Beauvericin and Moniliformin
Gregor Kos and Rudolf Krska
Chemistry
Beauvericin (CAS [26048-05-5]) is a mycotoxin produced by several Fusarium species and has also been isolated from Beauveria bassiana. It is a bioactive cyclodepsipeptide, which contains three D-a-hydroxy-isovaleryl and N-methyl-L-phenylalanyl residues in alternating sequence.
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| Figure 1: Structural formula of beauvericin |
Extraction
Extraction of beauvericin from cereal and corn samples takes place using an acetonitrile-water mixture by blending the sample. Alternatively Shephard et al. extracted the homogenised sample with methanol and (after evaporation and re-dissolving in mobile phase) injected the extract directly into the LC-MS without any further clean up and resulting in an LOD of 8 µg/kg. Filtering is usually included in all extraction procedures. Sometimes a defatting step using an extraction with hexane in the procedure, which however results in a considerable loss of BEA, is included. In order to obtain reasonable recoveries, the defatting step should be replaced by an appropriate clean-up (Josephs et al., 1999).
Clean-up
Liquid-liquid-partitioning with dichloromethane is often combined with SPE columns. Josephs et al. (1999) reported a Mycosep #224 and Silica SPE column clean up suitable for the µg/kg range and a recovery of 96.4%. Subsequent elution was performed with chloroform/2-propanol (97:3). The use of C18 clean-up columns has also been reported.
Separation and Detection
Separation and detection were performed by Josephs et al. using an HPLC-DAD system with a reversed phase C18 column and an acetonitrile-water mixture as mobile phase. 192 nm and 209 nm were used as detection wavelengths. LC-MS (Shephard et al.) and thermo spray MS systems have been used for detection and identification, and NMR Spectroscopy was used for identification purposes. Other separation and detection systems also include normal phase HPLC systems with silica columns.
High performance thin layer chromatography (HPTLC) has also been used as a means of separation using precoated silica gel 60 plates that were spotted with methanolic extracts. Mobile phases were toluene/acetone, chloroform/2-propanol and ethyl acetate/hexane. Detection was performed at 365 and 254 nm after colouring with iodine vapours with a detection limit in the mg/kg range.
It is important to note that so far no reliable and established methods exist for the determination of trace levels (lower µg/kg range) of BEA in cereals or similar complex matrices (in particular with respect to robustness and reproducibility). LC-MS is a promising and powerful tool for the determination and identification of BEA, but still subject to ongoing research. Disadvantages of LC-MS techniques are the high costs involved and the experience that is required.
References BEA
[1] Josephs, R.D., Krska, R., Schuhmacher, R., Grasserbauer, M., A Rapid Method for the Determination of the Fusarium Mycotoxin Beauvericin in Maize, Fresenius J. of Anal. Chem. 363, 130-131 (1999)
[2] Shephard, G.S., Sewram, V., Nieuwoudt, T.W., Marasas, W.F.O., Ritieni, A., Production of the Mycotoxins Fusaproliferin and Beauvericin by South African Isolates in the Fusarium Section Liseola, J.Agric. Food Chem., 47, 5111-5115 (1999)
[3] Logrieco, A., Moretti, A., Ritieni, A., Chelkowski, J., Altomare, C., Bottalico, A., Randazzo, G., Natural Occurrence of Beauvericin in Preharvested Fusarium subglutinans Infected Corn Ears in Poland, J. Agric. Food Chem., 41, 2149-2152 (1993)
[4] Ritieni, A., Moretti, A., Logrieco, A., Bottalico, A., Randazzo, G., Monti, S.M., Ferracane, R., Fogliano, V., Occurrence of Fusaproliferin, Fumonisin B1 and Beauvericin in Maize from Italy, J. Agric. Food Chem., 45, 4011-4016 (1997)
Chemistry
Moniliformin is the trivial name for 3-hydroxy-3-cyclobutene-1,2-dione, an anion usually occurring with potassium or sodium as a counter ion. (K/Na-salt of Semisquaric Acid), CAS [52591-22-7]. K or Na C4HO3, MW = 136.11 g/mol. It is a mycotoxin mainly produced by Fusarium species (F. fujikuroi, F. proliferatum, avenaceum, subglutinans). MON is toxic and phytotoxic.
