Determination Of Fumonisins Research Articles

Determination of fumonisins B<SUB>1</SUB> and B<SUB>2</SUB> in corn by high performance liquid chromatography with post-column derivatization method

Determination of fumonisins B<SUB>1</SUB> and B<SUB>2</SUB> in corn by high performance liquid chromatography with post-column derivatization method

Fungal contamination and determination of fumonisins and aflatoxins in commercial feeds intended for ornamental birds in Rio de Janeiro, Brazil

The purposes of this study were to determine the distribution of total mycobiota, to determine the occurrence of Aspergillus spp., Penicillium spp. and Fusarium spp. and to detect and quantify fumonisin B1 and aflatoxin B1 in birds' feedstuffs. Sixty samples from different commercial feeds were collected. Analysis of the total mycobiota was performed and total fungal counts were expressed as CFU g(-1). The isolation frequency (%) and relative density (%) of fungal genera and species were determined. Mycotoxins determination was carried out using commercial ELISA kits. The 48% of standard, 31% of premium and only 9% of super premium feed samples were found above of recommended limit (1 × 10(4) CFU g(-1)). Aspergillus (82%), Cladosporium (50%) and Penicillium (42%) were the most frequently isolated genera. Aspergillus niger aggregate (35%), Aspergillus fumigatus (28%) and Aspergillus flavus (18%) had the highest relative densities. Contamination with fumonisins was detected in 95% of total samples with levels from 0·92 to 6·68 μg g(-1), and the aflatoxins contamination was found in 40% of total samples with levels between 1·2 and 9·02 μg kg(-1). Feed samples contaminated with fumonisins and aflatoxins are potentially toxic to birds.

Occurrence of fumonisins in organic and conventional cereal-based products commercialized in France, Germany and Spain

A fast and effective method for quantitative determination of fumonisins B1 and B2 (FB1 and FB2) in different cereal-based products was developed. Fumonisins were extracted combining a solid liquid extraction (SLE) pre-treatment and immunoaffinity columns (IACs), which were applied for an effective clean-up. Consecutively, high pressure liquid chromatography (HPLC) coupled to hybrid triple quadrupole-linear ion trap mass spectrometer (QTrap®) was used for the separation, detection and quantification of targeted mycotoxins. The analytical method showed acceptable recoveries ranged from 89.7% to 99.1%, as well as a relative standard deviation (%, RSD) lower than 12%. A total of 1250 samples from France, Germany and Spain were analysed. The incidence of fumonisin B1 and B2 in organic samples were 11.4% and 11.3%, respectively. By contrast, the occurrence of conventional samples was 3.6% of FB1 and 3.5% of FB2. The highest values were obtained in Spanish samples of flour, as an example toasted corn, called gofio that was contaminated with 1201.7 and 1010.5 μg kg(-1) of FB1 and FB2, respectively.

Determination of fumonisins B1 and B2 in Chinese rice wine by HPLC using AQC precolumn derivatisation

Fumonisins are a group of naturally occurring mycotoxins produced by various Fusarium species that commonly infect maize and other cereals, including sorghum and rice. In this study a sensitive and selective method was developed for the determination of fumonisins B₁ and B₂ (FB₁ and FB₂) in Chinese rice wine. The method is based on high-performance liquid chromatography and fluorescence detection following precolumn derivatisation with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). FB₁ and FB₂ in Chinese rice wine were extracted and purified using strong anion exchange cartridges and derivatised with AQC at room temperature. The AQC derivatives were stable for 5 days. Optimal fluorescence was obtained at an excitation wavelength of 246 nm and an emission wavelength of 390 nm. Chromatography was performed using a C18 column and gradient elution at 1 mL min⁻¹ with methanol and 0.05 mol L⁻¹ phosphate buffer at pH 4. The limit of detection was 6 µg L⁻¹ for both FB₁ and FB₂. The method was successfully applied to the determination of FB₁ and FB₂ in Chinese rice wine, with recoveries of 87.5-94.5% being obtained. The established method was stable and sensitive for the determination of FB₁ and FB₂ in Chinese rice wine.

