Abstract
The type A trichothecenes T-2 and HT-2 toxins are toxic secondary metabolites produced by fungi of the Fusarium genus. Their occurrence in cereals, especially in oats, implies health risks for the consumer. Therefore, it is an important task to develop selective and sensitive methods for the analysis of T-2 and HT-2 toxins, and to undertake further studies on their stability and toxicity. Although most toxins are commercially available, their high prices are the limiting factor on the realization of these experiments. Thus, we developed a method for large-scale production of T-2 and HT-2 toxin as well as T-2 triol and T-2 tetraol. T-2 toxin was obtained in gram quantities by biosynthetic production with cultures of F. sporotrichioides. As HT-2 toxin was only formed as a by-product, and T-2 triol and T-2 tetraol were not generated, these compounds were produced by alkaline hydrolysis of T-2 toxin. Separation and isolation of crude toxins was achieved by fast centrifugal partition chromatography (FCPC), which is an efficient tool for the large-scale purification of natural products. Using this fast and yield effective technique, several hundred milligrams of HT-2 toxin, T-2 triol, and T-2 tetraol were obtained. Subsequent, HT-2 toxin and T-2 triol were used for the large-scale synthesis of isotope-labeled T-2 and HT-2 toxin, respectively. Using these standards, an isotope dilution-(ID)-HPLC-MS/MS method for the quantification of T-2 and HT-2 toxin in different matrices was developed.
Similar content being viewed by others
References
Asam S, Rychlik M (2006) Synthesis of four carbon-13-labeled type a trichothecene mycotoxins and their application as internal standards in stable isotope dilution assays. J Agric Food Chem 54:6535–6546.
Berthiller F, Schuhmacher R, Buttinger G, Krska R (2005) Rapid simultaneous determination of major type A- and B-trichothecenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 1062:209–216.
Biselli S, Hummert C (2005) 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. Food Addit Contam 22:752–760.
Cramer B, Bretz M, Humpf HU (2007) Stable isotope dilution analysis of the fusarium mycotoxin zearalenone. J Agric Food Chem 55:8353–8358.
Cramer B, Beyer M, Humpf HU (2009) Stable isotope labeled myvotoxins as standards for HPLC-MS/MS analysis - review and evaluation of published procedures for the introduction of stable isotopes into mycotoxins. In: Appell M, Kendra D, Trucksess M Mycotoxin (eds) Prevention and Control in Agriculture (in press).
Duffy MJ, Reid RS (1993) Measurement of the stability of T-2 toxin in aqueous solution. Chem Res Toxicol 6:524–529.
European Commission SCOF Opinion of the Scientific Committee On Food on Fusarium toxins, Part 5: T-2 TOXIN and HT-2 TOXIN, 2001 (http://europa.eu.int/comm/food/fs/sc/scf/out88_en.pdf, accessed date: 15/10/08) (2001).
European Commission SCOF Opinion of the Scientific Committee On Food on Fusarium toxins, Part 6: Group evaluation of T-2 toxin, HT-2 toxin, nivalenol and deoxynivalenol, 2002 (http://europa.eu.int/comm/food/fs/sc/scf/out123_en.pdf, accessed date: 15/10/08) (2002).
Hagler WM, Mirocha CJ, Pathre SV (1981) Biosynthesis of radiolabeled T-2 toxin by Fusarium tricinctum. Appl Environ Microbiol 41:1049–1051.
Haubl G, Berthiller F, Hametner C, Rechthaler J, Jaunecker G, Freudenschuss M, Krska R, Schuhmacher R (2007) Characterization of (13C24) T-2 toxin and its use as an internal standard for the quantification of T-2 toxin in cereals with HPLC-MS/MS. Anal Bioanal Chem 389:931–940.
Hietaniemi V, Kumpulainen J (1991) Contents of Fusarium toxins in Finnish and imported grains and feeds. Food Addit Contam 8:171–182.
