup step in sample preparation.

Janssens and Franken (Technical Laboratory Rotterdam, the Netherlands) analysed trichothecenes, fumonisins, aflatoxins and ochratoxin in feed after only one single extraction with acetonitrile-water.

We optimised, extended and validated their method for food, as mycotoxin limits for human consumption are lower. Special attention was paid to the matrix effect. The result on 13 mycotoxins was presented at the second World Mycotoxin Forum [START_REF] Spanjer | Single run LC-MS/MS analysis of mycotoxins subject to actual and upcoming EU legislation in one sample extract[END_REF]. Since then the number of mycotoxins in this method has been more then doubled. Latest additions were the Alternaria toxins [START_REF] Scussel | Determination of mycotoxins in fermented beverages, cork stoppers and fruit juices by liquid chromatography-tandem mass spectrometry[END_REF]) that even recently were analysed with thin layer chromatography by [START_REF] Centeno | Mycotoxins Produced by Fungi Isolated from Wine Cork Stoppers[END_REF]. The method has also been successfully applied to silage samples, as delivered by [START_REF] Driehuis | Mycotoxins in forage feeds for dairy cattle[END_REF], who presented their results elsewhere. It can also be adapted to the analysis of aflatoxin B 1 and ochratoxin A in baby food, for which lower legislative levels exist. This made it necessary to apply immunoassay cleanup again as to reach the necessary LODs. With the combined aflatoxin/ochratoxin column this could be done in one assay, as described for fluorescence detection by [START_REF] Chan | Simultaneous determination of aflatoxins and ochratoxin A in food using a fully automated immunoaffinity column cleanup and liquid chromatography-fluorescence detection[END_REF].

Materials and methods

Chemicals

Water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). Methanol (Merck KGaA, Darmstadt, Germany) and acetonitrile (Labscan Limited, Dublin, Ireland) were HPLC-grade. Formic acid (Merck) was analytical-reagent grade. All mycotoxins were purchased from Sigma-Aldrich (Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands). From all solid standards, individual stock standard solutions were prepared and stored in the freezer at -18°C. Stock standard solutions of all mycotoxins were made in methanol, except for OTA (in toluene/acetic acid), deoxynivalenol (DON, in acetonitrile), fumonisins (FB 1 , FB 2 , in 50% acetontrile/50% water, exceptionally stored in refrigerator, not freezer), ZEN (acetonitrile) and 3Ac-DON (acetonitrile), respectively. For aflatoxin B 1 , ochratoxin A, DON and zearalenone, purity checks are made by UV measurement since for these compounds molar absorptivities are known. As an independent check comparisons are made with measurements of standard solutions for aflatoxin B 1 , ochratoxin A, DON, zearalenone and fumonisins, as delivered by R-Biopharm Rhone Ltd. (West of Scotland Science Park, Unit 3.06 Kelvin Campus, Glasgow G20 0SP, Scotland) and Biopure Referenzsubstanzen GmbH. (Technopark 1, 3430 Tulln, Austria), since these are often used in routine analysis procedures and therefore available as well.

Preparation of standard solutions

From these individual stock standard solutions a stock-standard mixture was prepared in methanol, as to be used to prepare calibration curves in solvent and in matrix, as to determine ion suppression, recovery and limit of detection (LOD). Stock standard mixture solution: an appropriate volume (µl) of stock standard solution of each toxin was taken and transferred to a calibrated 200 ml flask and methanol was added to make to volume in order to obtain the following concentrations: aflatoxins (0.125 µg/ml each); ochratoxin A (0.250 µg/ml), fumonisins, zearalenone (ZEN), alfa-zearalanol (α-ZAL), beta-zearalanol (ß-ZAL), zearalanone (ZAN), alfa-zearalenol (α-ZEL); roquefortine, sterigmatocystin, cyclopiazonic acid, penicillic acid and citrinin (2.50 µg/ml each), ergotamin (5.00 µg/ml) and trichothecenes (DON, DAS, T2, HT2, 3 and 15 Ac-DON) all 6.25 µg/ml each. This stock standard mixture is also kept in the freezer at -18°C. Both stock standard solutions and stock standard mixture are stable for at least 2 years, when kept in the freezer at -18°C. This stability check was made by comparing the freshly made stock standard mixture with the former one. From the stock standard mixture, calibration solutions were prepared in 20% acetonitrile, by diluting 25,50,100,200,400 µl in volumes of 50 ml, resulting in concentration levels of 0.0625, 0.125, 0.25, 0.5 and 1.0 ng/ml for the aflatoxins, 0.125, 0.25, 0.5, 1.0 and 2.0 ng/ml for ochratoxin-A, 1.25, 2.50, 5.00, 10.0 and 20.0 ng/ml for fumonisins, zearalenone (ZEN), alfa-zearalanol (α-ZAL), betazearalanol (ß-ZAL), zearalanone (ZAN), alfa-zearalenol (α-ZEL); roquefortine, sterigmatocystin, cyclopiazonic acid, penicillic acid and citrinin, 2.50, 5.00, 10.0, 20.0 and 40.0 ng/ml for ergotamin and 3.125, 6.25, 12.5, 25.0, 50.0 ng/ml for trichothecenes (DON, DAS, T2, HT2, 3 and 15 Ac-DON) respectively.

Laboratory equipment

The usual laboratory equipment and following mixers: a high shearing mixer, Silverson model EX (Silverson Machines Ltd, Waterside, Chesham, UK), a mini shaker model KMS2 and a horizontal shaker model HS501/digital from IKA (IKA Werke GmbH & Co. KG, Staufen, Germany).

LC-MS system

The LC-MS/MS system consisted of a Waters Alliance 2695 separation module with a 100 µl injection loop (Waters, Milford, MA, USA) coupled to a Quattro Ultima triple quadrupole mass spectrometer (Waters-Micromass, Manchester, UK) equipped with an electrospray interface. A 20 µL aliquot is injected on an Alltima C 18 (150x3.2 mm, 5 µm) column (Alltech, Breda, the Netherlands) at 30°C column temperature. The gradient was composed of solvents A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at a flow rate of 0.3 ml/min (Table 1). The MS was operated in the positive mode at a capillary voltage of 2.5 kV, a desolvation temperature of 450°C, a desolvation gas flow of 600 L/h, a variable cone voltage, a cone gas flow of 100 L/h and nebuliser gas fully open, all nitrogen. For MRM the collision gas was argon at 0.8 bar on pressure regulator. Ionisation and MS-MS collision energy settings were optimised while continuously infusing the separate mycotoxin solutions (1 -5 µg/ml) at a flow rate of 5-10 µl/min by means of a syringe pump. For each mycotoxin the parent and two daughter ions were recorded, which are listed in Table 2.

The first is used for quantification and the second for confirmation. Quantification was done by matrix-matched standard calibration. All results were corrected for matrix-effect. Dwell time was 0.02 s. Masslynx and Quanlynx software was used for data acquisition and processing.

[Insert Table 2 about here] Extraction Dry milling: samples less then 1.5 kg are dry milled. Of the ground sample material 25 gram is mixed with 100 mL 80/20 v/v acetonitril-water and placed in the horizontal shaker during 2 hours. One millilitre of the clear extract is diluted with 3 ml water and mixed. Cloudy solutions were filtrated using a 0.45 µm membrane filter (Schleicher & Schull, Spartan 13). Samples larger then 1.5 kg are slurry mixed with water by means of a high shearing mixer [START_REF] Spanjer | Sample comminution for mycotoxin analysis: Dry milling or slurry mixing?[END_REF]). Usually to one part of sample, 1.5 parts of water is added. Other slurry-ratios of sample to water are possible. In such cases the slurry-amount for the extraction is adjusted. For extraction 50 grams slurry (1: 1.5) is taken to which 60 ml acetonitrile is added. This mixture is shaken for two hours in the horizontal shaker. 1 ml of the clear extract is diluted with 2.55 ml water and handled as described at the dry sample handling procedure. To investigate the matrix-effect, 7 standards were prepared in 20% acetonitrile as well as added to non-contaminated, i.e. blank matrix after extraction and injected 6 times each. Figs and raisins are extracted in 70% methanol-water instead of acetonitrile-water. This is due to the fact that the high sugar content of these matrices causes layer separation of the acetonitrile and water, a phenomenon that has been thouroughly investigated by [START_REF] Stroka | Investigation of various extractants for the analysis of aflatoxin in different food and feed matrices[END_REF]. Silage samples were taken by [START_REF] Driehuis | Mycotoxins in forage feeds for dairy cattle[END_REF] and delivered as a milled powder, for which they applied a freezedrying procedure, 5 g of which were extracted.

