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Journal of Chromatography A, 1170 (2007) 107–113

On-line preconcentration and chiral separation of propiconazole

by cyclodextrin-modified micellar electrokinetic chromatography
Wan Aini Wan Ibrahim ∗ , Dadan Hermawan, Mohd Marsin Sanagi
Separation Science Research Group, Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia,
81310 Skudai, Johor, Malaysia
Received 7 May 2007; received in revised form 21 August 2007; accepted 3 September 2007
Available online 12 September 2007

A method for the chiral separation of propiconazole using cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC)
with hydroxypropyl-␥-cyclodextrin (HP-␥-CD) as chiral selector is reported. The use of a mixture of 30 mM HP-␥-CD, 50 mM SDS,
methanol–acetonitrile 10%:5% (v/v) in 25 mM phosphate buffer solution was able to separate two enantiomeric pairs of propiconazole. Stacking-
and sweeping-CD-MEKC under neutral pH (pH 7) and under acidic condition (pH 3.0) were used as two on-line preconcentration methods to
increase detection sensitivity of propiconazole. Good repeatabilities in the migration time, peak area and peak height were obtained in terms of
relative standard deviation (RSD). A sensitivity enhancement factor of 100-fold was achieved using sweeping-CD-MEKC at acidic pH. This is
the first report on the separation of two pairs of propiconazole enantiomers and all the enantiomers of fenbuconazole and tebuconazole using
sweeping-CD-MEKC. The limit of detection (S/N = 3) for the three triazole fungicides ranged from 0.09 to 0.1 ␮g/mL, which is well below the
maximum residue limits (MRL) set by Codex Alimentarius Commission (CAC). Combination of solid-phase extraction (SPE) pretreatment and
sweeping-CD-MEKC procedure was applied to the determination of selected triazole fungicides in grapes samples spiked at concentration 10–40
times lower than the MRL established by the CAC. The average recoveries of the selected fungicides in spiked grapes samples were good, ranging
from 73% to 109% with RSD of 9–12% (n = 3).
© 2007 Elsevier B.V. All rights reserved.

Keywords: Propiconazole; Chiral triazole fungicides; Enantiomer separation; Capillary electrophoresis; CD-MEKC; On-line preconcentration; Stacking; Sweeping

1. Introduction Micellar electrokinetic chromatography (MEKC) has

become popular as a powerful technique for improving sepa-
Separation of enantiomers has become one of the most impor- ration efficiency not only of neutral analytes but charged ones
tant tasks of analytical chemistry especially in the field of by using CE instrument without any alteration. The advantages
pharmaceutical, clinical and agrochemical sciences, since it is of MEKC for enantiomeric separations are rapid method devel-
well known that a pair of enantiomers can display different opment, minimal use of expensive chiral reagents and provide
biological activities. Analytical methods used so far for the high-separation efficiencies. Two approaches can be used to
chiral separation include thin-layer chromatography [1,2], gas perform enantiomeric separation in MEKC, namely, the use
chromatography [3,4], high-performance liquid chromatogra- of chiral surfactants and the addition of cyclodextrins (CDs)
phy [5,6], supercritical fluid chromatography [7,8] and capillary to the micellar solution [14]. However, CE suffers from poor
electrochromatography [9]. However, a fast growing number concentration sensitivity when using UV detection because of
of studies are reported on the use of capillary electrophoresis the small injection volumes and narrow optical path length.
(CE) in chiral separation [10–13]. Capillary electrophoresis has Various alternatives have been developed such as the use of
shown to be a powerful separation technique for enantiomers laser-induced fluorescence [15] and electrochemical detection
compared to conventional chromatographic techniques. [16]. In addition, off-line concentration technique such as solid-
phase extraction (SPE) can be used as a clean-up method prior to
CE [17,18]. An attractive alternative is the use of on-line sample
∗ Corresponding author. Fax: +60 75566162. preconcentration techniques. This is a versatile and effec-
E-mail address: (W.A. Wan Ibrahim). tive way of increasing concentration sensitivity. Two on-line