Determination of Fumonisins B 1 , B 2 and Their Hydrolysed Metabolites in Bovine Milk by Liquid Chromatography-Tandem Mass Spectrometry

MAX column combined with high performance liquid chromatography-tandem mass spectrometry was used to determine fumonisins B1(FB1),FB2 and their hydrolysed metabolites in bovine milk.After 10 mL milk was diluted with 10 mL water,it was directly transferred to MAX column for purification.FB1 and FB2 were eluted with methanol and detected with HPLC-MS/MS in negative mode.The hydrolysed metabolites hydrolysed fumonisins B1(HFB1) and HFB2 were eluted with methanol containing 1% acetic acid and detected with HPLC-MS/MS in positive mode.The recoveries for four compounds were 76.4%-92.3% at fortification levels of 0.1-5 μg/L in bovine milk and relative standard deviation was in the range of 5.9%-12.5%.The LOD was 0.03 μg/L and LOQ was 0.1 μg/L for FB1,FB2 and their hydrolysed metabolites.This method is simple,sensitive,and repeatabilities with satisfied recoveries.

Determining Fumonisins in Corn by High Performance Liquid Chromato-graphy with Immunoaffinity Column Cleanup

Fumonisins are mycotoxins produced mainly by Fusarium verticillioides. It is frequently found that corn is contami-nated by fumonisins during its growth and storage. Epidemic study demonstrated that corn and corn-based products contaminated by fumonisins can cause equine leukoencephalomalacia and swine pulmonary oedema, also induce oesophageal cancer and neural tube defects of mankind. In this paper, a high performance liquid chromatography (HPLC) method with immunoaffinity column cleanup was established to determine the fumonisins B1 (FB1) and B2 (FB2) in corn. Meanwhile, the accuracy and reproducibility of the method were evaluated by statistical technique. The results showed that the linear ranges of FB1 and FB2 were 0.06–5.00 μg mL?1 and 0.04–2.50 μg mL?1, the recovery ranges were 76.6–93.8% and 77.9–93.4%, respectively. The quantification limit of FB1 and FB2 was 0.09 mg kg?1 and 0.06 mg kg?1, respectively. The coefficient of variation within-laboratory repeatability was below 5%, and that between-laboratories reproducibility was below 6%. Research results indicated that this method possessed good line-arity, accuracy, precision, sensitivity, repeatability within a laboratory and among laboratories. Therefore, this method was proved to be suitable for determination of fumonisins in corn. Applying this method, fumonisins in 310 specimens of corn were deter-mined. Results showed that total fumonisins (B1+ B2) ranged from 0.20 to 9.06 mg kg?1 for field corn, and from 0.21 to 6.10 mg kg?1 for stored corn. It is proposed that monitoring fumonisins’ level in corn should be reinforced to protect the health of man and livestock.

Analysis of fumonisins B1 and B2 in spices and aromatic and medicinal herbs by HPLC-FLD with on-line post-column derivatization and positive confirmation by LC-MS/MS

Fumonisins are produced by the fungus Fusarium verticillioides, which are known to cause fatal diseases in some animals and humans. Here, we describe a sensitive, reproducible and reliable analytical method for the quantitative determination of fumonisins B(1) (FB(1)) and B(2) (FB(2)) in 112 spices and aromatic and medicinal herbs marketed in China. This method is based on high performance liquid chromatography and fluorescence detection (HPLC-FLD) coupled to a new on-line post-column derivatization using ortho-phthaldialdehyde with 2-mercaptoethanol and immunoaffinity column clean-up. Under the optimized experimental conditions, a complete separation of FB(1) and FB(2) was obtained using a Synergi C(18) column and a gradient elution at 0.8 mL min(-1) with methanol and 0.1 M phosphate buffer at pH 3.15. The limits of detection for FB(1) and FB(2) were both 40 μg kg(-1). Good recoveries were found for spiked samples with FB(1) and FB(2), ranging from 82.34% to 98.16% for FB(1) and from 72.58% to 97.10% for FB(2), with relative standard deviation (RSD) < 7.0%. 5 spices, 11 aromatic herbs and 96 medicinal herbs including 93 normal samples and 19 visibly moldy samples, which were spoiled artificially, were analyzed. The results showed that 8 (42.1%) visibly moldy samples and 8 (8.6%) normal samples were contaminated with FB(1) at mean contents of 129.0 and 165.9 μg kg(-1), and with FB(2) at 1745.0 and 256.8 μg kg(-1), respectively. Positive confirmation of detected samples was performed by liquid chromatography tandem electrospray ionization mass spectrometry (LC-ESI-MS/MS), using a triple quadrupole analyzer and operated in the multiple reaction monitoring mode.

