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Enantioseparation of Imazalil and Monitoring Its Enantioselective Degradation in Apples and Soil using Ultra-high Performance Liquid Chromatography/Tandem Mass Spectrometry Runan Li, Fengshou Dong, Jun Xu, Xingang Liu, Xiaohu Wu, Xinglu Pan, Yan Tao, Zenglong Chen, and Yongquan Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00258 • Publication Date (Web): 06 Apr 2017 Downloaded from http://pubs.acs.org on April 7, 2017
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Enantioseparation of Imazalil and Monitoring Its Enantioselective Degradation
2
in
3
Chromatography/Tandem Mass Spectrometry
Apples
and
Soil
using
Ultra-high
Performance
Liquid
4 5
Runan Li, Fengshou Dong*, Jun Xu, Xingang Liu, Xiaohu Wu, Xinglu Pan, Yan Tao,
6
Zenglong Chen, Yongquan Zheng
7
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant
8
Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, P. R. China
9 10
∗
11
Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of
12
Agricultural Sciences, Beijing, 100193, P. R. China
13
Tel.: +86 10 62815938; fax: +86 10 62815938.
14
E-mail address:
[email protected] (F. Dong).
Correspondence: Prof. Fengshou Dong, State Key Laboratory for Biology of Plant
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ABSTRACT
25
Imazalil is a widely used systemic chiral fungicide that is still being employed as
26
a racemic mixture without distinguishing the difference between enantiomers, which
27
often leads to its inaccurate risk assessment. In this study, a robust and highly
28
sensitive chiral separation method was developed for imazalil enantiomers by
29
ultra-high performance liquid chromatography/tandem mass spectrometry and was
30
further applied to study the degradation dynamics of imazalil enantiomers in apples
31
and field soil at three sites in China. The baseline enantioseparation for imazalil was
32
achieved within 3.5 min on a Lux Cellulose-2 (CCMPC) column with acetonitrile
33
(ACN)/water (65:35, v/v) with a mobile phase at 0.5 mL/min flow rate and a column
34
temperature of 20°C. The limit of quantitation (LOQ) for each enantiomer was less
35
than 0.60 µg/kg, with a baseline resolution of approximately 1.75. The research
36
showed that (S)-(+)-imazalil degraded faster than (R)-(-)-imazalil in Gala apples,
37
whereas (R)-(-)-imazalil preferentially degraded in the Golden Delicious apple. No
38
significant enantioselectivity was observed in OBIR-2T-47 apples and field soils from
39
the three sites. Results of this study provide useful references for risk assessment and
40
the rational use of imazalil in further agricultural produce practice.
41
KEYWORDS: imazalil, enantioselectivity, apple, field soil, ultra-high performance
42
liquid chromatography tandem mass spectrometry (UPLC-MS/MS)
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INTRODUCTION
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Currently, chiral pesticides comprise over 40% of the pesticides in use in China,
48
with that percentage increasing as more complex structures are introduced and
49
utilized.1 Different enantiomers/stereoisomers of a chiral pesticide possess the same
50
physicochemical properties, however, they usually differ in biological properties (e.g.,
51
bioactivity, toxicity, metabolism, and degradation). Thus, understanding the different
52
fate of chiral pesticide enantiomers is essential for environmental risk assessment and
53
the rational application of chiral pesticides. Furthermore, many research results
54
illustrate that the stereoselectivity of pesticides can be affected by various
55
environmental factors, biological species, and uptake pathways. 2-8
56
Imazalil
57
belongs to the imidazole derivatives, has one chiral carbon atom and two enantiomers
58
(Figure 1). Imazalil is a systemic chiral fungicide that inhibits the biosynthesis of
59
ergosterin in fungi by interfering with lanosterol-14-α-demethylase (CYP51,
60
cytochrome P450-14DM), which indirectly leads to fungal cell death.9, 10 It is one of
61
the most widely employed post-harvest fungicides, and its use aids in the prevention
62
of pre-harvest fungal diseases of fruit, vegetables, and various crops. It is also used as
63
a broad-spectrum antimycotic drug in human and veterinary medicine.10, 11 Due to the
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extensive use of imazalil, it poses a potential threat to humans, animals and aquatic
65
environments.12 Therefore, it is essential to evaluate the risks of the two enantiomers
66
of imazalil in terms of its potential enantioselective residues. And the study of
67
pharmacokinetics degradation of imazalil enantiomers could provide significant data
(1-[2-(2,4-dichlorophenyl)-2(2-propenyloxy)ethyl]-1H-imidazole),
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to avoid toxic risks in agricultural products consuming.