JECFA (2001) Joint FAO/WHO expert committee on food additives (JECFA), Safety evaluation of certain food additives. WHO Food Additives Series 47:http://www.inchem.org/documents/jecfa/jecmono/v47je01.htm (accessed October 16, 2008).
Jestoi M, Ritieni A, Rizzo A (2004) Analysis of the Fusarium mycotoxins fusaproliferin and trichothecenes in grains using gas chromatography-mass spectrometry. J Agric Food Chem 52:1464–1469.
Klötzel M, Lauber U, Humpf HU (2006) A new solid phase extraction clean-up method for the determination of 12 type A and B trichothecenes in cereals and cereal-based food by LC-MS/MS. Mol Nutr Food Res 50:261–269.
Klötzel M, Gutsche B, Lauber U, Humpf HU (2005) Determination of 12 type A and B trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass spectrometry. J Agric Food Chem 53:8904–8910.
Königs M, Mulac D, Schwerdt G, Gekle M, Humpf HU (2009) Metabolism and cytotoxic effects of T-2 toxin and its metabolites on human cells in primary culture. Toxicology (in press). doi:10.1016/j.tox.2009.01.012.
Kotsonis FN, Ellison RA (1975) Assay and relationship of HT-2 toxin and T-2 toxin formation in liquid culture. Appl Microbiol 30:33–37.
Krska R, Baumgartner S, Josephs R (2001) The state-of-the-art in the analysis of type-A and -B trichothecene mycotoxins in cereals. Fresenius J Anal Chem 371:285–299.
Marasas WF, Yagen B, Sydenham E, Combrinck S, Thiel PG (1987) Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides. Appl Environ Microbiol 53:693–696.
Michaud DP, Karger BL, Vouros P (1988) The synthesis of deuterium labeled T-2 toxin. J Label Compd Rad 25:345–348.
Onji Y, Aoki Y, Yamazoe Y, Dohi Y, Moriyama T (1988) Isolation of nivalenol and Fusarenon-X from pressed barley culture by centrifugal partition chromatography. J Liq Chromatogr 11:2537–2546.
Royer D, Humpf HU, Guy PA (2004) Quantitative analysis of Fusarium mycotoxins in maize using accelerated solvent extraction before liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry. Food Addit Contam 21:678–692.
Sulyok M, Berthiller F, Krska R, Schuhmacher R (2006) Development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize. Rapid Commun Mass Spectrom 20:2649–2659.
Thompson WL, Wannemacher RW Jr (1986) Structure-function relationships of 12,13-epoxytrichothecene mycotoxins in cell culture: comparison to whole animal lethality. Toxicon 24:985–994.
Trebstein A, Seefelder W, Lauber U, Humpf HU (2008) Determination of T-2 and HT-2 toxins in cereals including oats after immunoaffinity cleanup by liquid chromatography and fluorescence detection. J Agric Food Chem 56:4968–4975.
Wallace EM, Pathre SV, Mirocha CJ, Robison TS, Fenton SW (1977) Synthesis of radiolabeled T-2 toxin. J Agric Food Chem 25:836–838.
Webb KS, Carter D (1997) The role of isotope dilution mass spectrometry in the development of tandem mass spectrometry for quantitative organic analysis. Rapid Commun Mass Spectrom 11:155–158.
Wei R, Strong FM, Smalley EB, Schnoes HK (1971) Chemical interconversion of T-2 and HT-2 toxins and related compounds. Biochem Biophys Res Commun 45:396–401.
Acknowledgment
We thank H. Luftmann for the exact mass measurements, U. Lauber from the CVUA Stuttgart for providing food samples, and the Ministry of Innovation, Science, Research and Technology of the State of North Rhine-Westphalia (EU-Ziel 2-program, NRW 2000-2006) for financial support.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Beyer, M., Ferse, I. & Humpf, HU. Large-scale production of selected type A trichothecenes: the use of HT-2 toxin and T-2 triol as precursors for the synthesis of d 3-T-2 and d 3-HT-2 toxin. Mycotox Res 25, 41–52 (2009). https://doi.org/10.1007/s12550-009-0006-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12550-009-0006-2