Simultaneous Enantioselective Determination of the Chiral Fungicide

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Article

Simultaneous Enantioselective Determination of the Chiral Fungicide Prothioconazole and its Major Chiral Metabolite Prothioconazole-desthio in Food and Environmental Samples by UltraPerformance Liquid Chromatography Tandem Mass Spectrometry Zhaoxian Zhang, Qing Zhang, Beibei Gao, Gaozhang Gou, Lianshan Li, Hai-yan Shi, and Ming-Hua Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02903 • Publication Date (Web): 27 Aug 2017 Downloaded from http://pubs.acs.org on August 27, 2017

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Journal of Agricultural and Food Chemistry

1

Simultaneous Enantioselective Determination of the Chiral Fungicide

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Prothioconazole and its Major Chiral Metabolite Prothioconazole-

3

desthio in Food and Environmental Samples by Ultra-Performance

4

Liquid Chromatography Tandem Mass Spectrometry

5

Zhaoxian Zhang†, Qing Zhang†, Beibei Gao†, Gaozhang Gou‡, Lianshan Li†, Haiyan

6

Shi†, Minghua Wang†*

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†

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

9

Invention and Application, Nanjing 210095, China

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

10

‡

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* Corresponding author (Tel.: + 86 025 84395479; Fax: + 86 025 84395672; E-mail

12

address: [email protected])

College of Science, Honghe University, Mengzi 661199, China

1

ACS Paragon Plus Environment


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ABSTRACT: An efficient and sensitive chiral analytical method was established for

14

the determination of the chiral fungicide prothioconazole and its major chiral

15

metabolite prothioconazole-desthio in agricultural and environmental samples using

16

ultra-performance liquid chromatography tandem mass spectrometry. The optical

17

rotation and absolute configuration of enantiomers were identified by optical rotation

18

detector and electronic circular dichroism spectra. The elution order of

19

prothioconazole and its chiral metabolite enantiomers was R-(+)-prothioconazole-

20

desthio,

21

prothioconazole. The mean recoveries from the samples was 71.8-102.0% with

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intraday relative standard deviations (RSDs) of 0.3-11.9% and interday RSDs of 0.9-

23

10.6%. The formation of prothioconazole-desthio was studied in soil under field

24

conditions and enantioselective degradation was observed for chiral prothioconazole.

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Remarkable enantioselective degradation was observed: R-prothioconazole degraded

26

preferentially with EF values from 0.48-0.37. Although prothioconazole-desthio is the

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most remarkably bioactive metabolite, no obvious enantioselective behavior was

28

observed in soil. These results may help to systematically evaluate prothioconazole

29

and its metabolites in food and environmental safety.

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KEYWORDS: Prothioconazole, Chiral metabolite, Enantioseparation, Absolute

31

configuration, UPLC-MS/MS.

S-(-)-prothioconazole-desthio,

R-(-)-prothioconazole

32

2


and

S-(+)-

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INTRODUCTION

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Chiral pesticides play a critical role in the control of pests and diseases in agricultural

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systems. The proportion of chiral pesticide has continually increased as more complex

36

compounds are introduced.1 Most chiral triazole fungicides have one or more chiral

37

centers, and thus two or more enantiomers. Although the enantiomers have identical

38

physical properties, their biological and physiological properties in chiral

39

environments can significantly differ.2-9 For example, the bactericidal activity of R-

40

diniconazole is higher than for S-diniconazole, but the activity profile was reversed

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when their ability to regulate plant growth was assessed.10 The activity of two

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triadimefon enantiomers is very low, but one of its reduction products ((1S, 2R)-

43

triadimenol) had higher bactericidal activity than other three enantiomers.11 In

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addition, (-)-hexaconazole was approximately six times more toxic than (+)-form to

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Scenedesmus obliquus.12 Furthermore, some chiral pesticides can degrade into

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different chiral metabolites that can exhibit more toxicity than the original

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xenobiotic.13 Sparling et al.14 reported that the corresponding oxide derivatives of

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diazinon, chlorpyrifos and malathion showed greater toxicity than the parent

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compound. To systematically assess chiral pesticide, both enantiomers and their chiral

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metabolites must be considered for risk assessment.

