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Food and Agricultural Immunology

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Determination of thiabendazole in potatoes by ELISA

Rodney J. Bushway a; Karin Larkin b; Brian Perkins a
a
Department of Food Science and Human Nutrition, University of Maine, Orono, USA b Envirologic Inc,
Westbrook, USA

Online Publication Date: 01 December 1997

To cite this Article Bushway, Rodney J., Larkin, Karin and Perkins, Brian(1997)'Determination of thiabendazole in potatoes by
ELISA',Food and Agricultural Immunology,9:4,249 — 255
To link to this Article: DOI: 10.1080/09540109709354955
URL: http://dx.doi.org/10.1080/09540109709354955

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 Food and Agricultural Immunology (1997) 9, 249-255

Determination of Thiabendazole in Potatoes by ELISA

RODNEY J. BUSHWAY,1 KARIN LARKIN2 AND BRIAN PERKINS1

1
Department of Food Science and Human Nutrition, 5736 Holmes Hall,
University of Maine, Orono, ME 04469-5736, USA; 2Envirologic Inc.
Westbrook, ME 04467, USA
Downloaded By: [University of Georgia] At: 22:25 29 June 2009

(Original manuscript received 3 March 1997; revised manuscript accepted 1 July 1997)

A method was developed to analyze thiabendazole (TBZ) in potatoes. Potatoes were

extracted in methanol for 3 min with a polytron. A 100-l aliquot was removed and
evaporated to dryness using air. The residue was dissolved in 1 ml of high-performance
liquid chromatography (HPLC) grade water before quantifying TBZ using an existing
antibody with a tube-formatted competitive enzyme-linked immunosorbent assay (ELISA).
Eight samples could be analyzed in 25 min without the use of complex and expensive
equipment. Indeed, samples could be analyzed at storage facilities. The detection limit for
this assay in potatoes was 3 ppb. Reproducibility and accuracy were good. Correlation of
TBZ concentration between HPLC and ELISA was 0.9729 for 78 samples.

Keywords: ELISA, thiabendazole, HPLC, fungicide

INTRODUCTION
Thiabendazole (TBZ), a pre- and post-emergence fungicide, is used extensively on potatoes
during storage to prevent dry rot by Fusarium species. TBZ belongs to the benzimidazole
class of pesticides which includes the fungicide benomyl.
There is a two-fold interest in measuring the quantity of TBZ in potatoes. First,
environmental groups are concerned because of its frequent use and the amount applied.
Second, potato growers need to know how much TBZ is present throughout storage in order
to ascertain its effectiveness.
Methods for analyzing TBZ are numerous. They are mostly high-performance liquid
chromatography (HPLC) procedures (Bushway et al, 1995; Bushway, 1996), although
there are some gas chromatography (GC) methods (Oishi et al, 1994). When analyzing any
substance, however, the best procedure is one that is accurate, rapid and inexpensive.
Enzyme-linked immunosorbent assay (ELISA) is the latest technique that encompasses
these qualities for analysis (Kaufman & Clover, 1991). Even though there have been some
ELISA procedures developed to analyze TBZ in potatoes (Brandon et al, 1993; Bushway
et al, 1994), the method described in this paper was a tube format that was much quicker,
and could be used in either the field or laboratory.

0954-0105/97/040249-07 © 1997 Carfax Publishing Ltd

 250 R. J. BUSHWAY ETAL.

MATERIALS AND METHODS

Samples
Potato samples were obtained from growers in northern and central Maine and supermarkets
in the Bangor, ME area.

Chemicals
TBZ pesticide standard (99%) was obtained from Cresent Chemical (New York, USA). All
solvents were HPLC grade and obtained from EM Science (Gibbstown, NJ, USA).

ELISA Materials
This study used Quantitubes from Millipore Corporation (Bedford, MA, USA). These tubes
were coated with a TBZ monoclonal antibody (MAb) from Dr David Brandon (USDA
Laboratory, Albany, CA, USA). Antibody production and conjugate chemistry was as
described by Brandon et al. (1992). Since this study, the commercial ELISA industry has
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undergone several changes. The TBZ Quantitubes can now be purchased from SDI
(Newark, DE, USA).

