Article pubs.acs.org/JAFC
Simultaneous Enantioselective Determination of the Chiral Fungicide Prothioconazole and Its Major Chiral Metabolite ProthioconazoleDesthio in Food and Environmental Samples by Ultraperformance Liquid Chromatography−Tandem Mass Spectrometry Zhaoxian Zhang,† Qing Zhang,† Beibei Gao,† Gaozhang Gou,‡ Lianshan Li,† Haiyan Shi,† and Minghua Wang*,† †
Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing 210095, China ‡ College of Science, Honghe University, Mengzi 661199, China S Supporting Information *
ABSTRACT: An efficient and sensitive chiral analytical method was established for the determination of the chiral fungicide prothioconazole and its major chiral metabolite prothioconazole-desthio in agricultural and environmental samples using ultraperformance liquid chromatography−tandem mass spectrometry. The optical rotation and absolute configuration of enantiomers were identified by optical rotation detector and electronic circular dichroism spectra. The elution order of prothioconazole and its chiral metabolite enantiomers was R-(+)-prothioconazole-desthio, S-(−)-prothioconazole-desthio, R-(−)-prothioconazole, and S-(+)-prothioconazole. The mean recoveries from the samples was 71.8−102.0% with intraday relative standard deviations (RSDs) of 0.3−11.9% and interday RSDs of 0.9−10.6%. The formation of prothioconazole-desthio was studied in soil under field conditions and enantioselective degradation was observed for chiral prothioconazole. Remarkable enantioselective degradation was observed: R-prothioconazole degraded preferentially with EF values from 0.48 to 0.37. Although prothioconazole-desthio is the most remarkably bioactive metabolite, no obvious enantioselective behavior was observed in soil. These results may help to systematically evaluate prothioconazole and its metabolites in food and environmental safety. KEYWORDS: prothioconazole, chiral metabolite, enantioseparation, absolute configuration, UPLC−MS/MS
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INTRODUCTION Chiral pesticides play a critical role in the control of pests and diseases in agricultural systems. The proportion of chiral pesticide has continually increased as more complex compounds are introduced.1 Most chiral triazole fungicides have one or more chiral centers and thus two or more enantiomers. Although the enantiomers have identical physical properties, their biological and physiological properties in chiral environments can significantly differ.2−9 For example, the bactericidal activity of R-diniconazole is higher than for S-diniconazole, but the activity profile was reversed when their ability to regulate plant growth was assessed.10 The activity of two triadimefon enantiomers is very low, but one of its reduction products ((1S, 2R)-triadimenol) had higher bactericidal activity than other three enantiomers.11 In addition, (−)-hexaconazole was approximately 6 times more toxic than (+)-form to Scenedesmus obliquus.12 Furthermore, some chiral pesticides can degrade into different chiral metabolites that can exhibit more toxicity than the original xenobiotic.13 Sparling et al.14 reported that the corresponding oxide derivatives of diazinon, chlorpyrifos, and malathion showed greater toxicity than the parent compound. To systematically assess chiral pesticide, both enantiomers and their chiral metabolites must be considered for risk assessment. Prothioconazole, (R,S)-(2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1,2-dihydro-3H-1,2,4-triazole-3-thione), 1 and 2 (Figure 1) is a broad spectrum systemic triazole fungicide © 2017 American Chemical Society
with prominent protective and curative properties that inhibits the C-14α-demethylase enzyme involved in the biosynthesis of fungal sterols.15 This fungicide has been widely used to control powdery mildew, Fusarium head blight, rusts, and sclerotium on
Figure 1. Chemical structures of prothioconazole and prothioconazoledesthio enantiomers. Received: Revised: Accepted: Published: 8241
June 23, 2017 August 25, 2017 August 27, 2017 August 27, 2017 DOI: 10.1021/acs.jafc.7b02903 J. Agric. Food Chem. 2017, 65, 8241−8247
Article
Journal of Agricultural and Food Chemistry
5 μm and 150 mm × 2 mm i.d., 3 μm, Lux Cellulose-1, 2 or 3, respectively) (Phenomenex, Guangzhou, China). A mixture of acetonitrile/water were used as the mobile phase with an injection volume of 5 μL. The chromatographic separation parameters, capacity factor (k), separation factor (α), resolution (Rs), and the isolation temperature (Tiso) were calculated to evaluate the effect of enantioseparation under different conditions, simultaneously. The enthalpy (ΔΔH°) and entropy (ΔΔS°) variation between enantiomers were also calculated using the following Van’t Hoff equations.16−18
corn, legume crops, and other economic crops. Prothioconazoledesthio, (R,S)-(2-(1-chlorocyclopropyl)-1-(2-chlorophenyl)-3(1,2,4-triazol-1-yl)-propan-2-ol), 3, and 4 (Figure 1) are major chiral metabolites of prothioconazole in crops and the environment. Prothioconazole and its metabolite have one chiral center, each. Until recently, prothioconazole has been produced and sold as a racemic mixture. Although prothioconazole was widely used to control plant disease, there are limited studies on the resolution of chiral prothioconazole and its chiral metabolites enantiomers,16 and enantiomeric analysis methods in agricultural food and environment samples have not been reported. Therefore, it is necessary to develop enantiomeric analysis methods to understand the stereoselective metabolism of prothioconazole in crops and the environment, which will be conducive to more accurate risk assessment. In this study, an efficient and reliable chiral analytical method was developed to determine prothioconazole and its chiral metabolites in agricultural products and environment samples using ultra performance liquid chromatography tandem QTRAP mass spectrometry (UPLC−MS/MS) with a Lux Cellulose-3 column. Variables related to separation of prothioconazole and its metabolites were investigated, including chiral stationary phases (CSPs), proportion of mobile phase, and column temperature. The optical rotation was determined by the HPLC tandem optical rotation detector. The absolute configurations of enantiomers were confirmed by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. The chiral stability of the four stereoisomers in different organic solvents and water was also discussed. The extraction procedures for target analytes were based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method. This is the first time that an effective chiral method has been established for the enantioselective analysis of prothioconazole and its major chiral metabolite in environmental and agricultural samples. This sensitive chiral analytical method was applied to investigate the enantioselective degradation and metabolism of prothioconazole in soil under field conditions.
