Article pubs.acs.org/JAFC
Enantioselective Analysis and Dissipation of Triazole Fungicide Penconazole in Vegetables by Liquid Chromatography−Tandem Mass Spectrometry Xinquan Wang, Peipei Qi, Hu Zhang, Hao Xu, Xiangyun Wang, Zhen Li, Zhiwei Wang, and Qiang Wang* State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, MOA Key Lab for Pesticide Residue Detection, Zhejiang Province Key Laboratory of Detection for Pesticide Residues and Control, Institute of Quality and Standard on Agricultural Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, People’s Republic of China ABSTRACT: Penconazole is a typical triazole fungicide, which is commonly used to control powdery mildew in vineyard and vegetable field. In this study, the enantioselective dissipation of penconazole in cucumber, tomato, head cabbage, and pakchoi was investigated by field experiments. A sensitive method for enantiomeric analysis of penconazole was established on the basis of the buffered QuEChERS sample preparation technique followed by reverse-liquid chromatography equipped with a TSQ Discovery triple quadrupole mass spectrometer and a Lux Cellulose-2 chiral column. Methanol and 2 mM ammonium acetate buffer solution containing 0.1% formic acid (70:30, v/v) were used as mobile phase at a 0.2 mL L−1 flow rate isocratic elution. The linearity, recovery, and precision of this method were also evaluated. Finally, the results of this study demonstrated that enantioselective dissipation occurred in head cabbage and pakchoi, with the preferential degradation of (−)-penconazole, and resulting in an enrichment of the (+)-penconazole residue in the two vegetables. However, the enantioselective behavior was not observed in cucumber and tomato. More importantly, this is the first report of enantioselective behavior of penconazole, and the result may provide useful information for the risk evaluation of penconazole in food and environmental safety. KEYWORDS: penconazole, enantioselective analysis, dissipation, vegetable, LC−MS/MS
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INTRODUCTION
enantiomer level, which resulted in lacking data during risk assessment. Penconazole, as the structure shown in Figure 1, is a typical triazole fungicide, and mainly applied on apples, grapes, and
Triazole fungicide has been one of the most important members in the fungicide family since the 1970s. Because of their excellent inhibition activity on fungal ergosterol biosynthesis, triazole fungicides are now widely applied on crops such as rice, wheat, barley, and orchard fruit to control fungus disease.1 However, with the extensive use of triazole fungicides, it also brings potential risk of environmental and food safety.2,3 Especially in recent years, with the development of chiral pesticide research,4,5 the concerns of triazole fungicides have been raised all over the world,6 and much research has been conducted to study the enantioselective behavior of the triazole fungicides.7 In previous studies, many researchers have reported the stereoselective behavior of some triazole fungicide, including the bioactivity,8−10 toxicity,9,11−14 racemization,15 and stereoselective degradation.16−20 Taking triadimenol enantiomers, for example, the order of the inhibition of gibberellin biosynthesis was found to be 1R, 2S > 1S, 2S > 1R, 2R > 1S, 2R, while the fungitoxicity was 1S, 2R > 1R, 2R > 1R, 2S > 1S, 2S.11 For the racemization, triadimefon enantiomers were the most representative, which might be racemized in protic solvents such as methanol and ethanol,21 and also in soils depending on pH value.15 In the field of stereoselective dissipation, researchers have studied several fungicides, such as flutriafol,18 epoxiconazole,19 cyproconazole,19 hexaconazole,12,20 myclobutanil,22 triadimefon,15 triadimenol,23 tebuconazole,16 and diniconazole,24 which exhibited obviously enantioselective dissipation in environmental samples (animals, plants, or soils). Unfortunately, in the present reports, there was little reference about the study on the environmental behavior of penconazole at the © 2014 American Chemical Society
Figure 1. Chemical structure of penconazole.