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Prothioconazole,

(R,S)-(2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-

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hydroxypropyl]-1,2-dihydro-3H-1,2,4-triazole-3-thione), 1 and 2 (Figure 1) is a broad

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spectrum systemic triazole fungicide with prominent protective and curative

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properties that inhibits the C-14α-demethylase enzyme involved in the biosynthesis of

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fungal sterols.15 This fungicide has been widely used to control powdery mildew,

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Fusarium head blight, rusts and sclerotium on corn, legume crops and other economic

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crop. Prothioconazole-desthio, (R,S)-(2-(1-chlorocyclopropyl)-1-(2-chlorophenyl)-33



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(1,2,4-triazol-1-yl)-propan-2-ol), 3 and 4 (Figure 1) is a major chiral metabolite of

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prothioconazole in crops and the environment. Prothioconazole and its metabolite

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have one chiral center, each. Until recently, prothioconazole has been produced and

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sold as racemic mixture. Although prothioconazole was widely used to control plant

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disease, there are limited studies on the resolution of chiral prothioconazole and its

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chiral metabolites enantiomers,16 and enantiomeric analysis methods in agricultural

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food and environment samples have not been reported. Therefore, it is necessary to

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develop enantiomeric analysis methods to understand the stereoselective metabolism

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of prothioconazole in crop and environment, which will be conducive to more

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accurate risk assessment.

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In this study, an efficient and reliable chiral analytical method was developed to

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determine prothioconazole and its chiral metabolites in agricultural products and

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environment samples using ultra performance liquid chromatography tandem QTRAP

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mass spectrometry (UPLC-MS/MS) with a Lux Cellulose-3 column. Variables related

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to separation of prothioconazole and its metabolites were investigated, including

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chiral stationary phases (CSPs), proportion of mobile phase and column temperature.

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The optical rotation was determined by HPLC tandem optical rotation detector. The

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absolute configurations of enantiomers were confirmed by comparing the

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experimental and calculated electronic circular dichroism (ECD) spectra. The chiral

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stability of the four stereoisomers in different organic solvents and water was also

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discussed. The extraction procedures for target analytes were based on the

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QuEChERS (quick, easy, cheap, effective, rugged and safe) method. This is the first

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time that an effective chiral method has been established for the enantioselective

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analysis of prothioconazole and its major chiral metabolite in environmental and

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agricultural samples. This sensitive chiral analytical method was applied to investigate 4


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the enantioselective degradation and metabolism of prothioconazole in soil under field

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conditions.

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MATERIALS AND METHODS

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Reagents and Materials. Prothioconazole and prothioconazole-desthio standards

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(≥98.1 purity) were acquired from the Alta Scientific Co., Ltd. (Tianjin, China).

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Enantiomers of prothioconazole and prothioconazole-desthio (≥99.39% purity) were

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obtained from the Chiralway Biotech Co., Ltd. (Shanghai, China). HPLC-grade

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acetonitrile was purchased from Tedia (Fairfield, OH). Ultrapure water was obtained

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by using MUL-9000 water purification systems (Nanjing Zongxin Water Equipment

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Co. Ltd., Nanjing, China). Mixed standard stock solutions of prothioconazole and

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prothioconazole-desthio and the individual enantiomers (1000 mg/L) were prepared in

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acetonitrile and stored at 4 °C. The Cleanert Florisil, Cleanert C18, PSA, Alumina-N

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and Alumina-A SPE (500 mg, 6 mL) were purchased from Agela Technologies

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(Tianjin, China).

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Chiral Separation of Prothioconazole and its Metabolite by UPLC-MS/MS.

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The enantiomeric separation and analysis of prothioconazole and its metabolite were

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performed on a model 30A Nexera UPLC system (Shimadzu, Kyoto, Japan) tandem

100

with a QTRAP6500 LC-MS/MS system (Sciex, Massachusetts, MA). The

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electrospray ionization source was used to quantitate prothioconazole and its

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metabolite with the following settings: ion source temperature, 550 °C; atomization,

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60 psi; auxiliary gas, 60 psi; air curtain gas, 40 psi; inlet voltage, 10 V; and outlet

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voltage, 12 V. Prothioconazole enantiomers were analyzed with negative ionization in

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full scan mode, prothioconazole-desthio was analyzed with positive ionization in full

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scan mode. The deprotonated molecular ion [M-H]- (m/z 342.2) was extracted for

5



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quantitative determination of prothioconazole enantiomers and [M+H]+ (m/z 312.2)

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was extracted for quantitative determination of prothioconazole-desthio enantiomers.