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ln k = −ΔH °/RT + ΔS°/R + ln Φ
(1)
ln α = −ΔΔH °/RT + ΔΔS°/R
(2)
Determination of Optical Rotation. The stereoisomeric optical rotation signals of enantiomers were measured by HPLC (Agilent, CA) coupled with optical rotation detector (ORD) (IBZ Messtechnik GmbH, Hannover, Germany) at 426 nm.19,20 The mixed standard solution of prothioconazole and prothioconazole-desthio dissolved in acetonitrile was measured on Lux Cellulose-3 with acetonitrile/water (40:60, v/v) as the mobile phase at the flow of 0.8 mL/min. Circular Dichroism Spectroscopy and ECD Calculations. Experimental electronic circular dichroism (ECD) spectroscopy for prothioconazole, and its metabolite enantiomers was performed using a J815 circular dichroism spectropolarimeter (Jasco, Tokyo, Japan) at room temperature in acetonitrile. The spectra were collected from 190 to 400 nm at a scan speed of 50 nm/min using a 0.1 cm quartz cell and an average of three scans. The experimental ECD spectra were drawn using Origin software (version 8.61). All computational procedure used Gaussian 09 W software.21 Initially, the stable conformation of prothioconazole and its chiral metabolites enantiomers was identified using the Monte Carlo MMFF94 molecular mechanics force field. The four lowest-energy (less than 6 kcal/mol) conformers of each enantiomer were selected and optimized without the symmetric restraint using density functional theory combined with the Becke, three-parameter, Lee−Yang−Parr (B3LYP) exchange-correlation functional with the basis set 6-31+G*. The ECD spectra of the lowest energy conformers was calculated using time-dependent density functional theory method with the same basis set. Tight, self-consistent field convergence standards were adopted in all calculations. The absolute configuration of pairs of enantiomers was determined by comparing the similarity of the experimental ECD spectra and predicted ECD spectra of the four lowest energy conformers.22−27 Chiral Stability. Clear epimerization occurs in methanol, acetonitrile, and water for some chiral triazole fungicides, such as triazolone.28,29 The 1 mg/L standard solutions of enantiomers of prothioconazole and its metabolite in methanol, acetonitrile, and water were placed in an incubator at 4 and 30 °C in the dark. The samples were analyzed using UPLC−MS/MS after filtration through a 0.22 μm nylon syringe filter at 0, 1, 3, 7, 14, 30, 60, 120, and 180 days. Sample Preparation. Extraction and Cleanup for Water. The 100 mL river water samples were slowly transferred to a C18 SPE cartridge pretreated with 6 mL of methanol followed by 6 mL of ultrapure water. Target compounds were eluted with 12 mL of methanol and collected. The eluate was evaporated to dryness with a rotary evaporator at 50 °C. The residue was dissolved in 1 mL of acetonitrile for UPLC−MS/MS analysis after filtration through a 0.22 μm filter membrane. Extraction and Cleanup for Soil, Cucumber, and Pear. A 20 g sample of soil, cucumber, or pear was weighted into a 100 mL Teflon centrifuge tube. Then, 10 mL of water (only for soil), 1 mL of cysteine hydrochloride solution (1g/L), and 40 mL of acetonitrile were added after 2 h at room temperature and mixtures were vortexed at high speed for 3 min, followed by sonication for 10 min. Subsequently, 3 g sodium chloride was added and the vortex step was repeated for 1 min. The centrifuge tube was centrifuged for 5 min at 3000 rpm. An aliquot (20 mL) of acetonitrile supernatant was concentrated to dryness under vacuum at 40 °C. The samples were dissolved in 12 mL of n-hexane and transferred to a Florisil-SPE cartridge that had been preleached with 5 mL of n-hexane. The Florisil SPE cartridge was rinsed with 6 mL of n-hexane, the column was eluted with 15 mL of n-hexane/acetone (98:2, v/v) and its metabolite was preferentially collected. The cartridge was rerinsed with 6 mL of
MATERIALS AND METHODS
