Journal of Pesticide. 104 (2017) 138-146 https://sites.google.com/site/photonfoundationorganization/home/pesticide-journal Original Research Article. ISJN: 6048-7014: Impact Index: 5.15

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Journal of Pesticide

Residue analysis of Fluopyram, Trifloxystrobin and itsmetabolite in fruits and vegetables by UHPLC-MS/MS Husham N.M. Hussana, XingangLiub*, Shasha Lib, FengshouDongb, Jun Xub, Yongquan Zhengb a b

Pesticides Analysis Laboratory, Agricultural Research Corporation, Sudan State Key Laboratory for Biology of Plant Diseases and Insect Pests- Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, P.R. China

Article history: Received: 17 October, 2016 Accepted: 18 October, 2016 Available online: 11 May 2017 Keywords: UHPLC-MS/MS; Residues Vegetables; Dissipation.

analysis;

Fungicides;

Fruits;

Corresponding Author: Prof. Liu Xin-gang Professor Email: liuxingang ( at ) caas ( dot ) cn Husham N.M. Hussan Dr. Shasha Li Ph.D Fengshou Dong Professor Jun Xu Professor Yongquan Zheng Professor

Abstract A simple analytical method for three fungicides (fluopyram, trifloxystrobin and its metabolite) in vegetables (tomato, and cowpea) and fruits (pear, grape,

apple, and watermelon) has been developed. The fungicides were extracted from six fruit and vegetable matrices using acetonitrile and subsequently cleaned up only using octadecylsilance(C18) as sorbent prior to HPLC-MS/MS analysis. The target compounds were achieved in less than 3.0 min using an electrospray ionization source in positive mode (ESI+). The method has been validated for six matrices spiked at 0.01, 0.1 and 1 mg kg-1 by matrix-matched standard. The average recoveries ranged from 71.7% to 116.4% with relative standard deviation (RSD) between 1.4 and 20% (n=5) for all analytes. The limits of detection (LODs) ranged from 0.2 to 2 µg kg-1, while the limits of quantification (LOQs) ranged from the 0.5 to 7 µg kg-1 in different matrices. The residue and dissipation dynamic of fluopyram in tomato at the field trials were investigated. The half-lives of fluopyram in tomato were2.8 -12.0 days. The terminal residues of fluopyram were all below the maximum residue limits (MRLs) value set by EU (0.9 mg/kg). Hence it was safe for the use of fluopyram and the results also could help to build MRLs of fluopyram in tomato in China. Citation: Husham N.M. Hussan, Liu Xingang,* Shasha Li, Fengshou Dong, Jun Xu, Yongquan Zheng., 2017. Residue analysis of Fluopyram, Trifloxystrobin and itsmetabolite in fruits and vegetables by UHPLC-MS/MS. Photon 104, 138-146 All Rights Reserved with Photon. Photon Ignitor: ISJN60487014D847211052017

1. Introduction Fungicides are a group of chemicals which are used to control fruits and vegetables from fungus attack (Dimitra et al., 2000). Fluopyram (N-{2-[3-chloro5-(trifluoromethyl)-2-pyridyl]ethyl}-α, α, αtrifluoro-o-toluamide) is a new broad-spectrum fungicide belonging to the subgroup of pyridinylethylbenzamides. It is biologically active against all the stages of fungal growth, from spore germination to spore production, and its activity spectrum includes several pathogens belonging to Ascomyctetes and Deuteromycetes, such as

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Botrytis spp., Sclerotinia spp. and Monilinia spp., on vegetable, pome and stone fruit crops (Hervéet al., 2010). Trifloxystrobin (TFS) is also a relative new broad spectrum fungicide. It is a synthetic derivative of the naturally occurring strobilurins found in several genera of wood decaying fungi. The mode of action of trifloxystrobin involves inhibition of mitochondrial respiration by blocking electron transfer in the electron transfer chain. The chemical structures of fluopyram, TFS and its metabolite are represented in Fig. (1).