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Separation of prothioconazole and its metabolite enantiomers was evaluated on

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chiral columns (the columns were 250 mm ×4.6 mm i.d., 5 µm and 150 mm × 2 mm

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i.d., 3 µm, Lux Cellulose-1, 2 or 3, respectively) (Phenomenex, Guangzhou, China).

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A mixture of acetonitrile/water were used as the mobile phase with an injection

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volume of 5 µL. The chromatographic separation parameters, capacity factor (k),

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separation factor (α), resolution (Rs) and the isolation temperature (Tiso) were

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calculated to evaluate the effect of enantioseparation under different conditions,

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simultaneously. The enthalpy (ΔΔH○) and entropy (ΔΔS○) variation between

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enantiomers were also calculated using the following Van’t Hoff equations.16-18

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Lnk = -ΔH○ / RT + ΔS○ / R + lnΦ

Eqn.1

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Lnα = -ΔΔH○ / RT + ΔΔS○ / R

Eqn.2

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Determination of Optical Rotation. The stereoisomeric optical rotation signals of

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enantiomers were measured by HPLC (Agilent, California, CA) coupled with optical

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rotation detector (ORD) ( IBZ Messtechnik GmbH, Hannover, Germany) at 426

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nm.19,20 The mixed standard solution of prothioconazole and prothioconazole-desthio

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dissolved in acetonitrile was measured on Lux Cellulose-3 with acetonitrile/water (40:

125

60, v/v) as the mobile phase at the flow of 0.8 mL/min.

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Circular Dichroism Spectroscopy and ECD Calculations. Experimental

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electronic circular dichroism (ECD) spectroscopy for prothioconazole and its

128

metabolite

129

spectropolarimeter (Jasco, Tokyo, Japan) at room temperature in acetonitrile. The

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spectra were collected from 190-400 nm at a scan speed of 50 nm/min using a 0.1-cm

enantiomers

was

performed

using

a

J815

6


circular

dichroism

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quartz cell and an average of three scans. The experimental ECD spectra were drawn

132

using Origin software (version 8.61).

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All computational procedure used Gaussian 09 W software.21 Initially, the stable

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conformation of prothioconazole and its chiral metabolites enantiomers was identified

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using the Monte Carlo MMFF94 molecular mechanics force field. The four lowest-

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energy (less than 6 kcal/mol) conformers of each enantiomer were selected and

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optimized without the symmetric restraint using density functional theory combined

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with the Becke, three-parameter, Lee–Yang–Parr (B3LYP) exchange-correlation

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functional with the basis set 6-31+G*. The ECD spectra of the lowest energy

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conformers was calculated using time-dependent density functional theory method

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with the same basis set. Tight, self-consistent field convergence standards were

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adopted in all calculations. The absolute configuration of pairs of enantiomers was

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determined by comparing the similarity of the experimental ECD spectra and

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predicted ECD spectra of the four lowest energy conformers.22-27

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Chiral Stability. Clear epimerization occurs in methanol, acetonitrile and water for

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some chiral triazole fungicides, such as triazolone.28,29 The 1 mg/L standard solutions

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of enantiomers of prothioconazole and its metabolite in methanol, acetonitrile and

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water were placed in incubator at 4 °C and 30 °C in the dark. The samples were

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analyzed using UPLC-MS/MS after filtration through a 0.22 µm nylon syringe filter

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at 0, 1, 3, 7, 14, 30, 60, 120 and 180 d.

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Sample Preparation. Extraction and clean up for water. The 100 mL river water

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samples were slowly transferred to a C18 SPE cartridge pretreated with 6 mL of

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methanol followed by 6 mL of ultrapure water. Target compounds were eluted with

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12 mL of methanol and collected. The eluate was evaporated to dryness with a rotary

7



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evaporator at 50 °C. The residue was dissolved in 1 mL of acetonitrile for UPLC-

156

MS/MS analysis after filtration through a 0.22 µm filter membrane.

