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Determination of T-2 Mycotoxin in Fusarium Strains by HPLC with Fluorescence Detector

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Determination of T-2 Mycotoxin in Fusarium Strains by HPLC with Fluorescence Detector Journal of Applied Biotechnology Reports Original Article Determination of T-2 Mycotoxin in Fusarium strains by HPLC with fluorescence detector Reza Kachuei1, Sassan Rezaie*2, Mohammad Hossein Yadegari3, Naser Safaie4, Abdol-Amir Allameh5, Mohammad-Ali Aref-poor6, Abbas-Ali Imani Fooladi7, Majid Riazipour8, Hasan Mirhaj Mohammad Abadi6 Abstract T-2 toxin is the most poisonous trichothecene produced by Fusarium species espe- 1. Molecular Biology Research Center, cially F.sporotrichioides. T-2 toxin is a biological contaminant in a number of agri- Baqiyatallah University of Medical Sciences, cultural commodities that can cause severe diseases among humans and animals and Tehran, Iran even lead to death. The aim of the current study is the analysis of T-2 mycotoxin in 2. Department of Medical Parasitology and My- Fusarium species by high-performance liquid chromatography (HPLC) combined cology, Faculty of Public Health, Tehran Univer- with florescence detection and derivatization with 1-antroylnitrile (1-AN). Totally, sity of Medial Sciences, Iran 11Fusariumisolates and reference strains were studied. The isolates were tested for 3. Department of Medical Mycology, Faculty of the T-2 toxin production after growing on rice substrate followed by using specific Medicine Science, Tarbiat Modares University, “Multisep 225 Trich Clean up columns” purification. In this study, T-2 toxin produc- Tehran, Iran tion was ranged from 197.05ug/kg to 8503.07ug/kg. This is the first study of T-2 4. Department of Plant Pathology ,Faculty of toxin analysis by HPLC-F in Iran. Agriculture ,Tarbiat Modares University ,Tehran , Iran 5. Department of Biochemistry, Faculty of Medicine Science, Tarbiat Modares University, Tehran, Iran 6. Department of Biology, Faculty of Basic sci- ences, Imam hossein University, Tehran, Iran 7. Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 8. Department of Medical Parasitology and Mycology, Faculty of Medicine, Baqiyatallah University of Medial Sciences, Iran * Corresponding Author Sassan Rezaie Department of Medical Parasitology and Mycol- ogy, Faculty of Public Health, Tehran University of Medial Sciences,Iran, Tel.: +9821 88008722. E-mail address: [email protected] Submission Date: 1/23/2014 Keywords: Fusarium, Trichothecene, T-2 toxin, HPLC-F Accepted Date:3/14/2014 Introduction Agricultural crop contaminations especially grains (e.g. Penicillium, Aspergillus and Fusariumspecies are three wheat, maize, barley and etc.) have been repeatedly report- major mycotoxin producers in the world. Among ed causing thousands of livestock and human deaths [6- fumonisins, zearalenone, and trichothecenes as important 12]. In the other hand, misusing toxins as biological weap- mycotoxins of Fusarium sp., trichothecenes are the most ons in the former Soviet Union (1974-1981) in Laeous, dispersed and the most diverse ones including 200 analogs Kambuj and Afghanistan, Vietnam War and probably in [1-2]. Based on their chemical characteristics and origina- Iran-Iraq war (1983-1984) show the great significance of tion, trichothecenes are classified into A, B, C, and D the concern [3, 13]. Amongst, F. sporotrichioides, is the groups. T-2 toxin, a member of the A-group trichothe- major source of T-2 toxin production. Some isolates in- cenes, is the most poisonous trichothecene (10 folds more cluding F. armeniacum, F. equeiseti, F.musarumand F. poisonous than B-group trichothecenes like deoxyniva- poae are able to produce T-2 too. Recently, F. langsethiae lenol). Chemically, T-2 toxin is insoluble in water but sol- is introduced as an important isolate for T-2 production uble in acetone, ethyl acetate, chloroform, ethanol, metha- [14, 15]. nol and propylene glycol. The toxin is stable in various Methodologically, T-2 analysis methods are divided into environmental conditions especially autoclaving. To inac- two classes; 1) screening methods such as Thin Layer tivate, the toxin should heat to 900 °F for 10 min or 500 °F Chromatography (TLC) & ELISA, and 2) analytical meth- for 30 min [1, 3-5]. ods like Gas chromatography (GC) and high-performance Worldwide, food poisoning of livestock and human with liquid chromatography (HPLC) methods [3]. HPLC meth- various reported epidermises along with toxin based bio- ods in some cases are combined with light detection meth- logical weapons are two major concerns in T-2 detection. ods like HPLC-RI (refractive index), UV detection, and Journal of Applied Biotechnology Reports, Volume 1, Issue 1, Winter 2014; 38-43 All rights reserved for official publication of Baqiyatallah university of medical sciences© Reza Kachuei. et al, Determination of T-2 Mycotoxinin Fusarium strains HPLC-F (fluorescence) detection systems to detect T-2 HPLC apparatus and analysis [16-18]. In this