Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Enantioseparation of Tefluthrin via a Box-Behnken Design and Its Stereoselective Degradation in Soil

Article abstract of DOI:10.1021/acs.jafc.9b04888

A simple and eco-friendly dispersive solid-phase extraction method coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the determination of the chiral pesticide tefluthrin in food and environmental samples. The response surface methodology was applied to optimize separation conditions. The elution order of tefluthrin enantiomers was Z-cis-(1S,3S)-(-)-tefluthrin and Z-cis-(1R,3R)-(+)-tefluthrin on a Lux Cellulose-1 chiral column was identified via a polarimeter and vibrating circular dichroism. The average recoveries in five matrices ranged from 76.9 to 107.6%, with intraday relative standard deviations (RSDs) less than 15.6% and interday RSDs less than 12.5% for two enantiomers. The enantioselective degradation was investigated via laboratory incubation experiments. Slightly enantioselective degradation was observed under aerobic conditions; (1S,3S)-tefluthrin degraded preferentially with the enantiomer fraction value of 0.57 at 120 days of incubation. No remarkable enantioselective degradation was observed under anaerobic and sterile conditions. It was the first time that pyrethroid pesticides were determined at the enantiomer levels via UPLC-MS/MS. This novel method was successfully applied for the enantioselective analysis of tefluthrin enantiomers in authentic samples, indicating its efficacy in investigating the environmental stereochemistry of tefluthrin in the food web and environment. It is of crucial importance to improve risk assessment and regulation of chiral pesticides in an agricultural system.

Full text of DOI:10.1021/acs.jafc.9b04888

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Agricultural and Environmental Chemistry
A novel LC-MS/MS method for enantioseparation tefluthrin via
Box-Behnken Design and its stereoselective degradation in soil
Yong Wen, Zhen Wang, Yingying Gao, Xuejun Zhao, Beibei Gao,
Zhaoxian Zhang, Lianshan Li, Zongzhe He, and MingHua Wang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04888 • Publication Date (Web): 26 Sep 2019
Downloaded from pubs.acs.org on September 29, 2019
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Page 1 of 33
Journal of Agricultural and Food Chemistry
1 A novel LC-MS/MS method for enantioseparation
2 tefluthrin via Box-Behnken Design and its
3 stereoselective degradation in soil
4
5
6
7
8
9
Yong Wen, Zhen Wang, Yingying Gao, Xuejun Zhao, Beibei Gao, Zhaoxian Zhang,
Lianshan Li, Zongzhe He, Minghua Wang*
Department of Pesticide Science, College of Plant Protection, State & Local Joint
Engineering Research Center of Green Pesticide Invention and Application, Nanjing
10 Agricultural University, Nanjing 210095, China.
11
12 *Corresponding author: M. Wang,
13 Address: College of Plant Protection, Nanjing Agricultural University, No. 1
14
Weigang Road, Nanjing, Jiangsu province, 210095, China.
15 E-mail: wangmha@njau.edu.cn
16 Tel: +86 25 84395479
17
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18 Abstract
19 A simple and eco-friendly dispersive solid-phase extraction (d-SPE) method coupled
20 with ultra-high-performance liquid chromatography tandem triple quadrupole mass
21 spectrometry (UPLC-MS/MS) was developed for the determination of the chiral
22 pesticide tefluthrin in food and environmental samples. The response surface
23 methodology (RSM) was applied to optimize separation conditions. The elution order
24 of
tefluthrin
enantiomers
was
Z-Cis-(1S,3S)-(-)-tefluthrin,
25 Z-Cis-(1R,3R)-(+)-tefluthrin on a Lux Cellulose-1 chiral column was identified via
26 polarimeter and vibrating circular dichroism (VCD). The average recoveries in five
27 matrices ranged from 76.9-107.6%, with intraday relative standard deviations (RSDs)
28 less than 15.6% and interday RSDs less than 12.5% for two enantiomers. The
29 enantioselective degradation was investigated via laboratory incubation experiments.
30 Slightly enantioselective degradation was observed under aerobic conditions:
31 (1S,3S)-tefluthrin degradation preferentially with the enantiomer fraction (EF) value
32 0.57 at 120 d of incubation. No remarkable enantioselective degradation was observed
33 under anaerobic and sterile conditions. It was the first time that pyrethroid pesticides
34 were determined on the enantiomers levels via UPLC-MS/MS. This novel method
35 was successfully applied for the enantioselective analysis of tefluthrin enantiomers in
36 authentic samples indicating its efficacy in investigating the environmental
37 stereochemistry of tefluthrin in food web and environment. It is of crucial importance
38 to improve risk assessment and regulation of chiral pesticides in agricultural system.
