Stereoselective Separation of the Fungicide Bitertanol Stereoisomers

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Stereoselective separation of the fungicide bitertanol stereoisomers by highperformance liquid chromatography and their degradation in cucumber Lianshan Li, Beibei Gao, Zhaoxian Zhang, mailun Yang, Xin Li, Zongzhe He, and MingHua Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04594 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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Journal of Agricultural and Food Chemistry

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Stereoselective separation of the fungicide bitertanol stereoisomers

2

by high-performance liquid chromatography and their degradation

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in cucumber

4 5

Lianshan Li, Beibei Gao, Zhaoxian Zhang, Mailun Yang, Xin Li, Zongzhe He,

6

Minghua Wang*

7

Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural

8

University; State & Local Joint Engineering Research Center of Green Pesticide

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Invention and Application, Nanjing, 210095, China

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∗Corresponding author. E-mail address: [email protected] (M. Wang)

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ABSTRACT

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Bitertanol is a widely used triazole fungicide and consisted of four stereoisomers. A

13

new HPLC method was developed for simultaneous analysis of the four stereoisomers

14

in apple, pear, tomato, cucumber, and soil. The mechanism of separation was

15

explained with molecular docking and effects of thermodynamic parameters on the

16

resolution. The absolute configuration and optical rotation of four stereoisomers were

17

confirmed by X-ray diffraction and HPLC tandem circular dichroism detection,

18

respectively. A good linearity (R2  0.999) was obtained for four stereoisomers in all

19

matrix-matched calibration curves in the range of 0.02-10 mg/L. The mean recoveries

20

of four stereoisomers in five matrices ranged from 74.6% to 101.0% with an intra-day

21

and inter-day relative standard deviation of 0.6% to 9.9%. Stereoselective degradation

22

of bitertanol in cucumber was observed: (1R,2S)-bitertanol and (1R,2R)-bitertanol

23

were preferentially degraded with EF values from 0.5 to 0.43 at 7 d and 0.42 at 5 d,

24

respectively. This research provides a useful tool for the analysis of bitertanol

25

stereoisomers.

26 27

Keywords: bitertanol, chiral separation, X-ray diffraction, stereoselective degradation

28

2



29

INTRODUCTION

30

Chirality is a common property in pesticides.1 Approximately 25% of commercial

31

pesticides were chiral over the world in 1996, and more than 40% in China in 2006.2,3

32

Triazole fungicides play an essential role in preventing and controlling fungal disease

33

through inhibiting14α-demethylase involved in the biosynthesis of fungal sterols.

34

Triazole fungicides are becoming the most popular fungicides and have attracted an

35

increasing attention since the 1970s due to their high potency.4,5 Currently, there are

36

31 commercial triazole fungicides, and 26 of those have 1 or 2 asymmetric carbon

37

atoms containing 1 or 2 pairs of enantiomers.6 Most chiral triazole fungicides are

38

marketed in racemic forms, except for diniconazole and uniconazole.

39

Different stereoisomers exhibit similar physicochemical properties in an achiral

40

environment, but different activities in the biological environment.7 Many papers

41

about enantioselective bioactivity, metabolism, degradation, and toxicity of chiral

42

triazole fungicides have been published in recent years.8-14 Bitertanol [(1RS,

43

2RS)-1-(biphenyl-4-yloxy)-3, 3-dimethyl-1-(1H-1, 2, 4-triazol-1 -yl) butan-2-ol] is an

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efficient triazole fungicide consisting of four stereoisomers (Supporting Information

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Figure S1) and was developed by Bayer in the 1970s.15,16 The ratio of four

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stereoisomers in the current commercial bitertanol is 4:4:1:1. Bitertanol has been

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frequently detected in food products and environmental samples due to frequent

48

applications over a long period of time.17,18 To date, the research about bitertanol was

49

mainly focused on residues and ecological toxicology. For instance, bitertanol

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significantly inhibits the activity of CYP3A4 and has potential androgenic-disrupting 3


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effects.19,20 Bitertanol can induce and inhibit rat cytochrome P450-dependent

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monooxygenases .21 The residue detection methods of bitertanol in baby food, food,

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and atmospheric samples were established using various technologies.22-25 However,

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the racemic bitertanol was used in the studies. The enantioselective bioactivity,

55

metabolism, degradation, and toxicity of each bitertanol stereoisomer have not been

56

reported.