vegetables to control powdery mildew. Nevertheless, due to the toxicity problem, it was inevitable that the residue of penconazole might affect the environmental safety and human health. In a recent toxicological study, the researchers found that penconazole could induce structural and functional testicular impairment on male albino rats.25 Worse more, there was evidence that penconazole was associated with endocrine disrupting mediated effects in human T-47D cells that strongly suggested a possible mode of action in thyroid carcinogenesis.26 In view of this, the residue of penconazole should be strictly Received: Revised: Accepted: Published: 11047
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Multiple reactions monitoring (MRM) mode was applied to determine the fragmentation of the protonated pseudomolecular ions of penconazole. The transitions m/z 284 > 159 and m/z 284 > 70 were used for quantification and confirmation, respectively. The corresponding collision energies were 15 and 20 eV. Elution Order Determination of Penconazole Enantiomers. The optical rotation dispersion (ORD) signal of (+) or (−) was used to distinguish the elution order of the enantiomers by an Agilent 1200 HPLC system equipped with a G1315B diode array detector (Wilmington, U.S.) and online CHIRALYSER-MP optical rotation detector (IBZ Messtechnik, Germany). The signal was received and processed by Agilent 1200 chemstation and N2000 SP1 chromatographic chemstation (Zhida, China) software. The chiral separation was performed under the same LC conditions as LC−MS/MS, but the injection volume was 20 μL and the flow rate was set at 1.0 mL/min with the UV detection at 230 nm. Sample Preparation. Samples were thawed previously at room temperature. A 15 g triturated vegetable sample was weighed into a 50 mL polypropylene centrifuge tube, and 15 mL of acetonitrile containing 1% of acetic acid was added. The mixture was shaken by a vortex mixer for 1 min and then sonicated for 15 min. Subsequently, 6 g of anhydrous magnesium sulfate and 1.5 g of anhydrous sodium acetate were added, and the tube was shaken vigorously by hand for 5 min and centrifuged at 6000 rpm for 5 min. A 1 mL aliquot of upper acetonitrile supernatant was transferred into a 2 mL centrifuge tube containing 50 mg of PSA sorbent, 50 mg of C18 sorbent, and 150 mg of anhydrous magnesium sulfate for cleanup. The tube was shaken by vortex mixer for 1 min and centrifuged at 6000 rpm for 5 min. A 0.5 mL portion of the upper extracts was transferred into a 2 mL centrifuge tube containing 0.5 mL of water. The resulting solution was filtered through a 0.22 μm Teflon syringe filter for LC−MS/MS analysis. Method Validation. A series of working standard solutions and matrix-matched calibration standard of (±)-penconazole for linearity of the method were prepared at 0.5, 1, 2.5, 5, 25, 100, and 500 μg L−1 for each enantiomer. The calibration curves were generated by plotting the peak area of quantification ion transition versus the concentration of each enantiomer. Linear regression analysis was performed using Microsoft Excel 2007, and the correlation coefficient was used to evaluate linearity of the method. The accuracy and precision of the method were investigated to evaluate its reliability. The blank vegetable samples obtained from the field experiment were fortified with standard solutions of each enantiomer at three levels (2.5, 50, and 500 μg kg−1). Recovery experiments were carried out with five replicates for each level, and fortified samples were equilibrated for 1 h at room temperature prior to extraction as described above. The precision of the method was investigated by the repeatability (intraday assays) and reproducibility (interday assays), expressed by relative standard deviation (RSD). The repeatability assays were measured in five replicates at three concentration levels on the same day, with the same instrument and by the same operator. The reproducibility assays were evaluated in five replicates at the earlier stated concentration levels on three different days, with the same instrument but by different operators. The limits of detection (LODs) for each enantiomer were calculated as 3 times the signal-to-noise (peak to peak) ratio of the quantitative ion transition by the lowest spiked analytes concentration assayed with five replicates. The limits of quantification (LOQs) were defined as the lowest concentration with acceptable accuracy for 70− 120% and precision for 15% variability.
controlled, and more comprehensive research should be conducted. In this Article, field experiments of penconazole were conducted, and then cucumber, tomato, head cabbage, and pakchoi samples were collected for analysis. A simple pretreatment method was adopted on the basis of buffered QuEChERS (quick, easy, cheap, effective, rugged, and safe) for the extraction, cleanup, and preconcentration of penconazole.27,28 The chiral analysis method of penconazole was established by liquid chromatography−tandem mass spectrometry (LC−MS/MS) equipped with a Lux Cellulose-2 chiral column. Finally, the result suggested that penconazole showed enantioselective dissipation in head cabbage and pakchoi, but not in the cucumber and tomato samples. The results in this Article might also provide some sufficient information on penconazole during the food and environment risk assessment.