39 Keywords
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40 Enantioseparation, Tefluthrin, Absolute configuration, Response surface methodology,
41 Enantioselective degradation
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42 Introduction
43 It is commonly assumed that the significance of molecular chirality is widely
44 recognized and discriminated in nature and artificial systems 1, 2. Synthetic pyrethroids
45 (SPs) are designed based on the structures of the pyrethrins, which are natural
46 insecticides isolated from chrysanthemum flowers. SPs contain a special chiral
47 structure and usually have two or more enantiomers; for example, bifenthrin should
48 have eight enantiomers, but it only contains two isomers in the actual production
49 process 3-5. Research indicated that different enantiomers of chiral pesticides
50 possessed the same physico-chemical properties but showed different characteristics
51 in biology and toxicology behaviors 6-8. Previous studies showed that
52 lambda-cyhalothrin underwent enantioselective biodegradation in lizards and
53 marine-derived fungi 9-12. The behavior of SPs in vivo and in vitro might provide
54 available evidence to better evaluate the environmental risk.
55
Tefluthrin (Figure 1), 2,3,5,6-tetrafluoro-4-methylbenzyl (1RS,3RS)-3-[(Z)-2-
56 chloro-3,3,3-trifluoroprop-1-enyl]-2,2 dimethylcyclopropanecarboxylate, is a broad
57 spectrum SPs insecticide that is a Na+ ion channel modulator that holds the axon of a
58 neuron open, resulting in paralysis and death. Tefluthrin has been wildly used to
59 control underground pests, lepidopteran pests, and coleopteran and health pests on
60 maize, cotton, and in the house. At same time, some countries and organizations have
61 set maximum residual limits (MRLs), for example, almost all fruits and vegetables
62 must be inferior to 0.05 mg/Kg in European Union. Furthermore, the MRL was set 0.5
63 mg/kg in kale and lettuce by Japan. Tefluthrin residue analytical methods focused on
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64 animal fat, children’s food and beeswax have been established 13-16. Tefluthrin
65 contains two chiral carbon atoms, double bond and cyclopropane; theoretically, there
66 are eight stereoisomers, but its main ingredients in production are Z-, cis-isomers that
67 only have two enantiomers. Until now, racemic tefluthrin was widely produced and
68 sold on the market. There was no report about its toxicity, bioactivity or
69 environmental behavior at enantiomer level. Therefore, it is necessary to develop a
70 reliable, rapid, and sensitive chiral analytical method to thoroughly understand
71 enantioselective behavior, which can provide accurate evidence of risk assessment.
72
Optimization of instrument conditions by traditional methods, in which one
73 factor is changed while other factors are kept constant, is monotonous,
74 time-consuming, and expensive, especially when many variables must be considered
75 18. Response surface methodology (RSM) has been widely used to design
76 optimization programs, build mathematical models, determine effective factors, study
77 interactions, and search for best conditions on the field of chemical technology,
78 biological pharmacy and medical and health work 19-21; it dismantles the barriers of
79 single-factor optimization. Box-Behnken design (BBD) was suitable for experimental
80 design of factors were less than 5 and levels were less than 3. Furthermore, number of
81 trials of BBD was less than Central Composite design (CCD) based on the same
82 factors. BBD was recommended owing to its economical advantage.
83
Soil play an important role in receiving and degrading organic pollutions. In this
84 study, the main objective was to develop an enantiomer analytical method of chiral
85 tefluthrin, and determine the enantioselective environmental behavior in soil. The
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86 established method was efficient, sensitive and accurate to determine tefluthrin in
87 food, vegetables and environmental matrices using UPLC-MS/MS with a Lux
88 Cellulose-1 column. RSM was used to optimized enantioseparation experiments
89 variables. The absolute configuration of tefluthrin enantiomers was confirmed by
90 comparing experimental and calculated the vibrating circular dichroism (VCD)
91 spectra. The extraction process was based on the QuEChERS (Quick, Easy, Cheap,
92 Effective, Rugged, Safe) method. This was the first time that an effective chiral
93 separation method has been established for analysis of tefluthrin enantiomers in fruit,
94 vegetables, and environmental samples, and the degradation was performed in soil
95 under different conditions on the enantiomers levels. This study could provide
96 scientific guidance for risk assessment and a basis for scientifically and rationally
97 studying the difference in enantiomers.