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Some studies and reports have been monitored for analysis of TFS in agricultural products including our lab (Xingang et al., 2010) Generally, The analytical methods of TFS in grapes and wine by liquid chromatographic with diode array detection and gas chromatographic were developed ( Abreu et al., 2006, Likas et al., 2007, Jose et al., 2007, Jyot et al., 2010). Figure 1: Chemical structures of Fluopyram, rifloxystrobin and its metabolite

It has been determined in foods by micellar electrokinetic capillary chromatography (Lilia et al., 2010). And it’s detection and degradation in banana (Soudamini et al., 2014), cotton seed (Jiaying et al., 2014), apple (S.K et al., 2013, Stanisław et al., 2013), wheat (Sarabjit et al., 2012) and vegetables (Armindo et al., 2012) by different method were reported. However, up to now for fluopyram, only one report has been monitoring this compound by GC-MS (Dong et al., 2014). To our knowledge, the simultaneously analytical method of residues of floupyram, TFS and its main metabolite in fruits and vegetables has not been studied. So, the aim of this paper was to develop a simple method and to validate a multi-residue analysis by UHPLC-MS/MS for the simultaneous analysis of fluopyram, trifloxystrobin and its metabolite in vegetables (tomato, and cowpea) and fruits (pear, grape, apple, and watermelon). The residue and dissipation dynamics of fluopyram in tomato were investigated. These data will help the government establish the MRL of fluopyram in tomato and provide guidance on the proper and safe use of this fungicide. 2. Experimental 2.1 Reagents and materials Analytical standards of fluopyram (purity, 99.4%), TFS (purity, 99.6%) and its metabolite (purity, 98.7%) were supplied from Bayer Crop Science, (Frankfurt, Germany). Analytical grade Ph ton

acetonitrile, hydrochloric acid and sodium chloride (NaCl) for pesticides residue analysis were purchased from Beijing Chemical Reagent Company (Beijing, China). Acetonitrile and formic acid (chromatography grade) were obtained from Honeywell International Inc. (New Jersey. USA). Anhydrous magnesium sulfate (MgSO4) was purchased from Sinopharm Chemical Reagent Co. Ltd (Beijing, China). The C18 sorbent from Angela Technologies Int. (Newark, DE, USA). 2.2 Field experiment design The field trails, including the dissipation experiments and residue experiments, were carried out both in Henan and Beijing, China in 2012. The experiment was designed according to “Guideline for Pesticide Residue Trials issued” by the Institute of the Control of Agrochemicals, Ministry of Agriculture (ICAMA), People’s Republic of China. In tomato field, the area of experiment plot was 30 m2 and each treatment was designed with three replicated plots. A buffer area (about 20 m distance) was maintained between the plots. The sprayer was a Jacto Heavy-Duty HD 400 (Agrolex, Singapore) with a nozzle diameter of 1.0 mm and operating pressure of 4 to 5 kg/cm2. In tomato field, the rate of fluopyram-tebuconazole (40%, SC) application in dissipation experiments was 375 grams of active ingredient per hectare (g a.i. ha-1) with one time spray. Representative tomato samples were collected in 0 (2 h after application), 1, 2, 3, 5, 7, 10 and 14 day after spraying of the pesticide. The degradation rate constant and halflife were calculated using the following equation: Ct=C0ekt, where Ct represents the concentration of the pesticide residue at the time of t, C0 represents the initial concentration after application and k is the dissipation degradation rate constant (days-1). The half-life (t1/2) was calculated from the k value for each experiment (t1/2=ln2/k). The terminal residue experiment was designed at two dosages of 250 g a.i. ha-1 (spray 2 times and 3 times) and 375 g a.i. ha-1 (spray 2 times and 3 times), respectively. The intervals of spraying were 7 days. A plot with the same size and clear water application was set as blank control. Representative 6-12 tomato samples were randomly collected at 3, 5 and 7 days after the last application in each plot. 2.3 Analytical procedure 2.3.1Preparationof standard Solution The standard stock solution of fluopyram (100 mg L-1), TFS (100 mg L-1) and its metabolite (100 mg L-1) were prepared in acetonitrile. The stock solutions were required for preparing standard curves (5.0, 1.0, 0.5, 0.1, 0.05, 0.01 mg L-1) by serial dilution. Correspondingly, matrix-matched standard solutions were prepared (5.0, 1.0, 0.5, 0.1, 0.05, 0.01 mg L-1) by adding the concentrated blank sample extract (tomato, and cowpea, pear,

