Food Additives & Contaminants
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Isolation and determination of zearalenone in rice cultures using liquid chromatography with diode array detection A. K. Shrivastava & A. A. Ansari To cite this article: A. K. Shrivastava & A. A. Ansari (1992) Isolation and determination of zearalenone in rice cultures using liquid chromatography with diode array detection, Food Additives & Contaminants, 9:4, 331-336, DOI: 10.1080/02652039209374078 To link to this article: http://dx.doi.org/10.1080/02652039209374078
Published online: 10 Jan 2009.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [NSTL]
Date: 05 November 2015, At: 17:12
FOOD ADDITIVES AND CONTAMINANTS, 1992, VOL. 9, NO. 4, 3 3 1 - 3 3 6
Isolation and determination of zearalenone in rice cultures using liquid chromatography with diode array detection A. K. SHRIVASTAVA and A. A. ANSARI University Department of Botany, Bhagalpur University, BhagaIpur-812007, India
Downloaded by [NSTL] at 17:12 05 November 2015
(Received 25 November 1991; revised 14 April 1992; accepted 3 June 1992) A rapid method is described for the isolation and determination of zearalenone (ZEN) produced by Fusarium spp., in moist rice culture. Following a simple solvent extraction using acetonitrile:water, the crude extract was defatted with hexane and diluted with methanol. The extract solution containing ZEN was evaporated to dryness, the residue dissolved in acetonitrile and diluted with water. The solution was analysed by liquid chromatography using a UV-diode array detector. The UV spectra and chromatographic data generated from the standard ZEN was stored in a computer and used to identify the toxin in a crude mixture. The purity of the separated peak and the amount of toxin in the crude mixture was determined. The present technique is fast and allows the acquisition of UV spectral information and chromatographic data of ZEN in a single chromatographic operation. Recovery of zearalenone added to the rice was 76-94%. Keywords: mycotoxins, zearalenone, Fusarium spp., analysis
Introduction
Zearalenone (ZEN) 6-(10 hydroxy-6-oxotrans-l undecyl)-/? resorcylic acid lactone, an oestrogenic mycotoxin produced by Fusarium graminearum and several other Fusarium species causes hyperoestrogenic syndromes in swine consuming feed contaminated with ZEN (Kurtz and Mirocha 1978). ZEN contamination appears to be a world-wide problem (Bennet and Shotwell 1979). Several methods for the extraction, clean-up, isolation, identification and confirmation of ZEN have been described (Mirocha et al. 1974, Shotwell et al. 1976, Ware and Thorpe 1978, Tanaka et al. 1985, Bagnaris 1986, Tanaka and Ueno 1989, Sugano et al. 1990). However, the methods described above were developed for determination of low levels of ZEN in cereals and other food products and require vigorous clean-up procedures. Liquid chromatography with diode array detection (LC-DAD) is a technique that is still uncommon in mycotoxin research (Selala et al. 1991). In a continuing research programme for rapid and accurate procedures that require less time and solvent than other methods without sacrificing analytical reliability, advantage was taken of recent developments in instrumental and analytical technology to improve upon the cumbersome methods described above. Our investigation was directed towards the determination of ZEN isolated from Fusarium spp. using the LC-DAD technique in combination with a computer for data capture, processing and evaluation. DAD-generated UV spectra and LC data for the standard toxin were stored as a library in the computer and used to identify the mycotoxin in crude mixtures. This technique has several advantages over previous methods: it saves 0265-203X/92 $3.00 © 1992 Taylor & Francis Ltd.