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
108 W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113

sample preconcentration techniques, sample stacking and two pairs of propiconazole enantiomers and all the enantiomers
sweeping are known to be effective techniques for enhancement of fenbuconazole and tebuconazole by CD-MEKC. The opti-
of the concentration sensitivity in MEKC [19–21]. mized on-line sample preconcentration-CD-MEKC procedure
Triazole fungicides represent the most important category of combined with SPE pretreatment was applied to the determi-
fungicides to date. They have excellent protective, curative and nation of selected triazole fungicides in grapes samples spiked
eradicant power towards a wide spectrum of crop diseases. Chi- at levels 10–40 times lower than the maximum residue limits
rality is expected to play a crucial role in the bioactivities of (MRL) set by Codex Alimentarius Commission (CAC) [26].
triazole fungicides. The enantiomeric separations of triazole-
type fungicides have been performed using CE procedures 2. Experimental
[22–25]. Mixtures of uniconazole and diniconazole enantiomers
were baseline resolved using 50 mM phosphate buffer (pH 6.5) 2.1. Chemicals and reagents
containing 5 mM carboxymethyl-␥-CD and a temperature of
50 ◦ C in less than 5 min [22]. Simultaneous chiral separation Propiconazole, tebuconazole and fenbuconazole were
of triadimefon and triadimenol [23] and chiral separation of obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany),
14 triazole fungicides (including propiconazole, tebuconazole, 2-hydroxypropyl-␥-cyclodextrin (HP-␥-CD) was obtained from
without fenbuconazole) [24] were successfully achieved using Sigma (St. Louis, MO, USA), sodium dodecyl sulfate (SDS)
sulfated-␤-cyclodextrin-mediated CE under acidic conditions. was obtained from Fisher Scientific (Loughborough, UK), and
However, the limit of detections (LODs) were not discussed. disodium hydrogen phosphate 12-hydrate was obtained from
Cyclodextrin-modified micellar electrokinetic chromatography Riedel-de Haen (Seelze, Germany). All other chemicals and sol-
(CD-MEKC) has also been employed to the enantiomeric vents were common brands of analytical-reagent grade or better,
and isomeric separation of seven commonly used pesticides and were used as received. Water was collected from a Millipore
(including propiconazole). However, only two diastereomers of Water Purification System (Molsheim, France). Fresh grapes
propiconazole were baseline separated in this study [25]. It is were obtained from local market in Skudai, Johor, Malaysia.
therefore of interest to study the CD-MEKC method for the enan- The stock solutions of the individual triazole fungi-
tiomeric separation of two pairs of propiconazole enantiomers cides were prepared in methanol in the concentration range
and the capabilities of two on-line preconcentration methods, i.e. 2000–4000 mg/L. Final dilutions (concentrations in the figures)
stacking- and sweeping-CD-MEKC were evaluated. Sweeping- were prepared by diluting the stock solution with various sam-
CD-MEKC at acidic pH was found to give the highest sensitivity ple solvents mentioned at the respective places. Sample solutions
(100-fold) and the method was applied to the chiral separation were prepared by diluting the stock solution with pure water for
of propiconazole enantiomers followed by the chiral separation normal stacking mode-CD-MEKC and with a buffer/separation
of two other triazole fungicides, i.e. fenbuconazole and tebu- solution without micellar phase for sweeping-CD-MEKC. The
conazole (Fig. 1). This is the first ever reported separation of separation solutions for CD-MEKC were prepared by dissolving

Fig. 1. Structures of selected triazole fungicides (*chiral centre).

