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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

1

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

15 16 17 18 19 20 21 22 23

ACS Paragon Plus Environment


24

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)

43 44 45 1


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INTRODUCTION

47

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

64

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),

2


which


68

to avoid toxic risks in agricultural products consuming.

69

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

3


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metabolizing pesticides.20 In contrast, non-enantioselective degradation was shown in

91

hydroponic solution studies as well as a study in soil under multiple conditions.14, 20

92

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



112

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

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in Henan, Gala in Shandong, and Golden Delicious in Liaoning. The mean

122

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

125

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

130

(without fungicide). Three representative apple samples (approximately 2000 g in

131

each sample) from each plot were collected on day 0 (2 h after application), and 1, 2,

132

4, 7, 10, 14, 21, and 28 days after treatment. Correspondingly, soil samples

133

(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

135

samples were put into polyethylene bags and transported to the laboratory. The apple

136

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.

139

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.

154

Apparatus and Chromatographic Conditions. The imazalil enantiomers were

155

chromatographically separated using a Waters ACQUITY UPLC system (Milford, 6



USA),

with

a

Lux

Cellulose-2

Page 8 of 37

156

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.

175

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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178

OECD guidance document by following criteria: linearity, stability, sensitivity

179

(including limit of detection (LOD) and the limit of quantitation (LOQ)), accuracy,

180

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.

201

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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216

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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346

(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



Page 18 of 37

368

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,

17


translocation,

Page 19 of 37


390

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

18



412

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


Page 20 of 37

Page 21 of 37


434

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


times;

SSE,

mass

signal


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

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460

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(38) Zhang, C.; Hu, X.; Luo, J.; Wu, Z.; Wang, L.; Li, B.; Wang, Y.; Sun, G., Degradation dynamics of

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(39) Khandelwal, A.; Gupta, S.; Gajbhiye, V. T.; Varghese, E., Degradation of kresoxim-methyl in soil:

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impact of varying moisture, organic matter, soil sterilization, soil type, light and atmospheric CO2 level.

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Chemosphere 2014, 111, 209-217.(40) Kumar, N.; Mukherjee, I.; Sarkar, B.; Paul, R. K., Degradation

571

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Hazard Mater. 2016, 321, 517-527.

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



588

Figure 4. EF values versus time plots of imazalil enantiomers in apple and soil in

589

three provinces.

590 591 592

27


Page 28 of 37

Page 29 of 37


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

28



Page 30 of 37

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).

29


Page 31 of 37


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.

30



Page 32 of 37

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

31


Page 33 of 37


GRAPHIC FOR TABLE OF CONTENTS

32



Figure 1. The chemical structure and chromatogram (A) and optical rotation (B) of imazalil enantiomers. 141x115mm (300 x 300 DPI)


Page 34 of 37

Page 35 of 37


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)



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)


Page 36 of 37

Page 37 of 37


Figure 4. EF values versus time plots of imazalil enantiomers in apple and soil in three provinces. 163x143mm (300 x 300 DPI)


Enantioseparation of Imazalil and Monitoring of Its ... - ACS Publications

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