Reagents and Materials. Prothioconazole and prothioconazoledesthio standards (≥98.1 purity) were acquired from the Alta Scientific Co., Ltd. (Tianjin, China). Enantiomers of prothioconazole and prothioconazoledesthio (≥99.39% purity) were obtained from the Chiralway Biotech Co., Ltd. (Shanghai, China). HPLC-grade acetonitrile was purchased from Tedia (Fairfield, OH). Ultrapure water was obtained by using MUL-9000 water purification systems (Nanjing Zongxin Water Equipment Co. Ltd., Nanjing, China). Mixed standard stock solutions of prothioconazole and prothioconazole-desthio and the individual enantiomers (1000 mg/L) were prepared in acetonitrile and stored at 4 °C. The Cleanert Florisil, Cleanert C18, PSA, Alumina-N, and Alumina-A SPE (500 mg, 6 mL) were purchased from Agela Technologies (Tianjin, China). Chiral Separation of Prothioconazole and Its Metabolite by UPLC−MS/MS. The enantiomeric separation and analysis of prothioconazole and its metabolite were performed on a model 30A Nexera UPLC system (Shimadzu, Kyoto, Japan) tandem with a QTRAP6500 LC−MS/MS system (Sciex, MA). The electrospray ionization source was used to quantitate prothioconazole and its metabolite with the following settings: ion source temperature, 550 °C; atomization, 60 psi; auxiliary gas, 60 psi; air curtain gas, 40 psi; inlet voltage, 10 V; and outlet voltage, 12 V. Prothioconazole enantiomers were analyzed with negative ionization in full scan mode, and prothioconazole-desthio was analyzed with positive ionization in full scan mode. The deprotonated molecular ion [M − H]− (m/z 342.2) was extracted for quantitative determination of prothioconazole enantiomers and [M + H]+ (m/z 312.2) was extracted for quantitative determination of prothioconazole-desthio enantiomers. Separation of prothioconazole and its metabolite enantiomers was evaluated on chiral columns (the columns were 250 mm × 4.6 mm i.d., 8242
DOI: 10.1021/acs.jafc.7b02903 J. Agric. Food Chem. 2017, 65, 8241−8247
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ions of m/z 100.1, 125.0, and 246.2 for fragmentation of [M − H]− (m/z 342.2) and product ions m/z 70.0 and 125.0 for fragmentation of [M + H]+ (m/z 312.2) were found in the mass spectra. The optimized MRM parameters for prothioconazole and its metabolite detection were as follows: the transitions m/z 342 > 100.1 were selected for quantification; m/z 342 > 100 and m/z 342 > 125 were applied for confirmation when the collision energy (CE) was set at 27 and 32 V for prothioconazole, respectively. For prothioconazole-desthio, m/z 312 > 70 was selected for quantification; m/z 342 > 70 and m/z 342 > 125 were applied for confirmation when the CE was set at 38 and 49 V. Optimization of Enantioseparation Conditions. Enantiomeric separation of chiral pesticide was based on several variables including the chiral stationary phase, mobile phase, flow rate and column temperature. Because the pairs of enantiomers could not be completely separated on Lux Cellulose-1, Lux Cellulose-2, and Lux Cellulose-3 (150 mm × 2 mm i.d., 3 μm), the separation of prothioconazole and its metabolite enantiomers was evaluated on Lux Cellulose-1, Lux Cellulose-2, and Lux Cellulose-3 (250 mm × 4.6 mm i.d., 5 μm) within the tolerable pressure range of the instrument and column. Perfect baseline separation of pairs of enantiomers was achieved on Lux Cellulose-3 (Figure 2A). The four stereoisomers were partially separated on Lux Cellulose-1 and could not be separated on Lux Cellulose-2. The composition of the mobile phase is a critical factor to achieve excellent chiral separation of enantiomers on UPLC− MS/MS. Acetonitrile and methanol are commonly used as the mobile phase for reverse phase. The proportion of acetonitrile (percentage of acetonitrile ranged from 35 to 55%) for chiral separation of prothioconazole and its metabolites were investigated on Lux Cellulose-3 chiral column. The resolutions (Rs) of pairs of enantiomers decreased significantly from 2.68 to 1.28 with an increased ratio of acetonitrile. Baseline separation of pairs of enantiomers was not achieved with a high percent of acetonitrile in the mobile phase (55:45, v/v). The retention time of enantiomers was more than 35 min using acetonitrile/water (35:65, v/v) as the mobile phase. Therefore, acetonitrile/water (40:60, v/v)
n-hexane/acetone (90:10, v/v) and then eluted with 15 mL of n-hexane/ acetone (75:25, v/v) for collecting prothioconazole. All the eluates were merged and evaporated to dryness with on a vacuum rotary evaporator at 40 °C. The residues were dissolved in 1 mL of acetonitrile and then filtered using a 0.22 μm nylon syringe filter for UPLC−MS/MS analysis. Method Validation. The specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision were used to evaluate the performance of the method. Blank control water, soil, cucumber, and pear samples were analyzed in quintuplicate to confirm the absence of interfering substances at the retention time of target chiral compounds. The linearity of solvent and different