W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113 109

appropriate amounts of SDS, HP-␥-CD, methanol and acetoni- the calibration curve along with the signal-to-noise ratio (S/N)
trile in phosphate buffers and adjusting the pH of the buffer as 3.
with phosphoric acid solution. All running buffers were filtered
through a 0.45 ␮m nylon syringe filter from Whatman (Clifton, 3. Results and discussion
3.1. Optimization of the MEKC conditions
2.2. Instrumentation
The chiral separation of propiconazole enantiomers was first
All electropherograms were obtained with the Agilent explored by MEKC using bile salts (sodium cholate and sodium
capillary electrophoresis system (Agilent Technologies, Wald- deoxycholate) as chiral surfactants. Effects of different bile salt
bronn, Germany), equipped with temperature control and diode types and concentrations on the enantiomeric separation of prop-
array detection (DAD). Separations were performed using an iconazole were explored to resolve the four enantiomers of
untreated fused silica capillary of 64.5 cm × 50 ␮m i.d. (with an propiconazole in this initial study. However, only two isomers
effective length of 56 cm to the detector window) obtained from of propiconazole were observed in these experiments (electro-
Polymicro Technologies (Phoenix, AZ, USA). Sample injec- pherogram not shown). Since the separation of the propiconazole
tion was performed hydrodynamically for 1–120 s at 50 mbar. enantiomers was not satisfactory, the use of an achiral ionic sur-
The detection wavelength used was 200 nm and the capillary factant, SDS, with HP-␥-CD as chiral selector was then explored.
temperature was maintained at 20 ◦ C. Data were collected and As the previous work by Wu et al. [24] and Penmetsa [25] has
processed on computer using ChemStation software (Agilent not been successful in separating the four stereoisomers of prop-
Technologies). The new capillary was conditioned by pass- iconazole, our work focused on the optimization process and
ing 1 M NaOH solution for 10 min followed by washing with applying the optimum conditions to two on-line preconcentra-
deionized water for 10 min and finally equilibrating with an tion methods, i.e. stacking and sweeping.
appropriate running buffer for 10 min. Between runs, the cap- The HP-␥-CD concentration was first varied in order to
illary was washed with 0.1 M NaOH, water, and run buffer for achieve the best separation of the two pairs of propiconazole
2 min each. All sample injections were performed in triplicate. enantiomers by CD-MEKC. Separation was obtained for two
pairs of propiconazole enantiomers using HP-␥-CD in the con-
2.3. Extraction procedure centration range 10–40 mM (with 50 mM SDS and 15% (v/v) of
methanol in 25 mM phosphate buffer solution, pH 7.0, the run-
A representative portion of sample (150 g of grapes) was ning voltage 30 kV and 20 ◦ C). It was found that the migration
chopped and homogenized. Five-gram portions of sample was times of the propiconazole enantiomers gradually shortened with
spiked with 250 ng each of fenbuconazole, tebuconazole and stepwise increase in the concentration of HP-␥-CD. This obser-
propiconazole, then placed into an Erlenmeyer flask and homog- vation is expected since with increasing HP-␥-CD concentration,
enized with 5 mL of methanol and 5 mL of water by sonication more of the analyte will be included in the HP-␥-CD phase and
for 15 min. The resulting suspension was filtered through a will be transported quickly towards the detector. The best reso-
Buchner funnel and the cake filter was washed twice with 10 mL lutions (Rs > 1.50) and peak efficiency (N > 200 000) were found
of deionized water. The filtrate was adjusted to a volume of to be at 30 mM HP-␥-CD. Based on this data, optimal concen-
100 mL with deionized water and passed under vacuum through tration for HP-␥-CD was set at 30 mM. This concentration was
a C18 solid-phase column, which was preconditioned with 10 mL employed in all subsequent investigations.
of methanol and 10 mL of deionized water. Fungicides were The effect of phosphate buffer concentration on the resolu-
eluted with 2× 5 mL of dichloromethane. The eluate was con- tion and efficiency of the propiconazole enantiomers was also
centrated under stream of nitrogen to dryness. Then it was evaluated at the optimal HP-␥-CD concentration (other condi-
reconstituted with 0.5 mL of buffer solution (without micellar tions were kept constant). An increase in the concentration of
phase). buffer from 10 to 50 mM caused a significant increase in the
migration time of all the propiconazole enantiomers. A 25 mM
2.4. Validation procedure phosphate buffer was selected for optimal concentration since it
yielded very good peak efficiencies (N > 200 000) with all Rs val-
The performance of the system was examined in terms of the ues greater than 1.50. This buffer concentration was employed
linearity, repeatability, increase in detection sensitivity or sensi- in all subsequent investigations.
tivity enhancement factor (SEF), and limit of detection (LOD). The effect of buffer pH on the chiral separation of propi-
The linearity of the optimized method was assessed by construct- conazole by MEKC was also investigated at basic/neutral pH,
ing the calibration curve of average peak areas (n = 3) against the ranging from 9.4 to 7.0. Resolution of all propiconazole enan-
concentration of standards (at the linear range). The repeatabil- tiomers was found to be greater than 1.20. The best separation
ity in the optimized method concerning the relative standard was found to be at pH 7.0 (Rs > 1.50). Based on this data, pH 7.0
deviation for peak area (RSD%, n = 3) was examined for the was employed in normal mode MEKC separation.
injection of the 1 ␮g/mL sample. SEF was calculated by com- The effect of SDS concentration was also evaluated in this
paring (peak area obtained with sweeping/peak area with usual study, ranging from 10 to 40 mM. At 10 and 25 mM SDS, prop-
MEKC injection) × dilution factor. The LOD was determined by iconazole enantiomers were only partly separated, while at 50
110 W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113