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grape, apple, and watermelon) to each serially diluted standard solution. All solutions were stored in a refrigerator in the dark at 4 °C and the working standard solutions underwent no degradation for 6 months. 2.3.2 Apparatus The mobile phase solvents were distilled and passed through a 0.22-µm prose size filter. HPLCgrade water obtained by purifying demineralized water in Milli-Q Integral 3 water (Millipore, Bedford, MA, USA).An ultrasonic water system bath (Model KH-500, Kunshan) a shaker (model HZQ-CA, Jintan, China) and acentrifuge (model Excelsius II, Fanen, Brazil) were used for sample preparation. Chromatographic separation was conducted on a Waters Acquity UHPLC binary solvent manager, an Acquity UHPLC manager, and an Acuity cartridge heater equipped with a BEH C18 column (2.1×50 mm, 1.7-µm particle size; Milford, MA, USA). This column is packed with a C18 reverse-phase bounded to an ethylenebridged hybrid (BEH) substrate. The mobile phases, which were composed of acetonitrile (A) and 0.1% (v/v) formic acid in water (B), were pumped at a flow rate of 0.3 mL min−1.Simultaneous separations were completed using a gradient profile of 0.0 min/10% A, 1.5 min/70% A, 2.5 min/90% A, 2.6 min/10% A, and 4.5 min/10% A,respectively. The injection volume was 3 µL. The column was kept at 40 °C to decrease theviscosity, and the temperature in the sample manager was set at 5 °C. All three fungicides were eluted within 4.0 min. Analysis of (fluopyram, TFS and its metabolite) fungicides compounds were conducted on a triple-quadrupole mass spectrometer (TQD,Waters Crop.) equipped with an electrospray ionization (ESI) source. The nebulizer gas was 99.95% nitrogen, and the collision was 99.999% argon. MS/MS detection was performed in positive ion mode and the monitoring conditions optimized for target compounds. The conditions were typically as follows: the capillary voltage was set at 3.0 kV, while the source temperature and desolvation temperature were held at 120 °C and 350 °C, respectively. The cone and desolvation gas were set at a flow of 50 and 500 Lh−1, respectively. Multireaction monitoring (MRM) was used for the detection of all pesticides with a dwell time of 50 ms. For UHPLC analysis, the Masslynx NT v 4.1 (Waters) software was used to process quantitative data obtained from calibration standards and from samples. 2.3.3 Sample preparation procedure The fruits (pear, grape, apple, and watermelon) and vegetables (tomato, cowpea) were obtained from supermarket located at Haidian region in Beijing. The matrices were not applied or contaminated by three fungicides, and they were put into Ph ton