Extraction of Potatoes for TBZ

Depending on the number of tubers in the sample, they were either processed using a food
processor or a silent cutter. This step was necessary to ensure the sample was homogeneous.
Once the sample had been made homogeneous, a 10-g sample was weighed into a 50-ml
polypropylene centrifuge tube followed by the addition of 20 ml of methanol. The sample
was polytroned for 3 min on high speed before centrifuging for 5 min at 5000 X g. The
supernant was used for both ELISA and HPLC analysis.

Preparation of Standards
A stock solution of TBZ was prepared at a concentration of 0.84 mg ml ~' in acetonitrile.
From the stock standard, an intermediate solution of 0.163 fig ml" 1 HPLC water was
made. Working standards for the ELISA analysis were prepared by removing 37.5, 62.5 and
187.5 ji\ of intermediate standard and diluting each aliquot to 5 ml with water. This yielded
standards containing 1.2, 2 and 6 ng ml" 1 TBZ.
TBZ standard for the HPLC was prepared by taking 10 fil of stock solution and adding
it to a total volume of 10 ml using 80% methanol-20% water. This gave a working standard
of 0.84 ng /il" 1 TBZ.

ELISA Analysis
Sample aliquots (100-1000 fil) were evaporated to dryness and the residue dissolved in
1 ml of water. The dissolving process was facilitated by using an ultrasonic bath. Sample
and standards (200 /il) were added to polystyrene tubes followed by 200 /A of enzyme
conjugate. The tubes were mixed and allowed to incubate for 15 min. As many as 10 tubes
could be run simultaneously. After 15 min, the contents of the tubes were dumped into a
sink and the tubes rinsed four times with tap water before blotting dry with paper towels.
Next, 500 /ri of substrate solution was added to each tube. After 10 min incubation at room
temperature, 300 ix\ of 1 M-HCI were added to each tube before reading at 450 nm using
either a plate reader or a spectrophotometer.

Analysis of TBZ by HPLC

A 50-/i\ aliquot of the methanol supernatant was injected into an HPLC system which
consisted of a Waters 510 pump (Waters Associates, Milford, MA, USA), a Valco
 DETERMINATION OF THIABENDAZOLE 251

pneumatic injector (VICI Instruments, Houston, TX, USA) containing a 50-/il loop, a
Waters 470 fluorescence detector and a Hewlett-Packard 3396 integrator (Avondale, PA,
USA).
The HPLC conditions were as follows: column, Ultracarb 30 ODS; mobile phase,
acetonitrile:water:methanol:monoethanolamine (200 + 275 + 75 + 0.1 ml); flow rate, 1 ml
min" 1 ; detector, fluorescence; wavelength, 305 nm excitation and 345 nm emission.

Recovery Study
Organic potatoes free of detectable amounts of TBZ were fortified with TBZ at the
following concentrations: 30, 60, 180, 500, 1000 and 10 000 ng g" 1 . The recovery study
was used to ascertain the accuracy of the immunoassay and the efficiency of the TBZ
extraction technique.

Reproducibility Study
Standards and potato samples were analyzed several times on the same day and on different
Downloaded By: [University of Georgia] At: 22:25 29 June 2009

days to determine intra- and inter-assay variations in the ELISA method.

RESULTS AND DISCUSSION

A typical ELISA standard curve for this TBZ tube format is shown in Figure 1. Even
though the standard curve is based on three points (concentrations of 1.2, 2.2 and 6.0 ppb),
it has been shown that the linearity range was 0.8-6.0 ppb. The 0.8 ppb concentration yields
a %B0 of 90 which is the highest %B0 that is considered acceptable for analytical work. The
IC50 (concentration of TBZ at a %B0 value of 50) was 4.8 ppb.
The ELISA detection limit for TBZ in potatoes was determined to be 3 ppb. In order to
obtain such a low detection limit, 1 ml of the supernatant was evaporated to dryness and
the residue was dissolved in 1 ml of water. The limit of quantification was ascertained as
12 ppb. This was accomplished by evaporating 0.3 ml of the supernatant and dissolving the
residue in 1 ml of water. These limits were determined according to the procedure
recommended by the American Chemical Society (ACS, 1980).

