Simultaneous Enantioselective Determination of Triazole Fungicide

Article pubs.acs.org/JAFC

Simultaneous Enantioselective Determination of Triazole Fungicide Flutriafol in Vegetables, Fruits, Wheat, Soil, and Water by ReversedPhase High-Performance Liquid Chromatography Qing Zhang, Mingming Tian, Meiyun Wang, Haiyan Shi, and Minghua Wang* Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Jiangsu Key Laboratory of Pesticide Science, Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing 210095, P. R. China ABSTRACT: A novel and effective method for enantioselective determination of flutriafol enantiomers in food and environmental matrices (cucumber, tomato, grape, pear, wheat, soil, and water) has been developed. The (R)-(−)-flutriafol was first eluted and measured from electronic circular dichroism spectra using a cellulose tris(3-chloro-4-methyl phenyl carbamate) chiral column. The mean recoveries from the samples ranged from 82.9% to 103.4%, with intraday relative standard deviations (RSD) of 2.2−8.3% and interday RSD of 3.4−7.9%. Good linearity (R2 ≥ 0.9989) was obtained for all analytes matrix calibration curves within the range of 0.1−10 mg/kg. The limits of detection for two enantiomers in the seven matrices were all below 0.015 mg/kg. The results show that the proposed method is convenient and reliable for the enantioselective detection of the flutriafol in the real samples and is applicable to the environmental stereochemistry of flutriafol in food and environmental matrices. KEYWORDS: flutriafol, chirality, enantioselective separation, stereoisomers, absolute configuration

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to become a groundwater contaminant.12 Since rac-flutriafol has been widely used, there may be an increased risk of secondary poisoning to humans and wildlife. The achiral determination of flutriafol in wheat and baby food has been published using liquid chromatography−tandem mass spectrometry (LC−MS/ MS).13,14 Wang et al. described an enantioselective separation of flutriafol using cellulose-tris(3,5-dimethylphenylcarbamate; CDMPC) for the first time, and baseline separation was achieved for the two enantiomers.15 The stereoselective degradation of flutriafol in rabbit was also investigated by a normal-phase HPLC. (R)-Flutriafol exhibited a shorter distribution half-life than the S-isomer in rabbits.16 To date, enantioselective determination of flutriafol enantiomers in environmental and food matrices is still not available because there are a great number of interferences presenting in the complex matrices (e.g., vegetables and wheat contain endogenous pigments and lipid compounds). Therefore, it is necessary to develop a truly effective enantioselective analytical method for determining the enantioselective bioactivities, toxicities, metabolism, and degradation behavior in the environment and food of the enantiomers of flutriafol.5 In this article, a complete enantioselective analysis method for flutriafol in environmental and food matrices was developed using reversed-phase high performance liquid chromatography (HPLC) on a cellulose chiral stationary phase, a cellulose tris(3-chloro-4-methyl phenyl carbamate). The extraction and purification procedure was based on modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) methods, using a homogenizer and ultrasound to improve the extraction efficiency of target compounds and adopting a Florisil-SPE

INTRODUCTION The significance of molecular chirality has been widely recognized in life sciences.1,2 Chiral pesticides are a subgroup of agrochemicals that contain at least one chiral center. Although stereoisomers have exactly the same physical and chemical characteristics, they behave drastically differently when exposed to a chiral environment. The chiral pesticides usually have different biological activities to target biological objects, enantioselective toxicity, and enantioselective degradation in the organism and environment.3,4 However, chiral pesticides are generally produced and used as racemates or mixtures of stereoisomers. Currently, the role of enantioselectivity in environmental safety is still not well understood for chiral pesticides.5 When racemic compounds are employed, about 50% or more of ineffective products will be included and added into the environment. These ineffective products may be less active as pesticides or even could be more toxic against nontarget organisms.6−8 In most cases, enantiomers are treated as only a single compound in conventional analysis.9 Therefore, traditional risk evaluations of chiral pesticides are incomplete and not reliable. Consequently, developing enantioselective separation and analysis method for chiral pesticides is of great importance to supply more accurate data for evaluating environmental risks and food safety. Flutriafol, [(R,S)-2,4-difluoro-α-(1H-1,2,4-triazol-1ylmethyl)benzhydryl alcohol (see Figure 1), is a broadspectrum chiral triazole fungicide used as a systemic foliar or seed treatment fungicide to control many plant diseases through inhibiting the C-14α-demethylase enzyme involved in the biosynthesis of fungal sterols.10 Previous studies showed that the (+)-isomer of flutriafol was more active than the (−)-isomer against both Alternaria solani and Alternaria mali.11 Like most other chiral pesticides, flutriafol is generally marketed and applied in its racemic form. Flutriafol is extremely persistent in soil, presenting high mobility potential, and likely © 2014 American Chemical Society