Rapid Determination of Fumonisins B1 and B2 in Corn by Liquid Chromatography-Tandem Mass Spectrometry with Ultrasonic Extraction

A simple, sensitive, and reliable analytical method is developed for the rapid determination of fumonisin B(1) and fumonisin B(2) in corn by high-performance liquid chromatography-positive electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). Fumonisin B(1) and fumonisin B(2) are extracted from corn with methanol-water (3:1, v/v) by means of ultrasonic extraction, and directly injected into an LC-MS-MS system after centrifugation. Fumonisin B(1) and fumonisin B(2) are separated on a Zorbax Eclipse XDB-C(18) column with a solution of methanol-water-formic acid as the mobile phase. The method is validated with respect to linearity, accuracy, precision, specificity, and stability. Moreover, the method was applied to real samples and demonstrated to be suitable for the determination of fumonisin B(1) and fumonisin B(2) in corn. The total time required for the analysis of one sample was ~30 min.

Simple Phosphate Buffer Extraction for the Determination of Fumonisins in Masa, Maize, and Derived Products

An accurate determination of fumonisins in masa can be troublesome because of difficulties often experienced in obtaining satisfactory recoveries. In this study, a new extraction method, effective both for masa and for maize and derived products, has been developed. The method extracts fumonisins using a 0.4 M phosphate buffer (PB) at pH 7.5, without any organic solvent; the purification of the extract is performed by an immunoaffinity column and the quantification by HPLC–MS/MS. The average recovery percentages were 95.5 ± 1.9% and 96.7 ± 2.1% for fumonisin B1 and B2, respectively. The limit of detection and of quantification for both fumonisins were 10 and 30 μg kg−1, respectively. For masa and tortillas, PB pH 7.5 gave significantly higher results when compared with AOAC and other published extraction methods; for maize flour, PB gave generally higher results, even if the difference with the other extraction methods was not always significant.

Comparison of methods and optimisation of the analysis of fumonisins B1 and B2 in masa flour, an alkaline cooked corn product

A comparison study of different extraction and clean-up procedures for the liquid chromatographic analysis of fumonisins B1 (FB1) and B2 (FB2) in corn masa flour was performed. The procedures included extraction (heat or room temperature) with acidic conditions or EDTA-containing solvents, and clean-up by immunoaffinity or C18 solid-phase extraction columns. Thereafter an analytical method was optimised using extraction with an acidic mixture of methanol–acetonitrile–citrate/phosphate buffer, clean-up through the immunoaffinity column and determination of fumonisins by liquid chromatography with automated pre-column derivatisation with o-phthaldialdehyde reagent. Recovery experiments performed on yellow, white and blue masa flours at spiking levels of 400, 800 and 1200 µg kg−1 FB1 and of 100, 200 and 300 µg kg−1 FB2 gave overall mean recoveries of 99% (±6%) for FB1 and 88% (±6%) for FB2. Good recoveries (higher than 90% for both FB1 and FB2) were also obtained with corn tortilla chips. The limits of quantification of the method (signal-to-noise ratio of 10) were 25 µg kg−1 for FB1 and 17 µg kg−1 for FB2. The method was tested on different commercial corn masa flours as well as on white and yellow corn tortilla chips, showing fumonisin contamination levels (FB1 + FB2) up to 1800 µg kg−1 (FB1 + FB2) in masa flour and 960 µg kg−1 in tortilla chips. Over 30% of masa flours originating from Mexico exceeded the European Union maximum permitted level.

