Enantioselective determination of triazole fungice tetraconazole by chiral high-performance liquid chromatography and its application to pharmacokinetic study in cucumber, muskmelon, and soils

Article abstract of DOI:10.1002/chir.21997

A simple chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of tetraconazole enantiomers in cucumber, muskmelon, and soils. Good separation was achieved at 20°C using cellulose tris-(4-methylbenzoate) as chiral stationary phase, a mixture of n-hexane and ethanol (90:10) as mobile phase at a flow rate of 0.8 ml/min. The assay method was linear over a range of concentrations (0.5-50 μg/ml) and the mean recoveries in all samples were more than 85% for the two enantiomers. The limits of detection for both enantiomers in plant and soil samples were 0.06 and 0.12 μg/g, respectively. Then, the proposed method was successfully applied to the study of enantioselective degradation of rac-tetraconazole in cucumber, muskmelon, and soils. The results showed that the degradation of two enantiomers of tetraconazole followed first-order kinetics and significantly stereoselective behavior was observed in cucumber, muskmelon, and Beijing soil. The preferential absorption and degradation of (-)-S-tetraconzole resulted in an enrichment of the (+)-R-tetraconazole residue in plant samples, whereas the (+)-R-tetraconazole showed a faster degradation in Beijing soil and the stereoselectivity might be caused by microorganisms. No stereoselective degradation was observed in Heilongjiang soil. Copyright

Full text of DOI:10.1002/chir.21997

CHIRALITY 24:294–302 (2012)
Enantioselective Determination of Triazole Fungice Tetraconazole by
Chiral High-performance Liquid Chromatography and Its Application
to Pharmacokinetic Study in Cucumber, Muskmelon, and Soils
1
JING LI,1 FENGSHOU DONG,1 JUN XU,1 XINGANG LIU,1 YUANBO LI,1 WEILI SHAN,2 AND YONGQUAN ZHENG
*
1Pesticide Residues and Environmental Toxicology Group, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Key laboratory of
Pesticide Chemistry and Application, Ministry of Agriculture, Beijing 100193, China
2Pesticide Residues and Environmental Toxicology Group, Institute of the Control of Agrochemicals, Ministry of Agriculture, Beijing 100125, China
ABSTRACT
A simple chiral high-performance liquid chromatography method with diode
array detector was developed and validated for stereoselective determination of tetraconazole
enantiomers in cucumber, muskmelon, and soils. Good separation was achieved at 208C using
cellulose tris-(4-methylbenzoate) as chiral stationary phase, a mixture of n-hexane and ethanol
(90:10) as mobile phase at a flow rate of 0.8 ml/min. The assay method was linear over a range
of concentrations (0.5–50 lg/ml) and the mean recoveries in all samples were more than 85%
for the two enantiomers. The limits of detection for both enantiomers in plant and soil samples
were 0.06 and 0.12 lg/g, respectively. Then, the proposed method was successfully applied to
the study of enantioselective degradation of rac-tetraconazole in cucumber, muskmelon, and
soils. The results showed that the degradation of two enantiomers of tetraconazole followed
first-order kinetics and significantly stereoselective behavior was observed in cucumber, musk-
melon, and Beijing soil. The preferential absorption and degradation of (2)-S-tetraconzole
resulted in an enrichment of the (1)-R-tetraconazole residue in plant samples, whereas the
(1)-R-tetraconazole showed a faster degradation in Beijing soil and the stereoselectivity might
be caused by microorganisms. No stereoselective degradation was observed in Heilongjiang
C
VV
soil. Chirality 24:294–302, 2012.
2012 Wiley Periodicals, Inc.
KEY WORDS: tetraconazole; enantioselective degradation; stereoselectivity; cucumber;
muskmelon; soil
INTRODUCTION
enantiomer to supply more accurate data for evaluating the
environmental risk and food safety.