Validation

Validation of the method is carried out for 13-24 mycotoxins in 7 different matrices. For the validation of the method, two extra standard solutions were prepared, by diluting 800 µl and 1600 µl of the stock standard mixture mixture in 50 ml 20% acetonitrile, resulting in two-and four times the highest concentration of the individual standard concentrations as mentioned before (i.e. the concentrations for the aflatoxins will be 2.0 and 4.0 ng/ml respectively). Three levels of contamination were prepared in matrix. Aflatoxins (1, 2 and 16 µg/kg each), OTA (2, 4 and 32 µg/kg), fumonisins B 1 and B 2 and ZON (20, 40 and 320 µg/kg each), trichothecenes DON, DAS, T-2, HT-2 and 3 Acetyl-DON (40, 80 and 640 µg/kg each). The three levels and a blank level were injected 6 times each in the LC-MS/MS system, resulting in 18 datapoints.

Ergot alkaloids

From a naturally contaminated rye sample (0.4% m/m) the ergot kernels were sorted out and processed according to the general sample preparation procedure. The extract was injected in the LC-MS system at parent scan mode at m/z 268, which represents the protonated lysergamide part of the molecule, a characteristic fragment for ergot alkaloids, as to investigate the presence of all possible natural constituents. After this experiment a number of ergot alkaloid standards were obtained, to add to the standard mixture. This lead to validation of ergotamin, ergocornin, ergocristin and α-ergocryptin in whole wheat flour at levels of 100, 1000 and 2000 µg/kg in the MRM mode of the multimethod.

Results

All chromatographic and mass spectrometric data are presented in table 2: parent and daughter ions, cone (V) and collision (eV) voltages, ratios and retention times (min) and references for fragment clarification. Validation data are given in tables 3-9 for peanut, pistachio, wheat, maize, cornflake, raisin and fig. The calibration curves are of the type Y = aX + b, with a weight factor 1/X and consisted of 7 data points.

[Insert Tables 3456789about here] The recoveries in several food types, which we gathered in 2005 and 2006 while applying the multimethod for analysis of routine samples, are listed in table 10.

[Insert Table 10 about here] Figure 1 gives an impression of peak shape of 26 mycotoxines as they appear in the chromatogram.

Figures 2 and3 show some examples of multi-mycotoxin measurements. Figure 2 gives an example of a maize sample as run with this multi-mycotoxin method. The following peaks could be identified: at formed in the field by Fusarium species. The presence of aflatoxin B 1 in this maize sample is an indication for the occurrence of heat stress during growth in the field [START_REF] Abbas | Aflatoxin and fumonisin contamination of commercial corn (Zea mays) hybrids in Mississippi[END_REF]. In Figure 3 an example is given of a buckwheat sample with zearalenone, ochratoxin A and sterigmatocystin levels around 35 µg/kg, and a trace of roquefortin C. Both examples show the advantage of the multimycotoxin method above single methods. In figure 4 an example is given of the measurement on a silage sample. Figure 5 shows the individual peaks of ergot alkaloids when running the LC-MS in the parent mode on an extract of naturally contaminated rye kernels, sorted out of an inspected lot.

[Insert Figures 12345about here]

Discussion

To separate these different type of mycotoxins a gradient is needed, which starts with a high percentage of water. Figure 1 gives an impression of 26 mycotoxins. It shows that for nivalenol it is not possible to obtain a chromatographic peak, even not at a level of 400 µg/kg and for citrinin and cyclopiazonic acid a slight tailing could not be prevented. In the ESI + mode the most prominent ion is the protonated molecular [M+H] + ion, the parent m/z. To validate the identity of the parent, these ions are fragmented into daughter ions with argon gas in the collision cell of the triple quadrupole.

Quantification is carried out on the primary transition. Each mycotoxin is confirmed by the second transition and the ratio between primary and secondary daughter ion is calculated. Differences in sensitivity between positive or negative electrospray were not investigated systematically in this study.

In literature several studies can be found on this topic. [START_REF] Razzazi-Fazeli | Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography-mass spectrometry with negative ion atmospheric pressure chemical ionisation[END_REF], 2002) considered the positive ion mode to be preferred for type A (e.g. T-2 and HT-2) and the negative mode for type B trichothecenes (e.g. nivalenol and DON). [START_REF] Lagana | Liquid chromatography/tandem mass spectrometry for the identification and determination of trichothecenes in maize[END_REF] proved this for trichothecenes in maize by data for the water/acetonitril/formic acid eluent. Biselli et al. (2004), [START_REF] Sulyok | Development and validation of a liquid chromatography/ tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize[END_REF] and Ren This doubles analysis time and gives raise to possible errors, i.e. combining the wrong runs for one sample. The main aim of this study was to end up with a single run multimethod for mycotoxins that are subject to present and upcoming EU food legislation. In the positive mode all EU limits for aflatoxin B 1 , ochratoxin A and DON were met. Therefore, it was preferred not to switch polarity, since T-2 and HT-2, the next important mycotoxins of interest, have distinctly more response in the positive mode (Razazzi-Fazeli et al., 2002). Another factor is that in an opposite mode mostly another buffer is preferred, i.e. formiate buffer, instead of formic acid, which does not make it even practical to switch polarity during a single LC run, even when it is technically possible in the MS. In a preliminary test we observed an increase in sensitivity for T2 and DAS, but a dramatically decrease for OTA when changing from formic acid to ammonium formiate buffer. This again led to the conclusion to maintain the positive mode in the multi-mycotoxin method for the entire run, since this mycotoxin is the next best regulated after the aflatoxins. The recent work of [START_REF] Delmulle | Development of a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 16 mycotoxins on cellulose filters and in fungal cultures[END_REF] confirmed this view.