Simultaneous determination of fumonisins B 1, B 2 and B 3 contaminants in maize by ultra high-performance liquid chromatography tandem mass spectrometry

The present work developed an analytical method for simultaneous determination of fumonisins B 1, B 2 and B 3 residues in maize by ultra high-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UHPLC–MS/MS) under the multiple reaction monitoring (MRM) mode, and especially focused on the optimization of extraction, clean-up, UHPLC separation and MS/MS parameters. The method involves addition of fumonisins isotope internal standards, extraction with a mixture of acetonitrile and water and clean-up with solid-phase extraction (SPE) cartridges before UHPLC–MS/MS analysis. A single-laboratory method validation was conducted by testing three different spiking levels for repeatability and recovery according to International Union of Pure and Applied Chemistry (IUPAC) guidelines. The LOQ of FB 1, FB 2 and FB 3 were 1.50, 1.65 and 0.4 μg kg −1, respectively, which were lower than the criteria of EU, USA and other countries regarding minimum residue limits of fumonisins in foods including baby foods and feedstuffs. Recoveries of three fumonisins ranged from 80.9% to 97.0% with RSD values of 2.4–11.1%.The advantages of this method include simple pretreatment, rapid determination and high sensitivity, and it fulfills the requirements for food analysis with respect to minimum residue limits of fumonisins in various countries.

A reversed phase high performance liquid chromatography method for the determination of fumonisins B 1 and B 2 in food and feed using monolithic column and positive confirmation by liquid chromatography/tandem mass spectrometry

The development of a reversed phase high performance liquid chromatography fluorescence method for the determination of the mycotoxins fumonisin B 1 and fumonisin B 2 by using silica-based monolithic column is described. The samples were first extracted using acetonitrile:water (50:50, v/v) and purified by using a C 18 solid phase extraction-based clean-up column. Then, pre-column derivatization for the analyte using ortho-phthaldialdehyde in the presence of 2-mercaptoethanol was carried out. The developed method involved optimization of mobile phase composition using methanol and phosphate buffer, injection volume, temperature and flow rate. The liquid chromatographic separation was performed using a reversed phase Chromolith ® RP-18e column (100 mm × 4.6 mm) at 30 °C and eluted with a mobile phase of a mixture of methanol and phosphate buffer pH 3.35 (78:22, v/v) at a flow rate of 1.0 mL min −1. The fumonisins separation was achieved in about 4 min, compared to approximately 20 min by using a C 18 particle-packed column. The fluorescence excitation and emission were at 335 nm and 440 nm, respectively. The limits of detections were 0.01–0.04 μg g −1 fumonisin B 1 and fumonisin B 2, respectively. Good recoveries were found for spiked samples (0.1, 0.5, 1.5 μg g −1 fumonisins B 1 and B 2), ranging from 84.0 to 106.0% for fumonisin B 1 and from 81.0 to 103.0% for fumonisin B 2. Fifty-three samples were analyzed including 39 food and feeds and 14 inoculated corn and rice. Results show that 12.8% of the food and feed samples were contaminated with fumonisin B 1 (range, 0.01–0.51 μg g −1) and fumonisin B 2 (0.05 μg g −1). The total fumonisins in these samples however, do not exceed the legal limits established by the European Union of 0.8 μg g −1. Of the 14 inoculated samples, 57.1% contained fumonisin B 1 (0.16–41.0 μg g −1) and fumonisin B 2 (range, 0.22–50.0 μg g −1). Positive confirmation of selected samples was carried out using liquid chromatography–tandem mass spectrometry, using triple quadrupole analyzer and operated in the multiple reaction monitoring mode.

Rapid Determination of Fumonisins in Corn-Based Products by Liquid Chromatography/Tandem Mass Spectrometry

A simple, fast, and robust method was developed for the determination of fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) in corn-based human food and animal feed (cornmeal). The method involves a single extraction step followed by centrifugation and filtration before analysis by ultra-performance liquid chromatographylelectrospray ionization (UPLC/ESI)-MS/MS. The LC/MS/MS method developed here represents the fastest and simplest procedure (<30 min) among both conventional HPLC methods and other LC/MS methods using SPE cleanup. The potential for high throughput analysis makes the method particularly beneficial for regulatory agencies and analytical laboratories with a high sample volume. A single-laboratory validation was conducted by testing three different spiking levels (200, 500, and 1000 ng/g for FB1 and FB2; 100, 250, and 500 ng/g for FB3) for accuracy and precision. Recoveries of FB1 ranged from 93 to 98% with RSD values of 3-8%. Recoveries of FB2 ranged from 104 to 108%, with RSD values of 2-6%. Recoveries of FB3 ranged from 94 to 108%, with RSD values of 2-5%.