The significance of molecular chirality in life sciences has
long been recognized since the separation of optical isomers
of tartrate by Pasteur.1,2 A lesser known fact is that many
pesticides also contain chiral structures and thus consist of
two or more enantiomers/stereoisomers.3 Chiral pesticides,
accounting for more than 25% among the frequently used
pesticides, have attracted increasing attention in the agro-
chemical research.4 As it is well known that pure enantiom-
ers from the same chiral compound have identical physical
and chemical properties, but they may differ dramatically in
biological activity, toxicity, and environmental fate when indi-
vidual enantiomers interact with other chiral molecules, such
as enzymes and biological receptors.5 The processes of
absorption, distribution, and degradation in organism and
environment are often enantioselective.6,7 Therefore, enantio-
selectivity is an important factor that should not be ignored
concerning the effect of chiral pesticides on ecosystem. How-
ever, in most cases, chiral pesticides are produced and used
as racemates or a mixture of stereoisomers, and the environ-
mental studies have historically neglected to determine the
adverse effects associated with particular enantiomers that
may persist in the environment.8 Therefore, the risk evalua-
tions of chiral pesticides residue in food and environmental
matrix based on the data obtained by traditional methods are
incomplete and nonspecific, if enantioselective behaviors
happened. Consequently, it is of great significance to develop
enantiomeric analysis methods of chiral pesticides and inves-
tigate the different environmental behavior of the individual
Tetraconazole, (6)-1-[2-(2,4-dichlorophenyl)-3-(1,1,2,2-tet-
rafluoroethoxy)propyl]-1H-1,2,4-triazole, is a broad spectrum
systemic triazole fungicide (Fig. 1). The compound was syn-
thesized as a novel demethylation inhibitor fungicide having
a triazole ring in its chemical structure and was selected for
its excellent fast-acting, curative, and prominent systemic ac-
tivity.9,10 Tetraconazole is effective in controlling a broad
spectrum of diseases such as powdery mildew and brown
rust on fruits, vegetables, and cereals. As most of triazole
fungicides, tetraconazole has a chiral carbon and consists of
two enantiomers. Its absolute configuration was already con-
firmed with right-optical (1) rotation of R-enantiomer and
left-optical (2) rotation of S-enantiomer.11,12 The (1)-R-enan-
tiomer of tetraconazole shows significantly greater fungicidal
activity than (2)-S-enantiomer.11–13 However, within our
existing knowledge, except for the study of enantioselective
bioaccumulation and biotransformation of tetraconazole in rain-
bow trout,14 few studies on the enantioselective environmental
Jing Li and Fengshou Dong have contributed equally to this work.
Contract grant sponsor: National Natural Science Foundation of China;
Contract grant number: 31071706.
*Correspondence to: Yongquan Zheng, Institute of Plant Protection, Chinese
Academy of Agricultural Sciences, Key Laboratory of Pesticide Chemistry
and Application, Ministry of Agriculture, Beijing 100193, China.
E-mail: yongquan_zheng@yahoo.com.cn
Received for publication 13 August 2011; Accepted 8 December 2011
DOI: 10.1002/chir.21997
Published online 25 January 2012 in Wiley Online Library
(wileyonlinelibrary.com).
C
VV
2012 Wiley Periodicals, Inc.

ENANTIOSELECTIVE DEGRADATION OF TETRACONAZOLE
295
Fig. 1. Structures of the enantiomers of tetraconazole (asterisks denote chiral position) and the UV (A) and optical rotation (B) chromatograms for the separa-
tion of tetraconazole enantiomers on the Chiralcel OD-RH column in Waters PrepLCTM 150 Semi Preparative HPLC system.
magnesium sulfate were analytical grade and from Sinopharm Chemical
behavior of tetraconazole enantiomers were done even
Reagent Beijing Company (Beijing, China). Stock solution of racemic
tetraconazole standard (200 lg/ml) was prepared in hexane. The pure
solvent solutions required for preparing a standard curve (0.5–50 lg/ml)
were prepared in hexane from the stock solution by serial dilution. All
solutions were protected against light with aluminum foil and stored in a
refrigerator at 2208C.
though the achiral analysis and the residue determinations
of tetraconazole racemate (not differentiating the two enan-
tiomers) were extensively studied.10,15–17
The objective of this research work was to develop and
validate the method of extraction of the enantiomers of tetra-
conazole from plant and soil matrix and determination by
high-performance liquid chromatography-chiral stationary
Plant Care and Fungicide Application
phase (HPLC-CSP) technology. Then, the enantioselective
Working areas for cucumber and muskmelon were prepared at the
behavior of tetraconazole in cucumber, muskmelon, and soils
experiment field in Chinese Academy of Agricultural Sciences (Shan-
was investigated by applying the developed analytical method.
dong and Henan, China). The fields were divided into 30-m2-sized blocks
and the buffer zone was set up between plots. Each field experiment
treatment was designed with three replicate plots for the control and the
dissipation rate study. The application rate in dissipation experiments
was 90 g (a.i.)/ha of 4% tetraconazole aqueous emulsion with one time
MATERIALS AND METHODS
Materials and Reagents
Analytical standard of rac-tetraconazole (purity: 98.4%, enantiomeric
ratio ([R]/[S]):0.99) was obtained from Institute for the Control of
Agrochemicals, Ministry of Agriculture, China. Acetonitrile, toluene,
hexane, ethanol, n-propanol, isopropanol, and n-butanol were LC solvent
purchased from Sigma–Aldrich (Steinheim, Germany). Purified water
was prepared using Milli-Q water purification system (Millipore Purifica-
tion Systems). Other reagents such as sodium chloride and anhydrous
spray. The foliar treatment was carried out on cucumber plants and
muskmelon in the growth stage 7.5 and 8.1, individually (according to
the Biologische Bundesanstalt, Bundessortenamt and CHemical Indus-
try (BBCH)-scale). Representative fruits of cucumber and muskmelon
were collected in 2 h, 1, 2, 3, 5, 7, 10, and 14 days after spraying (there
was no natural rainfall during the study period). Samples were put into
polyethylene bags and deep-frozen (2208C) until analysis.