332
A. K. Shrivastava and A. A. Ansari
time and eluent, improves identification and offers possibilities for checking the purity of chromatographic peaks. Materials and methods
Downloaded by [NSTL] at 17:12 05 November 2015
Culture conditions for production of zearalenone Twenty-two Fusarium isolates were tested for ZEN production. Corning glass conical flasks (150 ml) containing 10 g of rice (moisture content 45%) were stoppered with cotton plugs and autoclaved for 20 min at 121 °C twice with a 24-hour interval. After cooling, the flasks were inoculated with the spore suspension (1 ml) of Fusarium isolates. These rice cultures were incubated for a total period of 3 weeks, 2 weeks at 25°C and 1 week at 10°C. The cultures were harvested after 3 weeks, dried at 60°C for 36 h and subsequently powdered in a mixer and kept for extraction. Extraction Cultures (10 g) were extracted with 150 ml acetonitrile: water (3:1 v/v) and filtered through ordinary filter paper. The filtrate was defatted with 125 ml n-hexane; the w-hexane layer was discarded, the aqueous layer diluted with 100 ml methanol and evaporated to dryness on a rotary evaporator at 50°C. The dried extract was redissolved in 2 ml acetonitrile at room temperature and diluted with HPLC grade water and finally filtered through a Whatman no. 44 filter paper. The filtrate was collected in a 2 ml vial and sealed with a rubber top metallic rim cap using a hand crimper. Instrument The instrument specified is not exclusive. Any other suitable equipment may be used. A Hewlett-Packard HP 1090 A HPLC equipped with an HP 1040 A diode array detector coupled to an HP 9000 series 300 pascal work station with an HP 9153 C disk drive was used. Samples were injected through a Rheodyne valve fitted with 20 id sample loop and a six port valve controlling the solvent delivery system. Separation was carried out on a 200 x 4 • 6 mm Hypersil Cs column (Hewlett-Packard). Liquid chromatography-DAD programming The LC operating conditions were: solvent flow 1-5 ml/min consisting of watenacetonitrile (80:20 v/v); oven temperature ambient; maximum pressure 300 bar; stop time 20 min; injection volume 10 fi\. The instrument was stabilized for a suitable period at 1 • 5 ml/min flow rate and a diode array detector test was carried out with inbuilt systems. Further DAD signals and spectra were set to analyse at 236 ± 4, 274 ± 3, 315 ± 4 nm (±4 bandwidth) with reference wavelength at 550 nm with bandwidth 100, the DAD generated spectra were stored in the computer and about 200 spectral records were acquired during a single run. Peakwidth was 01100 min, sampling interval in DAD was 640 milliseconds and the spectrum range was 200-390 nm. Integration of the chromatogram comprises the following events: peak-width 0-100, threshold 0 (range—12 to 25), area reject 100 and shoulder off, concentration may be calculated using the external standard by area comparison method. During an LC run, the chromatogram was available at a controlled signal
Isolation and determination of zearalenone
333
(may be 236 ± 4 nm) on a window of the computer screen and UV spectra taken at 640 millisecond intervals were observed by another window on the same screen.
Downloaded by [NSTL] at 17:12 05 November 2015
Confirmation of ZEN DAD spectra appearing along with the chromatogram peak on the computer screen may be tentatively confirmed by comparing the UV spectra reported earlier (Cole and Cox 1981). Further confirmation was obtained by running the standard zearalenone (purchased from Sigma Chemicals, USA). Its purity was confirmed by TLC and spectrophotometry. The retention time and DAD spectra of the standard coincide with those of the putative ZEN peak from the extract. Recovery experiments Recovery of zearalenone was carried out by spiking control rice samples shown to be free of zearalenone. Spiked samples were prepared by adding standard ZEN solution in acetonitrile to the control samples in Erlenmeyer flasks, shaking thoroughly and letting the solution stand for at least 1 h at room temperature before extraction by the methods given above. Results and discussion
The proposed method was evaluated by analysing ZEN-free rice powder spiked with known amounts of ZEN and 22 Fusarium isolates grown on moist rice. ZEN was produced by Fusarium graminearum (4 isolates), F. oxysporum (2 isolates), F. moniliforme (2 isolates). The remaining isolates of F. moniliforme, F. oxysporum and F. equisiti did not produce toxin. The concentration ranged between 0#2 and !• 5 /ig/g of moist rice and the average recovery of ZEN was 76-94% (table 1). The lowest recovery was observed at a concentration of 10 ng/g. The 236 nm wavelength was preferred over the 274 nm and 315 nm wavelengths. At 315 nm wavelength the detectability was 3-25 times less than at 236 nm. Simplified extraction is required for achieving high precision between laboratories. Further, microprocessor control of HPLC facilitates the automation of sample application, solvent delivery, detection and integration also helps to increase accuracy and precision (Coker and Jones 1988). Figure 1 represents the chromatogram of a ZEN-free rice sample obtained under the above described experimental conditions, run at 236 nm, showing no major peak. Figures 2 and 3 present the chromatograms of a crude extract and of a standard of pure ZEN showing major peaks at 8-587 and 8*603 min in Fusarium cultured on moist rice and pure ZEN respectively. Further, the chromatogram of
Table 1. Recovery (%) of zearalenone spiked to rice powder. Zearalenone spiked (ng/g)
Rice powder Values
10
50
100
500
1000
76-5 (4-5)
90-0 (6-0)
93-0 (5-2)
90-0 (6-7)
94. 0
are mean (± standard determination.
deviation)
%
of
(3'• 5 ) triplicate
A. K. Shrivastava and A. A. Ansari
334
LC fl 23 6, it 550, 100
cr
e
50:
JM.162
1OO
:
0^ 2
8
6
it
10
Time (min. Figure 1. Chromatogram of ZEN-free rice extract.