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Extraction and clean up for soil, cucumber and pear. A 20 g sample of soil,

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cucumber or pear was weighted into a 100 mL Teflon centrifuge tube. Then, 10 mL

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water (only for soil), 1mL cysteine hydrochloride solution (1g/L) and 40 mL

160

acetonitrile were added after 2 h at room temperature and mixtures was vortexed at

161

high speed for 3 min, followed by sonication for 10 min. Subsequently, 3 g sodium

162

chloride was added and the vortex step was repeated for 1 min. The centrifuge tube

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was centrifuged for 5 min at 3000 rpm. An aliquot (20 mL) of acetonitrile supernatant

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was concentrated to dryness under vacuum at 40 °C.

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The samples were dissolved in 12 mL n-hexane and transferred to a Florisil-SPE

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cartridge that had been preleached with 5 mL n-hexane. The Florisil SPE cartridge

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was rinsed with 6 mL of n-hexane, the column was eluted with 15 mL of n-

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hexane/acetone (98:2, v/v) and its metabolite was preferentially collected. The

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cartridge was rerinsed with 6 mL of n-hexane/acetone (90:10, v/v) and then eluted

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with 15 mL of n-hexane/acetone (75:25, v/v) for collecting prothioconazole. All the

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eluates were merged and evaporated to dryness with on a vacuum rotary evaporator at

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40 °C. The residues were dissolved in 1 mL of acetonitrile and then filtered using a

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0.22 µm nylon syringe filter for UPLC-MS/MS analysis.

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Method Validation. The specificity, linearity, limit of detection (LOD), limit of

175

quantification (LOQ), accuracy, and precision were used to evaluate the performance

176

of the method.

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Blank control water, soil, cucumber and pear samples were analyzed in

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quintuplicate to confirm the absence of interfering substances at the retention time of

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target chiral compounds. The linearity of solvent and different matrix-matched 8


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calibration curves were determined at 5-500 µg/L and on the basis of the peak areas of

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target analytes in triplicate at six concentrations. The slope ratio of calibration curves

182

of standards in solvent and matrix-matched solutions was calculated to evaluate the

183

matrix effect.

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The matrix-dependent LODs and LOQs of pairs of enantiomers in agricultural

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products and environmental samples were determined at concentrations that produced

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signal-to-noise (S/N) ratios of 3 and 10, respectively.

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Spike and recovery experiments were used to evaluated the accuracy and precision

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of the method. Five replicate blank samples spiked with different concentrations (5,

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50 and 500 µg/kg) for agricultural products and environmental samples were prepared

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and incubated overnight. All spiked samples were prepared on three consecutive days

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and the enantiomers of prothioconazole and its metabolites were extracted and

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purified according to the procedure described above. The recoveries, intraday RSDs

193

and interday RSDs were used to evaluate accuracy and precision.

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Field experiments. Four plots of soil (30 m2) were selected in Nanjing, China,

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each plot had a buffer area of one-meter. A 40% suspension concentrate (SC) of

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prothioconazole was sprayed into three plots at a dosage of 337.5 g a.i/ha. The other

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plot was used as the control. Twelve representatively selected soil points were

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collected from each treatment at 2, 4, 8 and 12 h on day 0 and 1, 2, 3, 5, 7, 10, 14, 21,

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28 and 35 d after spraying. All of soil samples were mixed and stored at −20 °C until

200

analysis.

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RESULTS AND DISCUSSION

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MS analysis. MS/MS analyses were performed in multiple reaction monitoring

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(MRM) mode, measuring the fragmentation of prothioconazole and its metabolite.

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Higher abundant product ions of m/z 100.1, 125.0 and 246.2 for fragmentation of [M9



205

H]- (m/z 342.2) and product ions m/z 70.0 and 125.0 for fragmentation of [M+H]+ (m/z

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312.2) were found in the mass spectra. The optimized MRM parameters for

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prothioconazole and its metabolite detection were as follows: the transitions m/z 342 >

208

100.1 were selected for quantification; m/z 342 > 100 and m/z 342 > 125 were applied

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for confirmation when the collision energy (CE) was set at 27 and 32 V for

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prothioconazole, respectively. For prothioconazole-desthio, m/z 312>70 was selected

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for quantification; m/z 342 > 70 and m/z 342 > 125 were applied for confirmation

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when the CE was set at 38 and 49 V.