matrix-matched calibration curves were determined at 5−500 μg/L and on the basis of the peak areas of target analytes in triplicate at six concentrations. The slope ratio of calibration curves of standards in solvent and matrix-matched solutions was calculated to evaluate the matrix effect. The matrix-dependent LODs and LOQs of pairs of enantiomers in agricultural products and environmental samples were determined at concentrations that produced signal-to-noise (S/N) ratios of 3 and 10, respectively. Spike and recovery experiments were used to evaluated the accuracy and precision of the method. Five replicate blank samples spiked with different concentrations (5, 50, and 500 μg/kg) for agricultural products and environmental samples were prepared and incubated overnight. All spiked samples were prepared on three consecutive days and the enantiomers of prothioconazole and its metabolites were extracted and purified according to the procedure described above. The recoveries, intraday RSDs, and interday RSDs were used to evaluate accuracy and precision. Field Experiments. Four plots of soil (30 m2) were selected in Nanjing, China, each plot had a buffer area of one-meter. A 40% suspension concentrate (SC) of prothioconazole was sprayed into three plots at a dosage of 337.5 g a.i/ha. The other plot was used as the control. Twelve representatively selected soil points were collected from each treatment at 2, 4, 8, and 12 h on days 0 and 1, 2, 3, 5, 7, 10, 14, 21, 28, and 35 days after spraying. All of soil samples were mixed and stored at −20 °C until analysis.
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RESULTS AND DISCUSSION MS Analysis. MS/MS analyses were performed in multiple reaction monitoring (MRM) mode, measuring the fragmentation of prothioconazole and its metabolite. Higher abundant product
Figure 2. Chromatogram of prothioconazole and prothioconazole-desthio enantiomers on Lux Cellulose-3: (A) prothioconazole and prothioconazoledesthio enantiomers, (B) R-(+)-prothioconazole-desthio, (C) S-(−)-prothioconazole-desthio, (D) R-(−)-prothioconazole, and (E) S-(+)-prothioconazole. 8243
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Absolute Configuration of Prothioconazole and Prothioconazole-desthio Enantiomers. The predicted ECD spectra of four low-energy conformers are shown as dashed lines, and the experimental ECD spectra of enantiomers were drawn using continuous lines (Figure 4). The experimental ECD spectra of prothioconazole enantiomers most closely resembled the predicted ECD spectra of conformer 4, and the experimental spectra of prothioconazole-desthio enantiomers was similar to conformer 1. The absolute configuration of the prothioconazole and its metabolite enantiomers that eluted from the Lux Cellulose-3 column were determined by comparing experimental ECD to predicted ECD. Accordingly, peaks 1−4 of the chromatograms shown in Figure 2A were, respectively, assigned to R-prothioconazole-desthio, S-prothioconazole-desthio, R-prothioconazole, and S-prothioconazole. Stability of Enantiomers. The degradation and isomerization of enantiomers were not observed in methanol, acetonitrile, and water during the experiment period. There was no significant difference between the initial concentration and measured concentration at different sampling times of pairs of enantiomers in acetonitrile, methanol, and water. Optimization of Sample Preparation. Modified QuEChERS32 was applied for extraction of prothioconazole and its metabolites from agricultural products and environmental samples and providing a satisfactory result. The samples (cucumber and pear) contain complex matrixes consisting of chlorophyll, pigments, and high levels of sugar. To obtain better purification results, a variety of SPE columns (Cleanert-C18, Florisil, PSA, Alumina-N, and Alumina-A SPE) were evaluated to purify samples. C18-SPE was satisfactorily utilized for the water sample using methanol as an eluate. Florisil-SPE could efficiently remove pigments and interferences from soil and agricultural food samples. Because of the difference in the structure and property of prothioconazole and its metabolites, elution was repeated using mixed solvents with different polarities on the Florisil column. The pigments and interferences were not diminished and removed by PSA, Alumina-N, or Alumina-A SPE. Thus, C18-SPE and Florisil-SPE were most suited for the pretreatment of agricultural food and environmental samples with complex matrixes.