They ran 16 mycotoxins on a Micromass Quattro Micro triple quadrupole mass spectrometer. After investigations in both modes they found out that not all toxins were detectable in the negative mode. In the positive mode they achieved LOD values of 1, 2.3 and 50 µg/kg for aflatoxin B 1 , ochratoxin A and DON, respectively. They extracted mycotoxins directly from cellulose filters that were applied as indoor air collectors in buildings. Compared to food and feed this type of samples is apparently rather "clean" by itself, because even when the sample is mainly composed of water, there exists a matrix effect, as is shown by [START_REF] Hartmann | Quantification of estrogenic mycotoxins at the ng/L level in aqueous environmental samples using deuterated internal standards[END_REF] who analysed zearalenone in river water, drainage water and waste water effluent. [START_REF] Mohamed | Quantitative determination of five ergot alkaloids in rye flour by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF] concluded that positive ionisation mode also revealed a higher total ion current for all ergot alkaloids, with sensitivity increased up to 50-fold for ergotamine compared to the negative one. All together this confirmed us to stick at the choice we made for positive mode to be applied in general for mycotoxins. In all references but the one of [START_REF] Delmulle | Development of a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 16 mycotoxins on cellulose filters and in fungal cultures[END_REF] as mentioned in the above paragraph, some kind of cleanup by a cartridge or assay was applied. Using cleanup indirectly implies a restriction of the types of mycotoxins that can be analysed, as any cleanup will influence the presence of compounds in the finally extracted aliquot to be injected in a chromatographic system. It led to methods, which mostly were only suitable to handle Fusarium mycotoxins like trichothecenes, fumonisins and zearalenone derivatives [START_REF] Tuomi | Detection of polar and macrocyclic trichothecene mycotoxins from indoor environments[END_REF][START_REF] Razzazi-Fazeli | Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography-mass spectrometry with negative ion atmospheric pressure chemical ionisation[END_REF], 2002;Biselli et al., 2004;[START_REF] Klötzel | Determination of 12 type A and B trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass spectrometry[END_REF]Berthiller et al., 2005;[START_REF] Cavaliere | Determination of type B trichothecenes and macrocyclic lactone mycotoxins in field contaminated maize[END_REF][START_REF] Hartmann | Quantification of estrogenic mycotoxins at the ng/L level in aqueous environmental samples using deuterated internal standards[END_REF]. At our starting point with this multi-mycotoxin LC-MS method in 2001, we intended to analyze as many mycotoxins as possible, so to apply crude extracts only. Therefore ion suppression by the matrix components can be expected, as has been investigated basically by [START_REF] Nelson | Ion suppression in LC-MS-MS -a case study[END_REF]. In practice it is more common to prepare calibration curves in solvent and matrix, and calculate the matrix effect by comparison of the results. This was done first for quantification of diarrhetic shellfish toxins in scallops [START_REF] Ito | Matrix effect and correction by standard addition in quantitative liquid chromatographic-mass spectrometric analysis of diarrhetic shellfish poisoning toxins[END_REF], and the mycotoxin penitrem A in a food mixture [START_REF] Rundberget | Determination of Penicillium mycotoxins in foods and feeds using liquid chromatography-mass spectrometry[END_REF]. We did this in our first presentation for aflatoxin B 1 , ochratoxin A and DON in a peanut and a cornflake sample [START_REF] Spanjer | Single run LC-MS/MS analysis of mycotoxins subject to actual and upcoming EU legislation in one sample extract[END_REF]. These three mycotoxins were chosen because at that time they were already subject to EU legislation. The two matrices were chosen to investigate the difference in matrix effect for these mycotoxins, nevertheless the fact that it is rather weird to expect DON in a peanut sample. The presented graphs clearly indicated that the matrix effect depends on the analyte as well as on the matrix, which makes it obligatory to determine ion suppression for every single separate matrix-mycotoxin combination. Regarding the number of mycotoxins and matrices this would result in an enormous validation task. For practical reasons we focussed in this study on the most relevant combinations, which limited our choice to 13-24 mycotoxins in the 7 matrices peanut, pistachio, wheat, maize, cornflakes, raisins and figs. data in tables 3-9 it becomes clear that ion suppression in these matrices varies between a minor and a huge effect. For aflatoxin B 1 , all matrices showed a moderate matrix effect, except wheat, where this method revealed a matrix effect of around 50%. For ochratoxin A the effect was less. The opposite holds for DON. This mycotoxin has less matrix effect in wheat, but a huge matrix effect in peanuts, raisins and figs. It can generally be assumed that DON is absent in peanut, raisin and fig samples, however this does not hold for maize and cornflake samples. [START_REF] Sulyok | Development and validation of a liquid chromatography/ tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize[END_REF] found no matrix effect for aflatoxin B 1 , ochratoxin A and DON in wheat, but 80% signal decrease for aflatoxin B 1 in maize. They applied dry milling of samples and another type of MS/MS. Both factors will influence the matrix effect as well. Biselli and Hummert (2005) measured signal suppression up to 40% for DON and T-2 in ground soybeans. [START_REF] Hartmann | Quantification of estrogenic mycotoxins at the ng/L level in aqueous environmental samples using deuterated internal standards[END_REF] experienced 28-58% ion suppression for zearalenone, while going from river to waste water samples. Both examples underline the necessity to evaluate every matrix/mycotoxin combination again, even when it is mainly composed of water.

Compared to the application of HPLC after immunoaffinity cleanup this may look a more complicated and more time-consuming sample handling, but recently [START_REF] Castegnaro | Advantages and drawbacks of immunoaffinity columns in analysis of mycotoxins in food[END_REF] reported several drawbacks in this field as well. They detected false-positive presence of aflatoxin G 1 in maize, underestimation of ochratoxin A in some breakfast cereals and coffee and interfering compounds while applying fumonisin antibodies. They emphasized the point of necessary validation of every method using immunoaffinity for each type of matrix. From that perspective the application of matrixmatched calibration for LC-MS analysis is not more laborious at all and delivers above all the identification of the MS.

The fact that mycotoxins can be found in almost every food or feed sample, also complicates analysis in a sense of finding an appropriate internal standard. [START_REF] Tuomi | Detection of polar and macrocyclic trichothecene mycotoxins from indoor environments[END_REF] 2006) applied de-epoxy-deoxynivalenol as internal standards. The chemical structures of these compounds are all more or less related to mycotoxins, which will reveal similar chemical behaviour during extraction and analysis, but has the disadvantage that their complete absence in a naturally contaminated sample cannot be fully guaranteed. This is a drawback to the expected precision of the analysis, even when the methods are focused on Fusarium toxins only. It is known that verrucarol is likely to be present in samples of mouldy interior finishes from buildings with moisture problems [START_REF] Tuomi | Detection of polar and macrocyclic trichothecene mycotoxins from indoor environments[END_REF]. Its presence in food was questioned by [START_REF] Cavaliere | Determination of type B trichothecenes and macrocyclic lactone mycotoxins in field contaminated maize[END_REF], who for that reason chose 17 α-estradiol and penicillin, which nevertheless also can be present in nature. The same holds for the corticosteroid dexamethasone which was applied by [START_REF] Razzazi-Fazeli | Simultaneous determination of major Btrichothecenes and the de-epoxy-metabolite of deoxynivalenol in pig urine and maize using highperformance liquid chromatography-mass spectrometry[END_REF] when determining the major B-trichothecenes and the de-epoxy-metabolite of deoxynivalenol in pig urine and maize. This de-epoxy-DON is basically found in vivo in animals, but it can also be present in milk, as has been shown by [START_REF] Sörensen | Determination of mycotoxins in bovine milk by liquid chromatography tandem mass spectrometry[END_REF]. The only way to guarantee application of compounds that cannot be found in any food or feed sample is to apply isotope labelled isomers. [START_REF] Hartl | Rapid determination of fumonisin FB 1 in corn products by high-performance liquid chromatography-electrospray mass spectrometry[END_REF] applied deuterated fumonisin B 1 for analysis of this mycotoxin in corn. [START_REF] Rundberget | Determination of Penicillium mycotoxins in foods and feeds using liquid chromatography-mass spectrometry[END_REF] (Asam and Rychlik 2007). Basically this approach is the correct one, but when analysis has to be perfect, it implies the use of a labelled compound for every single mycotoxin of interest in the analytical method, as already noticed by Tuomi in 1998. But not all these compounds are commercially available yet. Since then carbon-13 labelled DON [START_REF] Häubl | Suitability of a fully 13 C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up[END_REF] became commercially available at a company that also supplies 13C labelled fumonisin B 1 and T-2 Toxin (www.biopure.at). The deuterated zearalenone as applied by [START_REF] Hartmann | Quantification of estrogenic mycotoxins at the ng/L level in aqueous environmental samples using deuterated internal standards[END_REF] can be obtained from RIVM (Bilthoven, The Netherlands). But running labelled compounds for every mycotoxin, as it should be, would cause a significant raise in analysis costs. The alternative, standard addition, has the same drawback, as one has to add all mycotoxins of interest at different levels to the same sample and analyse all these combinations, which multiplies analysis time and costs by the same factor. Altogether this led to the common practice to apply matrix-matched calibration in those cases when after application of the multi-method, one or more mycotoxins exceed the legal limit. This is done in this study as well. The mycotoxin of interest is added to blank matrix, which is diluted to the same extent as the sample. The analytical result is corrected for matrix-effect. For exact identification of a compound, both the ion ratio and retention time have to fit within the maximum tolerances.

