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Journal of Environmental Scienceand Health, Part B: Pesticides, FoodContaminants, and AgriculturalWastesPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/lesb20

Determination of T‐2 toxin in grainand grain products by HPLC and TLCGülden Z. Omurtag a & Duygu Yazicioğlu a

a Department of Pharmaceutical Toxicology, Faculty ofPharmacy , Marmara University , Haydarpaşa‐İstanbul, 81010,TurkeyPublished online: 14 Nov 2008.

To cite this article: Gülden Z. Omurtag & Duygu Yazicioğlu (2000) Determination of T‐2toxin in grain and grain products by HPLC and TLC, Journal of Environmental Science andHealth, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 35:6, 797-807, DOI:10.1080/03601230009373308

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J. ENVIRON. SCI. HEALTH, B35(6), 797-807 (2000)

DETERMINATION OF T-2 TOXIN IN GRAIN AND GRAIN PRODUCTSBY HPLC AND TLC

Key words: T-2 toxin, grain and grain products, HPLC, TLC

Gülden Z. Omurtag and Duygu Yazicioğlu

Department of Pharmaceutical Toxicology,Faculty of Pharmacy, Marmara University, 81010, Haydarpaşa- İstanbul, Turkey

ABSTRACT

The purpose of this study was to investigate the T-2 toxin contaminated grain

and grain products consumed especially by Turkish population. The T-2 toxin was

detected using the high performance liquid chromatography (HPLC) with UV

detector at 208 nm and the identify of T-2 was further confirmed by thin layer

chromatography (TLC). The recovery was 91 ±4.24% for corn flour fortified with

the known amount of T-2 toxin (1 ppm). The detection limits of T-2 toxin for the

HPLC and the TLC were 25 ng and 50 ng, respectively. A total of 30 commercially

available grain and grain product samples were analyzed. Two corn flour samples

were found to contain detectable levels of T-2 toxin at a level of 1.60 ppm and 4.08

ppm.

INTRODUCTION

The trichothecene (TCTC) mycotoxins, as a group of naturally occurring

contaminants of foods and feedstuffs and they have been detected in fungal

797

Copyright © 2000 by Marcel Dekker, Inc. www.dekker.com

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798 OMURTAG AND YAZÍCÍOGLU

contaminated grain and grain products. The T-2 toxin is one of the most toxic

TCTCs, which is (4ß, 15-diacetoxy-8a-(3- methylbutyryloxy) - 3a-hydroxy-12,

13-epoxytrichothec-9-ene) a naturally occurring toxic compound produced by

various species of the fungal genus Fusarium such as F. lateritium, F.

sporotrichioides , F.nivale, F. oxysporum, F. rigidisculum, F.roseum, F.solani,

F.tricinctum (Gentry and Cooper, 1983; Grost-Allman and Steyn,1979; Mann et al.,

1983; Yoshizawa et al., 1980). The TCTC mycotoxins are a group of toxic fungal

compounds characterised by a sesquiterpenoid structure with a double bond at the

C-9-C-10 position and an epoxide ring at the C-12-C-13 position. They are

produced predominantly by fungi in the class Deuteromycetes, e.g., Fusarium,

Myrothecium, and Trichothecium. Fusarium species such as F. sporotrichioides and

F.poae, which produce highly toxic type A TCTC mycotoxins, e.g. the T-2 toxin

(Parj et al., 1996). Fusaria, are widespread fungi on a variety of plants and in soil

throughout the cold and cold-temperate regions about 0°C to 10°C and they require

moisture in the range of 22 to 25% (Krüger, 1989). T-2 toxin is capable of growing

on agricultural products before harvest and during storage. Fusaria are field fungi

and grow at relatively high water activities (aw) of about 0.90 up to 1.0. If the grain

is dried adequately the growth of Fusaria is normally inhibited. When the drying

process is disturbed growth can continue on (Schwabe and Kramer, 1995). The T-2

toxin has been found in corn, barley, wheat, oats, rye and mixed feed in several

European countries (Gareis et al., 1989). Foodstuffs contaminated with these

materials can cause severe effects in humans and animals and it may result in death.