ZEN A
CM
CD
jCD
in f»
in
Li.
ci
1
—i—i
2
6
k
CD JOl
o o
\
e
500-
8~587
1OOO: ID cr
p 7-062 8
10
Time ( m i n ) Figure 2. Chromatogram of crude extract (120ng/10/il) showing ZEN peak at 8-587 min.
LC R 236,
ZEN t
550,100
800: 600-:
cr S 200-
8^603
3
If
in A
o 2
h
r
Downloaded by [NSTL] at 17:12 05 November 2015
LC R 236, ft 550 , 100
6
8
10
Time (min.) Figure 3. Chromatogram of standard ZEN (80 ng/10 ul) retention time at 8-603 min.
the culture extract contained additional peaks before and after the ZEN peak. No interfering compounds were eluted in the ZEN region, indicating no further cleanup step was necessary and identification could be made by a one step HPLC procedure. Peak start time and end time of the extract are very much within the elution time of standard. The one-line UV spectra recorded during elution at the retention time of the toxin provide a useful identification parameter. Using a LC-DAD software program the spectra were stored as a library file in the computer (figure 4). These spectra are practically identical to those which would be produced by a simple variable single wavelength detector on ZEN solutions in ethanol or methanol (Cole and Cox 1981).
Isolation and determination of zearalenone
250
300
Wavelength
335
350
(nm.)
Figure 4. UV spectrum of standard ZEN obtained by DAD kept in library file.
Downloaded by [NSTL] at 17:12 05 November 2015
lOOq
80: 60QJ O O
fuv
8.521 UV 8.587 Luv 8.657
A / \ / \
i+n-
to 20: 0-
Peok \ spectre motch-1000 \ 1 .
250
300
Wavelength
i
.
350 (n m. )
Figure 5. UV spectrum of ZEN presenting peak homogeneity.
DAD provides an indication of the peak homogeneity by comparison of the coincidence of UV spectra taken at different points on the eluting peak. In our experimental conditions spectra recorded at 8-521 min, 8*587 min and 8-656 min, i.e. upslope, apex and down slope respectively showed peak spectra match-1000 (figure 5). The value, which ranges between 0 and 1000, was calculated mathematically with the help of microprocessors by comparison of all the specified spectra of a peak. The match factor of the ZEN peak in the extract was of the maximum purity and comparable to that of the standard. The reliability of the UV spectrum was increased in combination with a second criterion such as retention time which also showed similarity. Using peak integration results from both the standard and the sample chromatogram the amount of each toxin in crude extracts was determined. The minimum detectable amount of ZEN was 0-5 ng per injection. The RP-HPLC method described here, based on DAD, can be applied for ZEN screening and the use of LC-DAD provides an additional useful aid for the isolation and determination of mycotoxins. The multichannel UV/Vis DAD offers rapid characterization of mycotoxins, eliminating the need for routine toxin confirmation tests. The ability of DAD to record and store spectral information at sequential time intervals under computer control during an elution makes this technique ideal for the efficient detection of fungal toxins from cultures. In this study it was possible rapidly to detect, separate and isolate fungal toxin with a minimum clean-up of the crude mixtures. The technique is extremely versatile as
336
A. K. Shrivastava and A. A. Ansari
data obtained from a single injection can be extensively manipulated to give a large amount of information. This improves detection and reduces the risk of misinterpreted data as chromatographic peaks can be clearly characterized and their spectral purity established. The method developed for the extraction and determination of zearalenone produced in moist rice cultures of Fusarium is rapid and requires only simple solvent extraction with no additional clean-up of extracts. Acknowledgements
We thank Prof. K. S. Bilgrami, FNA for laboratory facilities and encouragement. Financial assistance (F-3-39/86 SR-II) by University Grants Commission, New Delhi, India is also acknowledged.