1 2 3 4 5 7 8 9 10
THIABENDAZOLE (PPB)
FIG. 1. Standard curve for TBZ ELISA. For conditions see text.
 252 R. J. BUSHWAY ETAL.

TABLE 1. Reproducibility of the TBZ ELISA

Samples (ppb) %CV" intra-assay %CV* inter-assay

Standard (1.2) 2.5 2.2

Standard (2.0) 2.4 3.0
Standard (6.0) 3.4 4.2
Potato 1 (5) 5.7 21
Potato 2 (241) 11 16
Potato 3 (465) 2.2 15
Potato 4 (1443) 5.7 7.9
Potato 5 (1453) 7.9 16
Potato 6 (3653) 9.6 16
"Per cent coefficients of variation based on six determinations in 1 day
for both samples and standards.
*Per cent coefficients of variation based on 15 determinations over a
month for standards and six determinations for samples on six different
days, except for sample 5 which was five determinations.
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Limits for the HPLC method used in the comparison study were as follows: detection
limit, 1 ppb; limit of quantification, 5 ppb (ACS, 1980).
As with any analytical technique, precision both within and between days was extremely
important. Reproducibility results for TBZ standards and actual potato samples containing
TBZ are shown in Table 1. Three standard concentrations ranging from 1.2 to 6 ppb showed
excellent consistency with per cent coefficients of variation (%CVs) varying from 2.2 to
4.2. Actual samples (concentration range 5-3653 ppb) analyzed under the same conditions
as the standards showed intra-assay %CVs that were slightly higher than the standards,
indicating only a slight matrix effect especially when one considers that one of the samples
was at 5 ppb. Although the inter-assay %CVs were higher than the intra-assay values, they
were still sufficiently low to be considered good for a screening method.
It is also important to determine the efficiency of the extraction method and the accuracy
of the ELISA. Organic potato samples were fortified with 30, 60, 180, 500, 1000 and
10 000 ppb followed by extraction and quantification by ELISA. Recoveries ranged from
88 to 109% with %CVs varying from 6.3 to 14. These results demonstrated that the
extraction technique was efficient and that the ELISA was accurate (Table 2).
When developing ELISA methods, it is necessary to compare their results with a standard
analytical technique. In this case, HPLC was used as the comparison procedure. Potato
samples were from Maine, Idaho, Wisconsin and Washington. Results for the comparison
are given in Table 3 and Figure 2. The correlation coefficient was 0.9729 for 78 samples
while the equation was y = 0.856* -\— 6.38 indicating a slight high bias for ELISA.
However, these results indicate good agreement between both techniques especially

TABLE 2. Accuracy of TBZ for spiked

potato samples
TBZ added TBZ found
(ppb) (ppb) CV
30 29 14
60 56 11
180 178 11
500 442 7.0
1000 1090 6.3
10 000 9200 8.5
"Per cent coefficients of variation based
on 12 determinations done on 7 days;
recoveries ranged from 88 to 109%.
 DETERMINATION OF THIABENDAZOLE 253

TABLE 3. Comparison of ELISA and HPLC

methods for TBZ in potatoes

HPLC TBZ ELISA TBZ

Sample (ppb) (ppb)
Potato 1 573 813
Potato 2 521 750
Potato 3 141 213
Potato 4 188 263
Potato 5 225 344
Potato 6 851 1050
Potato 7 14 19
Potato 8 5 3
Potato 9 5 4
Potato 10 542 663
Potato 11 408 588
Potato 12 255 444
Potato 13 613 630
Potato 14 1070 1350
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Potato 15 760 950