study, HPLC-F detection of T-2 toxin from The HPLC system (Waters, USA, No. 2695) was connect- Fusarium strains is studied. ed to fluorescence scanning system of 474 integrated with EM Materials and Methods Chemicals Table 1. T-2 toxin production in standard strains and isolates of The reagents were analytical grade of Merck Chemicals studied strains. T-2 Production Recovery Co. Stock solution of T-2 was prepared by dissolving Fusarium Species amount (ppb) (%) commercial T-2 (Sigma Aldrich) in acetonitrile in 1000 ppm concentration (1µg/µl) and stored at -20. Other sol- F. sporotrichioides(VTT) 798.17 vent including methanol, chloroform, hexane, and etc. F. sporotrichioides (BBA) 8503.07 were from Merck, Germany Company. F. sporotrichioides 6370 Fungi and Culture preparation (MCR4333) Eleven standard strains, isolated from Iran, were studied F. sporotrichioides 513.24 (Table 1). A section of pure and proliferated colony on (MCR0043) PDA medium were added to sterile rice substrate (50 mg F. poae(MCR8485) 0 of rice in 50 mL sterile distilled water) and was incubated F. poae (MCR8486) 197.05 (27±1 °C for 5 days). Then 3 weeks was incubated at 10±2 °C according to literature [12]. Then growth medium was Fusariumcflangsethiae 5133.17 transferred onto a sterile aluminum foil and then was oven- F. tricinctum(isolated) 0 dried (45°C for 24-48 hours). The dry weight was pow- F. oxysporum (isolated) 0 dered by electronic grinder. Afterward the extract was pre- pared and used for further analysis. F. camptoceras (isolated) 0 Extract preparation F. compactum (isolated) 0 The grinded powder was mixed well. To each 25 gr of the powder was added 100 mL of acetonitrile solution and Spike1 (100ppb) 80.41 80.4 water in 84:16. Then, for 12 hours was shacked vigorously Spike2 (3000ppb) 1921.5 64 in >210 rpm. The achieved extract was fined twice by 9cm No.120 paper and then whatman paper 12.5cm No.1. The Power software. Reverse phase column was used for isola- fat of the extract was removed by hexane by adding 2:1 tion and the analytical column 18 reverse (200×4.6 mm, ratio of hexane to extract and shacked for 5-10 min. Sub- 5µm particles) (Waters, USA) combined by a guard filter sequently, the upper phase which included fat was re- (0.5µm, Merck, Germany). The mobile phase (acetonitrile- moved. The remained extract was used in rotary evapora- water in 65:35 v/v) had the flow rate of 2.5 ml/min. Exci- tor to dry it. Finally the powder was solved in 3mL of ace- tation and emission wavelengths of fluorescence were 381 tonitrile and water in 84:16 ratios. nm and 470 nm, respectively. Cleaning up After setting up HPLC apparatus with standard toxin, In order to cleaning up the extra contaminations of the sample toxin was injected each step to monitor the con- extract, special clean up columns (Multisep 225 Trich, stant and accuracy of the system. Quantitative analysis of Romer Company, USA) were used. After cleaning up, the the T-2 toxin in samples was performed by comparing solution was divided into 3 microtubes and then dried un- standard and sample peaks. For standard curve, four con- der the slow flow of N2 gas. Afterwards, 0.5 mL of centrations of 10, 50, 100 and 500 ppm of standard stock acetonitrile solvent and ethanol (3:1 ratio) was added and and pure residue of samples were dissolved in acetonitrile. mixed. Solvent was vaporized again and 0.5 mL chloro- Injection to HPLC performed after derivatization with 1- form and methanol (9:1 ratio) was added, mixed and AN dissolved in acetonitrile-water (80:20 v/v ratio). Deri- washed twice. Then 20 mL of solvent was used for rotary vatization reagents, DMAP and 1-AN, were injected to the evaporator to concentrate it in 2 mL of dichloromethane. system too. Then, the residue was dried under slow flow of N2 gas [18, Statistical analysis 19]. Percent of recovery was estimated as accuracy index. Per- Derivatization and HPLC determination cent of recovery was prepared for two spiking samples. 4-dimethyl amino pyridine (DMAP) (Sigma Aldrich) and For two samples including 5 gm of rice were added 0.5 1-anthroylnitrile (1-anthroylcyanide: 1-AN) (Wako, Japan) µg/gr of standard toxins 100bbp and 3000bbp, respective- powders were dissolved in toluene in 0.325 µg/µl and ly. After one hour, extractions was collected and analyzed. µg/µl 0.3 concentration, respectively. The prepared solu- Also, rice substrate was used as control sample for toxin tions were preserved at -20. DMAP (50 µl) was added to analysis. the residue and then derivatization reagent 1-AN was add- ed (50 µl) [17], mixed and heated to 50 °C for 15 min. Results After the residue was cooled on ice for 15 min and then In this study a fine peek of T-2 toxin indicating its pres- was dried under N slow circulation. Then residue was 2 ence underutilized analytical method was achieved. This solved in 100 µl acetonitrile and water (80:20ratio). 5 µl of peak was clearly defined and distinguishable from the oth- the achieved solution was used for HPLC analysis.
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