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MATERIALS AND METHODS
Chemicals and Materials. The analytical standard of (±)-penconazole (purity 99.0%) was purchased form Dr. Ehrenstorfer (Augsburg, Germany). The commercial product of penconazole-EC (10% of (±)-penconazole) was obtained from the Hangzhou Udragon Chemical Co. Ltd. (Zhejiang, China). Purified water was prepared by using a Milli-Q water purification system (Millipore Corp., Billerica, U.S.). LC grade methanol (CH3OH) and acetonitrile (CH3CN) were obtained from Merck (Darmstadt, Germany). Formic acid and ammonium acetate were obtained from TEDIA (Fairfield, U.S.). Acetic acid was obtained from Sigma-Aldrich (St. Louis, U.S.). Silicabased sorbents including C18 (40 μm particle size) and primary secondary amine (PSA) (40 μm particle size) were obtained from Agilent (Wilmington, U.S.). All other chemicals and solvents were of analytical grade and were purchased from commercial sources. Field Experiments. The vegetable (cucumber, tomato, head cabbage, and pakchoi) seeds purchased from Longda Seeds (Hangzhou, China) were sown in greenhouse conditions on 7 February 2012. The field experiments were conducted from March to May 2012 in Zhejiang Academy of Agricultural Sciences (Hangzhou, China). Every experiment treatment for a vegetable contained three replicate plots and a control plot without penconazole. The area of each plot was 30 (10 × 3) m2, and the buffer zone was set up between plots. These plots had never been treated with penconazole for more than 3 years. (±)-Penconazole 10% EC was applied as foliar spray at the dose of 47.6 g a.i. ha−1 (gram of active ingredient per hectare), dissolving in 2 L of water. Approximately 2 kg of vegetable samples was collected from nine randomly selected sampling points within each plot, at 0 (1 h after spraying), 1, 2, 3, 5, 7, 10, 14, and 21 days after spray. All vegetable samples were homogenized by a blender (Philips, China), and then stored at −20 °C until further analysis. Enantioselective LC−MS/MS Analysis. LC−MS/MS analyses were performed on a Surveyor liquid chromatograph equipped with a TSQ Discovery triple quadrupole mass spectrometer (Thermo Fisher Scientific, U.S.). Thermo Fisher Xcalibur software (version 2.0.7) was used for data quantitation and documentation. A satisfactory enantioseparation of penconazole was performed on a Lux Cellulose-2 chiral column (150 mm × 2.0 mm i.d., 3 μm, Phenomenex, U.S.) packed with cellulose tris (3-chloro-4-methylphenylcarbamate). The analysis was isocratic at 0.2 mL L−1 flow rate using mobile phase A (LC grade methanol) and mobile phase B (0.1% formic acid and 2 mM ammonium acetate in water) in a 70:30 v/v ratio. The column temperature was kept at 25 °C, and the autosampler temperature was set at 4 °C. The sample injection volume was 10 μL, and the total run time was 25 min. The ESI−MS/MS (electrospray ionization coupled with tandem mass spectrometry) was performed in positive ion mode, and the monitoring conditions were optimized for penconazole. The typical ESI source conditions were as follows: ion spray voltage, 4200 V; ion capillary temperature, 350 °C; sheath gas flow (N2), 35 (arbitrary) units; aux gas flow (N2), 8 U; collision gas pressure (Ar), 1.5 mTorr.
