Or ginaJ Fa e s

Quantitative Determination of Moniliformin in Vegetable Foods and Feeds Christa Jansen und Klaus Dose Institut ffir Biochemie, Johannes-Gutenberg-Universit~it, Johann-Joachim-Becherweg 30, D-6500 Mainz, Federal Republic of Germany

Quantitative Bestimmung von Moniliformin in pflanzliehen Nahrungs- und Futtermitteln

Zusammenfassung. Eine geeignete Analysenmethode zur Be- stimmung yon Moniliformin in Nahrungs- und Futtermit- teln wird beschrieben. Das Toxin wurde aus verschimmeltem Mais, Reis, Roggen, Hafer, Weizen und Gerste extrahiert. Der Extrakt wurde anschliel3end diinnschicht-chromatogra- phisch getrennt und Moniliformin mit N-Methylbenzthiazo- lon-2-hydrazon (MBTH), einem ffir den DC-Nachweis neuen Reagens, nachgewiesen. Das gebildete Moniliformin- derivat konnte bei 518 nm in Remission bestimmt werden. Mit Hilfe einer Eichkurve wurden die erhaltenen Daten quantitativ ausgewertet. Ein linearer Zusammenhang zwi- schen Substanzmenge und Peakfl~che besteht ffir den Be- reich yon 100-400 ng pro Fleck. Die Nachweisgrenze be- tr/igt nach dieser Methode 75 ng/Fleck.

Summary. A suitable and simple method for the quantitative determination of moniliformin in vegetable foods and feeds is described. The mycotoxin was extracted by Soxhlet extrac- tion with methanol from mouldy maize, rice, rye, oats, wheat and barley samples. Moniliformin was determined by thin- layer chromatography (TLC) using N-methylbenzthiazolon- 2-hydrazone (MBTH) as a new derivatization reagent for this mycotoxin. The moniliformin derivative was assayed at 518 nm. Quantification could be performed after calibra- tion. A linear relationship between mycotoxin amount and peak area was found from 100 to 400 ng/spot. The detection limit is 75 ng/spot.

Introduction

The mycotoxin moniliformin was discovered in 1973 by Cole et al. [2] as a product of Fusarium moniliforme and in 1974 by Springer et al. [12] as a product of Gibberella fujikuroi. Later, moniliformin was also found to be produced by F. moniliforme var. subglutinans, F. oxysporum, F. avena- ceum, F. concolor, F. equiseti, and F. semitectum.

The first indication of natural a occurrence of moniliformin was described by Thiel et al. [14].

Moniliformin has also been isolated as a sodium or potassium salt of 3-hydroxy-3-cyclobutene-l,2-dione (Fig. 1). A chemical synthesis of the free acid, also known

Offprint requests to: C. Jansen

Fresenius Z Anal Chem (1984) 319:60-62 �9 Springer-Verlag 1984

No,K Fig. 1. Moniliformin

as semisquaric acid, has been described by Hoffmann et al. [3]. The spectroanalytical parameters, isolation and purifica- tion [6, 10, 15] have been described in detail previously.

Several analytical methods for the determination of moniliformin based on Soxhlet-extractions, semiquantita- tive TLC, ion-exchange chromatography and high pressure liquid chromatography (HPLC) have been published earlier. But the quantitative recovery of added toxin, as well as the reproducibility and accuracy of these methods proved to be unsatisfactory.

We have therefore reconsidered numerous procedures and developed a relatively simple analytical method suitable for the determination of moniliformin in different agricul- tural products.

Experimental

Apparatus. Spectrophotometer model PMQ II (Carl Zeiss, Oberkochen, FRG), monochromator M4 Q III (Zeiss), photomultiplier (Zeiss), x-t recorder (Kipp & Zonen), TLC- Scanner (Camac), Waring Blendor (Waring Products Divi- sion, New Hartford, CT, USA); TLC plates with 0.25 mm layer of silica gel 60 (without fluorescence indicator) (Merck, Darmstadt, FRG).

Chemicals. 1% N-methylbenzthiazolon-2-hydrazone hydro- chlorid (MBTH) in methanol (EGA-Chemie, Karlsruhe, FRG). All chemicals were of p.a. grade.

Organisms. Fusarium moniliforme sp 955; F. oxysporum sp. 1028; F. avenaceum sp. 882.

Production of Moniliformin. 50 g different vegetable foods (maize, rice, rye, oats, barley and wheat) and 30 ml distilled water were autoclaved in I 1 Erlenmeyer flasks, inoculated

50 g sample (mouldy vegetable food)

grinding 2 min 150 ml CH2CL2

filtration I

extraction of the residue with 150 ml CHzCI2

magnetic stirrer, 15 min

filtrate I

residue 150 ml methanol I

150 ml I

defatting with cyclohexane 3 �9 50 ml (in cases of maize)

I MeOH-phase, evaporation

I residue redissolve with MeOH

I TLC

/ \ 1-dim. 2-dim.

quantitative analysis

Fig. 2. Flow diagram for the extraction and determination of moniliformin

with a spore suspension of Fusarium and cultivated at 22 ~ C for 2 - 30 days.

Extraction. 50 g of the mouldy sample (maize, rice, rye, oats, barley and wheat) was blended for 2 min with 150 ml dichloromethane. After filtration the residue was extracted a second time with 150 ml dichloromethane (15 min magnetic stirrer). Following the second filtration the residue was transferred to a Soxhlet apparatus and extracted for 5 h with 300 ml methanol. 150 ml of this extract were placed into a separatory funnel and defatted 3 x with 50 ml cyclohexane. The methanolic phase which contained moniliformin was evaporated to dryness under reduced pressure. The residue was redissolved with methanol (see Fig. 2).