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Optimization of Enantioseparation Conditions. Enantiomeric separation of

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chiral pesticide was based on several variables including the chiral stationary phase,

215

mobile phase, flow rate and column temperature. Because the pairs of enantiomers

216

could not be completely separated on Lux Cellulose-1, Lux Cellulose-2 and Lux

217

Cellulose-3 (150 mm × 2 mm i.d., 3μm), the separation of prothioconazole and its

218

metabolite enantiomers was evaluated on Lux Cellulose-1, Lux Cellulose-2 and Lux

219

Cellulose-3 (250 mm × 4.6 mm i.d., 5μm) within the tolerable pressure range of the

220

instrument and column. Perfect baseline separation of pairs of enantiomers was

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achieved on Lux Cellulose-3 (Figure 2A). The four stereoisomers were partially

222

separated on Lux Cellulose-1 and could not be separated on Lux Cellulose-2.

223

The composition of the mobile phase is a critical factor to achieve excellent chiral

224

separation of enantiomers on UPLC-MS/MS. Acetonitrile and methanol are

225

commonly used as the mobile phase for reverse phase. The proportion of acetonitrile

226

(percentage of acetonitrile ranged from 35-55%) for chiral separation of

227

prothioconazole and its metabolites were investigated on Lux Cellulose-3 chiral

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column. The resolutions (Rs) of pairs of enantiomers decreased significantly from

229

2.68-1.28 with an increased ratio of acetonitrile. Baseline separation of pairs of 10


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enantiomers was not achieved with a high percent of acetonitrile in the mobile phase

231

(55: 45, v/v). The retention time of enantiomers was more than 35 min using

232

acetonitrile/water (35: 65, v/v) as the mobile phase. Therefore, acetonitrile/water (40:

233

60, v/v) was selected as the eluent that provided good resolution and a shorter

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retention time for prothioconazole and its metabolite enantiomers.

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Column temperature affects the separation of enantiomers, primarily as a result of

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thermodynamic effect. The effects of column temperature on the chiral separation of

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prothioconazole and its metabolite enantiomers was investigated on the Lux

238

Cellulose-3 column using acetonitrile/water (40: 60, v/v) as the mobile phase. The

239

pairs of enantiomers had baseline separation at 15-35 °C. The capacity factors (k)

240

ranged from 3.63-9.01 and separation factor (α) was 1.11-1.22. An increase in the

241

column temperature offered shorter retention times but less efficient chiral separation.

242

To develop a satisfactory chiral separation method and to protect the column, a

243

column temperature of 25 °C was selected. The resolution (Rs) of prothioconazole

244

and its metabolites were satisfactory with the Rs 1.87 and 2.09.

245

The plots of lnk or lnα versus 1/T were fitted according to the Van’t Hoff equations

246

at 15-35 °C. Excellent linearity was obtained with correlation coefficients (R2) that

247

ranged from 0.9825-0.9965. These results demonstrate that the stationary phase

248

configuration and retention mechanism did not change in the experimental

249

temperature range.30,31 ΔΔH○ is defined as the interaction strength between the

250

enantiomers and stationary phase. The difference in the degree of freedom was

251

defined as ΔΔS○. The values of ΔΔH○ and ΔΔS○ were negative for prothioconazole

252

and its metabolites enantiomers, suggesting enthalpy driven separation. The resolution

253

of the pairs of enantiomers would increase as the column temperature decreases under

11



254

Tiso. This result clearly reveals that the thermodynamic process, enthalpy and entropy

255

change are closely related to the interaction of the solute with stationary phase.

256

Optical Rotation of Enantiomers of Prothioconazole and its Metabolite. The

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stereoisomeric optical rotation signals of prothioconazole and its metabolites are

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presented in Figure 3. Combining ORD (426 nm) with UV (220 nm), the optical

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rotation of prothioconazole and its metabolite was determined. The first compounds

260

eluted were prothioconazole-desthio enantiomers. The peak 1-4 were (+)-

261

prothioconazole-desthio, (-)-prothioconazole-desthio, (-)-prothioconazole and (+)-

262

prothioconazole, respectively.