Downloaded by [NSTL] at 17:12 05 November 2015
References BAGNARIS, R. W., 1986, High performance liquid chromatography method for the determination of zearalenone and zearalenol in grains and feeds. Journal of the Assocation of Official Analytical Chemists, 69, 894-898. BENNET, G. A., and SHOTWELL, O. L., 1979, Zearalenone in cereal grains. Journal of the American Oil Chemists Society, 56, 812-819. COKER, R. D., and JONES, B. D., 1988, Determination of mycotoxins, HPLC in Food Analysis, edited by R. Macrae, (London: Academic Press), pp. 336-373. COLE, R. J., and Cox, R. H., 1981, Handbook of Toxic Fungal Metabolites (New York: Academic Press), pp. 902-906. KURTZ, H. J., and MIROCHA, C. J., 1978, Mycotoxic Fungi, Mycotoxins, Mycotoxicoses. An Encyclopedic Handbook, edited by T. D. Wyllie and L. G. Morehouse (New York: Marcel Dekker, Inc.), Vol. 2, pp. 256-268. MIROCHA, C. J., SHAUERHAMER, B., and PATHRE, S. V., 1974, Isolation, detection and quantitation of
zearalenone in maize and barley. Journal of the Association of Official Analytical Chemists, 57, 1104-1110. SELALA, M. I., MUSUKU, A., and SCHEPENS, P. J. C., 1991, Isolation and determination of
Paspalitrem-type tremorgenic mycotoxins using liquid chromatography with diode array detection. Analytica Chimica Acta, 244, 1-8. SHOTWELL, O. L., GOULDEN, M. L., and BENNETT, G. A., 1976, Determination of zearalenone in corn,
collaborative study. Journal of the Association of Official Analytical Chemists, 59, 666-670. SUGANO, K., ISHIBASHI, T., and MATSUBARA, I., 1990, Determination of zearalenone in cereals by high
performance liquid chromatography. Shiryo Kenkyu Hokoku (Tokyo Hishiryo Kenaasho), 15, 12-21. TANAKA, T., HASEGAWA, A., MATSUKI, Y., LEE, U. S., and UENO, Y., 1985, Rapid and sensitive
detection of zearalenone in cereals by high performance liquid chromatography with fluorescence detection. Journal of Chromatography, 328, 271-278. TANAKA, T., and UENO, Y., 1989, Worldwide natural occurrence of Fusarium mycotoxins, nivalenol, deoxynivalenol and zeralenone. Mycotoxins and Phycotoxins, edited by S. Natori, K. Hashimoto and Y. Ueno (Amsterdam: Elsevier Science Publishers, BV), pp. 51-56. WARE, G. M., and THORPE, C. W., 1978, Determination of zearalenone in corn by high pressure liquid chromatography and fluorescence detection. Journal of the Association of Official Analytical Chemists, 61, 1058-1062.
ISSN: 0265-203X (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tfac19
Isolation and determination of zearalenone in rice cultures using liquid chromatography with diode array detection A. K. Shrivastava & A. A. Ansari To cite this article: A. K. Shrivastava & A. A. Ansari (1992) Isolation and determination of zearalenone in rice cultures using liquid chromatography with diode array detection, Food Additives & Contaminants, 9:4, 331-336, DOI: 10.1080/02652039209374078 To link to this article: http://dx.doi.org/10.1080/02652039209374078
Published online: 10 Jan 2009.