polyethylene bags. Vegetables and fruits samples were homogenized by Ultra–Turrax T25 Mixer. Vegetables and fruits (5g) were weighed into 50 mL centrifuge tube. 5 ml of acetonitrile was added to the samples, and the samples were shaken vigorously for 3 min by shaker to ensure that the solvent interacted well with the entire samples. The samples tubes were then stored in a refrigerator at -20°C for 20 min, after that NaCl (3g) and MgSO4 (2g) were added to the sample in the tube and vortexed immediately for 1 min and then the extracts were centrifuged for 5 min at 4000 rpm. Afterwards, a volume of 1.5 ml prepared aliquot sampled from upper layer into another 2ml centrifuge vial containing amount of C18 sorbent 50 mg. All the samples vortexed again for 1 min and then centrifuged for 5 min at 4000 rpm. Then, the upper layer was filtered using a 0.22 µm nylon syringe filter and transferred to an auto-sampler vial for chromatography injection. 2.4 Validation procedure To evaluate the performance of the developed method, the method was validated according to a conventional validation procedure that included the following parameters: linear range, limit of detection (LOD) and limit of quantitation (LOQ), accuracy, precision, and matrix effects. The blank samples from the matrix were analyzed toverify the absence of interfering species around the retentiontime of analytes.The linearity of the method was studied by linear regression analysis of the standard solution and the different matrices in triplicate at different concentrations ranging from 0.01 to 1 mgL-1. Satisfactory linearity's were obtained when the correlation coefficients (r2) were higher than 0.966 based on measurement of the analyte peak area for all compounds in all matrixes. The LOD for these three pesticides were considered to be the concentration that produced a signal-to-noise (S/ N) ratio of 3, and the LOQ was defined based on an S/ N ratio of 10; the LOQ was estimated from the chromatogram corresponding to the lowest point used in the matrix-matched calibration. Recovery assays were enforced to investigate the accuracy and precision of the method. Five replicates spiked samples (pear, grape, apple, watermelon, tomato, and cowpea) at different levels (shown in Table 3) were prepared on three different days. These pesticides were extracted and purified according to the above procedure. The precision in the conditions of repeatability, expressed as the RSD, was determined by the intra-day and inter-day assays. 3. Results 3.1: Optimization of chromatography UHPLC was optimized to achieve a minimal run time and optimal peak shapes. The three target

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compounds were separated using acetonitrile-water (containing 0.1% formic acid).As shown in Fig. 2, there were no interference peaks of blank samples around the retention time of the analytes. The analysis time of fluopyram, trifloxystrobin and its metabolite was shorter than 3.0 min. The compounds were eluted in the following order:

fluopyram (2.45 min), TFS (2.70 min) and its metabolite (2.60 min). In addition, these analysis times were shorter than the ones obtained during the GC-MS separation of these compounds (8 min), (Dong et al., 2014).

Figure 2: ULPC-MS/MS multiple-reaction ion MRM chromatography at 100µg l-1(A), apple untreated (B), and fortified apple sample at 10µg l-1(C).

3.2: Optimization of MS/MS In this study, positive mode and negative modewere tested for fluopyram, TFS and its metabolite. Full-scan and MS/MS mass spectra were obtained from the infusion of 5 mg L-1 standard solution of these compounds in acetonitrile/water including 0.1% formic acid (10: 90, v/v) at a flow rate of 10 mL min-1. ESI+ mode was selected for subsequent experiments becausefluopyram, TFS and its metabolite demonstrated higher responses in positive mode.

5-(trifluoromethyl) pyridine (m/z -208) and 2trifluoromethyl) benzaldehyde (m/z -173).

All compounds showed abundant [M+H] + ions, which were usually selected as precursor ions. Quantitation was conducted using the more abundant ion transitions, whereas the less ducted using the abundant ion transition was used for identification.The chemical formulas, molecular weights, cone voltage and collision energy were also listed in table 1. Figure 3 showed the product ion mass spectra of fluopyram. The fragments formed from fluopyram were lost 3-chloro-2-ethyl-

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Table 1: Experimental parameters and UHPLC-MS/MS conditions of thethree compounds in ESI+ mode. Molecular tR Ion source CV Quantification ion CE 1 Compound MW formula (min) (V) transition (eV) Fluopyram C16H11ClF6N2O 396.7 2.45 ESI+ 30 397.3→173 30 Trifloxystrobin C20H19F3N2O4 408.4 2.70 ESI+ 25 409.5→186 18 Trifloxystrobin / ESI+ C19H17F3N2O4 394.4 2.60 20 395.5→186 20 Acid

Diagnostic ion transition 397.3→208 409.5→206

CE 2 (eV) 25 15

Ion ratio 1.3 2.3

395.5→148

28

3.9

MW: molecular weight, CV: cone voltage, CE: collision energy, ion ratio = area of qualitative ion/area of quantificationion

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3.3: Precision and accuracy Recovery studies were performed to validate the UHPLC-MS/MS method by spiking the blank samples at three different concentration levels (0.01, 0.1, and 1 mg kg−1) and then analyzed in quintuplicate. The recoveries were calculated using the three-point matrix matched calibration curves. Only one type of sorbent C18was used in this work

to investigate the influences on recovery rate in six matrices. C18 was suitable to extract non-polar and medium-polar compounds from the polar samples. The mean recoveries of the target analytes at different fortification levels (0.01, 0.1, and 1 mg/kg) ranged from 71.7% to 116.4% with relative standard deviations (RSDs) of 1.4% - 20.0% showed in Table 3.

