Fumonisins are toxic secondary metabolites produced by Fusarium verticillioides (formerly F. moniliforme), F. proliferatum, F. fujikuroi and other less common Fusarium species, Alternaria alternata f. sp. Lycopersici, Aspergillus awamori and Aspergillus niger. A great number of researches have confirmed a significant relationship between FB1 contamination and risk of liver and oesophageal cancer, neural tube defects and a number of cases of acute mycotoxicosis in humans. In animals, it is associated with Porcine Pulmonary Edema, Equine Leukoencephalomalacia, hepatotoxicity and high toxicity to a variety of experimental animals (EFSA, 2018). After initial discovery, a great number of outbreaks of diseases in horses and pigs, availability of research data on fumonisin toxicity in humans and animals as well as data of their occurrence, resulted in evaluation of fumonisins by the IARC Monographs programme of the carcinogenic hazard to humans. Fumonisin B1 and B2 are classified as possibly carcinogenic to humans (Group 2B) (IARC, 1993, 2002; Yazar & Omurtag, 2008; JECFA 2011; FAO/WHO, 2017).
Fumonisins are long-chained poly-hydroxyl-amines that differ in chemical structure (variably hydroxylated and esterified with organic acids) that is considered responsible for their potential toxic effects. These highly polar compounds are easily soluble in water and other polar solvents. They can be present in samples as ‘hidden forms’ and/or “modified forms”. Their various reactive groups are capable of interaction with matrix components (i.e. matrix-associated and modified form). Potential presence of all these forms make detection of total fumonisin content in samples, as well as evaluation of their potential harmful effects on human and/or animal health, quite challenging and laborious task (Rychlik et al., 2014; Braun & Wink, 2018; EFSA, 2018). Fumonisin B (FB) series include the most toxic and most abundant naturally occurring mycotoxins: FB1, FB2, FB3 and FB4. They are structural analogs of sphingosine and sphinganine that inhibit ceramide synthases, disrupt sphingolipid metabolism causing diseases in plants and animals. The chemical structures of fumonisin B1, fumonisin B2, fumonisin B3 and fumonisin B4 are given in Fig. 1 (JECFA, 2001; Rheeder et al., 2002; Bartόk et al., 2006).
Fumonisins are common contaminants of cereals and cereal products (up to 100% of tested samples with the highest concentrations of 2.339 µg/kg). They are also found in rice, sorghum, beans, soya, tea, eggs, milk, meat and other food and feedstuffs worldwide, in various concentration ranges. It has been estimated that FB1 accounts for up to 80% of the total fumonisins produced (Griessler et al., 2010; Varga et al., 2010; Rodrigues & Naehrer, 2012; Scott, 2012; Streit et al., 2012; Logrieco et al., 2014; Li et al., 2015; Deepa & Sreenivasa, 2017; FAO/WHO, 2017; Braun & Wink, 2018; Gruber-Dorninger et al., 2019). In analysing the occurrence data of fumonisins by EFSA (2018), additional contribution of 60% of hidden forms has been documented. Among analysed samples, the majority data were on cereal grains, their products and by products (47%) of which the highest number of reported samples were for maize in the concentration range from 0.3 to 1.678.1 µg/kg (EFSA, 2018). It is important to point out that until recently, data on occurrence of fumonisins contained only information about FB1 and FB2 (90% of the samples analyzed). The actual contamination of all three FB series toxins is usually made by estimation. Updated Opinion of the Scientific Committee on Food on fumonisin B2, B2 and B3 has concluded that all three fumonisins have similar toxicological profiles and potencies. Therefore, new studies on occurrence are needed that monitor all three fumonisin analogues at the same time (EFSA, 2005; EFSA, 2018).
There are several screening and confirmatory, qualitative and quantitative analytical methods for detection of fumonisins including thin layer chromatography, gas-liquid chromatography (GLC), high performance liquid chromatography (HPLC), mass spectrometry, various enzyme-linked immunosorbent assays (ELISA) and molecular methods. The mass spectrometry methods are considered as the state-of-the-art in this area of research due to their high sensitivity and specificity for detection and quantification with limit as low as 0,001 µg/g (González-Jartín et al., 2021). Most methods are focused on detection of FB1. Use of liquid chromatography-mass spectrometry with electrospray ionization and ESI with tandem mass spectrometry enables detection of lesser-known fumonisin analogs that are not detected with other analytical techniques due to the necessary derivatization processes (Arranz et al., 2004; Bartόk et al., 2006; Musser and Plattner, 1997; Deepa and Sreenivasa, 2017; FAO/WHO, 2017; Janik et al., 2021). However, expensive equipment and high costs of its maintenance as well as specialized operators are still lacking in a great number of laboratories worldwide. Therefore, ELISA and HPLC methods are still often used for determination of fumonisins in food and feed. Since a great number of published data did not include mentioned three analogs, there is a need for evaluation of different methods for detection of three B series fumonisin analogs. Therefore, this research on fumonisins detection will provide new data on applicability and comparison of methods for determination of FB1, FB2 and FB3 and thus providing valuable data for comparison of similar research of other research groups.
The aim of this research was to evaluate the performance and applicability of both, the modified high-performance liquid chromatography method with post column derivatisation and fluorescence detection (HPLC-FLD) described by Sydenham et al. (1996) and commercial enzyme-linked immunosorbent assay (AgraQuant® Total fumonisin Assay 0.25/5.0, RomerLabs, Austria) for simultaneous determination of three B series fumonisin analogs (FB1, FB2 and FB3) in maize samples intended for use as animal feed in a wide range of concentrations.