was selected as the eluent that provided good resolution and a shorter retention time for prothioconazole and its metabolite enantiomers. Column temperature affects the separation of enantiomers, primarily as a result of thermodynamic effect. The effects of column temperature on the chiral separation of prothioconazole and its metabolite enantiomers was investigated on the Lux Cellulose-3 column using acetonitrile/water (40:60, v/v) as the mobile phase. The pairs of enantiomers had baseline separation at 15−35 °C. The capacity factors (k) ranged from 3.63 to 9.01 and separation factor (α) was 1.11−1.22. An increase in the column temperature offered shorter retention times but less efficient chiral separation. To develop a satisfactory chiral separation method and to protect the column, a column temperature of 25 °C was selected. The resolution (Rs) of prothioconazole and its metabolites were satisfactory with the Rs 1.87 and 2.09. The plots of lnk or ln α versus 1/T were fitted according to the Van’t Hoff equations at 15−35 °C. Excellent linearity was obtained with correlation coefficients (R2) that ranged from 0.9825 to 0.9965. These results demonstrate that the stationary phase configuration and retention mechanism did not change in the experimental temperature range.30,31 ΔΔH° is defined as the interaction strength between the enantiomers and stationary phase. The difference in the degree of freedom was defined as ΔΔS°. The values of ΔΔH° and ΔΔS° were negative for prothioconazole and its metabolites enantiomers, suggesting enthalpy driven separation. The resolution of the pairs of enantiomers would increase as the column temperature decreases under Tiso. This result clearly reveals that the thermodynamic process, enthalpy and entropy change are closely related to the interaction of the solute with the stationary phase. Optical Rotation of Enantiomers of Prothioconazole and Its Metabolite. The stereoisomeric optical rotation signals of prothioconazole and its metabolites are presented in Figure 3. Combining ORD (426 nm) with UV (220 nm), the optical rotation of prothioconazole and its metabolite was determined. The first compounds eluted were prothioconazole-desthio enantiomers. The peak 1−4 were (+)-prothioconazole-desthio, (−)-prothioconazole-desthio, (−)-prothioconazole, and (+)-prothioconazole, respectively.
Figure 3. Chromatogram of prothioconazole and prothioconazole-desthio with ORD and UV detector: (A) ORD signal and (B) UV signal. 8244
DOI: 10.1021/acs.jafc.7b02903 J. Agric. Food Chem. 2017, 65, 8241−8247
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Journal of Agricultural and Food Chemistry Method Validation. Specificity, Matrix Effect, Linearity, LOD, and LOQ. This method is specific, with no interference for prothioconazole and its metabolite in blank samples at the retention time. The responses of solvent standard calibration curves were compared to matrix-matched calibration curves using two-tailed paired t test with a probability of 95%. The P-values were 0.0219−0.0368 in water, soil, cucumber, and pear matrixes. The slope ratio of solvent calibration curves via matrix-matched calibration curves ranged from 0.8095 to 6.3868 as shown in Table 1. Signal suppression or enhancements of the four target compounds were typically observed in the all
matrix extracts, and the matrix-matched standard calibration curves were utilized for quantification. The data derived from matrix-matched calibration curves from 5 to 500 μg/kg are presented in Table 1, showing excellent linearity of all enantiomers with R2 ≥ 0.9909. The LODs were estimated at 0.0031−0.0087 μg/kg, and the LOQs were 0.0102−0.0290 μg/kg for enantiomers of prothioconazole. Correspondingly, the LODs and LOQs for the enantiomers of prothioconazole-desthio were estimated to be 0.0025−0.0075 μg/kg and 0.0083−0.025 μg/kg, respectively.
Figure 4. Predicted and experimental ECD spectra of prothioconazole and prothioconazole-desthio enantiomers: (A) R-prothioconazole-desthio, (B) S- prothioconazole-desthio, (C) R-prothioconazole, and (D) S-prothioconazole.