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The chiral analysis of imazalil has been conducted by high-performance liquid
70
chromatography (HPLC) and capillary electrophoresis (CE).13-25 HPLC is the most
71
popular and dominant method for separating chiral compounds.26 Imazalil underwent
72
complete separation on Lux Cellulose-2 (CCMPC), Lux Cellulose-3 (CTMB) and
73
CDMPC chiral stationary phases (CSPs) under normal phase (NP) conditions.16, 21
74
The analysis time for imazalil enantiomers under NP conditions usually exceeded 30
75
minutes requiring relatively large amounts of organic solvents. HPLC–MS/MS
76
detection was applied under reverse phase (RP) conditions which has higher
77
sensitivity, precision and specificity than NP-HPLC and HPLC-UV when analyzing
78
complex matrix samples. It was recently reported that imazalil enantiomers were
79
separated completely on un-derivatized β-cyclodextrin column by HPLC–MS/MS and
80
HPLC-UV19, 20, however, the sensitivity of the methods was not satisfactory. In our
81
study, the established method reduced by at least half of the analysis time and the
82
sensitivity was enhanced more than 30-fold compared with the former methods.
83
Imazalil was commonly employed as a racemic mixture until recently. Studies
84
have reported the (S)-(+)-enantiomer to demonstrate stronger fungicidal activity than
85
the (R)-(-)-enantiomer. In recent years, there has been limited research on the
86
enantioselective degradation of imazalil. Previous studies showed that the
87
(R)-(-)-enantiomer had a higher degradation rate than the (S)-(+)-enantiomer in
88
oranges.
89
compartments of aquatic plants, supporting the hypothesis that plants are capable of
18, 25
Enantioselective degradation was also found in various species and
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metabolizing pesticides.20 In contrast, non-enantioselective degradation was shown in
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hydroponic solution studies as well as a study in soil under multiple conditions.14, 20
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There is little concern about chiral determination and degradation on registered crops
93
of imazalil, such as apples. Here, we developed a sensitive, rapid, and high-specificity
94
UPLC-MS/MS method to investigate enantioselective degradation of imazalil in
95
apples and field soil of different geographic locations.
96
MATERIALS AND METHODS
97
Chemicals and Reagents. Racemic imazalil (99.2% purity for each enantiomer, 1:1
98
stereoisomer ratio) was obtained from China Standard Material Center (Beijing,
99
China). Chromatographic grade methanol (MeOH) and acetonitrile (ACN) was
100
purchased from Sigma-Aldrich (Steinheim, Germany). Chromatographic grade
101
ammonium acetate and formic acid were purchased from Tedia (Fairfield, OH, USA)
102
and Thermo Fisher Scientific (Waltham, MA, USA) respectively. Analytical grade
103
sodium chloride (NaCl), anhydrous magnesium sulfate (MgSO4) and ACN were
104
purchased from Beihua Fine-chemicals Co. (Beijing, PRC). Ultra-pure water was
105
obtained from a Milli-Q system (Bedford, MA, USA). Sorbents including Primary
106
secondary amine (PSA, 50 µm), octadecylsilane (C18, 50 µm), graphitized carbon
107
black (GCB, 120-400 mesh) and Florisil (120-400 mesh) were purchased from
108
Bonna-Agela Technologies (Tianjin, PRC). Uracil was purchased from Amresco
109
(Solon, OH, USA).
110
Standard stock solutions (100 mg/L) of rac-imazalil were prepared in pure ACN,
111
which was used as a stock solution to prepare the standard solutions used to obtain the 4
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calibration curves. Standard working solutions and matrix-matched standard solutions
113
at 10, 50, 100, 500, 1000 and 2000 µg/L for imazalil (5, 25, 50, 250, 500 and 1000
114
µg/L of each enantiomer) were serially diluted with pure ACN/water (65:35, v/v) and
115
blank matrix extraction, respectively. All solutions were wrapped with aluminum foil
116
and stored in the refrigerator in the dark at -20°C.