57

Therefore, it is necessary to establish an effective method for the analysis of

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bitertanol stereoisomers. The successful separation of racemate is fundamental to

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realize bitertanol at the level of stereoisomers. Some remarkable works have been

60

accomplished regarding the separation of bitertanol by various techniques. For

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example, Zhang et al. reported that 21 triazole fungicides, including bitertanol, were

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separated with high-performance liquid chromatography (HPLC) -tandem mass

63

spectrometry.26 Chai et al. separated bitertanol by normal-phase HPLC with two

64

cellulose-based chiral columns.2 However, no publication has been found about the

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absolute configuration and chiral analysis of bitertanol stereoisomers in the

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environmental and food samples.

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In this study, a reversed-phase HPLC method was established for simultaneous

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determination of bitertanol stereoisomers in vegetables, fruits, and soil after the

69

solid-phase extraction (SPE) cleanup of the extracts. The absolute configuration and

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optical rotation of bitertanol stereoisomers were confirmed. The stereoselective

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degradation in cucumber was determined.

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MATERIAL AND METHODS

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Chemicals and reagents. The bitertanol standard (≥97% purity) was provided by

75

Jiangsu Sword Agrochemicals Co., Ltd. (Jiangsu, China). The optical isomers

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(≥99.9%) were prepared by Chiralway Biotech Co., Ltd. (Shanghai, China).

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HPLC-grade acetonitrile and methanol were purchased from TEDIA (Fairfield, USA).

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Ultra-pure water was obtained using MUL-9000 water purification systems (Nanjing

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Zongxin Water Equipment Co. Ltd., Nanjing, China). Analytical grade sodium

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chloride, anhydrous sodium sulfate, and other reagents were acquired through

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commercial sources. The Cleanert Florisil on a cartridge (500 mg, 6 mL) was

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purchased from Agela Technologies (Tianjin, China).

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Stock standards (1000 mg/L) of individual stereoisomers were prepared in

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acetonitrile and a series of concentrations working standards 10, 5, 2, 0.5, 0.02 mg/L

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were prepared by diluted in acetonitrile. Corresponding, the matrix-matched standard

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solutions at 10, 5, 2, 0.5, and 0.02 mg/L were prepared by adding the standard

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working solutions of four stereoisomers to the control matrices (apple, pear, cucumber,

88

tomato, and soil). All standard working solutions were placed in the dark at 4 ºC.

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Chiral HPLC analysis. The separation was performed on an Agilent 1200 high

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perform liquid chromatograph (Agilent, California, USA) equipped an UV-detector

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and a chiral column Lux Cellulose-1 [cellulose tris (3,5- dime-thylphenylcarbamate)],

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250 mm×4.6 mm(i.d.), 5 μm] (Phenomenex, California, USA). The separation

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parameters were used to evaluate the effect of separation, including capacity factor k,

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separation factor α, and resolution factor Rs, which were expressed as follows: 5


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k=(tR-t0)/t0

(1)

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α=k1/k2

(2)

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Rs=2(t2-t1)/(w2+w1)

(3)

98 99

where tR is the retention time of enantiomers, t0 is the void time, k is the retention factor, and w represents the peak width at half height of stereoisomers.

100

A series of mobile phase compositions (Methanol/water and acetonitrile/water)

101

were used to explore the effect of mobile phase on the separation. Temperature

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influences the rate of the analyte sorption and desorption with the stationary phase

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and impacts the elution order of the stereoisomers.27,28 So, the impact of temperature

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on separation was discussed from 15 ºC to 35 ºC. The thermodynamic parameters

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were calculated according to the following equation of Van’t Hoff:

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lnk = -ΔH0 / RT + ΔS0 / R + lnΦ

(4)

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lnα = -ΔΔH0 / RT + ΔΔS0 / R

(5)

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The ΔH0 and ΔS0 represent the changes molecular enthalpy and molecular entropy

109

of bitertanol stereoisomers between the mobile phase and stationary phase,

110

respectively. T, R, and Φ represent absolute temperature, gas constant 8.314 J/(mol・

111

K) and phase ratio, respectively. ΔΔH0 and ΔΔS0 were the values of ΔH2-ΔH1 and

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ΔS2-ΔS1. If the Lnk and Lnα have a good linearity with 1/T, the value of -ΔH/R,

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(ΔS/R+lnΦ), -ΔΔH0 / R, and ΔΔS0 / R could be calculated from the slop and intercept

114

based on equations (4) and (5).

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Circular dichroism spectroscopy. The optical rotation and elution order of four

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stereoisomers were determined using HPLC (JASCO LC2000) with ultraviolet 6



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detector (JASCO UV2075) tandem CD (JASCO CD2095) detector at 254 nm. The

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mixed standard solution of four stereoisomers in acetonitrile was measured on Lux

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Cellulose-1 with acetonitrile/water (55: 45, v/v) as the mobile phase at the flow of 0.8

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mL/min.