Chirality DOI 10.1002/chir

296
LI ET AL.
TABLE 1. Selected physicochemical properties of the soils investigated
Particle size
Soil sample
Sand (%)
Slit (%)
Clay (%)
Total N (%)
Total P (%)
Total K (%)
pHa
Corg (%)
Beijing soil
Heilongjiang soil
15.12
16.75
45.09
51.13
39.79
32.12
0.08
0.19
0.08
0.21
0.97
1.81
7.82
5.86
1.16
3.81
aSuspension of soil in 0.01 M CaCl2, 1:2.5 (w/w).
Rs 5 2(t22t1)/(W1 1 W2). Where t was the retention time and t0 was the
void time at given conditions, k was retention factors, and W was base
widths (subscripts 1 and 2 represent the first and second eluted enan-
tiomers, respectively). Triplicate injections were made for the measure-
ments and the values were the mean of three injections. The samples
were analyzed using a mixture of hexane and ethanol (90:10) as mobile
phase. The flow rate was 0.8 ml/min. Chromatographic separation was
conducted at 208C and DAD detection at 225 nm.
Plant Sample Preparation
A 20 g aliquot of the sample was weighed into a 100-ml teflon centri-
fuge tube with screw caps, 20 ml of acetonitrile was added to the tube
and then homogenized at high speed for 1 min. A 6 g of anhydrous mag-
nesium sulfate (MgSO4) and 1.5 g of sodium chloride (NaCl) were
added, thereafter, the tube was capped and immediately vortexed vigo-
rously for 1 min and then centrifuged for 5 min at 2818g (4000 rpm).
Acetonitrile supernatant (15 ml) was applied to a Cleanert Pesti-Carb/
PSA, which had been formerly conditioned with 10 ml of the elution sol-
vent (acetonitrile/toluene, 3:1, v/v). The column was eluted with 12 ml
of elution solvent. After removal of the solvent in vacuo, the sample was
dissolved in 1 ml of hexane. After filtering thorough a 0.22-lm nylon
syringe filter, an aliquot (50 ll) was injected into HPLC.
To find out the elution order of tetraconazole enantiomers on OJ-H
chiral column, a Waters PrepLCTM 150 Semi Preparative HPLC Purifica-
tion system equipped with
Waters12489 UV/visible detector was applied for preparation of the indi-
vidual enantiomers of tetraconazole.
Waters12707 autosampler
a a
Waters1600 controller pump and
A
equipped with a 100-ll sample loop was used for sample injection. The
column effluent was fractionated using a Waters1 Fraction Collector III.
The signal was received and processed by EmpowerTM data software.
The enantiomers of tetraconazole were separated on a Chiralcel OD-RH
(cellulose tris-(3,5-dimethylphenylcarbamate)) chiral column (150 3
4.6 mm2 i.d., Daicel Chemical Industries, Japan) in reverse phase condi-
tion. The chromatographic separation was conducted at 208C. The mobile
phase applied was a mixture of acetonitrile and water (60:40 by volume),
and a flow rate at 0.3 ml/min and a detection wavelength at 210 nm. The
rac-tetraconazole was split into (1) and (2)-enantiomers. The elution
order of the two tetraconazole enantiomers in this system was distin-
guished by an online optical rotation dispersion (ORD) detector (JASCO
OR-2090 Plus, JASCO Corporation, Japan). Then, the two individual sepa-
rated enantiomers were injected and determined by Agilent 1100 HPLC to
find out their elution order on OJ-H chiral column.
Treatment and Incubation of Soils
The test soils from two different agriculture regions (0–15 cm horizon)
of China that had not been treated with tetraconazole in the last 5 years
were used in this study. Their major properties are shown in Table 1.
Air-dried soils at room temperature were sieved through a 2.0-mm sieve.