117
Field Trial and Sample Collection. The field trials were conducted according to the
118
guidelines for pesticide residue trials (NY/T 788–2004).28 Three kinds of working
119
areas for apples and soil were chosen in the major apple production areas (Henan,
120
Shandong, and Liaoning Provinces in China). The apple varieties were OBIR-2T-47
121
in Henan, Gala in Shandong, and Golden Delicious in Liaoning. The mean
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temperature was 25°C in Henan, 22°C in Shandong, 25°C in Liaoning during the
123
experiment. The working areas had no history of imazalil application. During the trial
124
period, the application of pesticides which have structures similar to imazalil were
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prohibited. The fields were divided into 30-m2-sized blocks and a buffer zone was set
126
up between plots. At each working area, the imazalil commercial product (20%
127
imazalil emulsion in water) was sprayed at a concentration of 375 mg/kg (1.5 × the
128
recommended dosage) on apple plants and the soil of three plots, which were treated
129
as three replicates in order to avoid random error. Another plot was used as the control
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(without fungicide). Three representative apple samples (approximately 2000 g in
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each sample) from each plot were collected on day 0 (2 h after application), and 1, 2,
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4, 7, 10, 14, 21, and 28 days after treatment. Correspondingly, soil samples
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(approximately 1000 g in each sample) were taken from a depth of 0–10 cm at the 5
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same increasing time intervals. Stones and plant debris were removed manually. All
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samples were put into polyethylene bags and transported to the laboratory. The apple
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samples were rinsed with distilled water to remove exterior impurities, chopped and
137
homogenized in an Ultra-Turrax homogenizer (IKA-Werke, Staufen, Germany). All
138
samples were kept at –20°C and analyzed as soon as possible.
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Sample Preparation. The extraction and purification procedures were based on
140
QuEChERS methodology, which has been widely used due to its advantages of
141
simplicity, effectiveness and flexibility.29, 30 The frozen samples were thawed at room
142
temperature. Aliquots of 10 g homogenized apple and 5 g sampled soil were weighed
143
separately into 50 mL PTFE centrifuge tubes with screw caps. Then, 10 mL ACN was
144
added (5 mL pure water was added additionally for the soil samples). The tubes were
145
shaken vigorously for 10 min in a CK-2000 high-throughput grinder (TH Morgan,
146
Beijing, China), at 1350 min−1. 1 g NaCl and 4 g anhydrous MgSO4 were added to the
147
mixture followed by an additional shaking for 5 min. Samples were centrifuged for 5
148
min at 2588×g followed by the transfer of the 1.5mL ACN supernatant to a
149
micro-centrifuge tube containing 50 mg PSA and 150 mg anhydrous MgSO4 (The
150
optimization method of sample extraction and purification was described in
151
Supplementary Information). The sample was vortexed for 1 minute and centrifuged
152
for 5 min at 2400×g. The resulting supernatant was filtered via a 0.22-µm nylon
153
syringe filter to prepare for UPLC-MS/MS analysis.
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Apparatus and Chromatographic Conditions. The imazalil enantiomers were
155
chromatographically separated using a Waters ACQUITY UPLC system (Milford, 6
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with
a
Lux
Cellulose-2
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MA,
(cellulose
157
tris-(3-chloro-4-methylphenylcarbamate) (CCMPC) column (150 mm×2.00 mm, 3µm
158
particle size) which was purchased from Phenomenex (CA, USA). The mobile phase:
159
solvent A (HPLC-grade ACN): solvent B (ultra-pure water) = 65:35(v/v), which was
160
pumped at a constant flow rate of 0.5 mL/min for 5 min and an injection volume of 5
161
µL. The temperatures of the column oven and sample vial holder were held at 20°C
162
and 5°C respectively. The detection of imazalil enantiomers were achieved using a
163
Xevo-triple quadrupole (Xevo-TQD) mass spectrometer (Waters Corp., Milford, MA,
164
USA) equipped with an electrospray ionization (ESI) source, operating in the positive
165
mode. The source parameters were set as follows: 3.0 kV capillary voltage, 150°C
166
source temperature, and 500°C desolvation temperature. The nebulizer gas was 99.95%
167
nitrogen and the collision gas was 99.99% argon at a pressure of 2×10−3 mbar in the
168
T-Wave cell. The flow rates of the cone gas and desolvation gas were held at 50 L/h
169
flow and 1000 L/h, respectively.