For the mycotoxins presented in table 10 can be concluded that recoveries fulfil the requirements as regulated by EC 401/2006. The fumonisins are missing, since these showed too low recoveries when applying the single extraction step as is done in this method. This is a common feature when apolar compounds like these are extracted with a large portion of acetonitrile, as reported likewise by [START_REF] Sulyok | Development and validation of a liquid chromatography/ tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize[END_REF]. Several experiments were done to improve the extraction efficiency for the fumonisins. The solvent/sample ratio was varied, the extraction solvent was acidified and the water content of the extraction solvent was increased, as to decrease the percentage of acetonitrile. The conclusion of these experiments was that increasing the water content of the extraction solvent was the most convenient way to improve extraction efficiency and thus recoveries for fumonisins and leave the multimethod unchanged for all other mycotoxins. A higher solvent/sample ratio increased the recovery, but has the disadvantage to repeat the extraction once again from the beginning. The addition of formic acid to the extract showed almost the same improvement, but this also means repetition of the extraction. When the measurement has to be repeated in order to improve the determination of fumonisins, it is most practical to use the first extract again for the quantification of the fumonisins. The first run in the multimethod gives a good qualitative estimate as to see whether legislative excess can be expected. If it turns out that fumonisins have to be quantified precisely, 100 ml of water is added to the original sample extract, shaken again for another hour and re-run with the LC-MS/MS. Using extra water in the extraction solvent yielded recoveries of 98% at RSD = 3.4 % for fumonisin B 1 and 90% at RSD = 2.8% for fumonisin B 2 (n=8), which fulfil the criteria of the EU regulation again. Since determination of fumonisins implies to re-run the extract a second time, less data are available when compared with the numbers in table 10. When quantifying fumonisins, a standard control sample is run in that series. This allows to judge on the performance for fumonisin analysis by evaluating the data of the measurements on the standard control sample instead. During last 1.5 year this revealed following data: means and RSD values of 1136 µg/kg and 8.3% for fumonisin B 1 and 235 µg/kg and 10.5% for fumonisin B 2 respectively, both at n=12.

To evaluate the performance of this multimethod, at first a comparison was made between measurement results obtained when aflatoxin B 1 was determined in peanut with LC-fluorescence (CEN method) and when analysed by the presented LC-MS method. Until now 128 data points resulted in mean and variance values of 2.2 µg/kg and 22.6% for CEN and 2.1 µg/kg and 22.0% for the LC-MS method respectively. From the t-test on these data (t-calculated = 0.98 and t-critical = 1.98) can be concluded that both methods do not differ significantly. In the mean time the method was applied in different laboratory proficiency tests [START_REF] Fapas® | Food Analysis Performance Assessment Scheme, Proficiency Testing Reports[END_REF]2006). The results for several rounds are listed in table 11. It illustrates that performance is good for the tested mycotoxin/matrix combinations, including fumonisin results, where we applied the procedure as described above.

[Insert Table 11 about here] Application as a single method is a good starting point to explore the prospects for the simultaneous determination of different mycotoxins in a sample. Finding more mycotoxins in one single sample has been done before, simply by applying more single methods to an extract, e.g. by [START_REF] Vargas | Co-occurrence of aflatoxins B 1 , B 2 , G 1 and G 2 , zearalenone and fumonisin B 1 in Brazilian corn[END_REF] who determined aflatoxins B 1 , B 2 , G 1 and G 2 , zearalenone and fumonisin B 1 in Brazilian corn, by [START_REF] Park | Natural co-occurrence of aflatoxin B 1 , fumonisin B 1 and ochratoxin A in barley and corn foods from Korea[END_REF] who investigated the natural co-occurrence of aflatoxin B 1 , fumonisin B 1 and ochratoxin A in barley and corn foods from Korea, by [START_REF] Eskola | Application of Manual and Automated Systems for Purification of Ochratoxin A and Zearalenone in Cereals with Immunoaffinity Columns[END_REF] who analysed ochratoxin A and zearalenone in cereals in Finland, by [START_REF] Pierard | Multi-mycotoxin determination methodology[END_REF] mycotoxins are not the most relevant ones when considering the EU legislation. With the multimycotoxin method presented here, the results as mentioned above could have been obtained after one run, as can be seen in figures 2 and 3. This also turned out in everyday practice where we found fumonisin B 1 in figs and ochratoxin A in pistachio in routine samples. The Italian Institute of Sciences of Food Production in Bari was the first to report the presence of toxigenic fungi, able to produce other mycotoxins then aflatoxin, in figs [START_REF] Logrieco | Epidemiology of toxigenic fungi and their associated mycotoxins for some Mediterranean crops[END_REF]. Two years later they reported the results of chemical analysis on rotten fig fruits, showing fumonisin contamination at a low level in some samples (Moretti et al., 2005). These finding are comparable with those of ochratoxin A in pistachio nuts. Bayman et al. (2002) reported the isolation of fungi with the ability to produce ochratoxin A and the British Food Standards Agency reported the actual analysis of it in a mycotoxin survey [START_REF] Matthews | Survey of nuts, nut products and dried tree fruits for mycotoxins[END_REF]. With this multi-method we also determined ochratoxin A in several nut types in 2006, apart from aflatoxin as well. The sensitivity of the method is very good when EU legislation is taken into account. Comparison of the LODs in tables 3-9 with the directive EC 1881/2006 reveals that they all can cope with the limits. Suylok et al. (2006) presented the only other method that is applied directly on the crude extract. Comparison of LODs for wheat in their table 6 with our data in table 5 shows some discrepancy for aflatoxin B 1 and ochratoxin A data. Both mycotoxins for which most legislation exists. Regarding the EC limit in wheat of 2 µg/kg for aflatoxin B 1 and 3 µg/kg for ochratoxin A, they reported LODs of 8 and 3.5 µg/kg respectively, where this method achieved 0.5 and 1 µg/kg. For Fusarium mycotoxins both methods reach the EC limits. The differences between the methods are twofold. They dilute 1 + 1 with eluent, i.e. by a factor 2 and their MS equipment is different, which implies another ionization chamber design. Both factors influence ion suppression and thus sensitivity.

Regarding other mycotoxins of interest it has already been proven by Biselli et al. (2004) and [START_REF] Kokkonen | Determination of selected mycotoxins in mould cheeses with liquid chromatography coupled to tandem with mass spectrometry[END_REF] that aflatoxin M 1 can be dealt with in the same way. For patulin this is impossible due to its small size and high polarity. As shown by [START_REF] Sulyok | Development and validation of a liquid chromatography/ tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize[END_REF] patulin can be run under the same LC-MS conditions, however, the extraction procedure is completely different when compared with the one for other mycotoxins. If patulin cannot be extracted from a sample, it is not useful to implement it in a method of analysis for routine samples. This conclusion was confirmed by [START_REF] Garon | Mycoflora and Multi-mycotoxin Detection in Corn Silage: Experimental Study[END_REF]. To determine patulin we apply the LC-MS method of [START_REF] Sewram | Determination of patulin in apple juice by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry[END_REF] as a single method. Regarding the ergot alkaloids we first got an impression of the presence of ergot alkaloids in a naturally contaminated sample. Figure 5 shows the major peaks in the chromatogram of an extract of Claviceps purpurea kernels, which were sorted out of a naturally contaminated lot of rye, while running the mass spectrometer in the parent mode. Five of these alkaloids could be identified as indicated. Epimerisation was not noticed, due to the fact that extraction is performed with 80/20 v/v % acetonitril-water, which is a neutral mixture. The eluent contains 0.1% formic acid, but retention time appeared to be too short to cause significant formation of epimers. Since alkaloids are LSD-type compounds it is difficult to get standards. At first we were only able to obtain ergotamin, ergocornin, ergocristin and α-ergocryptin, which we incorporated in this multi-mycotoxin method. Compared to the findings of others we missed ergonovin as detected by [START_REF] Mohamed | Quantitative determination of five ergot alkaloids in rye flour by liquid chromatography-electrospray ionisation tandem mass spectrometry[END_REF] and by [START_REF] Diers | Untersuchungen zum Einfluss von Mutterkornalkaloiden auf die Leistung und die Leberfunktion von Absatzferkeln[END_REF], who applied the name ergometrin. The variation in ergot alkaloid pattern in nature is well known, but few data exist. [START_REF] Diers | Untersuchungen zum Einfluss von Mutterkornalkaloiden auf die Leistung und die Leberfunktion von Absatzferkeln[END_REF] studied the 6 different ergot alkaloids as mentioned here, i.e. the 5 we found in the contaminated rye sample and ergometrin. She found variations for ergotamine content from 20-40% and 30-50% for ergocristin. These findings are slightly different from the data that are presented in the EFSA opinion on feed: ergocristine 31 % and ergotamine 17 % (EFSA, 2006).