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RESULTS AND DISCUSSION Enantioseparation and Identification Elution Order of Penconazole. Under the LC−MS/MS condition, the enantiomers of penconazole were separated completely on a Lux Cellulose-2 column (as shown in Figure 4). In this work, an online ORD was used to distinguish the elution order information on penconazole enantiomers based on the difference of the refractive index between left (−) and right 11048
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Table 1. Comparison of Matrix-Matched Calibration and Solvent Calibration (0.5−500 μg L−1) compound
matrix
(+)-penconazole
solvent cucumber tomato head cabbage pakchoi solvent cucumber tomato head cabbage pakchoi
(−)-penconazole
standard linear equation Y Y Y Y Y Y Y Y Y Y
= = = = = = = = = =
130131X 128910X 129063X 121242X 128443X 128074X 125365X 127859X 118915X 125778X
+ + + + + + + + + +
50683 23523 37715 47990 46901 51929 39602 39504 45003 37255
R2
slope of matrix/slope of solvent
0.9983 0.9998 0.9992 0.9986 0.9988 0.9982 0.9991 0.9989 0.9987 0.9987
0.991 0.992 0.937 0.987 0.979 0.998 0.928 0.982
Table 2. Recovery and Precision of the Method in Vegetable Samples mean recoveries (n = 15; %) (RSDa %, RSDb %) fortified level (μg kg−1) compound
matrixc
(+)-penconazole
C T H P C T H P
(−)-penconazole
2.5
50
89.5 (4.8, 85.9 (5.4, 98.9 (5.9, 78.5 (5.4, 90.3 (5.2, 88.3 (4.1, 102 (5.3, 7.4) 79.6 (6.5,
7.5) 6.6) 8.5) 10.4) 7.6) 6.2) 9.6)
98.7 79.4 86.5 82.2 98.0 80.4 86.8 84.6
(8.2, (5.7, (4.2, (2.8, (9.2, (4.1, (7.8, (4.9,
500 7.3) 8.2) 8.9) 9.2) 7.7) 7.8) 11.5) 6.3)
101 (7.6, 8.3) 100 (4.7, 5.3) 87.0 (3.6, 86.0 (5.1, 101 (8.0, 8.5) 97.4 (7.2, 89.3 (3.9, 87.9 (5.8,
8.5) 8.3) 8.4) 9.8) 6.0)
a
RSD represents the relative standard deviation of intraday (n = 5). bRSD represents the relative standard deviation of interday (n = 15). cC represents cucumber. T represents tomato. H represents head cabbage. P represents pakchoi.
(+) linearly polarized lights. The results showed that the first eluted enantiomer was confirmed as (+)-penconazole, while the second eluted enantiomer was (−)-penconazole under the LC condition described above. Method Development and Validation. The sample extraction and cleanup procedure for penconazole enantiomers in vegetables followed the buffered QuEChERS method. Validation of the proposed method included linearity, recovery, and precision. Good linear calibration curves for both enantiomers were obtained over the range of 0.5−500 μg L−1. The linear equations for each enantiomer in standard solutions and matrix-matched solutions are listed in Table 1. The matrix effect was calculated by comparing the slope of matrix-matched standard curve with the slope of solvent standard calibration curve.29 The slope ratios of matrix-matched to solvent-based calibration listed in Table 1 were in the range of 0.928−0.998, which implied no significant matrix effect for enantiomers of penconazole in the four vegetable matrixes. The recoveries of penconazole enantiomers in vegetable matrixes at three spiked levels ranged from 78.5% to 102%, and the RSDs of repeatability and reproducibility ranged from 2.8% to 11.5% (see Table 2). The results of recoveries and precisions obtained in the method validation portion proved that the method was efficient and reliable to determine the enantiomers of penconazole in the four vegetables with LC−MS/MS. The LODs for both enantiomers in the tested vegetables were estimated to be 0.5 μg kg−1, while the LOQs of enantiomers in vegetables were 2.5 μg kg−1 based on the lowest fortified level in the tested vegetables. Residues Dissipation of Penconazole in Cucumber and Tomato. Penconazole residue dissipation in cucumber and tomato was investigated under field conditions. In Figure 2, the data were plotted from cucumber and tomato as normalized