Submit your article to this journal
Article views: 6
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Download by: [NSTL]
Date: 05 November 2015, At: 17:12
FOOD ADDITIVES AND CONTAMINANTS, 1992, VOL. 9, NO. 4, 3 3 1 - 3 3 6
Isolation and determination of zearalenone in rice cultures using liquid chromatography with diode array detection A. K. SHRIVASTAVA and A. A. ANSARI University Department of Botany, Bhagalpur University, BhagaIpur-812007, India
Downloaded by [NSTL] at 17:12 05 November 2015
(Received 25 November 1991; revised 14 April 1992; accepted 3 June 1992) A rapid method is described for the isolation and determination of zearalenone (ZEN) produced by Fusarium spp., in moist rice culture. Following a simple solvent extraction using acetonitrile:water, the crude extract was defatted with hexane and diluted with methanol. The extract solution containing ZEN was evaporated to dryness, the residue dissolved in acetonitrile and diluted with water. The solution was analysed by liquid chromatography using a UV-diode array detector. The UV spectra and chromatographic data generated from the standard ZEN was stored in a computer and used to identify the toxin in a crude mixture. The purity of the separated peak and the amount of toxin in the crude mixture was determined. The present technique is fast and allows the acquisition of UV spectral information and chromatographic data of ZEN in a single chromatographic operation. Recovery of zearalenone added to the rice was 76-94%. Keywords: mycotoxins, zearalenone, Fusarium spp., analysis
Introduction
Zearalenone (ZEN) 6-(10 hydroxy-6-oxotrans-l undecyl)-/? resorcylic acid lactone, an oestrogenic mycotoxin produced by Fusarium graminearum and several other Fusarium species causes hyperoestrogenic syndromes in swine consuming feed contaminated with ZEN (Kurtz and Mirocha 1978). ZEN contamination appears to be a world-wide problem (Bennet and Shotwell 1979). Several methods for the extraction, clean-up, isolation, identification and confirmation of ZEN have been described (Mirocha et al. 1974, Shotwell et al. 1976, Ware and Thorpe 1978, Tanaka et al. 1985, Bagnaris 1986, Tanaka and Ueno 1989, Sugano et al. 1990). However, the methods described above were developed for determination of low levels of ZEN in cereals and other food products and require vigorous clean-up procedures. Liquid chromatography with diode array detection (LC-DAD) is a technique that is still uncommon in mycotoxin research (Selala et al. 1991). In a continuing research programme for rapid and accurate procedures that require less time and solvent than other methods without sacrificing analytical reliability, advantage was taken of recent developments in instrumental and analytical technology to improve upon the cumbersome methods described above. Our investigation was directed towards the determination of ZEN isolated from Fusarium spp. using the LC-DAD technique in combination with a computer for data capture, processing and evaluation. DAD-generated UV spectra and LC data for the standard toxin were stored as a library in the computer and used to identify the mycotoxin in crude mixtures. This technique has several advantages over previous methods: it saves 0265-203X/92 $3.00 © 1992 Taylor & Francis Ltd.
332
A. K. Shrivastava and A. A. Ansari
time and eluent, improves identification and offers possibilities for checking the purity of chromatographic peaks. Materials and methods
Downloaded by [NSTL] at 17:12 05 November 2015
Culture conditions for production of zearalenone Twenty-two Fusarium isolates were tested for ZEN production. Corning glass conical flasks (150 ml) containing 10 g of rice (moisture content 45%) were stoppered with cotton plugs and autoclaved for 20 min at 121 °C twice with a 24-hour interval. After cooling, the flasks were inoculated with the spore suspension (1 ml) of Fusarium isolates. These rice cultures were incubated for a total period of 3 weeks, 2 weeks at 25°C and 1 week at 10°C. The cultures were harvested after 3 weeks, dried at 60°C for 36 h and subsequently powdered in a mixer and kept for extraction. Extraction Cultures (10 g) were extracted with 150 ml acetonitrile: water (3:1 v/v) and filtered through ordinary filter paper. The filtrate was defatted with 125 ml n-hexane; the w-hexane layer was discarded, the aqueous layer diluted with 100 ml methanol and evaporated to dryness on a rotary evaporator at 50°C. The dried extract was redissolved in 2 ml acetonitrile at room temperature and diluted with HPLC grade water and finally filtered through a Whatman no. 44 filter paper. The filtrate was collected in a 2 ml vial and sealed with a rubber top metallic rim cap using a hand crimper. Instrument The instrument specified is not exclusive. Any other suitable equipment may be used. A Hewlett-Packard HP 1090 A HPLC equipped with an HP 1040 A diode array detector coupled to an HP 9000 series 300 pascal work station with an HP 9153 C disk drive was used. Samples were injected through a Rheodyne valve fitted with 20 id sample loop and a six port valve controlling the solvent delivery system. Separation was carried out on a 200 x 4 • 6 mm Hypersil Cs column (Hewlett-Packard). Liquid chromatography-DAD programming The LC operating conditions were: solvent flow 1-5 ml/min consisting of watenacetonitrile (80:20 v/v); oven temperature ambient; maximum pressure 300 bar; stop time 20 min; injection volume 10 fi\. The instrument was stabilized for a suitable period at 1 • 5 ml/min flow rate and a diode array detector test was carried out with inbuilt systems. Further DAD signals and spectra were set to analyse at 236 ± 4, 274 ± 3, 315 ± 4 nm (±4 bandwidth) with reference wavelength at 550 nm with bandwidth 100, the DAD generated spectra were stored in the computer and about 200 spectral records were acquired during a single run. Peakwidth was 01100 min, sampling interval in DAD was 640 milliseconds and the spectrum range was 200-390 nm. Integration of the chromatogram comprises the following events: peak-width 0-100, threshold 0 (range—12 to 25), area reject 100 and shoulder off, concentration may be calculated using the external standard by area comparison method. During an LC run, the chromatogram was available at a controlled signal
Isolation and determination of zearalenone
333
(may be 236 ± 4 nm) on a window of the computer screen and UV spectra taken at 640 millisecond intervals were observed by another window on the same screen.