and 75 mM SDS, two pairs of propiconazole enantiomers were

separated. The best resolutions (Rs > 1.50) and peak efficiencies
(N > 200 000) were obtained at 50 mM SDS. Based on this data,
optimal concentration chosen for SDS was 50 mM.
The effect of the addition of different organic modifier type
and percentages in the buffer solution on the resolution and
peak efficiency was also evaluated. In the absence of organic
modifier in the buffer solution, only three peaks of the prop-
iconazole enantiomers were observed (data not shown). Two
pairs of propiconazole enantiomers were baseline separated
with methanol and/or acetonitrile percentage ranging from
5% to 20% (v/v) and with mixed methanol–acetonitrile from
5%:5% to 10%:10% (v/v). Increasing organic modifier percent-
ages resulted in increasing migration time because of viscosity
increase. Optimal concentration chosen for organic modifier was
mixed methanol–acetonitrile 10%:5% (v/v). Under this condi-
tion, all Rs values were greater than 1.50 with peak efficiencies
greater than 400 000 and analysis times of less than 16 min was
obtained (Fig. 2a).

3.2. On-line sample preconcentration and CD-MEKC

under neutral conditions

In this present study, normal stacking mode and sweeping

techniques were applied to the CD-MEKC system to enhance
detection sensitivity. To apply the normal stacking mode to the
CD-MEKC system, the conductivity of the sample solution was
made lower than that of the micellar/separation solution. The
sample stock solution was then diluted with pure water. The
Fig. 2. Electropherograms of propiconazole enantiomers at neutral pH by (a)
sample injection time was optimized for stacking method and CD-MEKC (200 ␮g/mL), sample injection 1 s, at 50 mbar; (b) stacking-CD-
it was found that the best sample injection time was obtained MEKC (20 ␮g/mL), sample injection 15 s, at 50 mbar; (c) sweeping-CD-MEKC
at 15 s. Increasing sample injection time (higher than 15 s) (10 ␮g/mL), sample injection 90 s, at 50 mbar. Separation solution, 30 mM
resulted in decreasing peak resolutions while peak area was HP-␥-CD and 50 mM SDS in 25 mM phosphate buffer (pH 7.0) with
methanol–acetonitrile (10%:5%, v/v); capillary, 64.5 cm × 50 ␮m i.d. (effec-
not significantly increased. To apply the sweeping method to
tive length, 56 cm); applied voltage, 30 kV; detection wavelength, 200 nm UV;
the CD-MEKC system, the conductivity of the sample solu- temperature, 20 ◦ C.
tion (buffer solution without micellar phase) was adjusted to be
same as that of the separation solution. The sample injection
time was also optimized for sweeping method and it was found grams of propiconazole enantiomers by the normal stacking
that the best sample injection time was obtained at 90 s. Higher mode and the sweeping-CD-MEKC method at pH 7.0 are shown
injection time has been tried, but resolutions decrease with in Fig. 2b and 2c, respectively. As can be seen in Table 1, the
increasing injection times higher than 90 s. Typical electrophero- methods demonstrated a good linearity in the calibration curve

Table 1
Linearity, repeatability, LODs (S/N = 3) and sensitivity enhancement factor (SEF) for propiconazole enantiomers in the optimized CD-MEKC and on-line-CD-MEKC
methods under neutral condition (pH 7.0)
CD-MEKC Stacking-CD-MEKC Sweeping-CD-MEKC