Agreement seems to exist on the fact that ergometrine, ergosine, ergotamine, ergocornine, ergocryptine and ergocristine are the 6 most important ergot alkaloids. For this reason we will extend the method up to this number of indicator ergot alkaloids. To lower the limit of determination to the level of the EU limits for baby food (0.1 µg/kg for aflatoxin B 1 and 0.5 µg/kg for ochratoxin A (EC, 2004)) it was necessary to return to immunoaffinity cleanup. This was made easier by the introduction of a new type of immunoaffinity column composed of antibodies specific both for aflatoxins and ochratoxin A. The application is similar when compared to single mycotoxin analysis and is described by [START_REF] Chan | Simultaneous determination of aflatoxins and ochratoxin A in food using a fully automated immunoaffinity column cleanup and liquid chromatography-fluorescence detection[END_REF]. Unfortunately, these authors did not get lower than a limit of quantification of 0.2 µg/kg for both mycotoxins. Therefore, we adapted sample preparation with extra concentration steps. A test was done with a crisp bread sample at a level of 0.011 µg aflatoxin B 1 and 0.173 µg ochratoxin A per kilo. The corresponding standard deviation data were 0.0011 and 0.0220, with relative standard deviation (RSD) values of 10 and 13% (n=5). Due to the difficulties to extract spices and herbs the application of this combined assay column was also ideal to analyse these matrices properly on aflatoxin B 1 and ochratoxin A. This has the drawback of missing information on other mycotoxins, but for spices there is no legislation on other mycotoxins yet.

Unfortunately for babyfood all limits are set lower one by another, so these samples may need several assays again in the future. For silage this method yielded recoveries between 86 and 111 % for DON, aflatoxin B 1 and zearalenone at levels as those mentioned in tables 3-9. [START_REF] Garon | Mycoflora and Multi-mycotoxin Detection in Corn Silage: Experimental Study[END_REF] reported 65% recovery for DON at a level of 100-213 µg/kg, aflatoxin B 1 at a level of 4-34 µg/kg and zearalenone at 23-41 µg/kg. For citrinin, which we investigated partly in our validation study, they yielded 80% at a level of 4-25 µg/kg.

Conclusions

The multi-mycotoxin method as presented here has proven to be a good starting point for further development of sample analysis in one single run, without any cleanup. The EU limits are met for those mycotoxin-matrix combinations that are most commonly controlled in practice. The method gives relevant information about the presence of 33 mycotoxins in a run time of 30 min. Further research is planned regarding extraction of specific sample types, matrix effects, baby food limits and update of validation data. Changing the liquid chromatography system into an ultra performance liquid chromatography system is considered as well, as to cut analysis time further and to improve peak shape, especially for nivalenol. This might also lead to gain of sensitivity, as to reach lower, i.e. baby food, limits.

  al. (2007) dealt with this item by running a sample extract twice, both in positive and negative mode.

  applied reserpine, Cavaliere et al. (2005) nafcillin and Paepens et al. (2005) have chosen (2S, 3R)-2-aminododecane-1, 3-diol as standards, which compounds differ completely from mycotoxins, as to avoid intervention of naturally present compounds in a sample. However, this has the disadvantage of different behaviour of such compounds during extraction and the processes in the ion source of the mass spectrometer. On the other hand, Berger et al. (1999), Plattner et al. (2003), and Biselli et al. (2004) applied verrucarol, Berthiller et al. (2005) zearalanone, Ren et al. (2006) zearalenone and both Klötzel et al. (2005) and Delmulle et al. (

  [START_REF] Razzazi-Fazeli | Simultaneous quantification of A-trichothecene mycotoxins in grains using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry[END_REF] applied deuterated T-2 toxin as internal standard for determination of several Fusarium toxins.[START_REF] Lindenmeier | Quantification of Ochratoxin A in foods by a stable isotope dilution assay using high-performance liquid chromatography -tandem mass spectrometry[END_REF] reported the use of deuterated Ochratoxin A while analysing raisins.Asam and Rychlik (2006) synthesized four carbon-13 labelled type A-trichothecenes (T2, HT2, MAS and DAS) and applied these as internal in foods by LC tandem MS after cleanup on multifunctional columns. A year later they did the same for type B-trichothecenes (DON, 3 and 15 Ac-DON and fusarenon X) in foods and feeds

  who applied OTA and MycoSep cleanup to analyse wheat and by[START_REF] Domijan | Fumonisin B 1 , fumonisin B 2 , zearalenone and ochratoxin A contamination of maize in Croatia[END_REF] who determined fumonisins B 1 and B 2 , zearalenone and ochratoxin A of maize samples in Croatia. These studies are examples of investigations where not only Fusarium toxins are determined, but also other types like aflatoxin B 1 and ochratoxin A. In these studies, two or three single methods were applied to determine the specific mycotoxins of interest, which implied three different cleanup procedures and detection methods. Different from this approach,[START_REF] Aresta | Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples[END_REF] simultaneously determined ochratoxin A, cyclopiazonic, mycophenolic and tenuazonic acids in cornflakes by a single solid-phase microextraction. Unfortunately these

Figure 1 .

 1 Fig, Vilamoura, Portugal 16-20 May 2005.

Figure 2 .Figure 3 .

 23 Figure 2. LC-MS data of a maize sample containing aflatoxin B 1 (30 µg/kg) and fumonisins B 1 (4200 µg/kg), B 2 (1200 µg/kg) and B 3 . Top left, the chromatogram with 4 peaks; top right, and bottom left and right, the separate peaks characterised by the ion transitions of the MS as to prove their structure. The peak eluting before the fumonisin B 2 peak belongs to fumonisin B 3 .Since both fumonisins are isomers, they have the same transitions in the MS/MS spectra. Fumonisin B 3 could not be quantified, since there is no standard available.

Table 1 .

 1 ). Figure 4. LC-MS data of a freeze-dried mais silage sample containing several mycotoxins, i.e. DON (2500 µg/kg), zearalenone (240 µg/kg) and 15-Acetyl-DON (960 µg/kg). Figure 5. Chromatogram of ergot alkaloids extracted from a naturally contaminated rye sample with LC-MS in parent scan mode at m/z 268. Gradient programme of the LC system.

