Quantitation of Trichothecene Mycotoxins by Stable Isotope Dilution

Chapter 13

Quantitation of Trichothecene Mycotoxins by Stable Isotope Dilution Assays Downloaded via TUFTS UNIV on July 10, 2018 at 00:05:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Michael Rychlik and Stefan Asam Lehrstuhl für Lebensmittelchemie, Technische Universität München, München, Germany

Stable isotope dilution assays (SIDAs) for the simultaneous quantitation of type A and type B trichothecenes in cereal products were developed. Syntheses of [ C ]-diacetoxyscirpenol, [ C ]-monoacetoxyscirpenol, [ C ]-T-2 toxin, [ C ]-HT-2 toxin, [ C ]-3-acetyldeoxynivalenol, [ C ]-15acetyldeoxynivalenol, and [ C ]-4-acetylnivalenol were accomplished by complete [ C ]-acetylation of T-2 triol, scirpentriol, deoxynivalenol, and nivalenol, respectively, followed by careful alkaline hydrolysis of the peracetylated compounds. Samples were spiked with the synthesized internal standards and purified on multifunctional columns. A l l trichothecenes under study were quantified simultaneously within one liquid chromatography (LC)-mass spectrometry (MS) run using single and tandem MS detection. The method revealed good sensitivity with low detection and quantification limits along with excellent recovery data and good precision in inter-assay studies. Food samples were analysed using the developed SIDA and showed substantial contamination of oat products with T-2 toxin and HT-2 toxin. Diacetoxyscirpenol was detected rarely and monoacetoxyscirpenol and 4acetylnivalenol were not present in the analyzed samples. Further type B trichothecenes occurred more frequently and generally in higher concentrations than type A trichothecenes. The type B trichothecene deoxynivalenol was detectable in all cereal samples. Corn (maize) products did not only show the highest concentrations of deoxynivalenol, with values up to 300 μg/kg, but also distinct contamination with 15-acetyl13

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© 2008 American Chemical Society

Siantar et al.; Food Contaminants ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

253 deoxynivalenol, whereas 3-acetyldeoxynivalenol was pre dominantly found in oat products.

Trichothecene mycotoxins are produced by various fungi of the genus Fusarium, which infect cereals during growth and cause a characteristic disease pattern called Fusarium head blight in wheat and Gibberella ear rot in corn. These mycotoxins are tetracyclic sesquiterpenes bearing a spirocyclic epoxide moiety and are classified into four different types A, B, C, and D. Type A trichothecenes differ from those belonging to the type B by the absence of a carbonyl group at C-8 and hydroxylation at C-7. The type A group itself can be differentiated into two families namely the T-2 family with hydroxylation at C-8 and the scirpenol family, which is completely devoid of any functionalities at C8. Structures of common trichothecenes are shown in Figure 1. Due to the production of mycotoxins, infections of cereals with Fusarium present an acute hazard to the consumer. As trichothecenes are known to cause vomiting and hematoxic effects as well as immunosuppressive effects, a temporary tolerable daily intake (tTDI) value of 0.06 ¿ig/kg bw/d for the sum of T-2 toxin and HT-2 toxin, as well as a tTDI of 1 jig/kg bw/d and 0.7 ng/kg bw/d for the type B trichothecenes deoxynivalenol and nivalenol, respectively, has been suggested by the Scientific Committee on Food (SCF) (7). Therefore, trace concentrations of these compounds have to be controlled in foods, which requires sensitive and accurate methods for quantitation. Among the most reliable analytical methods, stable isotope dilution assays (SIDA) are becoming more common as reference methods to determine the "true" analytical value. In mycotoxin analysis, this concept already has been successfully applied to quantitation of patulin (2) and ochratoxin A (5). In the past, trichothecenes have commonly been analyzed by gas chromatography with either flame-ionization detection {4\ electron-capture detection (5), or mass spectrometric detection (6). Due to the polarity of the free hydroxyl moieties of trichothecenes, derivatization is necessary to afford volatile compounds for GC analysis. As this additional step in sample preparation is time consuming and may lead to increased analyte loss, direct analytical methods such as LC are generally preferred. However, LC-UV methods, which today are routinely applied to the determination of type B trichothecenes, cannot be used for type A trichothecenes due to lack of a UV-absorbing moiety in the molecular structure. As a consequence, methods for the determination of trichothecenes have been developed using LC with mass spectrometric (MS) detection (7). Due to losses during clean-up and ionization interferences of matrix compounds, the application of internal standards is often necessary during LCMS. For trichothecenes, the use of structurally similar internal standards such as