Linearity range (␮g/mL) 50–200 2.5–20 1–20

r2 0.9991–1.0000 0.9961–0.9973 0.9989–0.9999
RSD (%, n = 3)
Migration time 0.2–0.3 0.2–0.21 1.9–2.1
Peak area 2.0–5.8 1.9–2.2 3.6–6.0
Peak height 1.2–1.6 2.1–3.4 1.9–5.5
LOD (␮g/mL) 10 2.0 0.4
SEFarea – 5–6 34–38
SEFLOD – 5 25

Analysis conditions as in Fig. 2. SEFarea : (peak area obtained with on-line-CD-MEKC/peak area with usual MEKC injection) × dilution factor. SEFLOD :
W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113 111

(r2 > 0.996) and RSDs for migration time, peak height and peak
area were less than 6.0%. There is a 5-fold increase in the detec-
tion sensitivity for each propiconazole enantiomer in the normal
stacking mode CD-MEKC method (LOD = 2.0 ␮g/mL, S/N = 3)
and about 25-fold increase in the sweeping-CD-MEKC method
(LOD = 0.4 ␮g/mL) compared to CD-MEKC method at pH 7.0
(LOD = 10 ␮g/mL). Although these values are modest for the
analysis of real samples, the results clearly show the possibility
of improving the detectability by using stacking and sweeping
in CD-MEKC method at neutral pH.

3.3. CD-MEKC and sweeping-CD-MEKC under acidic


In order to achieve significantly lower LODs, CD-MEKC

separation in acidic buffer was then investigated. Firstly, effect
of acidic buffer pH was investigated ranging from 2.0 to 4.0.
It was found that the best separation was obtained at pH 3.0.
The effect of applied voltage was also evaluated in this study,
ranging from −20 to −30 kV and it was found that the best sep-
aration voltage was obtained at −25 kV (all Rs values greater
than 1.50 with shorter analysis time). Based on this data, acidic
buffer pH selected was pH 3.0 and the separation was performed
with negative power supply of −25 kV (average current 40 ␮A).
Other parameters are as in CD-MEKC method with neutral con-
dition (pH 7.0). The method was then applied to two other
triazole fungicides (tebuconazole and fenbuconazole). Electro-
pherogram of the selected triazole fungicide enantiomers by
CD-MEKC under acidic conditions (pH 3.0) is shown in Fig. 3a.
Sweeping technique was then applied to the CD-MEKC system Fig. 3. Electropherograms of the selected chiral triazole fungicides by CD-
MEKC at acidic pH (pH 3.0): (a) 50 ␮g/mL fenbuconazole (F), tebuconazole (T)
under acidic conditions to enhance detection sensitivity. The
and propiconazole (P); sample in separation solution, injected for 1 s, at 50 mbar
sample injection time was optimized for sweeping-CD-MEKC and (b) 1 ␮g/mL fenbuconazole (F), tebuconazole (T) and propiconazole (P).
method at pH 3.0 and it was found that the best sample injec- Sample in buffer solution without micellar phase, injected for 120 s, at 50 mbar;
tion time was obtained at 120 s. Increasing sample injection time separation voltage, −25 kV; 25 mM phosphate buffer (pH 3.0); other conditions
(higher than 120 s) resulted in significantly decreasing peak res- are as in Fig. 2.
olutions. Typical electropherogram of selected chiral triazole
fungicides employing the sweeping-CD-MEKC under acidic ment factor (SEF) and LODs for all selected chiral triazole fungi-
condition is shown in Fig. 3b. The increase in detection sen- cides are presented in Table 2. The results indicate that sweeping-
sitivity for each propiconazole enantiomer is about 100-fold in CD-MEKC method under acidic conditions is better than under
sweeping-CD-MEKC at pH 3.0 (LOD = 0.1 ␮g/mL) compared neutral conditions in term of achievable detection limit. The
to the conventional CD-MEKC. Linearity, sensitivity enhance- sweeping-CD-MEKC method under acidic conditions provides

Table 2
Linearity, repeatability, sensitivity enhancement factor (SEF) and LODs (S/N = 3) of the optimized sweeping-CD-MEKC method under acidic condition (pH 3.0)