concentrations (100C/C0) versus treatment time. Figure 2 shows that the residues of penconazole decreased quickly with time in cucumber and tomato when the (±)-penconazole was sprayed, and the dissipation processes followed first-order kinetics. The depletion rate constant were obtained by fitting the enantiomer of penconazole residue data from each experiment to the first-order kinetic equation ln(C/C0) = −kt, where C0 is the initial concentration of the enantiomer (μg kg−1), C is its concentration (μg kg−1) at time t (days), k is the depletion rate constant, and the corresponding half-lives (t1/2) were calculated as t1/2 = ln 2/k = 0.693/k.30 The depletion rate constant, half-life, and correlation coefficient (R2) of the two enantiomers are shown in Table 3, and the data show that the depletion rates of the two enantiomers in cucumber and tomato were similar to t1/2 ranging from 1.52 to 1.98 days. In this study, the enantiomeric ratio (ER) was used as a measure of the enantioselectivity of the residues dissipation of penconazole in vegetables. The ER value was defined as the concentration of the first eluting (+)-enantiomer divided by the concentration of the later eluting (−)-enantiomer.31 It was shown in Figure 3 that the ER values of (±)-penconazole were constant with time in cucumber and tomato. Student’s t-tests were made to compare the mean ER values of (±)-penconazole in the two vegetables with ER = 1 at a 95% confidence interval. The statistical test results indicated that the dissipation of penconazole enantiomers was not enantioselective either in cucumber (p = 0.17) or in tomato (p = 0.30). Residue Dissipation of Penconazole in Head Cabbage and Pakchoi. The residue dissipation of penconazole enantiomers in head cabbage and pakchoi was investigated under field conditions with (±)-penconazole foliage sprayed. Generally, the residues of both enantiomers of penconazole decreased quickly with time elapsed in the head cabbage and 11049
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Figure 2. Degradation of penconazole enantiomers in (A) cucumber, (B) tomato, (C) head cabbage, and (D) pakchoi. Normalized concentrations (100C/C0) are plotted versus treatment time (d). Note the faster degradation of the (−)-enantiomer. Plot of ln(ER) from (E) head cabbage and (F) pakchoi after treatment with (±)-penconazole versus treatment time showing a linear relationship.
Table 3. First-Order Depletion Rate Constant (k), Initial Concentration (C0), Half-Life (t1/2), and Correlation Coefficient (R2) for the Enantioselective Dissipation of Penconazole experiment cucumber tomato head cabbage pakchoi a
compound
k (day−1)
(+)-penconazole (−)-penconazole (+)-penconazole (−)-penconazole (+)-penconazole (−)-penconazole (+)-penconazole (−)-penconazole
0.4536 0.4547 0.3636 0.3491 0.2899 0.5497 0.2720 0.3632
C0 (μg kg−1)a 39.3 43.1 49.6 50.9 119.6 118.4 449.3 435.0
± ± ± ± ± ± ± ±
3.6 1.9 4.0 1.9 7.7 5.1 8.7 6.7
t1/2 (day)a
R2
± ± ± ± ± ± ± ±
0.9756 0.9667 0.9117 0.9025 0.9165 0.9895 0.9553 0.9861
1.53 1.52 1.90 1.98 2.39b 1.26b 2.55b 1.91b
0.07 0.09 0.12 0.06 0.05 0.11 0.03 0.07
Values represent the means ± SDs (n = 3). bSignificantly different from each other, p < 0.05 (paired t test).
performed, and the result suggested that the difference of the two enantiomers was significant (p < 0.05, student’s paired t test). In Figure 3C and D, the variation curve of ER value was plotted versus time from head cabbage and pakchoi, and it could be found that the ER values of penconazole consistently increased with time in both vegetables. Student’s t tests were made to compare the mean ER values of (±)-penconazole in the two vegetables with ER = 1 at a 95% confidence interval. The statistical test results indicated there was substantial enantioselectivity on the dissipation of (±)-penconazole in
pakchoi (see Figure 2), and the typical LC−MS/MS chromatograms are shown in Figure 4. In Figure 2C and D, the data were plotted from head cabbage and pakchoi as normalized concentration (100C/C0) versus time, and it was obvious that the second-eluted (−)-enantiomer dissipated more rapid than the other enantiomer, leading to residues enriched in (+)-form. By calculation, the depletion rate constant followed first-order kinetics and the half-life of each enantiomers is listed in Table 3, and these data also demonstrated that (−)-enantiomer (t1/2 = 1.26 and 1.91 days, respectively) degraded faster than (+)-enantiomer (t1/2 = 2.39 and 2.55 days, respectively) in the two tested vegetables. The statistical analysis was also 11050
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Figure 3. Enantiomeric ratio (ER) of penconazole residues in (A) cucumber, (B) tomato, (C) head cabbage, and (D) pakchoi.