	685179 100 %							13.83	3: MRM of 18 Channels ES+ TIC 6.62e5 18.89 15.38 16.70 18.86 19.08 14.83 17.33 19.36	685179 Sm ooth(Mn,2x1) 100	16.65 0.0 µg/l	Zearalenone	F3:MRM of 18 channels ,ES+ 319 > 187 5.575e+004
												20.25
	11												%
	685179									2: MRM of 22 Channels ES+
	100						11.17	12.38				TIC 5.52e5	4	13.86	14.19	15.36	15.99	17.07	18.02	m in
	%				9.88	10.37	13.82				685179 Sm ooth(Mn,2x1)	F3:MRM of 18 channels ,ES+ 319 > 185
	28 685179 5 100 3.24 3.41 5.00 4.29 %	7.50 6.61 8.66	1: MRM of 10 Channels ES+ r F 10.00 12.50 15.00 17.50 20.00 Time TIC 3.79e5 o 9.20	100 2 %	14.00	15.00	15.81 16.00	17.00 16.65	18.00	19.00	20.00	1.239e+005 m in
	685179 Smooth(Mn,2x1) 100 685179 Smooth(Mn,2x1) 1 3.26 % 100 1405.3 µg/l 4.17 4.19		5.50 DON	6.81	685179 Smooth(Mn,2x1) 100 r F1:MRM of 10 channels,ES+ P 297 > 231 6.582e+004 685179 Smooth(Mn,2x1) 14 F1:MRM of 10 channels,ES+ min 9.52 e % 3.32 2.78 3.71 3.91 4.66 4.04 4.90 5.40 5.77 5.79 5.82 e 100 297 > 249 3.49 3.55 1.315e+005 15-Acetyl-DON 5.87 5.92 6.65 6.10 7.15 7.83 7.78	F1:MRM of 10 channels,ES+ 339 > 279 1.481e+004 8.51 8.76 F1:MRM of 10 channels,ES+ min 7.96 8.53 962.0 µg/l 8.70 9.04 9.75 339 > 231 8.53 1.308e+004 962.0 µg/l
	% 3	3.00 3.18	4.00 3.92	5.00	6.00	7.00	8.00	9.00	% R 3.98 4.02 2.84 3.73 3.50 4.14 4.25 4.62 e 5.71 5.30 6.48 6.11 w 6.61 i v 3.00 4.00 5.00 6.00 13 min e 4.88	7.25 7.00	7.75 7.82 8.00	8.88 9.36 9.00	9.89	min
															O n
															l y

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Table 2 .

 2 Overview of all relevant information regarding the investigated mycotoxins: parent and daughter ions, cone (V) and collision (eV) voltages, ratios and retention times (min) and references for fragment clarification.

	Mycotoxin	Parent Primary Secondary Ratio Cone Coll. eV RT

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Table 3 .

 3 Validation results in peanut slurry (n=18)

												Page 40 of 50
	Compound Calibration curve		Cal. range Validation Recovery RSDr Max	Reporting	LOD Matrix-
		a	b	r 2	(µg/kg) levels (µg/kg)	(%)	(%) RSDr (%) limit (µg/kg) (µg/kg) effect (%)
	Aflatoxin B 1	48284	943 0.9932	1-64	1, 2, 16	118	5.2	20-30	1.0	0.5	-19
	Aflatoxin B 2	33041	1528 0.9930	1-64	1, 2, 16	119	10.9	20-30	1.0	0.5	-5
	Aflatoxin G 1 34812	1475 0.9930	1-64	1, 2, 16	117	9.1	20-30	1.0	0.5	4
	Aflatoxin G 2 20363	272 0.9944	1-64	1, 2, 16	114	13.6	20-30	1.0	0.5	21
	Ochratoxin A 63526	726 0.9955 2-128	2, 4, 30	91	12.7	20-30	2.0	0.5	-13
	DON	994	8113 0.9886 50-3200 50, 100, 780	96	11.4	12-18	150	75	-67
	Fumonisin B 1 33610 -3890 0.9914 10-640	10, 20, 160	26	61.7	15-23	20	5	18
	Fumonisin B 2 61988	4068 0.9970 10-640	10, 20, 160	41	46.6	15-23	20	5	-2
	Zearalenone	6086	2234 0.9903 20-1280 20, 40, 320	100	19.8	13-20	40	5	-7
	DAS	4456	617 0.9990 50-3200 50, 100, 780	118	8	12-18	100	10	-17
	T2-Toxin	3482	1998 0.9976 50-3200 50, 100, 780	117	8.1	12-18	100	10	-3
	HT2-Toxin	2165	798 0.9995 50-3200 50, 100, 780	126	8.3	12-18	100	10	-26
	3-Acetyl-DON 3277	100 0.9970 50-3200 50, 100, 780	119	8.5	12-18	100	10	-40

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	Table 4. Validation results in pistachio slurry (n=18)
	Compound	Calibration curve			Cal. Range Validation Recovery RSDr	Max	Reporting	LOD Matrix-
		a	b	r 2	(µg/kg) levels (µg/kg)	(%)	(%) RSDr (%) limit (µg/kg) (µg/kg) effect (%)
	Aflatoxin B 1	60609	-392 0.9979	1 -64	1, 2, 10	106	10.8 20 -30	1.0	0.5	32
	Aflatoxin B 2	33572	702 0.9967	1 -64	1, 2, 10	99	11.6 20 -30	1.0	0.5	29
	Aflatoxin G 1	26183	-947 0.9943	1 -64	1, 2, 10	93	9.4	20 -30	1.0	0.5	-4
	Aflatoxin G 2	15712	322 0.9940	1 -64	1, 2, 10	85	42	20 -30	1.0	10	31
	Ochratoxin A	16551	-414 0.9886 2 -128	2, 4, 20	102	28	20 -30	2.0	1.0	-7
	DON	212	90 0.9906 50 -3200 50, 100, 500	91	26	12 -18	100	50	15
	Fumonisin B 1	5869	-2017 0.9918 20 -1280	20, 40, 200	79	9.3	15 -23	20	10	-10
	Fumonisin B 2	11953	-3020 0.9885 20 -1280	20, 40, 200	86	10.3 15 -23	20	10	-1
	DAS	330	-776 0.9693 50 -3200 50, 100, 500	97	20.9 12 -18	50	25	-11
	T2-Toxin	102	5 0.9538 50 -3200 50, 100, 500	88	20.7 12 -18	100	50	-12
	HT2-Toxin	103	59 0.9646 50 -3200 50, 100, 500	83	27.0 12 -18	100	50	-25
	3-Ac-DON	286	274 0.9944 50 -3200 50, 100, 500	100	18.9 12 -18	100	50	11
	ZEN	903	21 0.9853 20 -1280	20, 40, 200	101	16.6 15 -23	20	10	7
	Penicillic acid	2834	-427 0.9959 20 -1280	20, 40, 200	107	7.5	15 -23	20	10	13
	Fusarenon-X	129	-57 0.9799 50 -3200 50, 100, 500	85	23.2 12 -18	100	50	24
	Ergotamin				40 -2560	40, 80, 400	98	9.5	15 -23	40	20	26
	Roquefortin	33076	47626 0.9925 20 -1280	20, 40, 200	101	11.0 15 -23	20	10	43
	ß-ZAL	2111	-830 0.9659 20 -1280	20, 40, 200	92	14.7 15 -23	40	10	-7
	α-ZAL	2332	-618 0.9628 20 -1280	20, 40, 200	96	19.7 15 -23	40	10	-3
	Citrinin	22476	1578 0.9993 20 -1280	20, 40, 200	53	17.7 15 -23	20	100	5
	ZAN	5129	-58 0.9739 20 -1280	20, 40, 200	94	17.2 15 -23	20	10	7
	Cyclopiazonic acid	6093	-7949 0.9872 20 -1280	20, 40, 200	59	25	15 -23	200	200	-32
	Sterigmatocystein	60056	59007 0.9974 20 -1280	20, 40, 200	82	14.1 15 -23	20	10	8
	α-ZEL	926	-282 0.9750 20 -1280	20, 40, 200	88	20.7 15 -23	40	40	-3

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Table 5 .