The moisture content of the test soils was adjusted to 25% and kept in
the dark at 25 (62)8C for 2 weeks activation period. After that period,
batches of 100 g soil (based on dry weight) were weighed into 250-ml
conical flasks. To avoid potential effects of solvents on the soil microbio-
logical activity, the volume of the application solution was limited to
1 ml. The tetraconazole stock solution was applied dropwise to give an
application rate equivalent to 5 mg/kg rac-tetraconazole for per gram
soil. After thorough mixing, the flasks were sealed with cotton-wool
plugs and stored at 25(62)8C in dark. During incubation, distilled water
was added at 1 day intervals to maintain the initial moisture. Fifteen
grams of the treated soil samples (based on dry weight) was removed
for analysis after incubation periods of 0 (2h), 1, 3, 5, 8, 16, 21, 28, 35,
42, and 64 days. Each incubation was carried out in triplicate.
Calibration Curves and Assay Validation
Matrix working standard rac-tetraconazole solutions (0.5–50 mg/l) for
linearities of the two enantiomers were prepared by diluted with blank
matrix. Calibration curves were generated by plotting peak area of each
enantiomer versus the concentration of the enantiomers. Linear regres-
sion analysis was performed using Microsoft1 Excel. The precision and
accuracy of the assay were obtained by comparing the theoretical con-
centration to the actual found concentration of each enantiomer spiked
in blank samples. The relative standard deviations (RSD 5 SD/mean)
were calculated at the calibration range. The intraday precision was
determined in five replicates at different concentrations on the same
day, and the interday precision was evaluated in three replicates at the
earlier concentrations on three different days. Recovery assay was per-
formed in blank plant and soil matrix over three concentration levels for
tetraconazole enantiomers. The samples were extracted, cleaned, and
determined as previously described. The limit of detection (LOD) for
each enantiomer was considered to be the concentration that produced a
signal-to-noise (S/N) ratio of 3, and the limit of quantification (LOQ) was
defined on S/N ratio of 10. The concentration of each enantiomer in
samples was calculated by calibration curves of corresponding enan-
tiomer using external standard method.
Extraction and Cleanup Procedures for Soil
A 15 g aliquot of the soil sample was weighed in a 50-ml teflon centri-
fuge tube with screw caps, and 2 ml of mill-Q water was added. After
soaking at room temperature for 60 min, 20 ml acetonitrile was added.
The mixture was homogenized and then salted out, and water was
removed with 4 g MgSO4 and 1 g NaCl as described earlier. A 15 ml of
acetonitrile supernatant was applied to a Supelclean Pesti-Carb, which
was formerly conditioned with 2 3 5 ml elution solvent (acetonitrile/to-
luene, 3:1, v/v). The column was eluted with 2 3 3 ml elution solvent.
After removal of the solvent in vacuo, the sample was dissolved in 1 ml
of hexane. After filtering thorough a 0.22-lm nylon syringe filter, an
aliquot (50 ll) was injected into HPLC.
Apparatus and Chromatographic Conditions
Chromatography in this experiment was performed using an Agilent
1100 series HPLC (Agilent Technology) equipped with a G1322A degas-
ser, G1311A pump, G1316A column compartment, G1329B automatic
injector, a 50-ll sample loop, and G1315B diode array detector (DAD).
The signal was received and processed by an Agilent Chemstation. The
enantiomers of tetraconazole were separated by HPLC with Chiralcel1
OJ-H (cellulose tris-(4-methylbenzoate)) chiral column (250 3 4.6 mm2,
5 lm, Daicel Company, Japan). Void times were determined using 1,3,5-
tri-tertbutyl benzene. Capacity factor (k), separation factor (a), and reso-
lutions (Rs) were calculated from the formula k 5 (t2t0)/t0, a 5 k2/k1,
Pharmacokinetic and Calculation
It was assumed that the degradation of the enantiomers in plants and
soil accorded with first-order kinetics. Corresponding rate constants k
was calculated according to Eq. 1. The starting point was the maximum
value of the concentration, and decreased in following days. The half-life
(T1/2, day) was estimated from Eq. 2.
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE DEGRADATION OF TETRACONAZOLE
297
TABLE 2. Influence of polar alcoholic on the enantiomeric separation of tetraconazole, including the retention factor of the first
eluted enantiomer (k1), the retention factor of the second eluted enantiomer (k2), separation factor (a), and resolution (Rs)
Percentage of
alcohol in hexane
k1
k1
a
Rs
k1
k1
a
Rs
Ethanol
n-Propanol
8.94
3.91
2.35
1.63
1.22
0.95
5
5.42
2.24
1.34
0.93
0.68
0.51
6.45
1.19
1.22
1.24
1.27
1.28
1.31
3.92
3.83
3.35
3.01
2.70
2.46
7.74
3.43
2.06
1.43
1.08
0.85
1.15
1.14
1.14
1.14
1.13
1.12
1.14
1.11
0.81
0.73
0.66
0.59
10
15
20
25
30
2.73
1.66
1.17
0.86
0.66
iso-Propanol
10.43
n-Butanol
5
7.22
3.14
1.87
1.27
0.93
0.71
1.44
1.44
1.45
1.46
1.47
1.49
6.57
5.05
4.13
3.57
3.14
2.83
8.59
4.41
2.69
1.88
1.41
1.12
12.11
6.26
3.79
2.62
1.98
1.57
1.41
1.42
1.41
1.40
1.40
1.39
2.76
2.01
1.63
1.38
1.25
1.12
10
15
20
25
30
4.54
2.70
1.85
1.36
1.06
Flow rate: 0.8 ml/min, wavelength: 225 nm, 208C, Chiralcel OJ-H chiral column in Agilent 1100 HPLC system.