Two serious cases of mycotoxicoses have been associated with the ingestion of food

or feed infected with F. sporotrichioides Sherb. The first one occurred in Russia

during the World War II. It is known as the "alimentary toxic aleukia", which was a

human disease involving leucopenia, necrotic angina, hemorrhage, and exhaustion

of bone marrow (Yagen and Joffe, 1976). The second one is the "bean-hulls

poisoning of horses" which was occurred in Japan (Ueno et al., 1972). In both cases

the strains of F. sporotrichioides, which were isolated from grains and beans, found

to produce the T-2 toxin and its derivatives, consistently (Visconti et al., 1985).

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T-2 TOXIN IN GRAIN PRODUCTS 799

Several methods have been used for the determination of the T-2 toxin, such as;

the HPLC (Cohen and Boutin-Muma, 1992; Kuronen, P., 1989; Maycock and Utley,

1985; McCormic et al., 1990; Sano et al., 1987; Schmidt et al., 1981; Stratton et al.,

1993; Yagen et al., 1986), the gas chromatography (Bata et al.; 1984; Gedloff et al.,

1984), the gas chromatography-mass spectrophotometry (GC-MS) (Pineiro et al.,

1996; Voyksner et al., 1987), thermospray liquid chromatography-mass

spectrometry (Rajakyla et al., 1987), enzyme-linked immunosorbent assay (ELISA)

(Gedloff et al., 1984; Stratton et al., 1993; Yoshizawa et al., 1982), the high

performance TLC (HPTLC) (Bata et al., 1984), polarography (Bata et al., 1984) and

the TLC. The detection on the TLC plates is achieved by dipping and spraying the

plates with sulphuric acid (Paster and Menasherov, 1988), aluminium chloride

(Baxter et al., 1983), p-anisaldehyde (Durackova et al., 1976; Filtenborg et al.,

1983), 4 (p-nitrobenzyl) pyridine, nicotinamide-2-acetylpyridine (Sano et al., 1982),

or Chromatographie acid (Baxter et al., 1983) followed by heating.

The purpose of this investigation was to determine the T-2 toxin contaminated

grain and grain products consumed especially by human. The Chromatographie

methods used in the analysis were the HPLC and the TLC. The HPLC method used

in this study was modified from Stratton et al. (1993). The origin of the samples was

from various parts of Turkey and they were collected from markets and street

bazaars existing in Istanbul. The T-2 toxin and the aflatoxin, both of them being

mycotoxin, the guideline appears for the aflatoxins (Omurtag et al., 1998), but

unfortunately the guideline does not accommodate the possibility that the T-2 toxin

might be present in the contaminated rations.

MATERIALS AND METHODS

Chemicals

A T-2 toxin standard was obtained from the Sigma (T-4887), aluminium chloride

anhydrous from the Sigma (A-7178) and other chemicals were the Merck products.

All solvents used were the HPLC grade. Standards and mobile phase were prepared

by using the daily water obtained from the Milli Q-RG (Millipore) system. Water

used for the other solutions was distilled in glass in the laboratory.

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800 OMURTAG AND YAZÍCÍOGLU

Preparation of Stock Solutions

The stock standard solution of the T-2 toxin was prepared at a concentration of

500 ug/ml in acetonitrile. The stock solutions of 25 ug/ml and 50 ug/ml in

methanol/water (55:45, v/v) were prepared from the stock standard solution. All

stock solutions were sealed and stored at -20° C when not in use.

Sample Preparation

Grain and grain product samples (50g) were placed in a Waring blender and

blended with 250 ml of acetonitrile/water (21:4, v/v) for 3 min in low speed. The

suspension was allowed to settle for 30 min and the liquid phase decanted and

filtered through the Whatman No :4 filter paper. Five-millilitre aliquots of the filtrate

were collected in glass tubes sealed with teflon-lined screw caps. The filtrate was

evaporated to dryness under N2 at 60°C and the residue stored at -20°C until needed

for cleanup. All glassware was decontaminated with 0.2 M sodium hypochloride,

washed with detergent, thoroughly rinsed, acid washed, and rinsed again with

distilled water prior to reuse.