Table 1. Linearity, LOD, and LOQ for Prothioconazole and Prothioconazole-desthio Enantiomers with Different Matrixes (5−500 μg/L) enantiomer
matrix
regression equation
R2
R-(+)-prothioconazole-desthio
solvent water soil cucumber pear solvent water soil cucumber pear solvent water soil cucumber pear solvent water soil cucumber pear
y = 28688x + 953 y = 23664x − 5033 y = 23439x − 2431 y = 25154x − 1260 y = 23703x + 2561 y = 28672x + 878 y = 23568x − 4546 y = 23577x − 2634 y = 24891x − 1157 y = 23210x + 3339 y = 1763x − 1747 y = 5613x − 8396 y = 6057x − 8441 y = 5641x − 11672 y = 11260x − 27753 y = 1737x − 1773 y = 5430x − 9116 y = 6346x − 8309 y = 6114x − 16338 y = 10939x − 25126
0.9998 0.9994 0.9999 0.9999 0.9995 0.9998 0.9996 0.9999 0.9999 0.9993 0.9945 0.9920 0.9949 0.9973 0.9974 0.9949 0.9929 0.9909 0.9927 0.9995
S-(−)-prothioconazole-desthio
R-(−)-prothioconazole
S-(+)-prothioconazole
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slope of matrix/slope of solvent
P
0.8249 0.8170 0.8775 0.8262
0.0242 0.0257 0.0283 0.0302
0.8220 0.8223 0.8681 0.8095
0.0297 0.0219 0.0221 0.0246
3.1838 3.4356 3.1997 6.3868
0.0289 0.0282 0.0368 0.0326
3.1260 3.6534 3.5199 6.2976
0.0285 0.0269 0.0364 0.0310
LOD (μg/kg)
LOQ (μg/kg)
0.0025 0.0055 0.0075 0.0070 0.0062 0.0029 0.0071 0.0071 0.0061 0.0062 0.0087 0.0067 0.0040 0.0045 0.0032 0.0058 0.0056 0.0056 0.0046 0.0031
0.0083 0.0183 0.0250 0.0232 0.0206 0.0092 0.0235 0.0235 0.0205 0.0206 0.0290 0.0222 0.0135 0.0152 0.0109 0.0195 0.0186 0.0188 0.0155 0.0102
DOI: 10.1021/acs.jafc.7b02903 J. Agric. Food Chem. 2017, 65, 8241−8247
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Journal of Agricultural and Food Chemistry
Figure 5. Variations of concentration and EF value during the degradation in soil: (A) concentration of prothioconazole and prothioconazole-desthio enantiomers and (B) EF value of prothioconazole.
Accuracy and Precision. The average recoveries of pairs of enantiomers were determined at three different concentrations (5, 50, and 500 μg/kg) in quintuplicate. The precision of this method was evaluated using relative standard deviation (RSD). The method provided high accuracy and precision, and the mean recoveries of prothioconazole enantiomers were 71.8−96.4% with 0.3−11.9% intraday RSDs and 1.5−10.6% interday RSDs. The recoveries for enantiomers of its metabolite ranged from 85.8 to 102.0% with 0.2−9.8% intraday RSDs and 0.9−7.1% interday RSDs, indicating that this method was able to provide accurate quantitative data for enantiomeric analysis of agricultural food and environmental samples. Enantioselective Dissipation of Prothioconazole and the Formation of Prothioconazole-desthio in Soil. The effectiveness of the chiral method was used to measure the degradation of prothioconazole and the formation of prothioconazole-desthio in soil under field conditions. The degradation of prothioconazole and prothioconazole-desthio in soil is shown in Figure 5A. Interestingly, both prothioconazole enantiomers were found to easily degrade in soil. The correlation coefficient of both enantiomers (R2) was above 0.9200 and the dissipation followed the first-order kinetics according to the following equation C = C0 e
−kt
identify the chirality of prothioconazole and its metabolites in soil, water, and agricultural samples. These results may be helpful to systematically evaluate the stereoselective behaviors of this chiral fungicide and its metabolites in the environment.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b02903. Effects of the flow rate, mobile phase compositions, and temperature on the enantioseparation parameters; Van’t Hoff equations and the thermodynamic parameters; stability of enantiomers; and typical chromatograms (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: + 86 025 84395479. Fax: + 86 025 84395672. E-mail:
[email protected]. ORCID
Minghua Wang: 0000-0001-5715-4981 Funding
This study was supported by the National Key Research and Development Program of China (Grant 2016YFD0200207).
(3)
Notes
The authors declare no competing financial interest.
The dissipation dynamics equations were C = 0.5322 e−1.281x (R2 = 0.9348) and C = 0.5938 e−1.478x (R2 = 0.9212) for R-prothioconazole and S-prothioconazole, respectively. The halflives of R-prothioconazole and S-prothioconazole were 0.48 and 0.60 days. The concentration of S-prothioconazole (0.074 mg/kg) was 1.72 times higher than the R-prothioconazole enantiomer (0.043 mg/kg) after 1 d of spraying. Remarkably enantioselective degradation was observed and the EF value was 0.48−0.37 (Figure 5B). R-prothioconazole showed preferential degradation in soil under field conditions. The formation of both prothioconazoledesthio enantiomers was detected during prothioconazole dissipation. The concentration of prothioconazole-desthio enantiomers increased steadily to a maximum concentration (0.288 mg/kg and 0.269 mg/kg for R-prothioconazole-desthio and S-prothioconazole-desthio, respectively) at 2 days and then decreased slowly to the end of the field experiment. The pair of enantiomers are shown in the same concentrations. As one of the most remarkably bioactive chiral metabolites, there was no obvious enantioselective behavior for prothioconazole-desthio in soil. The environmental degradation and metabolism of the chiral enantiomers in soil is important for the currently used chiral fungicides. This method provides a feasible way to positively
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ACKNOWLEDGMENTS We are grateful to Hu Zhang (Zhejiang Academy of Agriculture Sciences) and Bowen Tang (Xiamen University) for calculating the absolute configuration of prothioconazole and prothioconazole-desthio enantiomers by Gaussian 09 software.