C ¼ C0 eꢀkt
ð1Þ
ð2Þ
chiral resolutions was investigated from 5 to 358C using hex-
ane/isopropanol (95/5) mobile phase in this study (Table 3).
However, the final results in our investigation showed that
the retention and separation of tetraconazole enantiomers
increased as the column temperature increased. Considering
the influences of peak broadening at low temperature, the
optimal temperature in this experiment was 208C.
T1=2 ¼ ln2=k ¼ 0:693=k
The enantiomer fraction (EF) was used to measure the enantioselectivity
of the degradation of tetraconazole enantiomers in the plant and soil sam-
ples. The EF values defined range from 0 to 1, with EF 5 0.5 representing
the racemic mixture. EF was defined by equation as follows:
Determination of the Elution Order
EF ¼ peak areas of theðꢀÞ=½ðꢀÞ þ ðþÞꢁ
ð3Þ
For a chiral compound, the individual enantiomer corre-
sponded to a specific ORD signal at given wavelength and
thus ORD detector was frequently used to distinguish a pair
of enantiomers. In this work, two eluted enantiomers of tetra-
conazole were first determined by HPLC–ORD method on
Chiralcel OD–RH (cellulose tris-(3,5-dimethylphenylcarba-
mate)) Chiral column (150 3 4.6 mm2 i.d., Daicel Chemical
Industries, Japan) and distinguished by ORD signals ((1) or
(2)). The wavelength of 210 nm was selected in the HPLC
method due to the maximum absorption. It was found that
the ORD spectra of the two enantiomers were approximately
opposite to each other along the axis with the first eluted
one was (1) and the second was (2) in the effective
wavelength range (Fig. 1). To find out the absolute configu-
RESULTS AND DISCUSSION
Optimization of Enantioseparation Conditions
Mobile phase plays the most important role for enantio-
meric separations in terms of efficiency, retention, and resolu-
tion of enantiomers. Therefore, investigation of the mobile
phase composition and column temperature should be always
included in the optimization of enantioseparation conditions.
In this work, chiral separation of the two enantiomers was
performed using n-hexane-polar organic alcohols mobile
phase. Effects of ethanol, n-propanol, isopropanol, and n-buta-
nol modifiers and their volume content on the separation of
tetraconazole were investigated and listed in Table 2. The two
enantiomers of tetraconazole could achieve baseline separa-
tions using all the modifiers. Isopropanol gave the best reso-
lution compared with other alcohols and n-propanol gave the
worst separation. The optimal resolution was obtained using
5% isopropanol with the Rs value of 6.57. The results indicated
that the organic modifiers competed with the solutes for the
interactions with the CSP in some cases. The polarities and
viscosities of the alcohols may be not the only factor influenc-
ing the chiral recognitions, and the structure of the alcohols
may also have some effects on the stereoselectivity by alter-
ing the environment of the chiral site.
TABLE 3. Effect of temperature on the enantiomeric
separation of tetraconazole, including the retention factor of
the first eluted enantiomer (k1), the retention factor of
the second eluted enantiomer (k2), separation factor (a), and
resolution (Rs)
Temperature (8C)
k1
k2
a
Rs
5
4.452
3.913
3.496
3.103
2.717
2.399
2.203
6.689
5.821
5.157
4.489
3.876
3.361
3.044
1.503
1.488
1.475
1.447
1.427
1.401
1.382
3.11
3.72
4.33
4.93
5.37
5.77
5.87
10
15
20
25
30
35
Temperature is another significant factor both for optimiza-
tion of the chiral separations and investigation of the mecha-
nisms of chiral recognition. Generally, an increase in column
temperature often causes a reduction in retention and worse
resolution.18 The influence of column temperature on the
Flow rate: 0.8 ml/min, wavelength: 225 nm, n-hexane/isopropanol 5 95:5,
Chiralcel OJ-H chiral column in Agilent 1100 HPLC system.