254

Type A trichothecenes:

R

Ri

R

HT-2 toxin

OH

OH

OAc

OCOC4H9

T-2 toxin

OH

OAc

OAc

OCOC4H9

Monoacetoxyscirpenol

OH

OH

OAc

H

Diacetoxyscirpenol

OH

OAc

OAc

H

2

R4

3

Type B trichothecenes:

Deoxynivalenol 3-Acetyldeoxynivalenol 15-Acetyldeoxynivalenol 4-Acetylnivalenol

Ri OH OAc OH OH

R H H H OAc 2

R3

OH OH OAc OH

Figure 1. Structures of common trichothecenes.


255 deepoxy-deoxynivalenol, neosolaniol, and verrucarol (7) has been reported. Regarding the ideal structure for internal standards, there is general consensus that the use of isotopically labelled internal standards not only can compensate best for losses of analytes during sample preparation but also for any kind of matrix effect. Therefore, the aim of the present study was to synthesize stable isotopically labelled analogues of trichothecenes and to develop SIDAs for the simultaneous quantitation of these mycotoxins.

Materials and Methods Stable isotope labeled trichothecenes used as internal standards were synthesized by [ C ]-acetylation as described recently (8, 9). In brief, [ C ]-T-2 toxin and [ C ]-HT-2 toxin were obtained by alkaline hydrolysis of [ C ]-T-2 3,4,15 triacetate, which itself was prepared by complete acetylation of T-2 triol with [ C ]-acetic anhydride in dry pyridine. Analogously, [ C ]monoacetoxyscirpenol ([ C ]-MAS) and [ C ]-diacetoxyscirpenol ([ C ]-DAS) were prepared from scirpentriol, [ C ]-4-acetylnivalenol ([ C ]-4-AcNIV) from nivalenol, and [ C ]-3-acetyldeoxynivalenol ([ C ]-3-AcDON) and [ C ]-15acetyldeoxynivalenol ([ C ]-15-AcDON) from deoxynivalenol (DON). Identity and purity of all synthesized compounds were verified using NMR spectroscopy, LC-MS, and LC-MS/MS experiments before they were used as internal standards. [ C ]-Deoxynivalenol was obtained from Biopure Referenzsubstanzen GmbH (Tulln, Austria). Analysis of trichothecenes was performed using LC-MS. LC separation prior to MS was achieved on a Finnigan Surveyor Plus LC System (Thermo Electron Corp., Waltham, MA) using a 150 x 2 mm i.d., 4 urn Synergi Polar-RP column (Phenomenex, Aschaffenburg, Germany) as the stationary phase. The mobile phase consisted of variable mixtures of formic acid (0.1 %, solvent A) and acetonitrile acidified with formic acid (0.1 %, solvent B). A linear gradient was programmed starting with 5 % B, which was held for 5 min and then increased to 30 % B within 15 min. Immediately afterwards the concentration of B was raised to 60 % within 2 min and then increased more slowly to reach 100 % B 30 min after injection. 100 % B was held for 10 min before returning to initial conditions. The subsequent equilibration time between two runs was set to 15 min. Flow rate was 0.2 mL/min and the injection volume was 10 \iL. The LC was coupled to a triple-quadrupole mass spectrometer Finnigan TSQ Quantum Discovery (Thermo Electron Corp.). The ion source was operated in the electrospray ionization (ESI) positive mode resulting in protonated molecular ions (as for type B trichothecenes) or sodium adduct ions (as for type A trichothecenes). Most trichothecenes were quantified by tandem MS in the selected reaction monitoring (SRM) mode using characteristic transitions from the parent ion. The only exceptions were DAS and MAS, which did not show 13