Received: Revised: Accepted: Published: 2809

December 19, 2013 March 5, 2014 March 10, 2014 March 10, 2014 dx.doi.org/10.1021/jf405689n | J. Agric. Food Chem. 2014, 62, 2809−2815

Journal of Agricultural and Food Chemistry

Article

Figure 1. Chromatograms and mass spectra of flutriafol enantiomers: (A) HPLC chromatogram of racemic flutriafol; (B) chromatogram of (R)flutriafol. (C) chromatogram of (S)-flutriafol; (D) mass spectra of (R)-flutriafol; (E) mass spectra of (S)-flutriafol. obtained from Daicel (Shanghai, China). Stock standard (500 mg/L) of each enantiomer was prepared in methanol. All solutions were protected against light and stored at −20 °C. The Cleanert Florisil and Cleanert C18 (500 mg, 6 mL) were purchased from Agela Technologies (Tianjin, China). The samples were comminuted and blended by FeiGe DL-5200B centrifuge (Shanghai, China), Joyoung Soymilk Maker (Hangzhou, China), and Jinyi SK-1 Ultra-Turrax homogenizer (Jiangsu, China). Apparatus and Chromatographic Conditions. The chromatographic separation of the enantiomers of flutriafol was performed on an Agilent 1200 HPLC system (Agilent, USA) with chiral Lux Cellulose-2 (cellulose tris(3-chloro-4-methyl phenyl carbamate), 250 mm × 4.6 mm id)) (Phenomenex, China) using a mixture of acetonitrile and water (40:60, v/v) as the mobile phase at a flow rate of 0.8 mL/min with the injection volume of 20 μL. The column was kept at 30 °C, with a UV detection at 210 nm. Capacity factor (k), separation factor (α), and resolution factor (Rs) were calculated from the formulas

column to remove pigments and fatty acids from extracts. To the best of our knowledge, the current report is the first time to present the enantioselective analysis of flutriafol in environment and food samples (cucumber, tomato, grape, pear, wheat, soil, and water). The time-dependent density functional (TDDFT) methodology was applied to predict the electronic circular dichroism (ECD) spectra of flutriafol. The absolute configuration of flutriafol enantiomers was measured through the combination of experimental and predicted ECD spectra. This analytical method developed was validated by its application to the real samples.

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MATERIALS AND METHODS

Chemical and Materials. The standard of rac-flutriafol (>97.3% purity) was obtained from the China Shanghai Pesticide Research Institute (Shanghai, China). HPLC-grade acetonitrile was purchased from Fisher Scientific (Shanghai, China). Purified water was obtained by using MUL-9000 water purification systems (Nanjing Zongxin Water Equipment Co. Ltd., China). The mobile phase was filtered through a 0.22 μm filter membrane (Tengda, Tianjin, China). Sodium chloride, anhydrous sodium sulfate, acetonitrile, acetone, and hexane of analytical grade were purchased from commercial sources. A stock solution of racemic flutriafol standard (200 mg/L) was prepared in acetonitrile−water (60/40, v/v). Standard working solutions of rac-flutriafol at 0.2, 0.5, 1, 2, 5, 10, and 20 mg/L were prepared in acetonitrile/water (60/40, v/v) from the stock solution by serial dilution. Accordingly, the matrix-matched standard solutions were obtained at 0.2, 0.5, 1, 2, 5, 10, and 20 mg/L concentrations of rac-flutriafol by adding appropriate amounts of standards to the control matrix extract. Enantiomers of flutriafol (purity ≥97.0%) were