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ABBREVIATIONS USED UPLC−QTRAP-MS/MS, ultraperformance liquid chromatography tandem QTRAP mass spectrometry; CSP, chiral stationary phases; QuEChERS, quick, easy, cheap, effective, rugged and safe; k, capacity factor; α, separation factor; Rs, resolution; Tiso, isolation temperature
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REFERENCES
(1) Ulrich, E. M.; Morrison, C. N.; Goldsmith, M. R.; Foreman, W. T. Chiral pesticides: identification, description, and environmental implications. Rev. Environ. Contam. Toxicol. 2012, 217, 1−74. (2) Wang, M.; Zhang, Q.; Cong, L.; Yin, W.; Wang, M. Enantioselective degradation of metalaxyl in cucumber, cabbage, spinach and pakchoi. Chemosphere 2014, 95, 241−246.
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DOI: 10.1021/acs.jafc.7b02903 J. Agric. Food Chem. 2017, 65, 8241−8247
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Journal of Agricultural and Food Chemistry (3) Pérez-Fernández, V.; García, M.; Marina, M. L. Chiral separation of agricultural fungicides. J. Chromatogr. A 2011, 1218, 6561−6582. (4) Buerge, I. J.; Bächli, A.; Joffrey, J. P. D.; Müller, M. d.; Spycher; Poiger, S. T. The chiral herbicide beflubutamid(I): isolation of pure enantiomers by HPLC, herbicidal activity of enantiomers, and analysis by enantioselective GC-MS. Environ. Sci. Technol. 2012, 47, 6806−6811. (5) Xu, P.; Diao, J.; Liu, D.; Zhou, Z. Enantioselective bioaccumulation and toxic effects of metalaxyl in earthworm Eisenia foetida. Chemosphere 2011, 83, 1074−1079. (6) Tian, M.; Zhang, Q.; Hua, X.; Tang, B.; Gao, B.; Wang, M. Systemic stereoselectivity study of flufiprole: Stereoselective bioactivity, acute toxicity and environmental fate. J. Hazard. Mater. 2016, 320, 487−494. (7) Lewis, D. L.; Garrison, A. W.; Wommack, K. E.; Whittemore, A.; Steudler, P.; Melillo, J. Influence of environmental changes on degradation of chiral pollutants in soils. Nature 1999, 401, 898−901. (8) Dong, F.; Cheng, L.; Liu, X.; Xu, J.; Li, J.; Li, Y.; Kong, Z.; Jian, Q.; Zheng, Y. Enantioselective analysis of triazole fungicide myclobutanil in cucumber and soil under different application modes by chiral liquid chromatography/tandem mass spectrometry. J. Agric. Food Chem. 2012, 60, 1929−1936. (9) Tao, Y.; Dong, F.; Xu, J.; Liu, X.; Cheng, Y.; Liu, N.; Chen, Z.; Zheng, Y. Green and sensitive supercritical fluid chromatographic− tandem mass spectrometric method for the separation and determination of flutriafol enantiomers in vegetables, fruits, and soil. J. Agric. Food Chem. 2014, 62, 11457−11464. (10) Kurihara, N.; Miyamoto, J.; Paulson, G. D.; Zeeh, B.; Skidmore, M. W.; Hollingworth, R. M.; Kuiper, H. A. Pesticides Report 37. Chirality in synthetic agrochemicals: Bioactivity and safety consideration. Pure Appl. Chem. 1997, 69, 1335−1348. (11) Dong, F.; Liu, X.; Zheng, Y.; Cao, Q.; Li, C. Stereoselective degradation of fungicide triadimenol in cucumber plants. Chirality 2010, 22, 292−298. (12) Huang, L.; Lu, D.; Zhang, P.; Diao, J.; Zhou, Z. Enantioselective toxic effects of hexaconazole enantiomers against Scenedesmus Obliquus. Chirality 2012, 24, 610−614. (13) Cullen, J. W. The metabolism of xenobiotic chemicals. J. Chem. Educ. 1987, 64, 396. (14) Sparling, D. W.; Fellers, G. Comparative toxicity of chlorpyrifos, diazinon, malathion and their oxon derivatives to larval Rana boylii. Environ. Pollut. 