98 Experimental
99 Reagents and Materials.
100 The tefluthrin (purity≥95.5%) was purchased from JinyuChem Co., Ltd. (Weifang,
101 China). A pair of isomers (purity≥98%) was prepared by the Chiralway Biotech Co.,
102 Ltd. (Shanghai, China). HPLC grade methanol and acetonitrile were acquired from
103 Merck (Darmstadt, Germany), and ultrapure water was purchased from Hangzhou
104 Wahaha Group Co., Ltd. (Hangzhou, China). MS-grade ammonium formate was
105 acquired from CNW Technologies Inc. (California, USA). Sorbents including primary
106 secondary amine (PSA, 40-63 μm), C18 (40-63 μm), and graphitized carbon black
107 (GCB, 38-128 μm) were obtained from ANPEL Laboratory Technologies Inc.
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108 (Shanghai, China). All other chemical reagents were purchased from commercial
109 sources. Standard solutions of racemic tefluthrin and enantiomers were prepared in
110 HPLC-grade methanol and stored in the dark at 4 °C.
111 Chiral separation and MS conditions
112 The enantioseparation and analysis of tefluthrin were performed on a Waters
113 ACQUITY UPLCTM system (Milford, MA, USA) tandem triple quadrupole mass
114 spectrometer (Waters Corp., Milford, MA, USA) with an electrospray ionization (ESI)
115 source using Lux Cellulose-1 chiral column (150 mm × 4.6 mm i.d., 3 μm,
116 Phenomenex, USA). The mixture of solvent A (5 mM ammonium formate in
117 methanol) and solvent B (5 mM ammonium formate in water) in 83:17 (v/v) used as
118 mobile phase with flow rate 0.23 mL min-1 at 30 °C.
119
MS analyses were carried out in multiple reaction monitoring (MRM) mode
120 employing ESI in positive mode with capillary voltage 3.6 kV; source temperature
121 and desolvation temperature were 120 °C and 300 °C, respectively. The 99.95%
122 nitrogen was used as cone gas flow at 50 L h-1 and desolvation gas flow at 800 L h-1.
123 The 99.99% argon was used as collision gas was with a pressure of 2 × 10−3 mbar in
124 the T-Wave cell. The characteristic product ions arising from ammonium adduct
125 [M+NH4]+ (m/z 436 > 177) was extracted for quantitative and qualitative
126 determination of tefluthrin enantiomers with the cone voltage and collision energy set
127 as 20 v and 34 v, respectively. The Masslynx NT v.4.2 (Waters, USA) software was
128 used to collect and analyze the data.
129 Separation condition optimization
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130 To study the effect of single factors on the chiral separation, flow rate, temperature,
131 and mobile composition were tested individually while keeping other instrument
132 conditions at a constant level. The separation parameters, including capacity factor (k),
133 separation factor (α), and resolution (Rs), were calculated to evaluate the effect of
134 enantioseparation under different conditions. The enthalpy (ΔΔH°) and entropy (ΔΔS°)
135 variation between enantiomers were also calculated using the following Van’t Hoff
136 equations.
137 ln k = −ΔH°/RT + ΔS°/R + ln Φ
138 ln α = −ΔΔH°/RT + ΔΔS°/R
(1)
(2)
139 An RSM design was carried out to explain interaction effects between single factors
140 and multifactors on enantioseparation and define an optimal formulation. It is well
141 known that BBD gets the upper hand over the traditional “one-factor-at-a-time
142 optimization experiments”. In this study, BBD was used to optimize the separation
143 conditions for Rs and retention time of tefluthrin enantiomers using the Design-expert
144 8.0.6 trial software. Based on the single factor experimental results, a BBD with three
145 factors and three levels (including five replicates at center point) was carried out. A
146 total of 17 randomized experiences were run in quintuplicate in the central point (See
147 the Supporting Information Table S1). The process can be described by the quadratic
148 model using equation:
149 Y=b0 + b1X1 + b2X2 + b3X3 + b11ꢀꢁꢂ + b22ꢀꢂꢂ + b33ꢀꢃꢂ+ b12X1X2 + b13X1X3 + b23X2X3 (3)
150 where Y stands for the predicted response, bn are quadratic coefficients, and X1, X2, X3
151 are the studied variables.
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152 Determination of specific optical rotation.
153 The specific optical rotation of tefluthrin enantiomers was measured by Autopol Ⅳ
154 polarimeter (Rudolph Research Analytical., NJ USA) at 589 nm. Each enantiomer
155 dissolved in methanol with a concentration of 0.01g mL-1 was determined at 20 °C.