k = (t − t0)/t0

α = k 2 / k1 Rs = 2(t 2 − t1)/(W1 + W2) where t is the retention time, t0 is the void time at given conditions, and k is the retention factor. Parameter k1 is the capacity factor of the first eluted enantiomer: (t1 − t0)/t0. k2 is the capacity factor of the second eluted enantiomer: (t2 − t0)/t0. W1 and W2 are the corresponding baseline peak widths for first and second eluted enantiomers. Triplicate determination was made for the measurement of the mean values. 2810

dx.doi.org/10.1021/jf405689n | J. Agric. Food Chem. 2014, 62, 2809−2815


Article

Circular Dichroism Spectroscopy and ECD Calculations. The absolute configuration determination of the flutriafol enantiomers was carried out using a Jasco J815 circular dichroism spectropolarimeter (Tokyo, Japan) at room temperature in methanol. The spectrum was collected over a wavelength range of 200−400 nm at scan speed of 100 nm/min. A quartz cell with a path length of 0.5 cm was used for the scan, and the average of three scans was reported. The geometry of the flutriafol enantiomers was optimized and predicted by Gaussian 09 software using density functional theory (DFT) at a level of B3LYP/631+G* in methanol with a conductor-like polarizable continuum model (CPCM) solvent model.17,18 Extraction and Cleanup for Water. Water (100 mL) samples spiked at three concentration levels were prepared by the addition of appropriate amounts of standard solutions of racemic flutriafol to tap water. The C18 SPE was preconditioned using 6 mL of methanol followed by 6 mL of purified water. 100 mL of the water sample was slowly uploaded to a SPE cartridge at a flow rate of 2 mL/min, and the effluent was discarded. 10 mL of methanol was used to elute target analytes and collected. The eluate was evaporated to dryness using a rotary evaporator (30 °C). The residue was redissolved in 1 mL of acetonitrile−water (40:60, v/v) and filtered through a 0.22 μm filter membrane for HPLC analysis. Extraction and Cleanup for Vegetables and Fruits. A 20 g minced sample was added into a 100 mL Teflon centrifuge tube and spiked with different concentrations of flutriafol standard solutions. The tubes containing the spiked samples were vortexed for 30 s and allowed to stand overnight at room temperature to distribute the pesticide evenly. 40 mL of acetonitrile was added to the tube and then homogenized at high speed for 3 min, followed by sonication for 10 min. Subsequently, 6 g of anhydrous magnesium sulfate (MgSO4) and 1.5 g sodium chloride (NaCl) were added. The tube was vortexed vigorously for 2 min and then centrifuged for 5 min at 4,000 rpm. The supernatant acetonitrile (20 mL) was evaporated to dryness and redissolved in 5 mL of a mixture of hexane−acetone (98:2, v/v) followed by cleanup with Florisil-SPE conditioned with 4 mL of hexane. The SPE cartridge was rinsed with 6 mL of hexane−acetone (95:5, v/v) and eluted with 5 mL of hexane−acetone (85:15, v/v) three times sequentially. All eluates were collected and evaporated to dryness under vacuum at 40 °C. The sample was dissolved in 1 mL of acetonitrile−water (40:60, v/v) and filtered through a 0.22 μm filter for HPLC analysis. Extraction and Cleanup for Wheat and Soil. A 15 g sample of soil or wheat was weighed into a 100 mL Teflon centrifuge tube and spiked with different concentrations of flutriafol standard solutions. The samples were allowed to stand overnight at room temperature. Then, 5 mL of water and 30 mL of acetonitrile were added to the tube and then homogenized at high speed for 3 min, followed by sonication for 10 min. The mixture was homogenized, and 4 g of anhydrous MgSO4 and 1 g of NaCl were added. The tube was vortexed vigorously for 2 min and centrifuged for 5 min at 4,000 rpm. The acetonitrile (15 mL) was concentrated to dryness under vacuum at 40 °C. The residue was transferred with 5 mL of hexane−acetone (98:2, v/v) to a FlorisilSPE column conditioned with 4 mL of hexane. The SPE cartridge was rinsed with 6 mL of hexane−acetone (95:5, v/v) and eluted with 5 mL of hexane−acetone (85:15, v/v) three times sequentially. All eluate was evaporated to dryness with a vacuum rotary evaporator at 40 °C and dissolved in 1 mL of acetonitrile−water (40:60, v/v) for HPLC analysis. Method Validation. The specificity, linearity, limit of detection (LOD) and limit of quantification (LOQ), matrix effect, accuracy, precision, and stability were applied to evaluate the performance of the method. A total of 35 blank samples, five samples of each matrix, were applied to verify the absence of interfering substances. The linearity of calibration curves was evaluated based on the peak areas of the standard solutions and the different matrices at seven concentrations ranging from 0.1 to 10 mg/L in triplicate. The linearity was determined by linear regression analysis of both standard solution and matrix-matched calibration curves.