Cleanup of the Extracts

The sample extract residue was first dissolved in 10 ml of n-hexane. This was

extracted 3 times with 3.5 ml of acetonitrile/water (21:4, v/v). The acetonitrile/water

fractions were pooled and evaporated to dryness under N2 at 60° C. The residue,

dissolved in 10 ml of chloroform, was loaded onto a 6 ml (500mg) C18 configuration

solid phase extraction (SPE) column (JT Baker 7189-06) conditioned with 5 ml of

chloroform. The entire T-2 toxin was eluted from the column with 10 ml chloroform

and evaporated to dryness in water bath at 60° C under a stream of nitrogen.

Procedure for the HPLC

The residue was dissolved with 1.0 ml of methanol/water (55:45, v/v) and

filtered through a Millipore filter type (0.45um). An aliquot (20, 40 and/or 100/il)

was injected into the HPLC apparatus and analyzed in triplicate. The areas of the

peaks were used for quantification.

The high performance liquid chromatography (Waters Corporation, U.S.A.) was

a combination of a Model 510 pump with a Model 481 variable-wavelength UV

detector. Injector was Rheodyne 7725 sample injector-100^1 accessory. A reverse

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T-2 TOXIN IN GRAIN PRODUCTS 801

phase NovaPak C\% column was used (3.9x150 mm, 4-fim particle size) with a

/¿Bondapak Qs (part no: WAT 0088070) guard column of identical composition

(Waters). Data Station was a Unicam 4880 Chromatography Data Handling System.

Mobile phase was methanol/water (55:45, v/v); wavelength was 208 nm; flow

rate was 1.0 ml/min, chart speed was 0.25 cm/min and column temperature was

ambient. The mobile phase was filtered through a Millex HV Millipore (0.45 urn)

and degassed by Waters In-Line degasser. The standard curves of T-2 toxin (ÍR=9.1

min) were linear between the concentration ranges of 62.5 and 1000 ng. The linear

regression line for T-2 toxin was found to be y=2.165*-25.63 (/=0.9991). Figure 1

shows the HPLC chromatograms of T-2 toxin standard, sample extract and

contaminated sample extract.

Procedure for the TLC

The plates were activated at 110° C for 2 hours. 25/il and 50/d of sample extracts

were obtained in the same way as given in the HPLC procedure. The sample

extracts and the reference standards of the T-2 toxin between 0.05 to 1.0 ng were

spotted 2 cm from the bottom of the aluminium silica gel foil (Merck 5553). The

chromatogram was developed with toluene/ethyl acetate/90% aqueous formic acid

(5:4:1). The plate was dried, lightly sprayed with 20% aluminium chloride reagent

(aluminium chloride in ethanol/water, 15:85 v/v), heated for 5 min at 110° C then

plate was examined under an ultraviolet (UV) lamp at 366 nm. The T-2 toxin did

not react with aluminium chloride. H2SO4 (25% in water) was sprayed and heated at

110° C for 10 min. After cooling, the plate was re-examined under an UV lamp at

366 nm and visualised. The spots including the T-2 toxin were yellow-green.

RESULTS AND DISCUSSION

All the analyses were carried out by the HPLC and only T-2 toxin contaminated

samples were verified with the TLC. In this study, 30 grain and grain products were

provided from various parts of Turkey and they were bought from markets existing

in Istanbul. A small number of samples (6.66%) had detectable levels of the T-2

toxin. All through the trademark samples no T-2 toxin was detected and complete

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802 OMURTAG AND YAZÍCÍOGLU

g

oo

ooo

O Wo a° 3s I

FIGURE 1a) HPLC chromatogram of T-2 toxin standard (125ng), b) HPLC chromatogram of asample extract, c) HPLC chromatogram of a T-2 toxin contaminated sample extract.

list of the sample results are given in Table 1. The T-2 toxin was detected in two no

name samples of corn flour. The measured levels by the HPLC and the TLC, were

1.60 ppm, 4.08 ppm and 1.90 ppm, 3.30 ppm, respectively (see Table 1). Most of

the samples (76.66%) were from unknown origins and the rest being from the

known origins from various cities of Turkey. The detection limits of the T-2 toxin

for the HPLC were 25 ng and 50 ng for the TLC. The detection limit of T-2 toxin

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T-2 TOXIN IN GRAIN PRODUCTS 803