170
Multiple reaction monitoring (MRM) was applied to MS analyses of imazalil
171
with a dwell time of 163 ms per ion pair. The concrete MS/MS parameters were
172
optimized as follows: the cone voltage of imazalil was set to 30 V, a m/z 297.1 was
173
selected as the precursor ion, m/z 159.0 and 69.0 were chosen as the quantitative and
174
qualitative ions when collision energy was set to 23 V and 19 V, respectively.
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Masslynx NT v.4.1 (Waters Corp.) software was used to collect and analyze the
176
obtained data.
177
Method Validation and Calculation. The method was validated according to the 7
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OECD guidance document by following criteria: linearity, stability, sensitivity
179
(including limit of detection (LOD) and the limit of quantitation (LOQ)), accuracy,
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precision and matrix effect.31 The blank samples (apple, soil, ACN) were analyzed to
181
verify the absence of interfering substances with approximate retention times (RT) of
182
the analytes. The evaluation of the linearity of the analytical curves was performed by
183
analyzing standard working solutions and matrix-matched standard solutions in
184
triplicate at six concentrations, ranging from 10 to 2000 µg/L. The matrix-induced
185
signal suppression/enhancement (SSE) was evaluated by the slope ratio of the
186
matrix-matched
187
matrix-dependent LOD and LOQ are defined as the concentrations with a
188
signal-to-noise (S/N) ratio of 3 and 10 respectively, which were estimated from the
189
chromatogram in accordance with the spiked apple and soil samples at the lowest
190
concentration. The recovery assays were conducted to investigate the accuracy and
191
precision of the method. Five replicates of the spiked samples (soil and apple) at three
192
different levels (20, 200, 2000 µg/kg) were prepared on three different days.
calibration
curve
to
the
standard
working
curve.
The
193
The samples were prepared using the procedures in the Sample Preparation
194
section. The precision was determined by the intra-day and inter-day assays which
195
was expressed as the relative standard deviation (RSD). The stability of the stock
196
solutions (in the pure ACN/water (65:35, v/v) solvent and in the matrix) and spiked
197
samples were tested monthly by the injection of a newly prepared working solution
198
and all of the samples used in the stability tests were stored at -20 °C. The results of
199
the stability test samples were compared with statistics obtained from the newly 8
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prepared samples using Student’s paired t-test at 95% probability.
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Data Calculation and Analysis. The separation parameters of the imazalil
202
enantiomers including the retention factor (k’), the selectivity factor (α), the resolution
203
(Rs) and the enantiomeric fraction (EF) as measurements of enantioselectivity of
204
degradation were calculated as follows32: k' =
t-t0 t0
(1)
α = k2 ⁄k1 Rs = 1.177× EF =
(2) t2 -t1 w1 ⁄2+ w2 ⁄2
(3)
A+
A+ +A-
(4)
205
These are very standard equations used for evaluating chromatographic conditions.
206
Where t is the retention time (RT) and t0 (t0 = 0.79 min, determined using uracil)
207
under the chromatographic conditions mentioned above, k1 and k2 are retention factors
208
of the first and second eluted enantiomer separately, w/2 means the peak width at half
209
height, and A+ and A- are equivalent to the concentrations of the (+) and (-)
210
enantiomers. The EF values range from 0 to 1 and EF=0.5 means the racemate. In
211
addition, the degradation rate constants (K) and half-life (T1/2) of the imazalil
212
enantiomers in apples and soil were estimated according to the first-order kinetic
213
equations32:
214 215
C = C0 e-Kt
(5)
T1⁄2 = ln2⁄K= 0.693⁄K
(6)
where C0 and C are the concentrations of the enantiomers at time 0 and t separately. The Van’t Hoff equations were used to calculate the thermodynamic parameters 9
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to
elucidate
the
thermodynamic
effects
and
recognition
217
enantioseparation. These equations are shown as follows32:
mechanism
∆H° ∆S° + +lnϕ RT R ∆∆H° ∆∆S° lnα = + R RT
lnk = -
of
(7) (8)
218
where ∆H° and ∆S° are the changes in standard enthalpy and entropy of the analyte
219
between the mobile and stationary phase. ∆∆H° and ∆∆S° are the differences
220
∆H°2-∆H°1 and ∆S°2-∆S°1, respectively. R is the ideal gas constant (8.314
221
J·mol-1·K-1), T is absolute temperature, and ϕ is the phase ratio. Linear equations of
222
lnk versus 1/T and lnα versus 1/T were obtained. The intercepts were ∆S°/R+lnϕ and
223
∆∆S°/R, respectively.