Chirality DOI 10.1002/chir

298
LI ET AL.
Fig. 2. HPLC chromatograms for the separation of tetraconazole enantiomers on the Chiralcel OJ-H chiral column in Agilent 1100 HPLC system. (A): HPLC
chromatogram and mass spectra of (1) tetraconazole; (B): HPLC chromatogram and mass spectra of (2) tetraconazole; (C): HPLC chromatogram of racemic tet-
raconazole. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
rations of the eluted enantiomers on OJ-H chiral column, the
individual optical pure enantiomers of tetraconazole obtained
by preparative chromatography were injected and deter-
mined by Agilent 1100 HPLC. As can be seen from Figure 2
that (2)-tetraconazole was first eluted and (1)-tetraconazole
was subsequently eluted on OJ-H column and the Gas chro-
matography-mass Spectrometry (GC–MS) chromatograms of
the two enantiomers of tetraconazole were also given. The
resolved peaks of the two optical pure enantiomers were
found to contain the same mass spectra representative of the
intact racemic tetraconazole molecule.
soils including linearity, precision, recovery, and LOQ (the
results were summarized in Tables 4 and 5) showed the
method was efficient and reliable.
Enantioselective Degradation of Tetraconazole
in Cucumber and Muskmelon
Under field conditions, the concentration of tetraconazole
enantiomers in cucumber and muskmelon were both
attained maximum at 2 h after foliar treatment (Figs. 3A and
3B). Then, the concentrations of the two enantiomers
decreased gradually with time. Thereby, the regressive deg-
radation equations were established with maximum concen-
tration of enantiomers in plants regarded as degradation
starting point. Corresponding degradation kinetics of
enantiomers were shown in Table 6, the datum showed that
Validation of the Method
Validation of the developed method for determination of
the tetraconazole enantiomers in cucumber, muskmelon, and
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE DEGRADATION OF TETRACONAZOLE
299
TABLE 4. Linearities, calibration data and limits of detection of tetraconazole enantiomers in different sample matrixes (cucumber,
muskmelon, Beijing soil, and Heilongjiang soil)
Standard calibration curve
Enantiomers
Matrix
Calibration range (lg/g)
Slope
Intercept
R2
LOD (lg/g)
(1)-R-tetraconazole
Cucumber
Muskmelon
Beijing soil
Heilongjiang soil
Cucumber
Muskmelon
Beijing soil
0.5–50
0.5–50
0.5–50
0.5–50
0.5–50
0.5–50
0.5–50
0.5–50
52.561
50.517
49.364
51.511
52.808
50.597
49.098
51.888
210.93
3.64
0.9997
0.9997
0.9996
0.9992
0.9996
0.9996
0.9995
0.9986
0.06
0.06
0.12
0.12
0.06
0.06
0.12
0.12
5.14
210.75
213.14
5.97
5.57
216.58
(2)-S-tetraconazole
Heilongjiang soil
degradation of two enantiomers of tetraconazole ((2)-S and
(1)-R) in cucumber and muskmelon followed first-order
kinetics (r 5 0.951429879), and different half-life among of
tetraconazole enantiomers was found.
cucumber and muskmelon. The similar preferential enrich-
ment of higher activity enantiomer in plants has also been
reported.19 Some pioneer research work reported that
plant enzyme system play an important role in the enantio-
selective degradation, accordingly, it was possible that
corresponding enzyme system was restrained by higher
activity enantiomer,18–20 and which might result in accumu-
lation of (1)-R-tetraconazole in cucumber and muskmelon.
Nevertheless, further research should be carried out to
clarify whether there are underlying processes of stereose-
lective adsorption and degradation or enantiomeric trans-
formation in plants.
Preferential dissipation of (2)-S-tetraconazole was deter-
mined in cucumber and muskmelon after foliar treatment,
which resulted in plants enriched with (1)-R-tetraconazole
(Figs. 3A and 3B). As shown in Table 6, the half-lives of degra-
dation between (2)-S-tetraconazole and its optical antipode
were significantly different (P < 0.05, Student’s paired t-test)
in cucumber (T1/2 was 2.89 and 3.55 days) and muskmelon
(T1/2 was 6.13 and 7.14 days). The EF value in cucumber was
nearly 0.5 at first day after treatment, and decreased steadily
in the following days, and it dropped to 0.422 at 7 days after
treatment (Fig. 4A). Figure 4A shows that the EF value in
muskmelon decreased gradually from 0.503 to 0.402 after 5
days and then increased to 0.449 on the 10th day. The (2)-S-
enantiomer was preferentially metabolized by muskmelon and
it could not be detected after 14 days. These results indicated
there was substantial stereoselectivity on dissipation of rac-tet-
raconazole in cucumber and muskmelon.