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X-ray crystallography. Single-crystal X-ray diffraction analysis of four

122

stereoisomers was carried out on a Bruker SMART APEX II CCD diffractometer with

123

graphite-monochromated Cu-Ka radiation (λ=1.54178 Å) at 150(2) K. Data reduction

124

and absorption corrections were performed with the SAINT and SADABS software

125

packages, respectively. The structures were resolved by direct methods and refined by

126

the full matrix least-squares based on F2 using the SHELXL-2016 program package.29

127

The non-hydrogen atoms were refined anisotropically. All hydrogen atoms were

128

placed at the calculated positions and refined as riding on the parent atoms.

129

Molecular docking. The three-dimensional structures of the stereoisomers of

130

bitertanol and the CSPs were developed, and the energy was minimized using Yasara

131

molecular docking software. The stereoisomers were docked in the structures of the

132

CSPs using Yasara molecular docking software. The results were sorted by binding

133

energy, and more positive energy indicates stronger binding. The optimal result was

134

chosen for further analysis.30-32

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Sample preparation. Extraction. Approximately 10 g of samples (apple, pear,

136

cucumber, tomato, and soil) were weighed into a 250 mL conical flask, 5 mL water

137

and 30 mL acetonitrile was added and shaken 1 h in the ZQLY-180 thermostatic

138

oscillator (Shanghai, China) with 250 rpm/min at 25 ºC. The liquid phase was 7


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transferred into a 100 mL cylinder containing 2 g NaCl, then shaken for 1 min and

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allowed to stand for 30 min. A half of acetonitrile portion was dehydrated through 5 g

141

of anhydrous Na2SO4 which followed by evaporating to dryness at 45 °C.

142

Cleaning up the soil samples. The residue was dissolved in 4 mL n-hexane/acetone

143

(98:2, v/v) and transferred into a Florisil SPE pretreated with 6 mL n-hexane. The

144

column was rinsed with 5 mL n-hexane/acetone (95:5, v/v) and eluted by 12 mL

145

n-hexane/acetone (88:12, v/v). The eluant was collected and evaporated at 45 ºC. The

146

extractive was dissolved with 1 mL acetonitrile and filtered through a 0.22-μm nylon

147

syringe filter for HPLC analysis.

148

Cleaning up the vegetables and fruits samples. The residue was dissolved in 4 mL

149

n-hexane/acetone (98:2, v/v) and transferred into an SPE column pretreated with 6 mL

150

n-hexane/acetone (88:12, v/v). The column was washed with 5 mL n-hexane/acetone

151

(93:7, v/v) and eluted by 12 mL n-hexane/acetone (88:12, v/v). The eluant was

152

collected and evaporated to dryness at 45 ºC, then dissolved with 1 mL acetonitrile

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and filtered through a 0.22 μm nylon syringe filter for HPLC analysis.

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Method validation. The linearity, matrix effect, precision, accuracy, limit of

155

detection (LOD) and limit of quantitation (LOQ) were used to evaluate the feasibility

156

of the method for simultaneous determination of four bitertanol stereoisomers in fruits,

157

vegetables, and soil matrices. Linearity was assessed by the linear regression of

158

bitertanol stereoisomer peak areas versus the concentration. The matrix effect should

159

be ignored in the slope ratios of matrix/solvent ranging from 0.90 to 1.10. If the slope

160

ratios of the matrix/solvent were below 0.90 or higher than 1.10, a matrix suppression 8



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or enhancement effect should be concerned. The LOD and LOQ were defined as the

162

concentration that produced a signal-to-noise of (S/N) 3 and 10.

163

Accuracy and precision were estimated by recoveries, inter-day RSDs and intra-day

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RSDs of five spiked samples replicated at three concentrations over a continuous

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three days. The standard solutions were spiked to the control samples of apple, pear,

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tomato, cucumber, and soil at 0.02, 0.05, and 0.2 mg/kg for each isomer. The spiked

167

samples were shaken for 1 min and overnight. The extraction and cleanup were

168

performed as described above. The racemization and transformed of the stereoisomers

169

in the sample processing was evaluated by separate recovery experiment for each

170

stereoisomer in five matrices.