156 Confirmation of absolute configuration
157 Experimental infrared spectroscopy and VCD spectroscopy for tefluthrin enantiomers
158 were performed using Bruker FTIR Vertex50 spectrometer equipped with a Bruker
159 PMA50 VCD module (Karlsruhe, German) at room temperature. The spectra were
160 collected with a total acquisition time of 5 h (5 times × 1 h) using a path length of 0.1
161 mm potassium bromide plates with a resolution of 4 cm-1.
162
Geometry optimizations and IR and VCD spectra calculations for tefluthrin
163 enantiomers were performed via density functional theory using the B3LYP function
164 with the 6-311+G (2d,p) basis set 17,18. Frequency calculations were carried out at the
165 same level to confirm that the stationary points are the minima with zero imaginary
166 frequency. The different conformations of tefluthrin were searched on the
167 conformation bearing the lowest free energy, and the VCD calculation was performed.
168 To create the final spectra, each intensity of the line spectra obtained by the DFT
169 calculations were convoluted with a Lorentzian band shape with half-width at
170 half-height of 4 cm−1. All calculations were performed with the Gaussian 09 software
19
171 package . The absolute configuration of a pair of enantiomers was confirmed by
172 comparing the similarity of the experimental VCD spectra and calculated VCD
173 spectra.
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174 Chiral Stability
175 To exclude the epimerization occurrence of chiral SPs insecticides in acetonitrile,
176 methanol, and water, the enantiomer standard solution of 1 mg L-1 in water, methanol,
177 and acetonitrile was stored in 4 °C and 25 °C for 180 d. The samples were collected at
178 0, 1, 3, 7, 14, 30, 60, 120, and 180 days, and filtered using a 0.22-μm nylon syringe
179 filter before injecting the UPLC-MS/MS analysis.
180 Sample Preparation.
181 The soil sample was collected from a depth of 0-15 cm from a paddy field in Nanjing,
182 China. The physicochemical property of test soil was summarized in the Table S2.
183 The soil was thoroughly blended, air-dried, and sieved. The apple, pear, lettuce, and
184 tomato were purchased from the organic food window of the local supermarket, and
185 minced by a homogenizer. The samples were verified to be free of tefluthrin.
186 Extraction and Clean up for soil, apple, and pear.
187 The extraction process was based on the QuEchERs method. A.mple (soil, apple, and
188 pear) was weighed into 50 mL polytetrafluoroethylene (PTEF) centrifuge tube, and
189 then 5 mL water and 10 mL acetonitrile were added. The tube was immediately
190 capped and shaken by high-speed vortex mixer at 2500 rpm for 5 min and
191 ultrasonication for 10 min. The 3 g sodium chloride and 1 g anhydrous magnesium
192 sulfate were added into the mixture, the tube was again shaken for 3 min, and the
193 samples were centrifuged at 4000 rpm for 5 min, 5 mL of the upper layer was added
194 into a 15 mL centrifuge tube containing 100 mg GCB and 1 g anhydrous magnesium
195 sulfate. The tube was shaken vigorously for 1 min and centrifuged at 4000 rpm for 5
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196 min. Next, 2 mL of the upper layer was transferred into a 4 mL centrifuge tube and
197 dried with nitrogen. Finally, the residual was dissolved in 1 mL mobile phase and
198 filtered using a 0.22-μm nylon syringe filter before injecting for the UPLC-MS/MS
199 analysis.
200 Extraction and Clean up for tomato and lettuce.
201 The process was similar to extraction and cleanup for the above samples, a 10 g
202 matrix (tomato and lettuce) was weighed into a 250 mL flask, and a mixture of 10 mL
203 water and 20 mL acetonitrile was added. The flask was shaken for 1 h in an oscillation
204 table at 250 rpm before the liquid mixture was transferred into a 100 mL graduated
205 cylinder with stopper containing 2 g sodium chloride. The supernatant acetonitrile (6
206 mL) was transferred into a 15 mL centrifuge tube containing 100 mg GCB and 1 g
207 anhydrous magnesium sulfate. The tube was vigorously shaken for 1 min and
208 centrifuged for 5 min at 4000 rpm. The 4 mL of liquid supernatant was evaporated to
209 dryness at 40 °C, and then the residual was dissolved in 1 mL of the mobile phase and
210 filtered using a 0.22 μm nylon syringe filter before injecting the UPLC-MS/MS
211 analysis. The typical chromatograms of blank and spiked samples were shown in
212 Figure S1.
213 Method Validation.
214 The specificity, LOD, LOQ, accuracy, and precision were used to evaluate the
215 performance of the method. Control samples (soil, apple tomato, pear, and lettuce)
216 were analyzed to confirm the absence of interfering substances at the retention time of
217 target chemicals. The linearity was verified for both solvent and matrix-matched
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218 calibration curves at seven concentrations ranging from 10 to 1000 μg L-1. The
219 linearity of the solvent and the different matrix-matched calibration curves were
220 determined based on the peak areas and the concentrations of target analytes. The
221 equation of matrix effect was defined as
the slope of matrix matched curve - the slope of solvent curve
222 Matrix effect (%) =
× 100%.