 5 Validation results in wheat slurry (n=18)

												Page 42 of 50
	Compound Calibration curve		Cal. range Validation Recovery RSDr Max	Reporting	LOD Matrix-
		a	b	r 2	(µg/kg) levels (µg/kg)	(%)	(%) RSDr (%) limit (µg/kg) (µg/kg) effect (%)
	Aflatoxin B 1	64120	314 0.9728	1-64	1, 2, 10	87	14.9	20-30	1.0	0.5	-53
	Aflatoxin B 2	65165	153 0.9887	1-64	1, 2, 10	92	9.8	20-30	1.0	0.5	-32
	Aflatoxin G 1 71493	490 0.9877	1-64	1, 2, 10	92	9	20-30	1.0	0.5	-24
	Aflatoxin G 2 40235	376 0.9887	1-64	1, 2, 10	90	19	20-30	1.0	0.5	5
	Ochratoxin A 49176	1666 0.9736 2-128	1.5, 3, 15	94	16.3	20-30	2	1	-32
	DON	2811	1769 0.9977 50-3200 25, 50, 750	91	12.8	12-18	40	20	-23
	Fumonisin B 1 25656	1127 0.9957 10-640	10, 20, 160	59	24.9	15-23	20	10	16
	Fumonisin B 2 25581	5659 0.9912 10-640	5, 10, 150	51	57.4	15-23	20	10	-7
	Zearalenone	7373	-6392 0.9778 20-1280 25, 50, 500	80	41.6	13-20	40	5	-29
	DAS	2345	-1824 0.9884 50-3200 25, 50, 500	35	29.7	12-18	100	5	-10
	T2-Toxin	733	554 0.8896 50-3200 25, 50, 500	38	57.5	12-18	100	20	-39
	HT2-Toxin	353	159 0.9864 50-3200 25, 50, 500	84	41	12-18	100	50	-65
	3-Acetyl-DON 2232	3485 0.993 50-3200	5, 10, 150	115	24.2	12-18	100	50	-14

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Table 6 .

 6 Validation results in maize slurry (n=18)

	Compound	Calibration curve			Cal. Range Validation Recovery RSDr	Max	Reporting	LOD Matrix-
		a	b	r 2	(µg/kg) levels (µg/kg)	(%)	(%) RSDr (%) limit (µg/kg) (µg/kg) effect (%)
	Aflatoxin B 1	59401	-456 0.9958	1 -64	1, 2, 10	93	13.4 20 -30	1.0	0.5	17
	Aflatoxin B 2	40546	1506 0.9949	1 -64	1, 2, 10	87	27	20 -30	2.0	1.0	21
	Aflatoxin G 1	35053	385 0.9926	1 -64	1, 2, 10	99	21	20 -30	2.0	1.0	-1
	Aflatoxin G 2	20208	-302 0.9972	1 -64	1, 2, 10	108	14.7 20 -30	1.0	0.5	15
	Ochratoxin A	31924	275 0.9822 2 -128	2, 4, 20	81	11.9 20 -30	2.0	1.0	11
	DON	1238	722 0.9934 50 -3200 50, 100, 500	104	12.2 12 -18	50	50	15
	Fumonisin B 1	12726	-584 0.9970 20 -1280 20, 40, 200	20	72	15 -23	200	100	-17
	Fumonisin B 2	29590	-2798 0.9960 20 -1280 20, 40, 200	46	7.7	15 -23	200	100	-2
	DAS	1707	34 0.9954 50 -3200 50, 100, 500	104	7.4	12 -18	50	25	8
	T2-Toxin	1204	1253 0.9925 50 -3200 50, 100, 500	102	10.5 12 -18	50	25	13
	HT2-Toxin	718	769 0.9966 50 -3200 50, 100, 500	100	11.5 12 -18	50	25	20
	3-Ac-DON	1292	407 0.9939 50 -3200 50, 100, 500	102	17.8 12 -18	100	25	21
	ZEN	3301	638 0.9836 20 -1280 20, 40, 200	100	10.9 15 -23	20	10	12
	Penicillic acid	12869	-624 0.9958 20 -1280 20, 40, 200	103	6.5	15 -23	20	10	12
	Fusarenon-X	592	87 0.9952 50 -3200 50, 100, 500	97	17.2 12 -18	100	50	11
	Ergotamin	5560	8253 0.9982 40 -2560 40, 80, 400	100	10.6 15 -23	40	20	45
	Roquefortin	71857	159492 0.9889 20 -1280 20, 40, 200	99	7.9	15 -23	20	10	46
	ß-ZAL	16972	4905 0.9905 20 -1280 20, 40, 200	103	7.6	15 -23	20	10	15
	α-ZAL	17681	3076 0.9942 20 -1280 20, 40, 200	94	26	15 -23	20	10	11
	Citrinin	27106	47666 0.9813 20 -1280 20, 40, 200	13	105 15 -23	200	100	6
	ZAN	17496	6787 0.9867 20 -1280 20, 40, 200	101	6.7	15 -23	20	10	11
	Cyclopiazonic acid	16140	-6998 0.9787 20 -1280 20, 40, 200	19	105 15 -23	200	200	-177
	Sterigmatocystein	55332	48561 0.9868 20 -1280 20, 40, 200	109	10.9 15 -23	20	10	-18
	α-ZEL	3117	1597 0.9899 20 -1280 20, 40, 200	100	21.9 15 -23	40	20	17

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Table 7 .

 7 Validation results in dry milled cornflakes (n=18)

												Page 44 of 50
	Compound Calibration curve		Cal. range Validation Recovery RSDr	Max	Reporting	LOD Matrix-
		a	b	r 2	(µg/kg) levels (µg/kg)	(%)	(%) RSDr (%) limit (µg/kg) (µg/kg) effect (%)
	Aflatoxin B 1	27735	214 0.9983	1-64	1, 2, 16	101	12.6	20-30	1.0	0.5	-19
	Aflatoxin B 2	24749	787 0.9973	1-64	1, 2, 16	106	8	20-30	1.0	0.5	-6
	Aflatoxin G 1	6928	218 0.9916	1-64	1, 2, 16	99	27.6	20-30	3.4	1.7	-1
	Aflatoxin G 2	12810	189 0.9976	1-64	1, 2, 16	107	16.4	20-30	1.0	0.5	-5
	Ochratoxin A 37687	332 0.9909 2-128	2, 4, 30	108	9.4	20-30	2.0	1.0	-1
	DON	1177	284 0.9971 50-3200 50, 100, 780	91	7.2	12-18	150	75	-34
	Fumonisin B 1 19434	2940 0.9988 10-640	10, 20, 160	58	13.8	15-23	20	5	10
	Fumonisin B 2 41920	4697 0.9989 10-640	10, 20, 160	88	6.5	15-23	20	5	10
	Zearalenone	2954	826 0.9877 20-1280	20, 40, 320	108	6.3	13-20	40	15	-6
	DAS	4690	-319 0.9993 50-3200 50, 100, 780	104	4.6	12-18	100	10	-18
	T2-Toxin	3325	2325 0.9996 50-3200 50, 100, 780	111	3.7	12-18	100	10	-5
	HT2-Toxin	1931	244 0.9993 50-3200 50, 100, 780	110	4.9	12-18	100	10	-14
	3-Acetyl-DON 4171	-486 0.9994 50-3200 50, 100, 780	102	6.4	12-18	100	10	-23

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Table 8 .