171

Field experiment. The degradation test of bitertanol on cucumber was carried out

172

in Nanjing, China. The trial blocks were divided into 30 m2 and isolated by buffer

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zone with three replicate plots. 40% WP of bitertanol was used as foliar spray at 800 g

174

(a.i.)/ha. The cucumber samples were collected in 2 h, 1, 2, 3, 5, 7, 10 days after

175

spraying. All samples were separately homogenized and stored at -20 °C. The

176

concentrations of four bitertanol stereoisomers were detected by HPLC.

177

The first-order kinetics equation was used to estimate the degradation kinetics of

178

the four stereoisomers in cucumber. The enantiomeric fraction (EF) was used to

179

evalute the enantioselectivity of the bitertanol stereoisomer. The half-lives of each

180

stereoisomer and EF were calculated using the following equations:

181

C=C0e-kt

(6)

182

T1/2=ln2/k

(7) 9


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EFA=C(1R,2S)/C(1R,2S) + C(1S,2R)

(8)

184

EFB=C(1R,2R)/C(1R,2R) + C(1S,2S)

(9)

185

where C represents the concentrations of the analyte at time t, and the k is the

186

degradation rate constant. Cisomer is the concentration of the specified stereoisomer.

187 188

RESULT AND DISCUSSION

189

Chromatographic condition optimization. Mobile phase optimization Although,

190

both acetonitrile/water and methanol/water were used as the mobile phase in the

191

isocratic reversed-phase HPLC method, the baseline separation was obtained only

192

with acetonitrile/water as the mobile phase. Furthermore, a series of mobile phase

193

compositions were investigated by changing the proportion of acetonitrile from 55%

194

to 80%. When the acetonitrile percentage increased, the stereoisomers retention time

195

shortened, the peak width narrowed, and the resolutions decreased significantly.

196

Additionally, it was difficult to obtain the baseline separation when acetonitrile was

197

below 55% within an acceptable retention time. Baseline separation of four

198

stereoisomers was achieved with at the acetonitrile/water (55:45, v/v) as the mobile

199

phase with 0.8 mL/min at 25 °C, and all other parameters were constant. The

200

resolution of the first two enantiomers was 3.32, while the latter two enantiomers

201

were 9.28 with a retention time within 25 min (Figure S2 and Table S1). Bitertanol

202

had better solubility in acetonitrile than water and methanol. Therefore, acetonitrile

203

had a stronger power of eluting the analytes from the stationary phase. Zhang et al.

204

also found that acetonitrile presents a stronger eluting strength than methanol for 10



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bitertanol, baseline separation of bitertanol was obtained with acetonitrile and 0.1%

206

formic acid aqueous solution as mobile phase with 30 min. 30

207

Effect of temperature on chiral separation. The effects of temperature on bitertanol

208

enantiomers separation were carefully conducted from 15 ºC to 35 ºC on the Lux

209

Cellulose-1 chiral columns (Figure 1). When the temperature fell below 25 ºC, a more

210

efficient chiral separation was obtained, but the retention time increased. However,

211

increasing temperature shortened the retention time, but decreased the separation

212

efficiency. Finally, 25 ºC was used as the optimal temperature to separate bitertanol

213

stereoisomers. The results regarding k, Rs, and α are shown in the Table S2. The lnk′

214

and lnα had a good linearity with 1/T in the temperature range of 15 ºC to 35 ºC with

215

R2>0.989. The thermodynamic parameters indicated that (1R,2S)-(+)-bitertanol and

216

(1R,2R)-(+)-bitertanol had a weaker binding force with stationary than (1S,2R)-

217

(-)-bitertanol and (1S,2S)-(-)-bitertanol, respectively. The ΔΔH1 and ΔΔS1 were -1.24

218

kJ/mol and -3.53 J/(mol.K), showing that enthalpy drove the separation of

219

(1R,2S)-(+)-bitertanol and (1S,2R)-(-)-bitertanol. In contrast, ΔΔH02 and ΔΔS02 were

220

0.26 kJ/mol and 2.4 J/(mol.K), indicating that the separation of (1R,2R)-(+)-bitertanol

221

and (1S,2S)-(-)-bitertanol was driven by entropy (Table 1). The binding force between

222

stationary and stereoisomers will become weaker with the temperature increase, and

223

the stereoisomers are more easily eluted from the stationary phase. According to Chai

224

research,

225

chromatographic conditions. However, the result showed that the ∆∆H0 values were

226

negative and the ∆∆S0 values were positive, so, enthalpy and entropy contribute

0

bitertanol

stereoisomers

were

separated

11


0

under

normal-phase

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together to the separation of two enantiomers separation in normal-phase

228

chromatography with Lux-2 and Lux-3 chiral column.2

229

Absolute configuration and optical rotation of stereoisomers. Crystallization

230

was utilized to obtain four single-crystals of bitertanol stereoisomers at a low

231

temperature, and single-crystal X-ray analysis is shown in Figure 2. The details of

232

data collection, structure refinement, and crystallography are summarized in Table 2.