Thin-Layer-Chromatography. The extracts were separated with the following solvent systems: (A) chloroform/meth- anol (3 : 2); (B) butanol(D/acetic acid water (4: / : 5); (C) toluene/acetone/methanol (5: 3 : 2).

For a first separation (screening: usually more than 200 ng moniliformin/spot), system A was used. Monili- formin standards were applicated on the TLC plate in amounts of 100, /50, 200, 300, 400 rig/spot. After drying the chromatogramm was sprayed with a 1% solution of MBTH followed by heating for 20 rain at 140~ At extremely high toxin concentrations a tailing occurred. In these cases the extract was diluted with methanol (1:5) or (1:10). Small amounts of moniliformin (100-300 rig/spot) were always determined by two-dimensional TLC, with solvent system B in the first and C in the second direction.

Quantitative Determination. The MBTH derivative on the TLC plates was assayed in remission at a wavelength of

E o

LL

30

20

10

/ /o

/ I I 1 I t I I

100 200 300 400 500 600 700

Fig. 3. Calibration curve c (ng)

Table 1. Recovery of moniliformin added to rice

Added amounts of Recovery moniliformin to (%) 50 g rice

1 mg 97 700 gg 95 500 gg 91.5 300 gg 60 100 gg 51

518 nm. Signal registration was performed by a x-t recorder. The area (product of peak hight and peak half-width) was proportional to the mycotoxin amount. By calibration with toxin standards a quantitative determination of monili- formin in the samples could thus be achieved.

Results and Discussion

The chromatographic systems described in "Experimental" were successfully applied to the separation of moniliformin. The MBTH derivative of moniliformin showed purple col- our on a white underground. All other compounds of the extract were coloured yellow.

The detection limit for moniliformin after conversion into this derivative was 75 ng/spot. In quantitative analysis (remission at 518 nm) the limit was 100 ng/spot. A linear peak area/toxin relationship was found from 100 to 400 ng/ spot. A calibration curve is shown in Fig. 3. For recovery experiments, quantities of 1 mg, 700, 500, 300 and 100 gg moniliformin were added to 50 g substrate. The results are summarized in Table 1. In all fungi-inoculated cultures the rate of moniliformin production was highest after 10 to 15 days at 220 ~ C. The dependance on time and substrate is shown in Fig. 4. The present method for the quantitative

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Oricjina| Papers

15

gl0 LO

/ / . .U- - x

5 tz f o

'

1 10 20 30 t Cdl

Fig. 4. Moniliformin production in dependence on time and sub- strate (Fusarium oxysporum sp 1028 at 22~ 1, rice; 2, rye; 3, maize; 4, barley; 5, oats; 6, wheat

determinat ion of monil i formin is simple and efficient. The in t roduct ion of M B T H as a reagent for conversion of monil i formin into a coloured derivative is seen as a special advantage.

Acknowledgements. This research was supported by the Bundesmini- ster ffir Jugend, Familie und Gesundheit (412-6080-3/19). We thank Prof. Dr. Leistner, Kulmbach, for providing Fusarium strains.

R e f e r e n c e s

1. Burmeister HR, Ciegler A, Vesonder RF (1979), Appl Environ Microbiol 37 : 11 - 13

2. Cole RJ, Cutler M, Doupnik B, Reckham JC (1973) Science 179:1324-1326

3. Hoffmann RW, Bressel U, Gelhaus J, H/iuser H (1971) Chem Ber 104:873-885

4. Kriek NPJ, Marasas WFO, Steyn PS (1977) Food Cosmet Toxicol 15: 579- 587

5. Kriek NPJ, Marasas WFO, Thiel PG (1981) Food Cosmet Toxicol 19: 447-456

6. Lansden JA, Clarkson RJ, Neely WC, Cole RJ, Kirksey JW (1974) J Assoc Off Anal Chemists 57:1392

7. Marasas WFO, Leistner L, Hofmann G, Eckard C (1979) Eur J Appl Mierobiol Biotechnol 7 : 289-- 305

8. Marasas WFO, Kriek NPJ, Wiggens VN, Steyn PS (1979) Phytopathology, 69:1181 - 1185

9. Rabie CJ, Marasas WFO, Thiel PG, Lfibben A, Vleggaar R (1982) Appl Environ Microbio143 : 517- 521

10. Rabie CJ, Lfibben A, Louw A, Steyn PS (1978) J Agric Food Chem 26: 376- 379

11. Scharf HD (1980) In: Oxocarbons. The mycotoxin Monili- formin and related substances

12. Springer JP, Clardy J, Cole RJ, Kirksey JW, Hill RV, Carlson RH, Isidor JL (1974) J Am Chem Soc. 96:2267-2268

13. Steyn M, Thiel PG, Schalkwyk G (1978) J Assoc Off Anal Chemists 61 : 578 - 580

14. Thiel PG, Meyer CJ, Marasas WFO (1982) J Agric Food Chem 30: 308- 312

15. Thiel PG (1981) The determination of Moniliformin by high performance liquid chromatography. In: Krogh P (ed) Pro- ceedings of the 4th IUPAC Symposium of Mycotoxins and Phycotoxins, 1979. Pathotox, Illinois

Received November 25, 1983

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