263

The Absolute Configuration of Prothioconazole and Prothioconazole-desthio

264

Enantiomers. The predicted ECD spectra of four low-energy conformer are shown as

265

dashed line and the experimental ECD spectra of enantiomers was drawn using

266

continuous line (Figure 4). The experimental ECD spectra of prothioconazole

267

enantiomers most closely resembled the predicted ECD spectra of conformer 4 and

268

the experimental spectra of prothioconazole-desthio enantiomers was similar to

269

conformer 1. The absolute configuration of the prothioconazole and its metabolite

270

enantiomers that eluted from the Lux Cellulose-3 column were determined by

271

comparing experimental ECD to predicted ECD. Accordingly, peak1-4 of the

272

chromatograms shown in Figure 2A were respectively assigned to R-prothioconazole-

273

desthio, S-prothioconazole-desthio, R-prothioconazole and S-prothioconazole.

274

Stability of Enantiomers. The degradation and isomerization of enantiomers were

275

not observed in methanol, acetonitrile and water during over experiment period. There

276

was no significant difference between the initial concentration and measured

277

concentration at different sampling time of pairs of enantiomers in acetonitrile,

278

methanol and water. 12


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Optimization of Sample Preparation. Modified QuEChERS32was applied for

280

extraction of prothioconazole and its metabolites from agricultural products and

281

environmental samples and providing a satisfactory result. The samples (cucumber

282

and pear) contain complex matrices consisting of chlorophyll, pigments, and high

283

levels of sugar. To obtain better purification results, a variety of SPE columns

284

(Cleanert-C18, Florisil, PSA, Alumina-N and Alumina-A SPE) were evaluated to

285

purify samples. C18-SPE was satisfactorily utilized for the water sample using

286

methanol as an eluate. Florisil-SPE could efficiently remove pigments and

287

interferences from soil and agricultural food samples. Due to the difference in the

288

structure and property of prothioconazole and its metabolites, elution was repeated

289

using mixed solvents with different polarities on the Florisil column. The pigments

290

and interferences were not diminished and removed by PSA, Alumina-N or Alumina-

291

A SPE. Thus, C18-SPE and Florisil-SPE were most suited for the pretreatment of

292

agricultural food and environmental samples with complex matrices.

293

Method Validation. Specificity, Matrix effect, Linearity, LOD and LOQ. This

294

method is specific, with no interference for prothioconazole and its metabolite in

295

blank samples at the retention time. The responses of solvent standard calibration

296

curves were compared to matrix-matched calibration curves using two-tailed paired t-

297

test with a probability of 95%. The P-values were 0.0219-0.0368 in water, soil,

298

cucumber and pear matrices. The slope ratio of solvent calibration curves via matrix-

299

matched calibration curves ranged from 0.8095-6.3868 as shown in Table 1. Signal

300

suppression or enhancements of the four target compounds were typically observed in

301

the all matrix extracts and the matrix-matched standard calibration curves were

302

utilized for quantification.

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The data derived from matrix-matched calibration curves from 5-500 μg/kg are

304

presented in Table 1, showing excellent linearity of all enantiomers with R2 ≥ 0.9909.

305

The LODs were estimated at 0.0031-0.0087 µg/kg, and the LOQs were 0.0102-0.0290

306

µg/kg for enantiomers of prothioconazole. Correspondingly, the LODs and LOQs for

307

the enantiomers of prothioconazole-desthio were estimated to be 0.0025-0.0075 µg/kg

308

and 0.0083-0.025 µg/kg, respectively.

309

Accuracy and precision. The average recoveries of pairs of enantiomers were

310

determined at three different concentrations (5, 50 and 500 µg/kg) in quintuplicate.

311

The precision of this method was evaluated using relative standard deviation (RSD).

312

The method provided high accuracy and precision, and the mean recoveries of

313

prothioconazole enantiomers were 71.8-96.4% with 0.3-11.9% intra-day RSDs and

314

1.5-10.6% inter-day RSDs. The recoveries for enantiomers of its metabolite ranged

315

from 85.8-102.0% with 0.2-9.8% intra-day RSDs and 0.9-7.1% inter-day RSDs,

316

indicating that this method was able to provide accurate quantitative data for

317

enantiomeric analysis of agricultural food and environmental samples.