Figure 4. Representative chromatograms of (A) (±)-penconazole standard (50 μg L−1), (B) extract from head cabbage at 7 d after treatment with (±)-penconazole, and (C) extract from pakchoi at 7 d after treatment with (±)-penconazole.
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Applications, Mechanisms of Action; Lyr, H., Ed.; Longman Scientific and Technical: Harlow, 1987; pp 205−231. (2) Crofton, K. A structure-activity relationship for the neurotoxicity of triazole fungicides. Toxicol. Lett. 1996, 84, 155−159. (3) Filipov, N. M.; Lawrence, D. A. Developmental toxicity of a triazole fungicide: consideration of interorgan communication. Toxicol. Sci. 2001, 62, 185−186. (4) Liu, W.; Gan, J.; Schlenk, D.; Jury, W. A. Enantioselectivity in environmental safety of current chiral insecticides. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 701−706. (5) Zhang, Q.; Wang, C.; Zhang, X.; Jin, D.; Huang, C.; Zhao, M. Enantioselective aquatic toxicity of current chiral pesticides. J. Environ. Monit. 2012, 14, 465−472. (6) Garrison, A. W. Probing the enantioselectivity of chiral pesticides. Environ. Sci. Technol. 2006, 40, 16−23. (7) Sekhon, B. S. Chiral pesticides. J. Pestic. Sci. 2009, 34, 1−12. (8) Yang, L.; Li, S.; Li, Y.; Gao, R. Bioactivity of triazole fungicide enantiomers. Chin. J. Pestic. Sci. 2002, 4, 67−70. (9) Dong, F.; Li, J.; Chankvetadze, B.; Cheng, Y.; Xu, J.; Liu, X.; Li, Y.; Chen, X.; Bertucci, C.; Tedesco, D. Chiral triazole fungicide difenoconazole: absolute stereochemistry, stereoselective bioactivity, aquatic toxicity, and environmental behavior in vegetables and soil. Environ. Sci. Technol. 2013, 47, 3386−3394. (10) Kurihara, N.; Miyamoto, J.; Paulson, G.; Zeeh, B.; Skidmore, M.; Hollingworth, R.; Kuiper, H. Chirality in synthetic agrochemicals: bioactivity and safety consideration. Pure Appl. Chem. 1997, 69, 1335− 1348. (11) Burden, R. S.; Carter, G. A.; Clark, T.; Cooke, D. T.; Croker, S. J.; Deas, A. H.; Hedden, P.; James, C. S.; Lenton, J. R. Comparative activity of the enantiomers of triadimenol and paclobutrazol as inhibitors of fungal growth and plant sterol and gibberellin biosynthesis. Pestic. Sci. 1987, 21, 253−267. (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) Cheng, C.; Huang, L.; Diao, J.; Zhou, Z. Enantioselective toxic effects and degradation of myclobutanil enantiomers in scenedesmus obliquus. Chirality 2013, 25, 858−864. (14) Kenneke, J. F.; Mazur, C. S.; Kellock, K. A.; Overmyer, J. P. Mechanistic approach to understanding the toxicity of the azole fungicide triadimefon to a nontarget aquatic insect and implications for exposure assessment. Environ. Sci. Technol. 2009, 43, 5507−5513. (15) Li, Z.; Zhang, Y.; Li, Q.; Wang, W.; Li, J. Enantioselective degradation, abiotic racemization, and chiral transformation of triadimefon in soils. Environ. Sci. Technol. 2011, 45, 2797−2803. (16) Wang, X.; Wang, X.; Zhang, H.; Wu, C.; Wang, X.; Xu, H.; Wang, X.; Li, Z. Enantioselective degradation of tebuconazole in cabbage, cucumber, and soils. Chirality 2012, 24, 104−111. (17) Li, J.; Dong, F.; Cheng, Y.; Liu, X.; Xu, J.; Li, Y.; Chen, X.; Kong, Z.; Zheng, Y. Simultaneous enantioselective determination of triazole fungicide difenoconazole and its main chiral metabolite in vegetables and soil by normal-phase high-performance liquid chromatography. Anal. Bioanal. Chem. 2012, 404, 2017−2031. (18) Shen, Z.; Zhang, P.; Xu, X.; Wang, X.; Zhou, Z.; Liu, D. Genderrelated differences in stereoselective degradation of flutriafol in rabbits. J. Agric. Food Chem. 2011, 59, 10071−10077. (19) Buerge, I. J.; Poiger, T.; Mueller, M. D.; Buser, H.