Potato 16 383 470
Potato 17 255 .330
Potato 18 613 925
Potato 19 1380 2056
Potato 20 28 30
Potato 21 57 57
Potato 22 167 183
Potato 23 360 483
Potato 24 741 1180
Potato 25 7307 10 208
Potato 26 60 28
Potato 27 509 440
Potato 28 2300 2860
Potato 29 155 167
Potato 30 2270 2720
Potato 31 8600 7700
Potato 32 12 4
Potato 33 208 230
Potato 34 1106 1488
Potato 35 361 455
Potato 36 1061 1348
Potato 37 2772 3500
Potato 38 343 418
Potato 39 389 440
Potato 40 274 295
Potato 41 182 250
Potato 42 319 425
Potato 43 335 450
Potato 44 274 325
Potato 45 456 519
Potato 46 304 325
Potato 47 426 613
Potato 48 212 313
Potato 49 502 813
Potato 50 8 12
Potato 51 440 483
Potato 52 780 980
Potato 53 1090 1100
Potato 54 1140 1100
Potato 55 1080 1080
Potato 56 917 1010
Potato 57 1130 1100
Potato 58 1150 1200
 254 R. J. BUSHWAY ETAL.

TABLE 3.—continued
HPLC TBZ ELISA TBZ
Sample (ppb) (ppb)
Potato 59 300 311
Potato 60 1150 1200
Potato 61 1150 1440
Potato 62 157 154
Potato 63 138 138
Potato 64 603 718
Potato 65 3430 3430
Potato 66 67 65
Potato 67 328 374
Potato 68 45 63
Potato 69 149 244
Potato 70 23 25
Potato 71 811 930
Potato 72 941 777
Potato 73 941 738
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Potato 74 12 5
Potato 75 219 236
Potato 76 1106 1443
Potato 77 350 460
Potato 78 2772 3567

1 /LUUU-
Z
Q.
Q.
%^ 10000-
LU •
I

o 8000 -
M
<
Q
6000 -
LU
a •
HIA

4000 -

< 2000 -
—
LU o ^
0 1000 2000 3000 4000 5000 6000 7000 8000 9000
HPLC THIABENDAZOLE (PPB)
FIG. 2. Comparison of HPLC and ELISA for the determination of TBZ in potatoes.

considering ELISA is supposed to be a screening procedure. Thus, for monitoring TBZ in

potatoes, ELISA is a rapid, accurate, sensitive, reproducible and very cost-effective technique.
Furthermore, being a tube assay, this ELISA is easily adapted for field analysis which could
be very important to potato growers and/or processors.

ACKNOWLEDGEMENT
This paper is no. 2126 of the University of Maine Agricultural Experiment Station.
 DETERMINATION OF THIABENDAZOLE 255

REFERENCES
ACS (1980) Guidelines for data acquisition and data quality evaluation in environmental chemistry,
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ELISA for thiabendazole in liver, Journal of Agricultural and Food Chemistry, 40, 1722-1726.
BRANDON, D. L., BINDER, R. G., BATES, A. H. & MONTAGUE, W. C. JR (1993) Analysis of thiabendazole in
potatoes and apples by ELISA using monoclonal antibodies, Journal of Agricultural and Food
Chemistry, 4 1 , 996-999.
BUSHWAY, R. J. (1996) Complementation of direct injection high-performance liquid chromatography and
enzyme-linked immunosorbent assay for the analysis of thiabendazole in fruit juices and concentrates,
Journal of Chromatography, A, 754, 431-435.
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Official Analytical Chemists, 11, 1243-1248.
KAUFMAN, B. M. & CLOVER, M. JR (1991) Immunoassay of pesticides, Journal of the Association of Official
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OISHI, M., ONISHI, K., KANO, I., NAKAZAWA, H. & TANABE, S. (1994) Capillary gas chromatographic determi-
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