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| Figure 2: Structural formula of moniliformin |
The main challenge in the determination of MON is its ionic character that makes it difficult to detect the toxin with satisfying recovery rates.
Extraction
Janssen and Dose used a Soxhlet extraction (taking several hours) with methanol and a defattening step with cyclohexane before separation and detection. The use of acetonitrile:water mixtures (7:3) with subsequent filtering has also been reported. With TBAH (tetra-butyl ammonium hydroxide - TBAH, 20% in water and potassium di-hydrogen orthophosphate, pH=7 with NaOH) an ion pair with MON is generated that is subjected to a clean up procedure. Water is also used as an extraction solvent, usually with 1% tetrabutyl-ammonium hydrogen sulfate.
Clean-up
The relatively high LODs achieved with TLC (see below) were reduced by purification and derivatisation step to levels of 100 µg/kg and a recovery rate of 76 % (samples spiked with 2 mg/kg MON). Extrelut™ columns used as a clean-up procedure improved the recovery rates. SAX and C18 columns or combinations thereof have also been used for MON clean up.
Separation and Detection
TLC has been used for visualising MON by quenching or alternatively by a reaction with 2,4-dinitrophenylhydrazine (2,4-DNP), Ninhydrin or n-methylbenzthiazolon-2-hydrazone (MBTH). The limits of detection of TLC methods are rather high usually in the 1 µg/kg range and highly dependent on the clean-up procedure.
Ion-pair-reversed-phase-HPLC analysis with UV detection at 227 nm used by Shepherd et al. gave recovery rates in the range of 70-80 % by use of TBAH as ion-pair reagent in reversed phase HPLC analysis. Further improvements were obtained by the introduction of a derivatisation reaction with 1,2-diamino-4,5-dichlorobenzene (DDB) followed by HPLC-FLD analysis.
GC/MS detection after derivatisation with N-methyl-N-(tertiary butyl-dimethylsilyl)-trifluoracetamide (MTBSTFA) results in a compound with a MW of 453 g/mol and a characteristic mass spectrum.
An LC-MS method employing triethylamine as ion-pairing reagent for the determination of MON in culture material and naturally contaminated maize samples is described by Sewram et al.. Mass spectrometric detection of moniliformin was accomplished following atmospheric pressure chemical ionization (APCI) to yield the deprotonated molecular ion [M-H](-) at m/z 97. The MON response was found to be linear over the injected range 10 ng to 700 ng and a detection limit of 10 ng was attainable at a signal-to-noise (S/N) ratio of 4.
References MON
[1] Janssen, C., Dose, K., Quantitative Determination of Moniliformin in Vegetable Foods and Feeds, Fresenius Zeitschrift Analytische Chemie, 319, 60-62 (1984)
[2] Shepherd, M.J., Gilbert, J., Method for the Analysis in Maize of the Fusarium Mycotoxin Moniliformin Employing Ion-pairing Extraction and High Performance Liquid Chromatography, Journal of Chromatography, 358, 415-422 (1986)
[3] Lew, H., Chelkowski, J., Pronczuk, P., Edinger, W., Occurrence of the Mycotoxin Moniliformin in Maize (Zea mays L.) Ears Infected by Fusarium subglutinans (Wollenw. & Reinking) Nelson et al., Food Additives and Contaminants, 13, 3, 321-324 (1996)
[4] Sewram, V., Nieuwoudt, T.W., Marasas, W.F.O., Shephard, G.S., Ritieni, A., Determination of the Mycotoxin Moniliformin in Cultures of Fusarium subglutinans and in Naturally Contaminated Maize by High Performance Liquid Chromatography-Atmospheric Pressure Chemical Ionization Mass Spectroscopy, J. of Chromatography A, 848, 185-191 (1999)
[5] Josephs, R.D., Development, Application and Characterisation of Analytical Methods for the Determination of Agriculturally Important Fusarium Mycotoxins, PhD thesis, IFA-Tulln, Tulln, Austria (1999)