-R. Influence of pH on the stereoselective degradation of the fungicides epoxiconazole and cyproconazole in soils. Environ. Sci. Technol. 2006, 40, 5443−5450. (20) Wang, X.; Zhang, H.; Xu, H.; Wang, X.; Wu, C.; Yang, H.; Li, Z.; Wang, Q. Enantioselective residue dissipation of hexaconazole in cucumber (Cucumis sativus L.), head cabbage (Brassica oleracea L. var. caulorapa DC.), and soils. J. Agric. Food Chem. 2012, 60, 2212− 2218. (21) Li, C.; Zhang, Y.; L, L.; W, W.; L, J. The seperation and transformation of triazole fungicides. Chin. J. Anal. Chem. 2010, 38, 37−40.
head cabbage (p = 0.005) and pakchoi (p = 0.016) under the field experiments. The dissipation processes of penconazole enantiomers in the test vegetables followed first-order kinetics with a depletion rate constant of k(+) for the (+)-enantiomer, and a depletion rate constant of k(−) for the (−)-form; ER may be expressed as an equation of time (t) in the following relationship:32,33 ER t = [+]/[−] = ER 0 × e{k(−)− k(+)} = ER 0 × eΔk
(1)
where ER0 is the initial ER value at 1 h after spraying with (±)-penconazole in head cabbage and pakchoi, [+] and [−] are the concentrations of (+)-enantiomer and (−)-enantiomer at time t, respectively, and Δk is the difference from ER0 over time. The above relationship can be further expressed in a linear form after logarithmic transformation of ER: ln(ER t) = ln(ER 0) + Δk
(2)
Equation 2 indicated a linear relationship between ln(ERt) and time (t), and we showed the data plotted in Figure 2E and F, which could be used to determine the rate difference Δk and thus enantioselectivity. The Δk calculated from these plots for head cabbage and pakchoi is 0.2599 and 0.0911 d−1, respectively. The results also suggested that greater enantioselectivity of penconazole occurred in head cabbage than that in pakchoi. In the present study, the dissipation of the two enantiomers of penconazole was investigated in the four test vegetables. The results showed that the (−)-penconazole degraded faster than the (+)-isomer in head cabbage and pakchoi, which meant enantioselectivity happened. However, no obvious enantioselectivity of dissipation behavior existed in cucumber and tomato. Some studies16,17,20,23,24 verified that other triazole fungicides, such as hexaconazole, tebuconazole, and myclobutanil, exhibited stereoselectivity of degradation in cucumber or tomato. Many researchers 34−38 indicated that whether enantioselective behavior occurred or not may be related to the metabolic enzyme system in plant during the process of chiral pesticide biotransformation. Further research should be done to identify the metabolic enzyme systems related to penconazole biotransformation in vegetables. Meanwhile, research concerning the bioactivities and toxicities of individual enantiomer of penconazole also should be conducted to provide adequate information for further food safety risk evaluation of penconazole enantiomers.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 86-571-86404355. Fax: 86-571-86401834. E-mail:
[email protected]. Funding
This study was supported by the National High Technology Research and Development Program of China (The 863 Program, Grant No. 2011AA100806), and the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201303088). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Buchenauer, H. Mechanism of action of triazolyl fungicides and related compounds. In Modern Selective Fungicides: Properties, 11052