(4)
the slope of solvent curve
223
The matrix-dependent LODs and LOQs of tefluthrin enantiomers in the fruits,
224 vegetables, and environmental samples were determined at concentrations that
225 produced signal-to-noise (S/N) ratios of 3 and 10, respectively.
226
The recovery experiments were employed to deduced the accuracy and precision
227 of the method. The control samples in quintuplicate were spiked with three different
228 concentrations (0.05, 0.5, 2.5 mg kg-1) of five matrices, vortexed for 30 s and
229 incubated overnight. All spiked samples were prepared for three consecutive days and
230 tefluthrin enantiomers were extracted and purified according to the above procedure.
231 The recoveries, intraday RSDs, and interday RSDs were used to evaluate the accuracy
232 and precision.
233 Stereoselective degradation in soil
234 Incubation experiments were performed separately under aerobic, anaerobic and
235 sterile conditions. The experiments of racemate was to study the degradation tendency
236 and the enantiopure isomers treatments were used to evaluated the enantiomerization.
237
The sterile soil sample was prepared by sterilized two periods of 60 min, 121 °C
19
238 moist heat sterilization with a 24 h interval between autoclave treatments.
The
239 spiked concentration of racemate was 2 mg kg-1 and 1 mg kg-1 for enantiopure
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240 tefluthrin.
Aerobic and sterile incubation experiments were performed in 50 mL PTEF
241
242 centrifuge tubes covered with air-permeable, sterile cotton plugs. Accurately
243 measured 5 g of the soils into PTEF centrifuge tubes. The water content was adjusted
244 60% using deionized water (contain 0.2% sodium azide solution for sterile conditions)
245 of the saturation holding capacity and incubated at 25 °C in the dark was referenced
246 Pan 20 and Zhang 21. Triplicate samples were conducted at 0, 1, 3, 7, 14. 21, 35, 60, 90,
247 120 d for tefluthrin, and immediately transferred into a freezer (-20 °C) and stored for
248 later analysis.
249
In anaerobic incubation experiments, deionized water, previously purged with N2
250 to remove O2, was added to each tube to form a 1 cm water layer on surface, and then
251 the tubes were filled with N2 and sealed immediately. Finally, the soil samples was
252 treated the same as those described in under aerobic conditions.
253
The first-order kinetic Eq. (5) was used to declare the degradation kinetic of
254 tefluthrin and enantiomers. The half-life (T1/2) was calculated with the following Eq.
255 (6), and EF value was defined in Eq. (7).
256 Ct = C0e-kt
(5)
(6)
(7)
257 T1/2 = ln2/k = 0.693/k
258 EF = [R] / ([R]+[S])
259 where C0 and Ct are the initial concentration and the concentration at time in soil,
260 respectively. k is the degradation rate constant. [R] and [S] are the contration of the
261 (1R,3R)- and the (1S,3S)-enantiomers, respectively.
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262 Results and discussion
263 MS Analysis
264 Experiments were performed to optimize the MS/MS conditions for tefluthrin, which
265 forms abundant adduct ions in the mass spectra, such as [M+Na]+, [M+K]+, or
266 [M+NH4]+ , when undergoing ionization in the positive mode. The use of mobile
267 phase containing ammonium formate helped to favor the formation of [M+NH4]+ for
268 tefluthrin. Higher abundant product ions of m/z 177 for fragmentation of [M+NH4]+
269 (m/z 436 ) were excavated by manual tuning mode. The [M+NH4]+ (m/z 436 > 177)
270 was selected as quantitative and qualitative determination ions of tefluthrin
271 enantiomers, in agreement with the literature 14-16. Different buffer concentrations (1,
272 2, 5 and 10 mM) were investigated, and the MS signal response was the highest at 5
273 mM.
274 The specific optical rotation and absolute configuration of tefluthrin enantiomers
275 The specific optical rotations were determined as Peak 1 [α]2D0 = -16.6° (methanol,
276 c=1.13) and Peak 2 [α]2D0 = + 16.8° (methanol, c=1.15). As shown in Figure 2, the
277 dashed lines indicated the calculated VCD spectra of the conformation bearing the
278 lowest free energy, and the experimental VCD spectra of enantiomers were drawn
279 using continuous lines. The experimental VCD spectra of tefluthrin enantiomers better
280 agreed with the calculated VCD spectrum of (1S,3S) and (1R,3R). Therefore, the
281 elution order of tefluthrin on the Lux Cellulose-1 was determined as
282 Z-(1S,3S)-cis-(-)-tefluthrin and Z-(1R,3R)-cis-(+)-tefluthrin. VCD would affect the
283 tendency of definiting absolute configuration for its advantage of giving more
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284 information.