Enantioselective Degradation of Tetraconazole in Soil
Under laboratory conditions, the degradation of the two
enantiomers of tetraconazole in soil followed first-order
kinetics. The degradation kinetics of (1)-R- and (2)-S-enan-
tiomers in two different types of soil are shown in Table 6. It
was observed that (1)-R-tetraconazole degraded faster than
its optical antipode in Beijing soil. The half-lives of degrada-
tion between the two enantiomers were significantly different
(P < 0.05, Student’s paired t-test) in Beijing soil (T1/2 was
70.71 and 92.40 days for (1)-R- and (2)-S-enantiomers,
respectively). Figure 4C shows that the EF value of the
tetraconazole in Beijing soil was always above 0.5 indicating
Previous study has revealed that the (1)-R-enantiomer
of tetraconazole shows significantly greater fungicidal activ-
ity than (2)-S-enantiomer,11 and the results in our investi-
gation showed that the (1)-R-enantiomer was enriched in
TABLE 5. Intraday and interday precision and accuracy results
Intraday (n 5 5)
(1)-R-tetraconazole (2)-S-tetraconazole
Interday (n 5 3)
(1)-R-tetraconazole
(2)-S-tetraconazole
b
Spiked level
(mg/kg)
Recovery
(%)
RSDra
(%)
Recovery
(%)
RSDr
(%)
Recovery
(%)
RSDR
(%)
Recovery
(%)
RSDR
(%)
Sample
Cucumber
0.2
0.5
1
0.1
0.5
1
0.5
1
5
88.9
95.9
90.5
85.9
91.7
93.4
89.9
92.7
88.8
91.2
88.9
92.5
3.5
3.6
1.2
4.1
3.6
5.4
3.2
2.9
1.9
1.7
2.1
3.1
87.3
96.7
90.5
86.8
90.9
92.9
91.3
90.2
89.4
91.9
89.2
92.9
4.0
3.3
1.6
4.9
3.8
6.5
2.7
3.5
1.3
2.1
3.1
2.9
90.2
93.2
91.7
88.6
91.4
92.7
91.0
91.7
90.4
92.4
90.2
93.7
3.4
2.4
3.0
5.3
6.3
1.1
0.9
4.1
3.1
1.7
2.2
2.0
90.7
94.9
92.0
87.9
91.6
93.1
92.5
92.3
91.4
93.0
90.4
93.2
5.6
3.4
1.9
4.0
4.4
2.5
1.3
3.0
2.9
2.5
2.4
1.9
Muskmelon
Beijing soil
Heilongjiang
soil
0.5
1
5
aRSDr 5 Measured by comparing SD (standard deviation) of the recovery percentage of spiked samples run on the same day.
bRSDR 5 Measured by comparing SD of the recovery percentage of spiked samples for three different days by three operators.
Chirality DOI 10.1002/chir

300
LI ET AL.
Fig. 3. Degradation curves (concentration vs. time curves) of tetraconazole enantiomers in cucumber (A), muskmelon (B), Beijing soil (C), and Heilongjiang
soil (D) (n 5 (1)-R-tetraconazole, ^ 5 (2)-S-tetraconazole); Values represent the average 6 standard deviation. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
enantioselectivity occured. It increased from 0.50 at the first
day to the 0.542 on the 64th day after treatment (Fig. 5). As
for Heilongjiang soil, the results from Table 5 and Figure 4D
showed that the two enantiomers of tetraconazole degraded
at similar rates in 64 days, which indicated that there was
no stereoselectivity on dissipation of rac-tetraconazole in
Heilongjiang soil.