318

Enantioselective Dissipation of Prothioconazole and the Formation of

319

Prothioconazole-desthio in Soil. The effectiveness of the chiral method was used to

320

measure the degradation of prothioconazole and the formation of prothioconazole-

321

desthio in soil under field conditions. The degradation of prothioconazole and

322

prothioconazole-desthio in soil is shown in Figure 5A. Interestingly, both

323

prothioconazole enantiomers were found to easily degrade in soil. The correlation

324

coefficient of both enantiomers (R2) was above 0.9200 and the dissipation followed

325

the first-order kinetics according to the following Eq.:

326

C=C0e-kt

Eq.3

14


Page 15 of 30

327


The dissipation dynamics equations were C=0.5322e-1.281x (R2=0.9348) and

328

C=0.5938e-1.478x

329

respectively. The half-lives of R-prothioconazole and S-prothioconazole were 0.48

330

and 0.60 d. The concentration of S-prothioconazole (0.074 mg/kg) was 1.72 flod

331

higher than the R-prothioconazole enantiomer (0.043 mg/kg) after 1 d of spraying.

332

Remarkably enantioselective degradation was observed and the EF value was 0.48-

333

0.37 (Figure. 5B). R-prothioconazole showed preferential degradation in soil under

334

field condition. The formation of both prothioconazole-desthio enantiomers was

335

detected during prothioconazole dissipation. The concentration of prothioconazole-

336

desthio enantiomers increased steadily to a maximum concentration (0.288 mg/kg and

337

0.269

338

respectively) at 2 d, and then decreased slowly to the end of the field experiment. The

339

pair of enantiomers are shown in the same concentration. As one of the most

340

remarkably bioactive chiral metabolites, there was no obvious enantioselective

341

behavior for prothioconazole-desthio in soil. The environmental degradation and

342

metabolism of the chiral enantiomers in soil is important for the currently used chiral

343

fungicides. This method provides a feasible way to positively identify the chirality of

344

prothioconazole and its metabolites in soil, water and agricultural samples. These

345

results may be helpful to systematically evaluate the stereoselective behaviors of this

346

chiral fungicide and its metabolites in the environment.

mg/kg

(R2=0.9212)

for

for

R-prothioconazole

R-prothioconazole-desthio

and

and

S-prothioconazole,

S-prothioconazole-desthio,

347 348

ASSOCIATED CONTENT

349

Supporting Information

350

The effects of the flow rate (Table S1), mobile phase compositions (Table S2) and

351

temperature (Table S3) on the enantioseparation parameters. Van’t Hoff equations 15



Page 16 of 30

352

and the thermodynamic parameters (Table S4). Stability of enantiomers (Figure S1)

353

and typical chromatograms (Figure S2).

354

AUTHOR INFORMATION

355

Corresponding Author

356

*Tel: +86 25 84395479. Fax: +86 25 84395479. E-mail: [email protected].

357

Funding

358

This study was supported by the National Key Research and Development Program of

359

China (2016YFD0200207).

360

Notes

361

The authors declare no competing financial interest.

362

ACKNOWLEDGEMENT

363

We are grateful to Hu Zhang (Zhejiang Academy of Agriculture Sciences) and Bowen

364

Tang

365

prothioconazole and prothioconazole-desthio enantiomers by Gaussian 09 software.

366

ABBREVIATIONS USED

367

UPLC-QTRAP-MS/MS, ultra-performance liquid chromatography tandem QTRAP

368

mass spectrometry; CSP, chiral stationary phases; QuEChERS, quick, easy, cheap,

369

effective, rugged and safe; k, capacity factor; α, separation factor; Rs, resolution; Tiso,

370

isolation temperature.

371

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372

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configuration

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comparisons of experimental ECD and ORD spectra with DFT calculations.

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structures used for quantum mechanical predictions of chiroptical properties.

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Wang, Q.; Wang, M. Enantioselective analysis and degradation studies of 19



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Food Chem. 2014, 62, 2809-15.

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theory calculations of optical rotation and electronic circular dichroism. J. Am.

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separation and determination of triadimefon and triadimenol in wheat, straw, and

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[29] Liu, W.; Gan, J. J.; Lee, S.; Werner, I. Isomer selectivity in aquatic toxicity and

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[30] Ding, H.; Grinberg, N.; Thompson, R.; Ellison, D. Enantiorecognition

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Chromatogr. Related Technol. 2000, 23, 2641-2651.