The matrix-dependent LOD and LOQ were determined using the blank and calibration standards of the vegetables, fruits, wheat, soil, and water matrices. The LOD and LOQ for enantiomers of flutriafol were considered as concentrations in a sample matrix resulting in peak areas with signal-to-noise (S/N) ratio of 3 and 10, respectively. The recoveries were carried out to investigate the accuracy and precision of the method. Soil and water samples from trial plots were located in the Nanjing region in Jiangsu. Other samples were purchased from regional retailers. Five replicates of spiked sample at three different levels from 0.004 to 1 mg/kg were prepared on three consecutive days. The flutriafol was extracted and purified according to the above procedure. The average recoveries and intraday and interday relative standard deviations (RSD) were used to evaluate accuracy and precision. It has been reported that some kinds of chiral pesticides, such as pyrethroids, could undergo epimerization under certain conditions.19 Two optically pure flutriafol enantiomers were determined under the same conditions to evaluate the stability of the flutriafol enantiomers during analysis and storage in solvent and matrices. Stability of the stock solutions and the spiked sample matrix-matched standard was tested monthly, and all the samples used in the stability test were stored at −20 °C. The results of the stability tests were compared with those concluded from the freshly prepared samples using Student’s t test (P < 0.05).

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RESULTS AND DISCUSSION Chromatographic Condition Optimization. A number of the chiral stationary phases (CSPs) were used for chiral separations by HPLC in various chemical fields. The polysaccharide-based CSPs, especially cellulose- and amylasebased polysaccharide columns with higher recognition abilities, are the most used.20,21 Zhang et al. reported that the two enantiomers of flutriafol could be baseline separated on polysaccharide-based CSPs by reversed-phase HPLC−MS/ MS.22 Ying et al. reported that the flutriafol enantiomers were separated using Chrialcel OD and Chrialcel OJ column by normal phase HPLC.23 Despite chiral separation, the retention time was not less than 25 min in all reports. In this study, the two enantiomers of flutriafol were completely separated with the Lux Cellulose-2 column under reversed-phase conditions using acetonitrile−water (40:60, v/v) as the mobile phase at a flow rate of 0.8 mL/min with the good resolution factor (Rs) of 1.82. The consumption of solvents was reduced within 20 min. The methanol and acetonitrile may induce different effects for the separation of the flutriafol on the same column. The flutriafol enantiomers were separated with all Rs higher than 1.54 using a mixture of acetonitrile and water as mobile phase at the rate of 65:35 to 30:70 (v/v). When the percentage of acetonitrile in the eluent increased, a decrease in the retention factors was observed and a worse resolution was obtained. Therefore, a solvent system mixture of acetonitrile−water (40:60, v/v) was used as mobile phase at a flow rate of 0.8 mL/ min based on retention time and satisfactory separation (Figure 1). The effect of column temperature on the chiral separation also was evaluated. The result showed a slightly better separation and better peak shape at 30 °C. The Absolute Configuration and Elution Order Determination of Flutriafol Enantiomers. The each enantiomer was stereochemically determined by circular dichroism (CD) spectroscopy. The two CD curves were almost mirror imaged, and the curves of the predicted ECD and experimental ECD were very similar (Figure 2). The absolute configuration of enantiomers was measured through the combination of experimental and predicted ECD spectra. The configurations of the flutriafol enantiomers that eluted from the 2811



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matrix-matched calibration curves within the range of 0.1−10 μg/mL for each enantiomer of flutriafol are summarized in Table 1. Table 1 indicates the slopes, P values, and correlation Table 1. Comparison of Matrix-Matched Calibration and Solvent Calibration calibration (matrix)

Figure 2. The predicted and experimentally measured ECD spectra of flutriafol enantiomers: (A) predicted ECD spectra; (B) experimentally measured ECD spectra.