224
Statistical analysis was performed using SAS (version 9.2, SAS Institute, Beijing,
225
China), the difference were considered statistically significant when P value was
0.05, Student’s paired t-test) between
338
newly prepared samples and stability test samples under the pure ACN/water (65:35,
339
v/v) solvent and matrix storage. Imazalil standard solutions were injected five times to
340
examine the stability of measured values of EF and the EF values were 0.500±0.003
341
(n = 5).
342
Practice Application on Enantioselective Degradation of Imazalil in Apple and
343
Soil. Degradation in apple. The concentrations of the two enantiomers gradually
344
decreased in apples after foliage spraying of 20% imazalil emulsion in water (Figure
345
S3). The degradation kinetics equations generally followed first–order kinetics 15
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(R2 = 0.6335-0.8749), as shown in Table 3. The enantiomeric fractions (EFs) of
347
imazalil enantiomers in apples on three sites at different days were also calculated
348
(Figure 4). The EF values initially range from 0.488 to 0.502 at 2 h after rac-imazalil
349
was sprayed on apple trees in three sites and there was no significant difference
350
compared with EF = 0.5 (P > 0.05, one sample t-test for mean).
351
Different degradation trends of the two enantiomers of imazalil were observed in
352
different sites. The half-life (T1/2) is an important indicator of pesticide efficacy and
353
pollution which was calculated and evaluated for the enantioselective degradation of
354
the two enantiomers of imazalil.36 The half-lives of (S)-(+)-imazalil and
355
(R)-(-)-imazalil were 5.06±0.24 days and 5.08±0.17 days in Henan province,
356
7.17±0.09 days and 7.51±0.05 days in Shandong province, and 12.20±0.18 days and
357
11.75±0.20 days in Liaoning province, respectively. In Henan province, with EF
358
values from 0.488 (after 2 h) to 0.487 (after 21 day), the difference between
359
(S)-(+)-imazalil and (R)-(-)-imazalil was not statistically significant by comparing the
360
T1/2 between enantiomer pair of three replicates (P > 0.05, Student’s paired t-test). In
361
Shandong province, the EF values changed from 0.498 at the beginning to 0.466 on
362
day 28, and the half-lives of degradation between (S)-(+)-imazalil (7.17 day) and
363
(R)-(-)imazalil (7.51 day) were significantly different (P < 0.05, Student’s paired
364
t-test), whereas in Liaoning province, the EF value gradually declined from 0.502
365
(after 2 h) to 0.520 (after 28 day), and the increase in T1/2 was significant (P < 0.05,
366
Student’s paired t-test). The results suggest that (S)-(+)-imazalil preferentially
367
undergoes degradation as compared to (R)-(-)-imazalil in Gala apples in Shandong 16
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province. However, the opposite was observed in the Golden Delicious apple in
369
Liaoning province. No significant difference was observed in enantioselective
370
degradation of the OBIR-2T-47 apple in Henan province. The present work is the first
371
to demonstrate different enantioselective trends of imazalil enantiomers in apples of
372
different varieties at different geographic locations, which was ignored in previous
373
studies. For instance, the (R)-(-)-imazalil enantiomer was found to be degraded more
374
quickly than the (S)-(+)-imazalil enantiomer in oranges and enantioselective
375
degradation was also found in different species and compartments of aquatic plants18,
376
20,
377
geographic locations had not previously been reported.