Determination of Fumonisins in Milled Corn Grains Using HPLC-MS

A new method for determination of fumonisins in corn samples was developed and validated. The mycotoxins were extracted by a mixture of methanol-acetonitrile-water (1:1:2, v/v/v) and determined on a liquid chromatograph with mass spectrometric detection. The separation was performed on Zorbax XDB-C(18) column (150 x 4.6 mm; 5 microm) with a Metaguard ODS-2 precolumn (30 x 4.6 mm; 5 mum) using gradient elution with mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate (adjusted by acetic acid to pH 3.0). For detection of (M+H(+)) ions, a quadrupole mass spectrometer in single ion monitoring mode was applied. Developed method showed very good linearity in a tested range of concentration. Detection limit is 62.0 microg FB(1)/kg and 58.5 microg FB(2)/kg of maize grains. Because the detection limits lie under the maximum permitted EU levels, the method is suitable for determination of fumonisins in milled corn grains.

A reliable isotope dilution method for simultaneous determination of fumonisins B1, B2 and B3 in traditional Chinese medicines by ultra‐high‐performance liquid chromatography‐tandem mass spectrometry

A reliable isotope dilution method for simultaneous determination of fumonisin B1, fumonisin B2 and fumonisin B3 in traditional Chinese medicines by ultra-high-performance LC-MS/MS was developed, and a special focus was placed on the optimization of extraction, cleanup, ultra-high-performance LC separation and MS/MS conditions. Homogenized samples were extracted by 50% acetonitrile aqueous solution and purified with MultiSep 211 Fum columns. A linear gradient mobile phase, consisting of water containing 0.2% formic acid and acetonitrile/methanol (50:50 v/v) and an Acquity UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) were employed to obtain the best resolution of the analytes for the positive ESI(+) analysis. The established method was validated by determining the linearity (R(2)≥0.9991), sensitivity (LOQ, 0.08-0.16 ng/mL), recovery (88.2-113.3%) and precision (RSD≤12.3%). Finally, the validated method was successfully applied to the determination of fumonisins in different traditional Chinese medicines. Of 35 samples, 18 were contaminated with fumonisins. The mean levels (incidence) of fumonisin B1, fumonisin B1 and fumonisin B1 in positive samples were 8.55 (94.4%), 4.88 (77.8%) and 0.52 μg/kg (33.3%), respectively. Based on the contaminant situations, a possible association between the contamination levels and the selected herbs was clarified in this study.

Determination of fumonisins B1 and B2 in maize-based baby food products by HPLC with fluorimetric detection after immunoaffinity column clean-up

A method for the determination of fumonisin B1 (FB1) and B2 (FB2) in different commercial maize-based products for infants and young children was developed and tested in a limited validation study involving 3 laboratories. The method used extraction at 55 °C with an acidic mixture of methanol-acetonitrile-phosphate/citrate buffer, clean-up through immunoaffinity column and fumonisin determination by high performance liquid chromatography with automated pre-column derivatisation with o-phthaldialdehyde. Recovery experiments were performed at five spiking levels in the ranges of 80-800 µg/kg FB1 and 20-200 µg/kg FB2. Mean recoveries ranged from 83 to 97% for FB1 and from 61 to 78% for FB2. Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 5 to 12% for FB1 and from 8 to 13% for FB2, whereas relative standard deviation for between-laboratory reproducibility (RSDR) ranged from 6 to 10% for FB1 and from 9 to 16% for FB2. The limit of quantification of the method (signal to noise ratio of 6) was 2.8 µg/kg for FB1 and 2.2 µg/kg for FB2. Fumonisins were found in 6 out of 19 maize-based baby foods obtained from the Italian retail market at levels up to 53 µg/kg.

Widespread Occurrence of the Mycotoxin Fumonisin B2 in Wine

Fumonisins are important mycotoxins because they are suspected to cause human and animal toxicoses by the consumption of contaminated corn-based food and feeds. However, with the discovery of fumonisin production in grapes by Aspergillus niger , wine may also be a fumonisin-containing commodity. In the present study, we have developed a simple and quantitative cation-exchange-based purification method for the subsequent isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of fumonisins in wine. A comparative study of seven different solid-phase extraction (SPE) columns showed that polymeric mixed-mode reversed-phase (RP) cation-exchange columns were superior to classic silica-based cation and mixed-mode cation-exchange columns. A total of 77 wine samples from 13 countries were subsequently tested, and surprisingly, 18 (23%) were found to contain fumonisin B(2) in the range of 1-25 microg/L. These findings were further confirmed by immunoaffinity purification and re-analysis of the positive cation-exchanged extracts.