285 Sample Extraction and Purification
286 The QuEChERS method is a major development for sample preparation in pesticide
287 residue analysis. Inorganic buffer salts play an irreplaceable role in this whole process;
288 for example, anhydrous magnesium sulfate is soluble in water and prompts
289 partitioning of the pesticides into the organic phase. The application of adsorbents in
290 the cleanup procedure makes the QuEChERS method an attractive alternative
291 approach 22. PSA, C18, and GCB were used to remove the interfering substances, and
292 the experiments indicate that after purification with C18 and PSA, and dispersive
293 solid-phase extraction (d-SPE), poor recovery (<60%) was obtained for lettuce
294 samples. Based on the higher recovery, GCB was selected to purify the lettuce matrix
295 in place of C18 and PSA. The typical chromatograms were shown in Figure S1.
296 Method validation
297 Specificity, linearity, LOD, LOQ, and Matrix Effect
298
The specificity of the proposed method was detected by preparing blank samples
299 according to the above procedures, and no interference was shown at the retention
300 time. Good linearities (R2 ≥0.9903) were obtained for enantiomers; LODs and LOQs
301 for tefluthrin enantiomers are summarized in Table 1. The LODs for tefluthrin
302 enantiomers were estimated at 0.0029~0.0067 mg kg-1, and the corresponding LOQs
303 were 0.0101~0.0227 mg kg-1.
304
One of the major drawbacks in pesticide analysis is that the response can be
305 suppressed or enhanced by the presence of matrix components. Table 1 also shows the
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306 matrix effect for each enantiomer of tefluthrin. The results show that significant
307 matrix enhancement effects existed between (1R,3R)-tefluthrin and (1S,3S)-tefluthrin
308 enantiomers. As a result, a calibration was performed for each enantiomer using the
309 external matrix-matched standards to eliminate the matrix effect and to obtain more
310 realistic results in the 5 matrices.
311 Precision and Accuracy
312
The recoveries and RSDs of tefluthrin enantiomers in quintuplicate at three
313 concentrations and on three consecutive days are shown in Table 2. From Table 2, the
314 proposed method possesses satisfactory mean recoveries values (76.9%–107.6%) and
315 precision with all RSDs values below 15.6%. For (1S,3S)-tefluthrin and
316 (1R,3R)-tefluthrin, the mean recoveries ranged from 78.8% to 107.6% and 76.9% to
317 105.4%, respectively, with the intraday RSDs of 0.64% to 15.6% (n=5) and 1.7% to
318 10.7% (n=5). The results indicate that this method can reach expectation of precision
319 and accuracy for the enantiomeric analysis of tefluthrin in 5 matrices.
320 Stereoselective degradation of tefluthrin in soil
321 Stereoselective degration in soils under aerobic conditions
322 The dissipation of tefluthrin in paddy soil was studyed under aerobic conditions
323 (Figure 3a). The degradadation of different isomers followed the first-order kinetic
324 model (R2 = 0.9716-0.9737), and the half-lives, k and R2 are listed in Table 3. The
325 half-lives of (1S,3S)-tefluthrin and (1R,3R)-tefluthrin were 63.4 ± 5.3 and 77.6 ±
326 4.5 d, respectively. During the incubation, the degradation rate of (1R,3R)-tefluthrin
327 tend to be slower than (1S,3S)-tefluthrin. The calculated EF value was used to
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328 evaluate the stereoselective dissipation, EF slightly increased after 60 d (Figure 3d).
329 The results indicate that (1S,3S)-tefluthrin were preferentially degraded under aerobic
330 conditions. The concentration of (1R,3R)-tefluthrin was higher than (1S,3S)-tefluthrin
331 in soil under aerobic condition would lay the foundation for understanding the toxicity
332 for terrestrial biota.
333 Stereoselective dissipation in soils under anaerobic conditions
334 The concentration of isomers decreased about 15%-18% and the degradation all
335 followed the first-order kinetics (0.962-0.9794), k and half-lives were summarized in
336 Table 3. Notably, the degradation rate of tefluthrin isomers under anaerobic
337 conditions (Figure 3b) was slower than aerobic conditions. The half-lives of two
338 stereoisomers were 351.7 ± 13.5 and 357.2 ± 9.6 d, was 6 times than under aerobic
339 conditions. At the same time, the EF value was shown in Figure 3d to evaluate the
340 stereoselective dissipation, EF values changed around 0.5 during the whole incubation,
341 indicating that no significant enantioselectivity was observed under anaerobic
342 conditions and anaerobic bacteria made a contribution to degradation.