TABLE 1The T-2 Toxin Results in Grain and Grain Products, by HPLC and TLC

Samples

Fresh cornCanned corn

Frozen cornCorn flour

Corn flakes

Corn snacks

CipsCornstarchPop corn

Wheat flour

Wheat flakes

SourceBought fromStreet-Bazaar

M*K*

L*Street-Bazaar

MarketStreet-BazaarStreet-BazaarStreet-Bazaar

H*Street-BazaarStreet-Bazaar

J*Market

H*Street-Bazaar

A*B*C*D*E*G*D*F*

Street-BazaarMarket

H*N*p*A*

OriginUO

Giresun§

UOGiresun§

UOUO

Giresuns

Giresun5

Black Sea coast

Adapazan5

Samsun§

UO

HPLC(ppm)NDNDND

NDNDNDND1.60NDNDND4.08NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDND

TLC(ppm)

-----

1.90-

3.30-----------

------

* : Various Trade Marks, UO: Unknown Origin, ND: Not Detected,5 : A City in Turkey

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804 OMURTAG AND YAZÍCÍOGLU

was taken as two times of the noise. The recovery of the T-2 toxin was 91 ±4.24%

and it was performed using corn flour with known amount of the T-2 toxin (1 ppm).

The recovery results were expressed as the mean of five different manipulations

including the extraction from the same sample. The coefficient of variation was

4.66%. Rf value on the silica gel TLC of the T-2 toxin was 0.48.

The incidence of the T-2 toxin contaminated samples is generally below 10%

and most frequently at levels less than 0.1 ppm (WHO, 1990). Rather high levels of

the T-2 toxin findings were, e.g., 25 ppm T-2 toxin in barley (Puls and Greenway,

1976), and 38.9 ppm T-2 toxin in peanuts (Bhavanishankar and Shantha, 1987). Out

of 15 different species of Fusarium only two isolates of F. sporotrichioides were

able to produce T-2 toxin and HT-2 toxin (Visconti et al., 1985). F. sporotrichioides

was found to be the predominant fungus in approximately 2 % of corn ears damaged

by Fusarium species, before harvest during 1984 and 1985 in Poland (Chelkowski

et al., 1987). The randomly selected grain samples (spring and winter wheat, 2-row

and 6-row barley) from Nova Scotia were tested in 1989 and 11% of them had

detectable levels of T-2 toxin (between 0.16 and 0.31 ppm) (Stratton et al., 1993).

CONCLUSION

The HPLC is one of the most popular methods due to the identification of

mycotoxins. Analysis of the T-2 toxin by the HPLC with UV detection is difficult

due to its low absorption intensity and one needs to use low wavelengths. In this

study the wavelength was 208nm. However the HPLC procedure used in the present

study yielded a good recovery for the T-2 toxin. There is also a lack of legislation

on the acceptable levels of TCTCs in grain and grain products since no limits were

given for the TCTC toxins except deoxynivalenol (Canada, Russia, and the United

States) (Stratton et al., 1993). The T-2 toxin levels in food commodities are

generally <0.5 ppm, but are somewhat higher in animal feeds (Jelinek et al. 1989).

In our research two samples out of thirty samples had detectable levels of the T-2

toxin and they were 4.08 ppm, 1.60 ppm (the HPLC results) in corn flour.

Protection of human food supplies from the mycotoxin contamination, a special care

must be given from the beginning point of production till to the very end point of

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T-2 TOXIN IN GRAIN PRODUCTS 805

the consumption. For example, food and feedstuff supplies must be stored such that

mold growth will be prevented on the farm, in the storage facilities, in the markets

and in the houses. Considering the toxic effects of the T-2 toxin if a critical upper

limit in foods consumed by the human is going to be set, we may recommend the

value of 0.5 ppm. As a consequence, if the legal loophole is filled in the future, it

will be an important attempt to keep the contamination of TCTCs out of the food

and feed supply.

ACKNOWLEDGEMENT

The results presented in this study were obtained during an investigation

supported by the Research Fund of Marmara University, project number 1999-SA-

5.

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