233

Combining single-crystal X-ray analysis with the spectrogram of CD and UV (Figure

234

3) showed that the elution order of four enantiomers was (1R,2S)-(+)-bitertanol,

235

(1S,2R)-(-)-bitertanol, (1R,2R)-(+)-bitertanol and (1S,2S)- (-)-bitertanol, respectively.

236

This study has significance for confirming and oriented synthesis of high-efficient

237

stereoisomers for bitertanol in future research.

238

Molecular docking. The research of the interactions between the analytes and the

239

CSPs could explore the mechanisms of stereoisomer separation. The stereoisomers

240

were separately docked into the CSPs of the Lux-1 column. Binding energy for

241

(1R,2S)-(+)-bitertanol, (1S,2R)-(-)-bitertanol, (1R,2R)-(+)-bitertanol and (1S,2S)-(-)-

242

bitertanol were 5.3Cc, 5.4BCc, 5.6Bb, and 6.0Aa kcal/mol, respectively. In this research,

243

two kinds of forces were mentioned including π-π stacking and hydrogen bonds. The

244

π-π stacking was formed between the phenyl of bitertanol and the 3,5-dimethyl phenyl

245

of CSPs. Hydrogen bonds were formed between the 1,2,4-triazole in bitertanol and

246

the carbonyl of CSPs. The bond lengths and energies were also different. The higher

247

binding energy and shorter lengths may have contributed to easier combinations

248

between the CSPs and bitertanol stereoisomers, which resulted in the different elution 12



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orders. Beyond that, the steric hindrance, dipole-dipole interactions, and van der

250

Waals force may have contributed to the binding energy. The stereo matching between

251

the chiral cavity of CSPs and stereoisomers was also different, which is an important

252

factor to affect the chiral separation (Figure 4). Xie et al reported that hydrogen bonds

253

were the major factor in the separation of amide herbicides by AY-H

254

[amylasetris-(5-chloro-2-methylphenylcarbamate)],

255

((S)-1-phenylcarbamate)], OD-H [cellulose tris-(3, 5-dimethylphenylcarbamate)] and

256

OJ-H [cellulose tris-(4-methylbenzoate)] columns.32

AS-H

[amylasetris

257

Linearity, matrix effect, LOD, and LOQ. Four stereoisomers of bitertanol

258

showed a good linearity in solvent and five matrices in the range of 0.02-10 mg/kg

259

with the R2 > 0.999. The results indicated that no noticeable matrix effect was found

260

for four stereoisomers in soil, pear, apple, and tomato. However, a weak matrix

261

enhancement effect was found in cucumber for all stereoisomers in the range from

262

15.3% to 18.8%. The LOD and LOQ for four stereoisomers in five matrices were 2.2

263

-11.5 µg/kg and 7.2 -38 µg/kg, respectively (Table S3).

264

Accuracy and precision. An excellent accuracy and precision of four

265

stereoisomers were obtained in five matrices. There was no racemization and

266

transformation of the stereoisomers in different matrices during the sample processing

267

step (Figure S3). The mean recoveries ranged from 74.6% to 101.0 % with 0.7–9.9 %

268

of intra-day (n=5) RSD and 0.6-7.1% of intre-day (n=15) RSD for bitertanol

269

stereoisomers (Table S4), which showed good accuracy and precision and could apply

270

to the simultaneous determination of four enantiomers of bitertanol in soil, vegetables, 13


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and fruits.

272

Stereoselective degradation in cucumber. The dissipation of four stereoisomers

273

in cucumber followed first-order kinetics (R2, 0.9303-0.9954) and half-lives of

274

(1R,2S)-, (1S,2R)-, (1R,2R)-, (1S,2S)-bitertanol were 4.4, 5.1, 4.0, and 4.8 d (Figure

275

S4). The representative chromatogram is shown in Figure S5. The results indicated

276

that (1R,2S)-bitertanol and (1R,2R)-bitertanol were preferentially degraded in

277

cucumber and the half-lives of A-bitertanol (the first pair of enantiomers

278

((1R,2S)-bitertanol and (1S,2R)-bitertanol)) and B-bitertanol (the second pair of

279

enantiomers ((1R,2R)-bitertanol and (1S,2S)-bitertanol)) were all significantly

280

different (P

Stereoselective Separation of the Fungicide Bitertanol Stereoisomers

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