25
,
but
the
difference
between
samples
of
different
varieties
and
378
As no enantioselectivity occurred in the different types of field soil in different
379
geographic locations (described in the next part), the physicochemical properties of
380
field soils appear to have no effect on the enantioselective degradation of imazalil in
381
apples. We hypothesize that the three trends of enantioselective degradation of
382
imazalil in apples of three sites may be caused by the different apple varieties. As
383
reported in several studies, different trends of enantioselective degradation of
384
pesticides occurred in different species of plants under the same application mode,
385
and
386
metabolism may affect the enantioselectivity of pesticides. Furthermore, the plants’
387
functional enzymes play an important role in enantioselective transformation of chiral
388
pesticides.37 Therefore, future research should be conducted to investigate these
389
factors which exert an influence on chiral recognition and degradation of imazalil
many factors
include enantioselective
biotransformation,
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enantiomers in different varieties of apples. In addition, other possible reasons for
391
different tendency of enantioselective degradation in apples were environmental
392
factors, for example temperature, wind speed, sunlight intensity, moisture, etc.
393
However, the mean temperatures of three sites have no significant difference during
394
the experiment phase, other factors should also be explored during future study.
395
Degradation in soil. The concentration of imazalil enantiomers in soil decreased with
396
elapsed time under the same test conditions. The EF values of the three provinces
397
were close to 0.5 (P > 0.05, one sample t-test for mean) at 2 h after application of
398
rac-imazalil to soil and the degradation equations of the two enantiomers followed
399
first–order kinetics (R2 = 0.6956-0.8221) which are listed in Table 3. Half-lives of
400
(S)-(+)-imazalil were 19.25±1.34 days, 10.37±0.13 days and 21.86±0.80 days in
401
Henan, Shandong and Liaoning provinces, respectively. Values for (R)-(-)-imazalil
402
were 19.97±1.38 days, 10.50±0.15 days and 21.66±0.79 days in the above three
403
provinces, respectively. There appears to be no significant difference in degradation
404
half-lives between (S)-(+)-imazalil and (R)-(-)-imazalil using Student’s paired t-test at
405
95% probability. Moreover, the EF values were all nearly at 0.5 at different days after
406
treatment (Figure 4). The result of non-enantioselective degradation of imazalil
407
enantiomers in field soils was
408
laboratory conditions.14 Similarly, prior research showed that EF values of imazalil
409
enantiomers in hydroponic solutions remained stable throughout an incubation period
410
of 24 days.20 In addition, the degradation rates of imazalil enantiomers in Shandong,
411
Henan and Liaoning province were performed in descending order. The degradation
similar to that of a previous study of soils under
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dynamics of pesticides in soil, influenced by many factors, would provide significant
413
references towards their environmental risk assessment and rational application. The
414
mean temperatures of three sites during the experiment had no significant difference
415
which eliminated the temperature effect. Previous research showed that half-lives was
416
closely related to physicochemical properties of soils including organic matter, pH,
417
soil texture, etc.38 In our research, the half-lives of imazalil in soil decreased when the
418
organic matter content of the soil increased, indicating that the organic matter played
419
a crucial role in degradation dynamics of imazalil. The higher organic matter content
420
may be lead to increasing of microbial population in soil, therefore the dissipation rate
421
of imazalil accelerated when organic matter content increased.39 The physicochemical
422
properties of field soils from three provinces is shown in Table 4. In addition, some
423
studies showed that soil moisture, soil sterilization, light, atmospheric CO2 level could
424
affect the dissipation of pesticides39, 40, and these factors should be evaluated in future
425
study.
426
In this study, a rapid and sensitive detection method of imazalil enantiomers in
427
apples and soil was established using UPLC-MS/MS. The method was applied by
428
analyzing the degradation of (S)-(+)-imazalil and (R)-(-)-imazalil in apples and field
429
soil. Resulted in no clear trend of enantioselectivity of fungicide imazalil, exhibiting
430
various trends in different varieties of apples at various sites of growth. The
431
degradation process showed no enantioselectivity in field soil from these three sites.
432
Moreover, T1/2 of imazalil enantiomers were related to the physicochemical properties
433
of field soils. The results could provide information about enantioselective behaviors 19
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and risk assessment of the chiral fungicide imazalil.
435
AUTHOR INFORMATION
436
Corresponding Author
437
*Tel.: +86 10 62815938. Fax: +86 10 62815938.
438
E-mail:
[email protected].