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256 reproduciblefragmentationand, therefore, were determined by single stage MS in the single ion monitoring mode. Cereal based food samples were purchased from local markets, finely ground, and thoroughly homogenized. About 3-5 grams of homogenous material was weighed in an Erlenmeyer flask and labelled standards were added. Subsequently, trichothecenes were extracted by a mixture of water-acetonitrile (16:84, v/v, 15 mL) followed by filtration and clean-up over MultiSep® 225 Trich cleanup columns (Romer Labs Inc., Union, USA). The purified extracts were evaporated at 60 °C in vacuo, redissolved in mobile phase for LC-MS analysis (100 \xL), membrane filtered, and subjected to LC-MS/MS (9). For matrix-assisted external calibration, homogenous samples of oat flakes devoid of mycotoxins were spiked with solutions of DON and T-2 toxin at different concentration levels and analyzed in triplicate as described above. The same solutions of DON and T-2 toxin were used for direct external calibration after dilution with water. To determine analyte losses during sample preparation labelled internal standards were added to a series of samples at different stages of analysis, i.e., after filtration, after column clean-up, and after evaporation. Analysis of each stage was performed in triplicate.

Results and Discussion 13

To date, [ C ]-DON is the only trichothecene mycotoxin that is commercially available as a labelled internal standard. To develop SIDAs for all other relevant trichothecenes (Figure 1) we synthesized these compounds as isotopologues using carbon-13 labelling of the acetyl moieties. The four labelled type A trichothecenes and [ C ]-4-AcNIV were easily obtained by alkaline hydrolysis of the peracetylated compounds. The preferred formation of the desired mono- and di-acetates out of a number of potential isomeric compounds is due to the fact that they are more stable under alkaline conditions than the respective isomers. In a like manner, alkaline hydrolysis of [ C ]-DON-3,15-diacetate resulted in only one monoacetate, namely the more stable [ C ]-3-AcDON isomer. In contrast to this, direct [ C]-acetylation of DON by using sub-stoichometric amounts of [ C ]-acetic anhydride yielded mainly [ C ]-15-AcDON and [ C ]-DON-3,15-diacetate along with unreacted DON, but only traces of [ C ]-3-AcDON (Figure 2A). Therefore, synthesis of both isomers was only possible after separating a reaction mixture containing [ C ]-15-AcDON and [ C ]-DON-3,15-diacetate using preparative LC and subsequent alkaline hydrolysis of the diacetate (Figure 2B). Using the seven synthesized isotopologic trichothecenes and [ Ci ]-DON, stable isotope dilution assays were developed applying LC-MS/MS. Appropriate LC conditions allowed the separation of all trichothecenes under study within one run (Figure 3). Chromatographic separation was essential to distinguish 15

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.0

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10.0

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[ C K3-Ac-DON

25.0 [min]

[ C h3,15-DON-diacetate

Figure 2. (A) Reaction of [ C ]-acetic anhydride with an excess of DON; (B) alkaline hydrolysis of[ C ]-DON-3,15-diacetate.