Downloaded by [NSTL] at 17:12 05 November 2015
Confirmation of ZEN DAD spectra appearing along with the chromatogram peak on the computer screen may be tentatively confirmed by comparing the UV spectra reported earlier (Cole and Cox 1981). Further confirmation was obtained by running the standard zearalenone (purchased from Sigma Chemicals, USA). Its purity was confirmed by TLC and spectrophotometry. The retention time and DAD spectra of the standard coincide with those of the putative ZEN peak from the extract. Recovery experiments Recovery of zearalenone was carried out by spiking control rice samples shown to be free of zearalenone. Spiked samples were prepared by adding standard ZEN solution in acetonitrile to the control samples in Erlenmeyer flasks, shaking thoroughly and letting the solution stand for at least 1 h at room temperature before extraction by the methods given above. Results and discussion
The proposed method was evaluated by analysing ZEN-free rice powder spiked with known amounts of ZEN and 22 Fusarium isolates grown on moist rice. ZEN was produced by Fusarium graminearum (4 isolates), F. oxysporum (2 isolates), F. moniliforme (2 isolates). The remaining isolates of F. moniliforme, F. oxysporum and F. equisiti did not produce toxin. The concentration ranged between 0#2 and !• 5 /ig/g of moist rice and the average recovery of ZEN was 76-94% (table 1). The lowest recovery was observed at a concentration of 10 ng/g. The 236 nm wavelength was preferred over the 274 nm and 315 nm wavelengths. At 315 nm wavelength the detectability was 3-25 times less than at 236 nm. Simplified extraction is required for achieving high precision between laboratories. Further, microprocessor control of HPLC facilitates the automation of sample application, solvent delivery, detection and integration also helps to increase accuracy and precision (Coker and Jones 1988). Figure 1 represents the chromatogram of a ZEN-free rice sample obtained under the above described experimental conditions, run at 236 nm, showing no major peak. Figures 2 and 3 present the chromatograms of a crude extract and of a standard of pure ZEN showing major peaks at 8-587 and 8*603 min in Fusarium cultured on moist rice and pure ZEN respectively. Further, the chromatogram of
Table 1. Recovery (%) of zearalenone spiked to rice powder. Zearalenone spiked (ng/g)
Rice powder Values
10
50
100
500
1000
76-5 (4-5)
90-0 (6-0)
93-0 (5-2)
90-0 (6-7)
94. 0
are mean (± standard determination.
deviation)
%
of
(3'• 5 ) triplicate
A. K. Shrivastava and A. A. Ansari
334
LC fl 23 6, it 550, 100
cr
e
50:
JM.162
1OO
:
0^ 2
8
6
it
10
Time (min. Figure 1. Chromatogram of ZEN-free rice extract.
ZEN A
CM
CD
jCD
in f»
in
Li.
ci
1
—i—i
2
6
k
CD JOl
o o
\
e
500-
8~587
1OOO: ID cr
p 7-062 8
10
Time ( m i n ) Figure 2. Chromatogram of crude extract (120ng/10/il) showing ZEN peak at 8-587 min.
LC R 236,
ZEN t
550,100
800: 600-:
cr S 200-
8^603
3
If
in A
o 2
h
r
Downloaded by [NSTL] at 17:12 05 November 2015
LC R 236, ft 550 , 100
6
8
10
Time (min.) Figure 3. Chromatogram of standard ZEN (80 ng/10 ul) retention time at 8-603 min.
the culture extract contained additional peaks before and after the ZEN peak. No interfering compounds were eluted in the ZEN region, indicating no further cleanup step was necessary and identification could be made by a one step HPLC procedure. Peak start time and end time of the extract are very much within the elution time of standard. The one-line UV spectra recorded during elution at the retention time of the toxin provide a useful identification parameter. Using a LC-DAD software program the spectra were stored as a library file in the computer (figure 4). These spectra are practically identical to those which would be produced by a simple variable single wavelength detector on ZEN solutions in ethanol or methanol (Cole and Cox 1981).