439
Notes
440
The authors declare no competing financial interest.
441
ABBREVIATIONS USED
442
UPLC-MS/MS,
443
spectrometry; HPLC, high-performance liquid chromatography; CE, capillary
444
electrophoresis; CSP, chiral stationary phase; NP, normal phase; RP, reverse phase;
445
MeOH, methanol; ACN, acetonitrile; PSA, primary secondary amine; C18,
446
octadecylsilane; GCB, graphitized carbon black; RCF, relative centrifugal force; ESI,
447
electrospray ionization; MRM, multiple-reaction monitoring; LOD, limit of detection;
448
LOQ,
449
suppression/enhancement; RSD, relative standard deviation; Rs, resolution; EF,
450
enantiomer fraction; T1/2, half-life; OR, optical rotations.
451
ACKNOWLEDGMENT
452
This work was financially supported by the National Key Research and Development
453
Program of China (2016YFD0200202). We would like to thank Dr. Michelle
454
McGinnis for language help in writing.
455
SUPPORTING INFORMATION
limit
ultra-high-performance
of
quantification;
liquid
RT,
chromatography-tandem
retention
20
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times;
SSE,
mass
signal
Journal of Agricultural and Food Chemistry
Page 22 of 37
456
Supporting Information Available: [Table S1-S2; Figure S1-S3.] This material is
457
available free of charge via the Internet at http://pubs.acs.org.
458 459
REFERENCES
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573 574 575 576
FIGURE CAPTIONS
577 578
Figure 1. The chemical structure and chromatogram (A) and optical rotation (B) of
579
imazalil enantiomers.
580
Figure 2. Influence of different factors on chiral separation of imazalil enantiomers:
581
(A1-B3) effects of three CSPs (1: Lux™ Amylose-2, 2: Lux Cellulose-1, 3: Lux
582
Cellulose-2) and two kinds of mobile phases (A: ACN, B: MeOH) on the
583
enantioseparation of imazalil; (C1-D5) comparison of different flow rates (0.2-0.5
584
mL/min) and column temperature (20-45°C) on the chiral separation of imazalil,
585
concerning RT, α and Rs.
586
Figure 3. Van’t Hoff plots (lnk and lnα versus 1/T) and equations for chiral separation
587
of imazalil enantiomers on a Lux Cellulose-2 column. 26
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588
Figure 4. EF values versus time plots of imazalil enantiomers in apple and soil in
589
three provinces.
590 591 592
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TABLES Table 1. Linear regression parameters of calibration curves and matrix effect of imazalil enantiomers. compounds (S)-(+)-imazalil
(R)-(-)-imazalil a
Matrix
regression equation
R2
slope ratioa
matrix effectb (%)
LODs (µg/kg)
LOQs (µg/kg)
solvent
y = 45.70x + 1495.21
0.9941
-
-
0.12±0.01
0.39±0.03
apple
y = 48.34x + 1015.00
0.9994
1.06
5.76
0.16±0.01
0.53±0.03
soil
y = 45.33x + 2288.25
0.9944
0.99
-0.81
0.16±0.01
0.53±0.03
solvent
y = 47.24x + 1,336.57
0.9957
-
-
0.13±0.01
0.43±0.04
apple
y = 49.00x + 806.33
0.9983
1.04
3.72
0.17±0.01
0.58±0.02
soil
y = 47.28x + 1919.24
0.9962
1.00
0.07
0.18±0.01
0.60±0.04
Slope ratio=matrix/ACN. Matrix effectb (%) = ((slope matrix-slope solvent)/ slope solvent) × 100
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Table 2. The recovery and RSDs of imazalil in apple and soil at different spiked levels. intra-day (n = 5) day 1
spiked compound
matrix
level
average
(µg/kg)
recoveries (%)
20 apple (S)-(+)-imazalil soil
apple (R)-(-)-imazalil soil a
76.4
day 2 RSDa (%) 1.0
average recoveries
inter-day (n = 15)
day 3 RSDa
(%) 78.6
(%) 3.9
average recoveries (%) 77.1
RSDa
RSDb
(%)
(%)
6.0
4.1
200
86.3
3.2
91.1
2.8
84.7
1.8
4.1
2000
85.7
1.4
85.2
2.0
86.7
2.1
1.9
20
83.1
5.8
86.1
3.4
88.1
2.9
4.6
200
89.5
1.2
92.7
3.4
87.1
3.0
3.7
2000
83.6
0.8
93.8
1.2
94.8
2.6
6.0
20
75.2
1.5
79.6
4.3
76.8
4.6
4.3
200
85.8
2.3
93.9
2.0
84.0
1.4
5.4
2000
84.5
1.6
85.7
2.9
86.3
2.6
2.5
20
83.4
6.2
81.3
1.6
87.6
3.9
5.2
200
89.5
0.8
88.7
4.0
85.0
3.7
3.7
2000
82.9
0.9
91.6
1.2
92.7
2.1
5.3
intra-day RSD (n = 5). b inter-day RSD (n = 15).