HPLC were determined based on the predicted ECD spectra by the TDDFT methodology. Accordingly, peak 1 and peak 2 of the chromatograms shown in Figure 1 were assigned to (R)flutriafol and (S)-flutriafol, respectively. Chang et al. had confirmed that (S)-flutriafol was (+)-flutriafol and the (R)flutriafol corresponded to (−)-flutriafol.24 As can be seen from Figure 1, (−)-flutriafol was first eluted under the above conditions, and the mass spectra of the two enantiomers of flutriafol are also provided. Sample Preparation. The QuEChERS method is a major development for sample preparation in pesticide residue analysis.25 The magnesium sulfate reduces the water phase and promotes the partitioning of the pesticides into the organic phase.26 The dispersive cleanup procedure of QuEChERS is faster and easier. However, some food colorings and lipidic soluble materials could not been removed by primary secondary amine (PSA), C18, and graphitized carbon black (GCB). It is not as effective as using SPE columns, and the QuEChERS method has a high requirement in the device.27−29 Flutriafol was extracted based on the modified QuEChERS method assisted with ultrasound extraction. The flutriafol was concentrated by 10 or 7.5 times, and a further cleanup step was improved using SPE columns. The Cleanert Florisil, Cleanert NH2, and Cleanert C18 were used for the cleanup procedure. The pigments and interferences were not efficiently removed and diminished by NH2SPE, whereas the C18-SPE was satisfactorily utilized for water sample and the Florisil-SPE could remove the visible pigments of vegetables, soil, and fruits. The chromatograms showed very clean baseline and separation. Methanol was selected as the eluting solvent in Cleanert C18 because of lower toxicity and higher efficiency for the analytes than acetonitrile.9 Specificity, Linearity, and Matrix Effect. No interference was detected at the retention time of flutriafol in blank samples of different matrices, and this showed the specificity of the method. Linear regression results of standard solution and

calibration range (mg/kg)

solvent soil

0.1−10 0.1−10

water

0.1−10

cucumber

0.1−10

tomato

0.1−10

pear

0.1−10

wheat

0.1−10

grape

0.1−10

solvent

0.1−10

soil

0.1−10

water

0.1−10

cucumber

0.1−10

tomato

0.1−10

pear

0.1−10

wheat

0.1−10

grape

0.1−10

regression equation

(R)-Flutriafol y = 80.27x + 1.11 y = 78.271x − 2.5071 y = 75.782x − 3.7409 y = 76.622x − 0.4451 y = 81.998x + 0.4045 y = 81.02x − 2.0206 y = 81.268x − 0.9632 y = 81.845x + 1.1147 (S)-Flutriafol y = 80.642x − 1.3033 y = 78.604x − 2.6554 y = 75.105x − 3.6976 y = 76.604x − 0.7979 y = 81.472x + 1.4445 y = 81.042x − 3.4951 y = 81.292x − 0.6155 y = 81.515x + 2.3394

R2

P value

0.9999 0.9993

0.0579

0.9996

0.823

0.9997

0.0832

0.9995

0.0889

0.9991

0.676

0.9989

0.187

0.9992

0.249

0.9999 0.9994

0.0525

0.9998

0.0946

0.9995

0.114

0.9997

0.117

0.9989

0.623

0.9990

0.176

0.9993

0.0721

coefficients of determination (R2) of both matrix-matched and standard solution calibration curves. Excellent linearities were observed for the enantiomers (R2 ≥ 0.9989), which were adequate for enantiomeric-specific quantitative trace analysis. The response of standard solution and matrix-matched calibration curves were compared to matrix-matched calibration curves using two-tailed paired t test with a probability of 95%. Table 1 showed that no significant matrix-induced enhancement or suppression effects were observed for the two enantiomers. The P values implied that the data between pure solvent and sample matrices were not significantly different (P > 0.05) and the matrix effect could be negligible. LODs and LOQs. The LODs for both enantiomers were estimated to be 0.001 mg/kg in water, 0.012 mg/kg in grape, cucumber, pear, and tomato, and 0.015 mg/kg in wheat and soil. The LOQs were estimated at 0.003−0.05 mg/kg for the two enantiomers based on five replicate extractions and analyses of seven spiked samples at low concentration levels. The low detection limits indicated that chiral HPLC was capable of the quantitation of flutriafol enantiomers in the food and environmental matrices. Accuracy and Precision. The analytical precision and accuracy, expressed with the RSD and the recoveries, was evaluated by spiking the blank samples at three different 2812