10.0

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[ C ]-15-Ac-DON

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[ C h3-Ac-DON

1 3

[ C h3,15-DON-diacetate

258 between the acetyl-DON isomers showing the same signal of the protonated molecule and the samefragmentationduring MS/MS. Positive electrospray ionisation transformed the type B trichothecenes into protonated ions and the type A trichothecenes into sodium adducts. Except for MAS and DAS all substances showed reproduciblefragmentationof the parent ions in MS/MS experiments. MAS and DAS did notfragmentand, therefore, had to be quantified with single stage MS. Validation of the developed method resulted in recoveries of 90 to 127 % depending on the toxin under study. The coefficient of variation in inter-assay precision studies (n = 3) ranged between 2.6 and 8.0 %, also differing between the single trichothecenes. Limits of detection (LOD) and limits of quantitation (LOQ) were determined by the method suggested by Hadrich and Vogelgesang (10% which considers also precision, matrix effects, and recovery of additions near the LOD. In this way, LODs spanned a range between 1 ng/kg (DAS) and 30 jig/kg (MAS) and LOQs were found to range between 4 ng/kg (DAS) and 80 ^g/kg(MAS). The advantages of SIDA became obvious when comparing the analytical results of a naturally contaminated sample (oat flakes) (a) using SIDA, (b) using external calibration, and (c) using external matrix-assisted calibration (Figure 4). External calibration resulted in about 50 % of the concentration of DON and about 30 % of the concentration of T-2 toxin compared to the values obtained using SIDA. This effect could be attributed either solely to incomplete recovery or to a combination with matrix dependent ion-suppressing effects in the ESI interface during LC-MS/MS analysis. To study these effects, labelled standards were added to an extract of oat flakes only after clean-up, which enabled calculation of the loss of analyte during sample preparation, whereas the labelled standards compensated for the ion-suppression in MS/MS analysis. The results of this experiment showed that during sample preparation the loss of DON was about 60 %, in contrast to T-2 toxin, which was recovered almost completely. Further studies (data not shown) revealed that this effect was not a consequence of the clean-up process itself in the first place, but rather of different extraction behaviours from the matrix with acetonitrile-water due to differing polarity of the analytes. From these results it could be concluded that at least in the case of oat flakes a remarkable ion-suppression did not occur for DON. However, the latter effect might be responsible for the low result of T-2 toxin in external calibration as it appears to be almost completely extracted from the matrix. Using external matrix-assisted calibration the content of DON correlated with the SIDA value indeed, but standard derivatization was poor in triplicate analysis. Furthermore the concentration of T-2 toxin determined by external matrix-assisted calibration was 25 % higher than the value obtained using SIDA, indicating signalinterference with matrix compounds during LC-MS/MS analysis. Taken together, these results clearly indicate that the use of stable isotope labelled standards is the method of choice to overcome analytical difficulties,


Figure 3. LC-MS run of eight trichothecenes (only traces of unlabelled comp. shown). Siantar et al.; Food Contaminants ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


120 n

SIDA past clean-up

External calibration

Matrix-assisted external calibration

Figure 4. Difference between analytical methods and effects of clean-up on the concentration ofDON and T-2 toxin.

SIDA

261 time consuming method optimization, and varying matrix effects to achieve accurate analytical results for trichothecene contamination of food. As an application of the developed SIDA a series of food samples obtained from local markets were analyzed for trichothecene mycotoxins. Regarding type A trichothecenes, we found high contamination of oat and oat products with T-2 toxin and HT-2 toxin especially, whereas MAS was not detectable and DAS was present only in traces (Table I). In oat flakes the sum of T-2 toxin and HT-2 toxin exceeded 50 fig/kg in most cases. DAS could be found in different agricultural commodities such as maize, spelt, and potatoes, but its concentrations were lower than 10 ng/kg in general. Type B trichothecenes occurred more frequent and generally in higher concentrations than type A trichothecenes. None of the analyzed wheat or maize samples was devoid of DON, which is in fact alarming, although the absolute levels did not exceed the legal limit for DON in the European Union. In maize, the concentration of 15-AcDON was sometimes higher than that of DON, pointing to the need for methods that can detect more than one mycotoxin simultaneously. Of special interest are the concentrations of type B trichothecenes in processed foods. In breakfast cereals DON was regularly found in amounts up to 280 ng/kg, but also 3-AcDON was detectable in 3 out of 11 samples. Whereas concentrations of DON were relatively low in bread, some samples of maize chips, cookies, and pasta were much more highly contaminated with DON and also with 3-AcDON and 15-AcDON (Table II). A bottom fermented (lager) beer made from barley contained DON only at levels of 8 |ig/kg, whereas in a top fermented beer made from wheat, DON could be quantified in concentrations up to 20 ¿ig/kg. Although the contamination level appears rather low, the total DON intake can be significant when considering the higher consumption of beer compared to other food such as maize chips or cookies. The bottom line is that contamination of cereal products with trichothecenes is still high. Often there is more than one trichothecene mycotoxin present in the food sample. For the protection of consumers it is therefore important to apply sensitive and reliable analytical methods that are able to detect different trichothecenes in varying food matrices simultaneously, together with a reasonable analytical effort. As we have demonstrated, SID As are very suitable to meet all the demands mentioned.