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Table 3. Degradation equations of imazalil enantiomers in apple and soil in different provinces of China. matrix
province Henan
apple
Shandong Liaoning Henan
soil
Shandong Liaoning
enantiomers
degradation equation
R2
T1/2 (days)a
Pb
(S)-(+)-imazalil
Ct = 79.311e-0.1369t
0.8749
5.06±0.24
1.0000
(R)-(-)-imazalil
Ct = 82.197e
-0.1364t
0.8671
5.08±0.17
(S)-(+)-imazalil
Ct = 308.14e-0.0967t
0.8510
7.17±0.09
(R)-(-)-imazalil
Ct = 313.26e-0.0923t
0.8446
7.51±0.05
(S)-(+)-imazalil
-0.560t
0.6335
12.20±0.18
Ct = 583.52e
-0.0364t
0.6509
11.75±0.20
Ct = 126.86e
-0.0360t
0.7084
19.25±1.34
(R)-(-)-imazalil
Ct = 125.55e
-0.0347t
0.6956
19.97±1.38
(S)-(+)-imazalil
Ct = 572.33e-0.0668t
0.8221
10.37±0.13
(R)-(-)-imazalil
Ct = 559.09e
-0.0660t
0.8140
10.50±0.15
(S)-(+)-imazalil
Ct = 818.88e
-0.0317t
0.7446
21.86±0.80
(R)-(-)-imazalil
Ct = 827.31e-0.0320t
0.7428
21.66±0.79
(R)-(-)-imazalil (S)-(+)-imazalil
Ct = 591.55e
0.0071c 0.0131c 0.2616 0.9024 0.1100
a
Values refer to the means±STDEVs (n = 3).
b
P values from degradation half-lives (T1/2) between (S)-(+)-imazalil and (R)-(-)-imazalil using Student’s paired t-test at 95% probability.
c
Statistical significant difference with P < 0.05.
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Table 4. Physicochemical properties of field soils from three provinces. soil type
site
pH
brown loam
Jiyuan, Henan
brown loam
Weifang, Shandong
sandy loam
Xingcheng, Liaoning
soil texture
organic matter (g/kg)
sand (%)
silt (%)
clay (%)
7.6
16.0
14.8
68.9
16.3
6.8
20.0
37.5
52.6
9.9
6.7
13.2
26.5
62.1
11.4
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GRAPHIC FOR TABLE OF CONTENTS
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Figure 1. The chemical structure and chromatogram (A) and optical rotation (B) of imazalil enantiomers. 141x115mm (300 x 300 DPI)
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Figure 2. Influence of different factors on chiral separation of imazalil enantiomers: (A1-B3) effects of three CSPs (1: Lux™ Amylose-2, 2: Lux Cellulose-1, 3: Lux Cellulose-2) and two kinds of mobile phases (A: ACN, B: MeOH) on the enantioseparation of imazalil; (C1-D5) comparison of different flow rates (0.2-0.5 mL/min) and column temperature (20-45°C) on the chiral separation of imazalil, concerning RT, α and Rs. 185x200mm (300 x 300 DPI)
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Figure 3. Van’t Hoff plots (lnk and lnα versus 1/T) and equations for chiral separation of imazalil enantiomers on a Lux Cellulose-2 column. 288x201mm (300 x 300 DPI)
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Figure 4. EF values versus time plots of imazalil enantiomers in apple and soil in three provinces. 163x143mm (300 x 300 DPI)
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