Fungicide Peaka Regression equationb r2 RSD peak area (%, n = 3) SEFarea c LODs (␮g/mL)

Fenbuconazole 1 A = 26.718 C − 2.9483 1 0.63 30 0.09

2 A = 26.120 C − 2.1827 0.9999 1.83
Tebuconazole 1 A = 32.445 C − 3.7379 0.9998 1.39 70 0.1
2 A = 23.667 C + 2.7312 0.9979 0.68
Propiconazole 1 A = 19.346 C − 1.9806 0.9999 0.14 100 0.1
2 A = 20.363 C − 2.5999 0.9999 1.66
3 A = 23.965 C − 3.4852 0.9999 1.84
4 A = 24.698 C − 4.0619 1 1.10

Conditions as in Fig. 4a.

a 1, 2, 3, and 4 are first, second, third, and fourth migrating enantiomers of triazole fungicides, respectively.
b Linear range: 0.5–5.0 ␮g/mL; A = peak area; C = concentration (␮g/mL).

area : (peak area obtained with sweeping/peak area with usual MEKC injection) × dilution factor.
112 W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113

Table 3
Percent recovery and repeatability of three selected triazole fungicides spiked in
grapes sample (amount of sample processed 5 g) by SPE-sweeping-CD-MEKC
method (pH 3.0) and MRL established by the Codex Alimentarius Commission
Fungicide Fortified Average RSD MRL
control (mg/kg) recovery (%) (%, n = 3) (mg/kg)

Fenbuconazole 0.05 109 11 1.0

Tebuconazole 0.05 73 9 2.0
Propiconazole 0.05 74 12 0.5

solubility of these fungicides in water and salting out effect with

sodium chloride might help to increase the recovery of these two
fungicides from the spiked grapes. A more comprehensive study
on the simultaneous chiral separation of triazole fungicides are
currently in progress.

4. Conclusion

This is first report on the enantiomeric separation of two

pairs of propiconazole enantiomers and all enantiomers of fen-
buconazole and tebuconazole by CD-MEKC using HP-␥-CD as
chiral selector. Stacking-CD-MEKC at neutral pH enhanced the
detection sensitivity of propiconazole 5-fold and sweeping-CD-
MEKC enhanced it 25-fold and finally sweeping-CD-MEKC
at acidic pH enhanced it 100-fold. The sweeping-CD-MEKC
method under acidic condition at pH 3.0 gave the best detection
sensitivity with a limits of detection (S/N = 3) for selected tria-
zole fungicide enantiomers ranged from 0.09 to 0.1 ␮g/mL, well
below the limit set by Codex Alimentarius Commission. The
Fig. 4. Electropherograms of the selected chiral triazole fungicides by sweeping- sensitivity enhancement of the three fungicides ranged from 30-
CD-MEKC at acidic pH (pH 3.0): (a) 1 ␮g/mL the selected triazole fungicide
standards and (b) spiked grapes sample after preconcentration by SPE. Sample in
to 100-fold. The average recoveries of the selected fungicide in
buffer solution without micellar phase, injected for 120 s, at 50 mbar; separation spiked grapes sample by combination of SPE pretreatment and
voltage, −25 kV; 25 mM phosphate buffer (pH 3.0); other conditions are as in sweeping-CD-MEKC at pH 3 were good for all the compounds,
Fig. 2. Peak assignment: F, fenbuconazole; T, tebuconazole; P, propiconazole; ranging from 73% to 109% with RSDs between 9% and 12%
and unknown peaks from the sample matrix. (n = 3).

lower limit of detection for the fungicides compared to a previ- Acknowledgements

ously reported study of enantiomeric separation of chiral triazole
pesticides by high-performance liquid chromatography [27]. The authors would like to thank Universiti Teknologi
Malaysia and the Ministry of Science, Technology and Innova-
3.4. Application of method tion (MOSTI), Malaysia, for financial support through the IRPA
grant project number 09-02-06-0035 EA158 and studentship for
The validity of the method was studied by using grapes sam- D. Hermawan.
ple spiked (0.05 mg/kg) with the selected triazole fungicides
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