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Journal of Agricultural and Food Chemistry
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(22) Zhang, H.; Wang, X.; Qian, M.; Wang, X.; Xu, H.; Xu, M.; Wang, Q. Residue analysis and degradation studies of fenbuconazole and myclobutanil in strawberry by chiral high-performance liquid chromatography−tandem mass spectrometry. J. Agric. Food Chem. 2011, 59, 12012−12017. (23) Dong, F.; Liu, X.; Zheng, Y.; Cao, Q.; Li, C. Stereoselective degradation of fungicide triadimenol in cucumber plants. Chirality 2010, 22, 292−298. (24) Wang, Q.; Qiu, J.; Zhou, Z.; Cao, A.; Wang, X.; Zhu, W.; Dang, Z. Stereoselective pharmacokinetics of diniconazole enantiomers in rabbits. Chirality 2009, 21, 699−703. (25) El-Sharkawy, E. E.; El-Nisr, N. A. Testicular dysfunction induced by penconazole fungicide on male albino rats. Comp. Clin. Pathol. 2013, 22, 475−480. (26) Perdichizzi, S.; Mascolo, M. G.; Silingardi, P.; Morandi, E.; Rotondo, F.; Guerrini, A.; Prete, L.; Vaccari, M.; Colacci, A. Cancerrelated genes transcriptionally induced by the fungicide penconazole. Toxicol. In Vitro 2014, 28, 125−130. (27) Lehotay, S. J.; Mastovska, K.; Lightfield, A. T. Use of buffering and other means to improve results of problematic pesticides in fast and easy method for residue analysis of fruits and vegetables. J. AOAC Int. 2005, 88, 615−629. (28) Wang, J.; Leung, D.; Chow, W. Applications of LC/ESI-MS/MS and UHPLC QqTOF MS for the determination of 148 pesticides in berries. J. Agric. Food Chem. 2010, 58, 5904−5925. (29) Gosetti, F.; Mazzucco, E.; Zampieri, D.; Gennaro, C. M. Signal suppression/enhancement in high-performance liquid chromatography tandem mass spectrometry. J. Chromatogr., A 2010, 1217, 3929−3937. (30) Martins, J. M.; Mermoud, A. Sorption and degradation of four nitro aromatic herbicides in mono- and multi-solute saturated/ unsaturated soil batch systems. J. Contam. Hydrol. 1998, 33, 187−210. (31) Garrison, A. W.; Schmitt, P.; Martens, D.; Kettup, A. Enantiomeric selectivity in the environmental degradation of dichlorprop as determined by high-performance capillary electrophoresis. Environ. Sci. Technol. 1996, 30, 2449−2455. (32) Diao, J.; Xu, P.; Wang, P.; Lu, D.; Lu, Y.; Zhou, Z. Enantioselective degradation in the sediment and aquatic toxicity to daphnia magna of the herbicide lactofen enantiomers. J. Agric. Food Chem. 2010, 58, 2439−2445. (33) Marucchini, C.; Zadra, C. Stereoselective degradation of metalaxyl and metalaxyl-M in soil and sunflower plants. Chirality 2002, 14, 32−38. (34) Gu, X.; Wang, P.; Liu, Y.; Zhou, Z. Stereoselective degradation of diclofop-methyl in soil and Chinese cabbage. Pestic. Biochem. Phys. 2008, 92, 1−7. (35) Wang, X. Q.; Jia, G. F.; Qiu, J.; Diao, J. L.; Zhu, W. T.; Lv, C. G.; Zhou, Z. Q. Stereoselective degradation of fungicide benalaxyl in soils and cucumber plants. Chirality 2007, 19, 300−306. (36) Liu, D.; Wang, P.; Zhu, W.; Gu, X.; Zhou, W.; Zhou, Z. Enantioselective degradation of fipronil in Chinese cabbage (Brassica pekinensis). Food Chem. 2008, 110, 399−405. (37) Lu, D.; Liu, D.; Gu, X.; Diao, J. L.; Zhou, Z. Stereoselective metabolism of fipronil in water hyacinth (Eichhornia crassipes). Pestic. Biochem. Phys. 2010, 97, 289−293. (38) 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. 2015, 60, 1929−1936.
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dx.doi.org/10.1021/jf5034653 | J. Agric. Food Chem. 2014, 62, 11047−11053