343 Stereoselective dissipation in soils under sterile conditions
344 During the incubation process, the concentration of tefluthrin slightly changed in
345 autoclaved soil compared with that under aerobic conditions. In sterile conditions,
346 10%-12% of tefluthrin isomers was degraded. This indicated that tefluthrin could be
347 degraded without microorganisms (Figure 3c). In addition, no stereoselective
348 dissipation was observed under these conditions (EF≈0.5, Figure 3d).
349
The microbial community plays an important contribution to degrade the
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350 contaminant in soil samples 23,24. In this study, the result indicated that the main
351 reason causing the dissipation of tefluthrin stereoisomers was the microbial activity
352 rather than abiotic factors. Interestingly, the degradation rate of tefluthrin isomers
353 under aerobic condictions was significantly faster than under other conditions. In
354 addition, the concentration of (1R,3R)-tefluthrin was slightly higher than that of the
355 (1S,3S)-tefluthrin under aerobic condition. It may be that the oxygen status affected
356 the makeup of microorganism population, hereby influenced the degradation rate and
357 stereoselectivity 20. Compared to aerobic condictions, autoclaved soil samples
358 exhibited emaciated degradation ability to tefluthrin. Although the underlying
359 processes are unknown, this result may be explained by the activation/inhibition of
360 enzymes 25-27. Therefore, increasing the microbial abundance may reduce the
361 environmental risks of tefluthrin. At the same time, this also lay the groundwork for
362 terrestrial environmental risk evaluation of tefluthrin stereoisomers.
363 Stability of tefluthrin stereoisomers
364 Incubating the enantiopure isomers under aerobic conditions was to study the
365 interconversion. During the 120 d of incubation, no enantioselective transformation
366 was observed. As shown in Figure S2, there was no significant change between the
367 initial concentration and measured concentration at different times of a pair of
368 enantiomers in acetonitrile, methanol, and water during the 6 months. The data
369 indicated that the chiral configuration was stable in soil and solvents.
370 Abbreviations Used
371 UPLC-MS/MS, ultraperformance liquid chromatography tandem mass spectrometry;
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372 RSM, response surface methodology; VCD, vibrating circular dichroism; RSD, relative
373 standard deviations; QuEChERS, quick, easy, cheap, effective, rugged and safe; k,
374 capacity factor; α, separation factor; Rs, resolution; EF, enantiomer fraction.
375 Acknowledgement
376 This study was supported by the National Key Research and Development Program of
377 China (2016YFD0200207). We are grateful to Fang Liu (Nanjing Agricultural
378 University) for calculating the absolute configuration of tefluthrin enantiomers using
379 Gaussian 09 software.
380 Supporting Information description
381 Effects of the flow rate, mobile phase compositions, and temperature on the
382 enantioseparation parameters; separation condition optimization via RSM with BBD;
383 stability of enantiomers; and anova of the regression model for the response variables.
384 Conflict of interest
385 The authors declare that they have no conflict of interest.
386
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475 Arabian J. Geosci. 2019, 12 (3), 95.
476
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477 Figure captions
478 Figure 1 The chemical structure of tefluthrin enantiomers and typical chromatograms.
479 Figure 2 Calculated VCD spectrum and experimental VCD spectrum.
480 Figure 3 Degradation of tefluthrin enantiomers in Nanjing soil. a. Rac-tefluthrin
481 under aerobic condition; b. Rac-tefluthrin under anaerobic condition; c. Rac-tefluthrin
482 under sterile condition; d. EF value of tefluthrin stereoisomers under three conditions.