References 1.

European Commission. Scientific Committee on Food (SCF). Opinion on Fusarium Toxins - Part 5: T-2 Toxin and HT-2 Toxin; Brussels, Belgium; 2001 ; http://europa.eu.int/comm/food/fs/sc/scf/out88_en.pdf (accessed: May, 2006)



10 1 2 1 1 1 1

Oat flakes

Oat kernels

Oat cookies

Maize flour

Maize grit

Spelt kernels

Potatoes

Sample

No. of Samples

Type A

0

0

0

1(~3)

0

0

0

0

0

0

0

5(40-100)

6(4-50) 1(~3)

HT-2 toxin

T-2 toxin

1(4)

1(10)

1(5)

1(~2)

2(~1)

0

2(~1)

DAS

No. ofPositive Samples (concentrationin fig/kg)

Table I. Concentrations of Trichothecenes in Different Cereal Products *)

Is*

as


2

(b) organically

2 1 2 3 1

(b) organically

Barley kernels

Oat kernels

Rye flour

Spelt kernels

0

0

3(20-50) 1 (40)

0

0

0.

0

0

0

0

2(10-20)

3 (30-50)

0

1(~6)

1(~4)

1(~4)

15-AcDON

1(~7)

3-AcDON

0

1(35)

2 (6 - 30)

4(8-160)

2(6-30)

12(35-300)

DON

No. ofPositive Samples (Concentration in fig/kg)

0 = concentration below limit of detection; ~ = concentration below limit of quantification. MAS and 4-AcNIV were not present in any analyzed sample.

4

(a) conventionally

Maize, grown

12

No. of Samples

(a) conventionally

Wheat, grown

Sample

TypeB


11 1 1 3 2 3 1 4 1

Breakfast cereals

Toasted bread

Whole wheat bread

Maize chips

Cookies

Pasta

Beer (bottom fermented)

Wheat beer

Baby formula

Sample

No. of Samples

1(15)

4(7-20)

1(8)

3(30-140)

2(15-80)

3 (30 - 320)

1(40)

1(45)

11 (15-280)

DON

0

0

0

1(~2)

1(20)

1(~2)

0

0

3(10-25)

3-AcDON

0

0

0

2(20-30)

0

1(35)

0

0

0

15-AcDON

No. of Positive Samples (Concentration in fig/kg)

Table II. Concentrations of Type B Trichothecenes in Processed Food

265 2. 3.

Rychlik, M.; Schieberle, P. J. Agric. Food Chem. 1998, 46, 5163-5169. Lindenmeier, M . ; Schieberle, P.; Rychlik, M . J. Chromatogr. A 2004, 1023, 57-66. 4. Schothorst, R. C.; Jekel, A. Food Chem. 2001, 73, 111-117. 5. Kotal, F.; Holadova, K.; Hajslova, J.; Poustka, J.; Radova, Z. J. Chromatogr. A 1999, 830, 219-225 6. Nielsen, K. F.; Thrane, U. J. Chromatogr. A 2001, 929, 75-87. 7. Berger, U.; Oehme, M . ; Kuhn, F. J. Agric. Food Chem. 1999, 47, 42404245. 8. Asam S., Rychlik, M . Eur. Food Res. Technol. 2006, 223, published online, DOI: 10.1007/s00217-006-0373-2 9. Asam S., Rychlik, M . J. Agric. Food Chem. 2006, 54, 6535-6546. 10. Hädrich, J.; Vogelgesang, J. Dtsch. Lebensm. Rundsch. 1999, 95, 428-436.


Quantitation of Trichothecene Mycotoxins by Stable Isotope Dilution

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