or reduced importance of biological degradation. It was
reported that tetraconazole is slowly degraded by micro-
organisms in soil matrix.22 Thus, the difference of the degra-
dation behavior of tetraconazole among the test soils in this
study may be explained that the different soil types may con-
tain different micro-organisms, which preferentially degrade
one enantiomer.6
Microbial decomposition can play an important role in ste-
reoselective metabolism of many chiral chemicals in soils.6,21
With regard to tetraconazole, the enantioselectivities
observed in Beijing soil suggests a biologically mediated
process. Conversely, the nonenantioselective dissipation in
Heilongjiang soil does not necessarily indicate the absence
Enantioselective degradation of chiral compounds is often
influenced by pH.23,24 According to Table 6, Figures 3 and 4,
the obvious enantioselectivity was found in Beijing soil, the
soil with the higher pH value of 7.82, whereas nonenantiose-
lective degradation was observed in Heilongjiang soil, the soil
with the lower pH value of 5.86. In several published study,
TABLE 6. Degradation equation of tetraconazole enantiomers in cucumber, muskmelon and soila
Test materials
Cucumber
Enantiomers
Degradation equationb
Related coefficient
Half-lives (days)*
(1)-R-tetraconazole
(2)-S-tetraconazole
(1)-R-tetraconazole
(2)-S-tetraconazole
(1)-R-tetraconazole
(2)-S-tetraconazole
(1)-R-tetraconazole
(2)-S-tetraconazole
Ct 5 0.2169e20.1951t
Ct 5 0.2151e20.2398t
Ct 5 0.1587e20.097t
Ct 5 0.1413e20.113t
Ct 5 3.8482e20.0098t
Ct 5 3.8151e20.0075t
Ct 5 4.1465e20.007t
Ct 5 4.1668e20.0068t
r 5 0.9671
r 5 0.9785
r 5 0.9879
r 5 0.9704
r 5 0.9773
r 5 0.9514
r 5 0.9557
r 5 0.9556
3.55c
2.89d
Muskmelon
7.14c
6.13d
Beijing soil
70.71c
92.40d
99.00c
101.91c
Heilongjiang soil
aThe starting point of regressive functions was the maximum value of the enantiomers concentration in cucumber, muskmelon, and soils.
bThe regressive functions were obtained based on the mean value of three replicates.
*Significant differenced (P < 0.05, Student’s paired t-test) are indicated with different alphabets in the same material.
Chirality DOI 10.1002/chir

ENANTIOSELECTIVE DEGRADATION OF TETRACONAZOLE
301
Fig. 4. EF value versus time curves of tetraconazole in cucumber (A), muskmelon (B), Beijing soil (C), and Heilongjiang soil (D). Values represent the aver-
age 6 standard deviation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Fig. 5. Typical chromatograms of R-(1)- and (2)-S-tetraconazole enantiomers in the Beijing soil extracted at Day 0 and Day 64 of incubation. [Color figure can
be viewed in the online issue, which is available at wileyonlinelibrary.com.]
correlations were observed between the stereoselectivity of
CONCLUSIONS
degradation and soil pH.24 Buerge et al. published results
This study showed an effective chiral analysis method of
for the triazole fungicides epoxiconazole and cyproconazole,
fungicide tetraconazole and the proposed method was suc-
cessfully applied to the study of stereoselective behaviors of
indicating a similar correlation between soil pH and enantio-
selectivity as for tetraconazole.
Chirality DOI 10.1002/chir

302
LI ET AL.
10. Khalfallah S, Menkissoglu-Spiroudi U, Constantinidou HA. Dissipation
study of the fungicide tetraconazole in greenhouse-grown cucumbers.
J Agric Food Chem 1998;46:1614–1617.
tetraconazole in cucumber, muskmelon, and soils. It was
observed that the degradation of the two enantiomers of tet-
raconazole in cucumber, muskmelon, and Beijing soil were
enantioselective. The (2)-S-tetraconazole showed a faster
degradation in plants, whereas the (1)-R-tetraconazole
showed a faster degradation in Beijing soil. The results may
help to understand the detailed environmental and biological
behavior of tetraconazole better. However, due to the current
lack of knowledge concerning the bioactivities and toxicities
of individual enantiomer of tetraconazole and the potential
ecotoxicological effects to benefit species, the possible risk
about this chiral fungicide are still not clear. Further studies
are needed to supply the detailed characters and determine
the exact nature of the enantioselective processes of this chi-
ral fungicide. This study showed an evidence of enantioselec-
tive behavior of a chiral pesticide and emphasized the impor-
tance to understand the detailed fate of the enantiomers so
as to make more accurate environment and human health
risk assessment.
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synthesis and biological activity of both enantiomeric forms of tetracona-
zole, a new antifungal triazole. J Agric Food Chem 1991;39:197–201.
12. Carelli A, Farina G, Gozzo F, Merlini L, Kelly SL. Interaction of tetracona-
zole and its enantiomers with cytochrome P450 from Ustilago maydis.
Pestic Sci 1992;35:167–170.
13. Gozzo F, Carelli A, Carzaniga R, Farina G, Arnoldi A, Lamb D, Kelly SL.
Stereoselective interaction of tetraconazole with 14a-demethylase in
fungi. Pestic Biochem Physiol 1995;53:10–22.
14. Konwick BJ, Garrison AW, Avants JK, Fisk AT. Bioaccumulation and
biotransformation of chiral triazole fungicides in rainbow trout (Onco-
rhynchus mykiss). Aquat Toxicol 2006;80:372–381.
15. Pan HJ, Ho WH. Determination of fungicides in water using liquid phase
microextraction and gas chromatography with electron capture detection.
Anal Chim Acta 2004;527:61–67.
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