483
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484 Table 1 Linear regression parameters, LOD, and LOQ for the tefluthrin enantiomers
485 in different matrices
Linear
Regression
equation
Matrix effect
(%)
LOD
LOQ
Compound Matrix range (μg
L-1)
R2
(mg kg-1) (mg kg-1)
solvent 10 ~ 1000 y=1853.4x-188.8 0.9992
tomato 10 ~ 1000 y=4290.2x+330.3 0.9986
(1R,3R)- lettuce 10 ~ 1000 y=4001.3x-539.2 0.9901
tefluthrin pear 10 ~ 1000 y=4692.9x+144.9 0.9936
apple 10 ~ 1000 y=3028.8x+426.4 0.9904
0.0029
0.0032
0.0045
0.0037
0.0038
0.0041
0.0036
0.0039
0.0032
0.0043
0.0067
0.0046
0.0101
0.0114
0.0146
0.0125
0.0137
0.0138
0.0126
0.0131
0.0117
0.0144
0.0227
0.0151
131.4
115.8
153.2
63.4
soil 10 ~ 1000 y=6341.3x-316.7 0.9935
242.1
solvent 10 ~ 1000 y=1846.9x-187.9 0.9997
tomato 10 ~ 1000 y=4327.5x+352.3 0.9993
(1S,3S)- lettuce 10 ~ 1000 y=3997.9x-507.4 0.9972
tefluthrin pear 10 ~ 1000 y=4731.2x+160.7 0.9941
apple 10 ~ 1000 y=3232.7x+412.3 0.9981
134.3
116.5
156.2
75.0
soil 10 ~ 1000 y=6452.3x-280.9 0.9966
249.4
486
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487 Table 2 Accuracy and precision of the method in the 5 matrices
Intraday (n=5)
Day1
Mean
Day2
Mean
Day3
Mean
Spiked
level
(mg kg-1)
Interday
(n=15)
Compound Matrix
RSD
RSD
RSD
RSD(%)
recovery
(%)
recovery
(%)
recovery
(%)
(%)
1.3
4.6
6.6
2.6
8.9
6.5
6.3
10.4
4.5
5.5
2.9
0.6
3.3
1.1
1.1
2.9
4.3
8.2
2.6
14.2
14.6
4.8
5.7
5.2
7.1
4.9
1.6
14.1
9.3
1.4
(%)
4.8
7.6
6.9
2.4
6.9
6.8
2.8
2.8
3.6
2.7
15.6
1.2
2.6
1.9
2.8
3.9
9.4
2.0
3.1
12.2
14.5
7.2
5.8
3.5
6.8
8.7
2.3
6.2
3.7
2.1
(%)
4.8
9.4
6.7
2.5
11.1
5.5
5.4
5.7
8.4
4.6
15.3
1.0
10.1
2.1
3.3
1.6
10.7
7.0
1.0
5.5
9.9
2.4
2.8
4.9
9.9
8.3
1.7
4.6
3.6
4.8
0.05
0.5
95.1
78.8
95.2
101.7
83.9
80.0
99.2
87.6
101.7
104.3
100.0
97.2
89.5
87.4
88.9
96.2
76.9
90.0
99.1
85.5
87.9
90.7
93.2
81.0
94.9
98.6
100.0
104.0
102.0
97.4
98.3
84.8
106.9
101.8
86.5
79.9
93.2
102.3
84.3
107.6
88.2
97.4
96.3
90.2
89.3
97.3
86.1
103.8
98.5
89.7
90.8
93.6
105.4
88.9
99.9
93.7
102.9
81.2
105.4
98.4
98.3
82.3
100.1
100.2
88.9
80.8
90.4
93.7
98.9
105.8
93.4
97.1
82.0
86.8
89.6
98.7
84.0
99.5
97.3
94.4
92.8
91.4
96.4
86.7
97.8
96.6
101.8
93.1
105.4
98.4
4.5
9.2
6.0
2.5
7.4
5.3
5.4
5.7
8.4
4.0
14.0
0.9
8.0
2.0
2.4
3.2
9.9
7.3
2.5
11.6
12.5
2.6
7.6
6.6
7.4
10.8
2.2
12.1
2.5
0.6
tomato
lettuce
2.5
0.05
0.5
2.5
0.05
0.5
(1S,3S) -
pear
Tefluthrin
2.5
0.05
0.5
apple
soil
2.5
0.05
0.5
2.5
0.05
0.5
tomato
lettuce
2.5
0.05
0.5
2.5
0.05
0.5
(1R,3R) -
pear
Tefluthrin
2.5
0.05
0.5
apple
2.5
0.05
0.5
soil
2.5
488
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489 Table 3 Parameters for the dissipation of tefluthrin stereoisomers in soil.
Incubation condition Isomers k (d-1) Half-lives (d)a
R2
EF (End point)
0.57
(1S,3S) 0.011
(1R,3R) 0.009
63.4 ± 5.3
77.6 ± 4.5
0.9737
0.9716
0.962
aerobic
(1S,3S) 0.00197 351.7 ± 13.5
(1R,3R) 0.00194 357.2 ± 9.6
0.49
anaerobic
0.9794
490 a Value represent mean ± SD.
491
492
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493
494 Figure 1 The chemical structure of tefluthrin enantiomers and typical chromatograms.
495
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