 8 Validation results in raisin slurry (n=18)

	Page 45 of 50
	Compound Calibration curve		Cal. range Validation Recovery RSDr Max	Reporting	LOD Matrix-
		a	b	r 2	(µg/kg) levels (µg/kg)	(%)	(%) RSDr (%) limit (µg/kg) (µg/kg) effect (%)
	Aflatoxin B 1	39637	-436 0.9857	1-64	1, 2, 16	95	11.3	20-30	1.0	0.5	6
	Aflatoxin B 2	23265	728 0.9951	1-64	1, 2, 16	110	12.9	20-30	1.0	0.5	14
	Aflatoxin G 1 33805	143 0.9970	1-64	1, 2, 16	98	8.4	20-30	1.0	0.5	44
	Aflatoxin G 2 12109	41	0.9956	1-64	1, 2, 16	103	13.5	20-30	2.0	1.0	19
	Ochratoxin A	5244	-29 0.9919 2-128	2, 4, 30	103	5.4	20-30	16.0	8.0	7
	DON	1306	-150 0.9985 50-3200 50, 100, 780	98	3.9	12-18	18	9	-40
	Fumonisin B 1 38186 -7381 0.9986 10-640	10, 20, 160	89	5.8	15-23	20	5	3
	Fumonisin B 2 47530	844 0.9987 10-640	10, 20, 160	87	4.8	15-23	20	1	2
	Zearalenone	61790	-805 0.9946 20-1280 20, 40, 320	95	4.4	13-20	40	2	1
	DAS	3693	-1256 0.9985 50-3200 50, 100, 780	92	4.9	12-18	100	20	-4
	T2-Toxin	2049	953 0.9963 50-3200 50, 100, 780	89	6.3	12-18	100	15	-5
	HT2-Toxin	1845	-310 0.9980 50-3200 50, 100, 780	94	7	12-18	100	15	-5
	3-Acetyl-DON 2421	-1083 0.9992 50-3200 50, 100, 780	98	5	12-18	100	10	-8

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Table 9 .

 9 Validation results in fig slurry (n=18)

	Compound Calibration curve		Cal. range Validation Recovery RSDr Max	Reporting	LOD Matrix-
		a	b	r 2	(µg/kg) levels (µg/kg)	(%)	(%) RSDr (%) limit (µg/kg) (µg/kg) effect (%)
	Aflatoxin B 1	2054	32	0.9879	1-64	1, 2, 16	84	11.7	20-30	1.0	0.5	-3
	Aflatoxin B 2	1503	-32 0.9820	1-64	1, 2, 16	77	12.9	20-30	8.0	4	-12
	Aflatoxin G 1	2658	2	0.9910	1-64	1, 2, 16	74	13.1	20-30	3.0	1.5	-4
	Aflatoxin G 2	978	-33 0.9893	1-64	1, 2, 16	91	14.8	20-30	10.0	5	-8
	Ochratoxin A 1667	-904 0.9867 2-128	2, 4, 30	74	12.8	20-30	14.0	7.0	-5
	DON	297	112 0.9961 50-3200 50, 100, 780	97	12.8	12-18	250	125	-47
	Fumonisin B 1 7313	15281 0.9954 10-640	10, 20, 160	64	36.4	15-23	30	15	1
	Fumonisin B 2 15924	-150 0.9958 10-640	10, 20, 160	76	17.3	15-23	20	5	-1
	Zearalenone	19473	-648 0.9953 20-1280 20, 40, 320	76	9.1	13-20	5	1	-11
	DAS	415	71	0.9958 50-3200 50, 100, 780	86	9.5	12-18	100	10	-2
	T2-Toxin	254	164 0.9900 50-3200 50, 100, 780	82	11.2	12-18	100	10	-11
	HT2-Toxin	362	-185 0.9938 50-3200 50, 100, 780	84	13.3	12-18	100	10	-14
	3-Acetyl-DON 564	291 0.9960 50-3200 50, 100, 780	82	10.2	12-18	100	10	-21

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Table 10 .

 10 Recovery data for different mycotoxins in several types of food.

			Food Additives and Contaminants	Page 48 of 50
	Mycotoxin	Recovery (%) RSD%	n
	Aflatoxin B 1	104	17	152
	Aflatoxin B 2	105	18	151
	Aflatoxin G 1	103	17	152
	Aflatoxin G 2	107	21	143
	Ochratoxin A	99	15	163
	Deoxynivalenol	101	18	150
	Diacetoxyscirpenol	108	16	153
	T2-Toxin	110	19	152
	HT2-Toxin	105	20	149
	3-Ac-Don	105	18	148
	ZEN	103	17	150
	Penicillic acid	101	17	153
	Fusarenon X	102	20	148
	Ergotamin	105	16	149
	Roquefortin	104	13	150
	ß-ZAL	107	17	152
	α -ZAL	104	14	147
	Citrinin	80	20	151
	ZAN	104	16	150
	Cyclopiazonic acid	85	25	150
	Sterigmatocystin	101	21	152
	α -ZEL	107	17	150

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Table 11 .

 11 FAPAS test results as obtained in the indicated rounds in 2005 and 2006, n.d. = not detected.

	Compound	Commodity	Reference Our result Accuracy z-score	Fapas
			(µg/kg)	(µg/kg)	(%)		report
	Aflatoxin B 1	fig	4.36	5.2	119	0.9	483
	Aflatoxin B 2	fig	1.62	2.1	130	1.4	483
	Aflatoxin G 1	fig	3.09	2.9	94	-0.3	483
	Aflatoxin G 2	fig	0.88	0.9	102	0.1	483
	Aflatoxins total fig	10.0	11.1	111	0.5	483
	Aflatoxin B 1	maize	4.69	6.2	132	1.5	489
	Aflatoxin B 2	maize	2.54	2.6	102	0.1	489
	Aflatoxin G 1	maize	3.27	2.5	76	-1.1	489
	Aflatoxin G 2	maize	1.67	< 1			489
	Aflatoxins total maize	12.1	11.3	93	-0.3	489
	Aflatoxin B 1	pistachio	3.53	3.7	105	0.2	490
	Aflatoxin B 2	pistachio	0.85	1.0	118	0.8	490
	Aflatoxin G 1	pistachio	2.73	2.9	106	0.3	490
	Aflatoxin G 2	pistachio	0.95	1.0	105	0.2	490
	Aflatoxins total pistachio	7.92	8.3	105	0.6	490
	Aflatoxin B 1	chili powder	6.89	6.1	89	-0.5	493
	Aflatoxin B 2	chili powder	0.49	< 1			493
	Aflatoxin G 1	chili powder	n.d.	n.d.			493
	Aflatoxin G 2	chili powder	n.d.	n.d.			493
	Aflatoxins total chili powder	7.53	6.1	81	-0.9	493
	Aflatoxin B 1	baby food	0.145	0.19	131	1.4	494
	Aflatoxin B 1	hazelnut	2.36	3.1	131	1.4	495
	Aflatoxin B 2	hazelnut	0.93	1.2	129	1.3	495
	Aflatoxin G 1	hazelnut	1.75	2.5	143	2.0	495
	Aflatoxin G 2	hazelnut	0.43	< 1			495
	Aflatoxins total hazelnut	5.39	6.8	126	1.2	495
	Ochratoxin A	baby food	0.89	0.81	91	-0.4	1734
	Ochratoxin A	paprika powder	56.1	60.8	108	0.4	1735
	Ochratoxin A	wine	1.8	2.2	122	1.0	1736
	Ochratoxin A	barley	8.44	7.1	84	-0.7	1739
	Ochratoxin A	dried vine fruit	8.3	9.4	113	0.6	1740
		(raisins)
	Ochratoxin A	baby food	0.60	0.66	110	0.4	1742
	Ochratoxin A	animal feed	8.03	9.3	116	0.7	1748

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Acknowledgements

Harm Janssens and Gerard Franken, Technical Laboratory Rotterdam (P.O. Box 893, 3000 AW Rotterdam, The Netherlands) are acknowledged for supplying the basis for this method, which they developed for feed. Frank Driehuis, NIZO Food Research (P.O. Box 20, 6710 BA Ede, The Netherlands), is acknowledged for supplying the freeze dried powdered silage samples. We also acknowledge our VWA colleagues Elma Bobeldijk and Yuk Lai Liu from the Laboratory in Amsterdam for performing most of the measurements on samples and the validation studies and Yvonne van der Laan from the Surveillance & Development department for retrieving references.

; Food and Consumer Product Safety Authority (VWA)Peter; Food and