Isolation and determination of zearalenone
250
300
Wavelength
335
350
(nm.)
Figure 4. UV spectrum of standard ZEN obtained by DAD kept in library file.
Downloaded by [NSTL] at 17:12 05 November 2015
lOOq
80: 60QJ O O
fuv
8.521 UV 8.587 Luv 8.657
A / \ / \
i+n-
to 20: 0-
Peok \ spectre motch-1000 \ 1 .
250
300
Wavelength
i
.
350 (n m. )
Figure 5. UV spectrum of ZEN presenting peak homogeneity.
DAD provides an indication of the peak homogeneity by comparison of the coincidence of UV spectra taken at different points on the eluting peak. In our experimental conditions spectra recorded at 8-521 min, 8*587 min and 8-656 min, i.e. upslope, apex and down slope respectively showed peak spectra match-1000 (figure 5). The value, which ranges between 0 and 1000, was calculated mathematically with the help of microprocessors by comparison of all the specified spectra of a peak. The match factor of the ZEN peak in the extract was of the maximum purity and comparable to that of the standard. The reliability of the UV spectrum was increased in combination with a second criterion such as retention time which also showed similarity. Using peak integration results from both the standard and the sample chromatogram the amount of each toxin in crude extracts was determined. The minimum detectable amount of ZEN was 0-5 ng per injection. The RP-HPLC method described here, based on DAD, can be applied for ZEN screening and the use of LC-DAD provides an additional useful aid for the isolation and determination of mycotoxins. The multichannel UV/Vis DAD offers rapid characterization of mycotoxins, eliminating the need for routine toxin confirmation tests. The ability of DAD to record and store spectral information at sequential time intervals under computer control during an elution makes this technique ideal for the efficient detection of fungal toxins from cultures. In this study it was possible rapidly to detect, separate and isolate fungal toxin with a minimum clean-up of the crude mixtures. The technique is extremely versatile as
336
A. K. Shrivastava and A. A. Ansari
data obtained from a single injection can be extensively manipulated to give a large amount of information. This improves detection and reduces the risk of misinterpreted data as chromatographic peaks can be clearly characterized and their spectral purity established. The method developed for the extraction and determination of zearalenone produced in moist rice cultures of Fusarium is rapid and requires only simple solvent extraction with no additional clean-up of extracts. Acknowledgements
We thank Prof. K. S. Bilgrami, FNA for laboratory facilities and encouragement. Financial assistance (F-3-39/86 SR-II) by University Grants Commission, New Delhi, India is also acknowledged.
Downloaded by [NSTL] at 17:12 05 November 2015
References BAGNARIS, R. W., 1986, High performance liquid chromatography method for the determination of zearalenone and zearalenol in grains and feeds. Journal of the Assocation of Official Analytical Chemists, 69, 894-898. BENNET, G. A., and SHOTWELL, O. L., 1979, Zearalenone in cereal grains. Journal of the American Oil Chemists Society, 56, 812-819. COKER, R. D., and JONES, B. D., 1988, Determination of mycotoxins, HPLC in Food Analysis, edited by R. Macrae, (London: Academic Press), pp. 336-373. COLE, R. J., and Cox, R. H., 1981, Handbook of Toxic Fungal Metabolites (New York: Academic Press), pp. 902-906. KURTZ, H. J., and MIROCHA, C. J., 1978, Mycotoxic Fungi, Mycotoxins, Mycotoxicoses. An Encyclopedic Handbook, edited by T. D. Wyllie and L. G. Morehouse (New York: Marcel Dekker, Inc.), Vol. 2, pp. 256-268. MIROCHA, C. J., SHAUERHAMER, B., and PATHRE, S. V., 1974, Isolation, detection and quantitation of
zearalenone in maize and barley. Journal of the Association of Official Analytical Chemists, 57, 1104-1110. SELALA, M. I., MUSUKU, A., and SCHEPENS, P. J. C., 1991, Isolation and determination of
Paspalitrem-type tremorgenic mycotoxins using liquid chromatography with diode array detection. Analytica Chimica Acta, 244, 1-8. SHOTWELL, O. L., GOULDEN, M. L., and BENNETT, G. A., 1976, Determination of zearalenone in corn,
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