Preparation of an in-house reference material containing fumonisins in Thai rice and matrix extension of the analytical method for Japanese rice.

Mycotoxin contamination in rice is less reported, compared to that in wheat or maize, however, some Fusarium fungi occasionally infect rice in the paddy field. Fumonisins are mycotoxins mainly produced by Fusarium verticillioides, which often ruins maize. Rice adherent fungus Gibberella fujikuroi is taxonomically near to F. verticillioides, and there are sporadic reports of fumonisin contamination in rice from Asia, Europe and the United States. Therefore, there exists the potential risk of fumonisin contamination in rice as well as the need for the validated analytical method for fumonisins in rice. Although both natural and spiked reference materials are available for some Fusarium mycotoxins in matrices of wheat and maize, there are no reference materials for Fusarium mycotoxins in rice. In this study, we have developed a method for the preparation of a reference material containing fumonisins in Thai rice. A ShakeMaster grinding machine was used for the preparation of a mixed material of blank Thai rice and F. verticillioides-infected Thai rice. The homogeneity of the mixed material was confirmed by one-way analysis of variance, which led this material to serve as an in-house reference material. Using this reference material, several procedures to extract fumonisins from Thai rice were compared. Accordingly, we proved the applicability of an effective extraction procedure for the determination of fumonisins in Japanese rice.

Determination of fumonisins in maize by HPLC with ultraviolet detection of o-phthaldialdehyde derivatives

Fumonisins are mycotoxins that are produced by various Fusarium species and occur naturally in maize and maize-based foods. Fumonisins are carcinogenic, causing liver cancer in rats, and are associated with oesophageal cancer and neural tube defects in humans. Analytical methods for individual fumonisin analogues in maize rely on reversed-phase high-performance liquid chromatographic (HPLC) separation after suitable extraction and clean-up. As fumonisins lack a useful chromophore or fluorophore, HPLC detection is achieved by suitable derivatization and sensitive, specific fluorescence detection. A widely used and validated method involves extract clean-up on strong anion exchange solid phase extraction cartridges and pre-column derivatization with o-phthaldialdehyde (OPA). However, many laboratories requiring infrequent fumonisin analysis are only equipped with HPLC with ultraviolet (UV) detection. A HPLC system equipped with both UV and fluorescence detectors connected in series was used to determine the extent to which UV offers an alternative to fluorescence detection in the above analytical method. Comparison of the detection systems using fumonisin standards indicated that fluorescence is about 20-times more sensitive than UV. Analysis of maize samples with differing fumonisin contamination levels indicated that, at fumonisin B1 levels above 1,000µg/kg, the two detection systems were comparable and gave repeatabilities equal or less than 10% on six replicate analyses. Although a sensitive fumonisin analysis requires fluorescence detection, UV may offer an alternative in certain circumstances.

Difficulties in fumonisin determination: the issue of hidden fumonisins

In this paper, the results obtained by five independent methods for the quantification of fumonisins B(1), B(2), and B(3) in raw maize are reported. Five naturally contaminated maize samples and a reference material were analyzed in three different laboratories. Although each method was validated and common calibrants were used, a poor agreement about fumonisin contamination levels was obtained. In order to investigate the interactions among analyte and matrix leading to this lack of consistency, the occurrence of fumonisin derivatives was checked. Significant amounts of hidden fumonisins were detected for all the considered samples. Furthermore, the application of an in vitro digestion protocol to raw maize allowed for a higher recovery of native fumonisins, suggesting that the interaction occurring among analytes and matrix macromolecules is associative rather than covalent. Depending on the analytical method as well as the maize sample, only 37-68% of the total fumonisin concentrations were found to be extractable from the samples. These results are particularly impressive and significant in the case of the certified reference material, underlying the actual difficulties in ascertaining the trueness of a method for fumonisin determination, opening thus an important issue for risk assessment.