2007, 147, 535−539. (15) Griffiths, K. M.; Bacic, A.; Howlett, B. J. Sterol composition of mycelia of the plant pathogenic ascomycete Leptosphaeria maculans. Phytochemistry 2003, 62, 147−153. (16) Zhang, H.; Qian, M.; Wang, X.; Xu, H.; Wang, M. HPLC−MS/ MS enantioseparation of triazole fungicides using polysaccharide-based stationary phases. J. Sep. Sci. 2012, 35, 773−777. (17) Péter, A.; Vékes, E.; Armstrong, D. W. Effects of temperature on retention of chiral compounds on a ristocetin a chiral stationary phase. J. Chromatogr. A 2002, 958, 89−107. (18) O’Brien, T.; Crocker, L.; Thompson, R.; Thompson, K.; Toma, P. H.; Conlon, D. A.; Feibush, B.; Moeder, C.; Bicker, G.; Grinberg, N. Mechanistic aspects of chiral discrimination on modified cellulose. Anal. Chem. 1997, 69, 1999−2007. (19) Qiu, J.; Dai, S.; Zheng, C.; Yang, S.; Chai, T.; Bie, M. Enantiomeric separation of triazole fungicides with 3-mm and 5-mm particle chiral columns by reverse phase high-performance liquid chromatography. Chirality 2011, 23, 479−486. (20) Tian, Q.; Bi, C.; Ren, L.; Wang, L.; Zhou, Z. The application of high performance liquid chromatography with circular dichroism detector in the chiral compounds. Chin. J. Anal. Chem. 2006, 34, 427− 432. (21) Frisch, M. J.; et al. Gaussian 09, revision a.02; Gaussian, Inc.: Wallingford, CT, 2009. (22) Ding, S.; Kolbanovskiy, A.; Durandin, A.; Crean, C.; Shafirovich, V.; Broyde, S.; Geacintov, N. E. Absolute configurations of DNA lesions determined by comparisons of experimental ECD and ORD spectra with DFT calculations. Chirality 2009, 21, E231−E241. (23) Ding, S.; Jia, L.; Durandin, A.; Crean, C.; Kolbanovskiy, A.; Shafirovich, V.; Broyde, S.; Geacintov, N. E. Absolute configurations of
spiroiminodihydantoin and allantoin stereoisomers: Comparison of computed and measured electronic circular dichroism spectra. Chem. Res. Toxicol. 2009, 22, 1189−1193. (24) Zhao, D.; Li, Z.; Cao, F.; Liang, M.; Charles, U.; Pittman, J. R.; Zhu, H. Revised absolute configuration of sibiricumin A: substituent effects in simplified model structures used for quantum mechanical predictions of chiroptical properties. Chirality 2016, 28, 612−617. (25) Zhang, H.; Wang, X.; Zhuang, S.; Jin, N.; Wang, X.; Qian, M.; Xu, H.; Qi, P.; Wang, Q.; Wang, M. Enantioselective analysis and degradation studies of isocarbophos in soils by chiral liquid chromatography-tandem mass spectrometry. J. Agric. Food Chem. 2012, 60, 10188−10195. (26) Zhang, Q.; Tian, M.; Wang, M.; Shi, H.; Wang, M. Simultaneous enantioselective determination of triazole fungicide flutriafol in vegetables, fruits, wheat, soil, and water by reversed-phase highperformance liquid chromatography. J. Agric. Food Chem. 2014, 62, 2809−15. (27) Stephens, P. J.; McCann, D. M.; Devlin, F. J.; Cheeseman, J. R.; Frisch, M. J. Determination of the absolute configuration of [32](1,4) barrelenophanedicarbonitrile using concerted time-dependent density functional theory calculations of optical rotation and electronic circular dichroism. J. Am. Chem. Soc. 2004, 126, 7514−7521. (28) Liang, H.; Qiu, J.; Li, L.; Li, W.; Zhou, Z.; Liu, F.; Qiu, L. Stereoselective separation and determination of triadimefon and triadimenol in wheat, straw, and soil by liquid chromatography-tandem mass spectrometry. J. Sep. Sci. 2012, 35, 166. (29) Liu, W.; Gan, J. J.; Lee, S.; Werner, I. Isomer selectivity in aquatic toxicity and biodegradation of cypermethrin. J. Agric. Food Chem. 2004, 52, 6233−6238. (30) Ding, H.; Grinberg, N.; Thompson, R.; Ellison, D. Enantiorecognition mechanisms for derivatized cellulose under reversed phase conditions. J. Liq. Chromatogr. Relat. Technol. 2000, 23, 2641−2651. (31) Qi, P.; Yuan, Y.; Wang, Z.; Wang, X.; Xu, H.; Zhang, H.; Wang, Q.; Wang, X. Use of liquid chromatography-quadrupole time-of-flight mass spectrometry for enantioselective separation and determination of pyrisoxazole in vegetables, strawberry and soil. J. Chromatogr. A 2016, 1449, 62−70. (32) Koesukwiwat, U.; Lehotay, S. J.; Mastovska, K.; Dorweiler, K. J.; Leepipatpiboon, N. Extension of the QuEChERS method for pesticide residues in cereals to flaxseeds, peanuts, and doughs. J. Agric. Food Chem. 2010, 58, 5950.
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DOI: 10.1021/acs.jafc.7b02903 J. Agric. Food Chem. 2017, 65, 8241−8247