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Table 2. Accuracy and Precision of the Proposed Method in the Seven Matrices (n = 5) intraday (n = 5) day 1 matrix soil

water

cucumber

tomato

pear

wheat

grape

soil

water

cucumber

tomato

pear

wheat

grape

spiked level (mg/kg)

av recoveries (%)

day 2 RSD (%)

0.02 0.1 0.5 0.004 0.02 0.1 0.02 0.1 0.5 0.02 0.1 0.5 0.02 0.1 0.5 0.05 0.25 1 0.02 0.1 0.5

88.5 90.2 91.1 98.2 98.5 95.1 92.5 91.1 85.3 90.8 92.1 85.6 92.2 85.4 85.8 92.6 90.4 89.8 91.9 85.1 90.7

6.6 4.2 5.6 3.2 3.9 2.2 4.6 4.1 5.2 4.2 2.5 2.9 5.1 2.4 3.8 4.3 3.1 2.9 3.4 3.5 4.8

0.02 0.1 0.5 0.004 0.02 0.1 0.02 0.1 0.5 0.02 0.1 0.5 0.02 0.1 0.5 0.05 0.25 1 0.02 0.1 0.5

92.7 94.9 90.5 97.1 97.1 94.1 96.1 94.2 84.6 101.4 94.5 86.7 91.1 87.1 84.2 95.8 93.2 90.8 95.2 87.7 87.1

5.2 4.8 6.7 6.2 5.2 2.6 3.8 3.9 5.9 5.8 2.2 3.6 4.3 6.3 7.2 5.1 2.8 8.3 7.5 2.5 5.8

av recoveries (%) (R)-Flutriafol 87.3 89.2 94.1 93.4 93.8 91.8 90.5 87.7 84.2 88.9 91.1 83.8 87.5 83.5 83.5 89.9 88.6 92.3 87.9 83.8 88.9 (S)-Flutriafol 89.3 91.8 93.5 92.1 94.3 90.9 94.3 92.7 83.1 95.4 92.4 84.2 88.3 84.2 82.9 90.7 87.9 90.8 92.1 84.8 85.9

concentrations with five replications on three consecutive days (Table 2). As shown in Table 2, the method presented satisfactory mean recoveries and precision for both enantiomers. The average recoveries ranged from 83.5% to 101.2% with intraday RSD 2.2−7.3% for (R)-flutriafol and ranged from 82.9% to 103.4% with intraday RSD 2.2−7.9% for (S)-flutriafol. The chromatograms of the flutriafol in the blank samples and the spiked pear at 0.5 mg/kg are shown in Figure 3. Further statistical analysis using one-way analysis of variance at 95% confidence limit showed no significant difference of interday and intraday assays. The results demonstrated that this method could achieve a satisfactory precision and accuracy for the enantiomeric analysis of flutriafol in food and environmental

day 3 RSD (%)

av recoveries (%)

RSD (%)

interday (n = 15) RSD (%)

4.3 3.9 2.5 2.7 4.3 3.1 5.1 3.7 3.6 3.8 2.9 3.4 3.2 3.5 6.9 5.3 4.8 3.4 3.2 2.8 3.3

91.5 92.3 90.5 99.4 101.2 98.2 95.3 90.5 87.8 96.1 88.3 87.8 95.2 89.3 88.9 94.1 93.1 87.1 94.3 88.9 92.1

3.8 4.3 4.1 5.6 4.9 3.6 5.3 4.9 4.2 5.3 3.3 3.5 6.5 5.1 4.9 7.1 4.7 3.8 4.3 2.9 4.4