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Use of liquid chromatography-quadrupole time-of-flight mass spectrometry for

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strawberry and soil. J. Chromatogr. A. 2016, 1449, 62-70.

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[32] Koesukwiwa, U.; Lehotay, S. J; Mastovska, K.; Dorweiler, K. J.; Leepipatpiboon,

474

N. Extension of the QuEChERS method for pesticide residues in cereals to

475

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21



Page 22 of 30

476

FIGURE CAPTIONS

477

Figure 1. Chemical structures of prothioconazole and prothioconazole-desthio

478

enantiomers.

479

Figure

480

enantiomers on Lux Cellulose-3: (A) prothioconazole and prothioconazole-desthio

481

enantiomers; (B) R-(+)-prothioconazole-desthio; (C) S-(-)-prothioconazole-desthio;

482

(D) R-(-)-prothioconazole; (E) S-(+)-prothioconazole.

483

Figure 3. Chromatogram of prothioconazole and prothioconazole-desthio with ORD

484

and UV detector: (A) ORD signal; (B) UV signal.

485

Figure 4. Predicted and experimental ECD spectra of prothioconazole and

486

prothioconazole-desthio

487

prothioconazole-desthio; (C) R-prothioconazole; (D) S-prothioconazole.

488

Figure 5. Variations of concentration and EF value during the degradation in soil: (A)

489

concentration of prothioconazole and prothioconazole-desthio enantiomers; (B) EF

490

value of prothioconazole.

2.

Chromatogram

of

prothioconazole

enantiomers:

(A)

and

prothioconazole-desthio

R-prothioconazole-desthio;

22


(B)

S-

Page 23 of 30


Table 1. The Linearity, LOD and LOQ for Prothioconazole and Prothioconazole-desthio Enantiomers with Different Matrices (5-500 µg/L).

enantiomer

R-(+)prothioconazoledesthio

S-(-)prothioconazoledesthio

R-(-)prothioconazole

slope of matrix/slope of solvent

matrix

regression equation

R

solvent

y=28688x+953

0.9998

water

y=23664x-5033

0.9994

0.8249

soil

y=23439x-2431

0.9999

cucumber

y=25154x-1260

pear

2

LOD(µg/kg)

LOQ(µg/kg)

0.0025

0.0083

0.0242

0.0055

0.0183

0.8170

0.0257

0.0075

0.0250

0.9999

0.8775

0.0283

0.0070

0.0232

y=23703x+2561

0.9995

0.8262

0.0302

0.0062

0.0206

solvent

y=28672x+878

0.9998

0.0029

0.0092

water

y=23568x-4546

0.9996

0.8220

0.0297

0.0071

0.0235

Soil

y=23577x-2634

0.9999

0.8223

0.0219

0.0071

0.0235

cucumber

y=24891x-1157

0.9999

0.8681

0.0221

0.0061

0.0205

pear

y=23210x+3339

0.9993

0.8095

0.0246

0.0062

0.0206

solvent

y=1763x-1747

0.9945

0.0087

0.0290

water

y=5613x-8396

0.9920

3.1838

0.0289

0.0067

0.0222

Soil

y=6057x-8441

0.9949

3.4356

0.0282

0.0040

0.0135

23


P


S-(+)prothioconazole

Page 24 of 30

cucumber

y=5641x-11672

0.9973

3.1997

0.0368

0.0045

0.0152

pear

y=11260x-27753

0.9974

6.3868

0.0326

0.0032

0.0109

solvent

y=1737x-1773

0.9949

0.0058

0.0195

water

y=5430x-9116

0.9929

3.1260

0.0285

0.0056

0.0186

Soil

y=6346x-8309

0.9909

3.6534

0.0269

0.0056

0.0188

cucumber

y=6114x-16338

0.9927

3.5199

0.0364

0.0046

0.0155

pear

y=10939x-25126

0.9995

6.2976

0.0310

0.0031

0.0102

24


Page 25 of 30


Figure 1

25



Figure 2.

26


Page 26 of 30

Page 27 of 30


Figure 3.

27



Figure 4.

28


Page 28 of 30

Page 29 of 30


Figure 5.

29



Graphic for table of contents

30


Page 30 of 30

Simultaneous Enantioselective Determination of the Chiral Fungicide

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