5.9 5.2 3.8 5.1 5.4 3.9 4.2 4.5 3.7 4.9 3.0 3.3 7.3 5.4 4.4 6.5 4.6 3.5 4.1 3.8 4.7

3.6 3.3 3.1 5.3 5.8 2.3 4.8 3.3 5.8 4.5 3.2 2.8 5.9 4.6 6.2 4.7 3.5 7.5 6.5 3.8 5.4

93.4 96.7 89.5 102.1 99.5 97.1 101.7 96.8 88.3 103.4 90.8 87.2 96.2 87.8 89.1 95.2 94.1 88.8 97.1 87.4 89.2

7.5 5.8 7.2 7.3 3.6 4.6 6.7 3.8 5.5 4.1 3.8 5.9 7.2 6.8 7.8 5.8 6.2 3.9 5.8 4.7 6.2

6.7 5.2 5.7 7.5 5.3 3.9 5.2 3.4 5.7 4.9 2.9 4.0 7.9 5.2 7.6 5.6 4.3 7.1 5.2 4.3 5.1

matrices. In addition, an evaluation of the stability of the two enantiomers was conducted, and no significant difference (P > 0.05) was observed under the solvent and matrix storage treatment as described in Materials and Methods. Application to Authentic Samples. The effectiveness of this method in measuring trace levels of rac-flutriafol was monitored by analyzing seven different samples obtained from field and local market (Nanjing, China). Each kind of sample was analyzed in quintuplicate. The results showed three positive pear samples with flutriafol residues in the range of 0.009−0.019 mg/kg. The enantiomers of flutriafol were not found in other real samples. The typical chromatograms of pear 2813



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Figure 3. The typical chromatograms of pear sample: (A) blank pear sample; (B) pear sample spiked with 0.5 mg/kg; (C) authentic pear sample.

Funding

are presented in Figure 3 with 0.0151 and 0.0158 mg/kg for (R)-flutriafol and (S)-flutriafol, respectively. In the present study, a simple and reliable enantioselective method using chiral HPLC for the simultaneous quantitative determination of flutriafol enantiomers in food and environment matrices (cucumber, tomato, grape, pear, wheat, soil, and water) has been successfully established and validated. Target compounds were extracted and purified based on the modified QuEChERS method. In addition, the absolute configurations and elution order of flutriafol enantiomers were determined by a combination of experimental and predicted ECD spectra. Adequate analytical performance characteristics were obtained in terms of linearity range and precision for both enantiomers of flutriafol in seven matrices. Satisfactory results were obtained in the real samples. The purpose of the current work was not only to establish a novel valid chiral HPLC method to separate the two enantiomers of the flutriafol but also to develop a method of quantitative determination of the two enantiomers of flutriafol in the food and environmental samples. The developed chiral HPLC method may be used to facilitate further studies in tracing the different toxicities, bioactivities, metabolism, and environmental behavior of each enantiomer, which could help minimize the hazards posed by flutriafol to humans, animals, and ecological health.

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This work was supported by the Special Fund for Agroscientific Research in the Public Interest (201203022) and the National “863” High-Tech Research Program of China (2011AA100806). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Hu Zhang (Zhejiang Academy of Agricultural Sciences) for calculating the absolute configuration of flutriafol enantiomers by Gaussian 09 software.

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REFERENCES

(1) Koeller, K. M.; Wong, C. Enzymes for chemical synthesis. Nature 2001, 409 (6817), 232−240. (2) Yoon, T. P.; Jacobsen, E. N. Privileged chiral catalysts. Science 2003, 299 (5613), 1691−1693. (3) 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. (4) Perez-Fernandez, V.; Garcia, M. A.; Marina, M. L. Chiral separation of agricultural fungicides. J. Chromatogr. A 2011, 1218 (38), 6561−6582. (5) 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 (3), 701−706. (6) Perez-Fernandez, V.; Garcia, M. A.; Marina, M. L. Chiral separation of metalaxyl and benalaxyl fungicides by electrokinetic chromatography and determination of enantiomeric impurities. J. Chromatogr. A 2011, 1218 (30), 4877−4885.

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Simultaneous Enantioselective Determination of Triazole Fungicide

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