Table 3: Recoveries (%) and RSD (%) for target compounds from different matrices in three spiked levels Matrix Tomato

Pear

Grape

Apple

Cowpea

Watermelon

Watermelon (peal)

Spiked level (µg /kg) 10 100 1000 10 100 1000 10 100 1000 10 100 1000 10 100 1000 10 100 1000 10 100 1000

Fluopyram Recovery RSDa 100.5 76.4 78.4 116.4 87.0 92.9 87.1 85.7 79.3 83.7 83.5 80.0 109.7 78.3 81.4 102.6 84.7 83.5 89.9 88.4 89.5

5.5 2.3 18.6 10.4 10.7 12.7 13.8 3.4 5.8 8.8 17.3 1.4 7.1 2.9 14.1 9.7 12.6 4.2 17.3 5.6 9.1

RSDb 7.3 3.2 9.5 16.7 8.6 3.0 11.8 6.8 4.8 15.8 5.1 3.0 9.3 4.0 3.6 3.6 7.1 2.7 9.8 7.8 7.1

AIntra-day (n = 5). b Inter-day (n = 15). 3.4: Matrix effects, Limits of detection (LODs) and quantitation (LOQs) Compared with those produced by solvent solutions of the target analytes, the matrix effects may affect response in a positive or negative way. This can greatly affect the method reproducibility and accuracy. In this study, the response of the solvent standard solution was compared with that of matrix matched standard solution. And the matrix effect could be calculated as follows: matrix effect (ME, %) = (slope of calibration curves in matrix−slope of calibration curves in solvent)/slope of calibration curves in solvent (×100%). The results showed that the matrix significantly affected (from -28.7 to 76.6) the response of the instrument

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Trifloxystrobin Recovery RSDa RSDb 85.0 77.2 85.9 89.0 93.8 91.0 98.6 84.1 83.9 93.9 78.9 82.6 95.8 80.2 78.4 103.4 85.3 79.4 98.0 89.2 78.3

10.6 5.8 18.4 19.3 19.0 15.6 19.3 4.1 3.9 4.1 11.5 1.9 3.0 3.4 12.9 8.9 12.0 8.5 2.4 14.8 13.8

1.5 3.3 9.8 14.2 6.0 2.8 10.1 4.0 3.1 12.8 6.1 4.2 9.4 6.7 3.6 17.4 9.9 5.0 2.4 14.8 13.8

Recovery 92.0 78.9 79.8 106.5 76.9 90.1 100.1 76.7 80.6 80.5 76.1 85.1 93.5 76.9 81.1 84.4 71.7 82.2 100.1 88.9 94.4

TFA RSDa

RSDb

14.9 4.2 12.7 10.4 3.0 20.0 16.1 5.7 3.1 13.0 8.3 2.3 10.4 2.2 11.5 9.4 9.9 16.4 12.4 15.9 11.9

12.1 7.5 8.7 16.8 3.3 3.2 12.7 5.8 4.3 12.1 5.8 4.3 14.1 3.3 4.4 15.0 5.8 3.0 3.3 13.9 18.0

(Table 2). Therefore, a calibration was performed for three target compounds using the external matrix-matched standards to eliminate the matrix effect and to obtain more realistic results in the samples. As shown in Table 2, the LODs for the three pesticides were estimated to be 0.3–2µg kg−1, and the LOQs for three fungicides were 1–7 µg kg−1. The reliability of the developed method was confirmed with authentic samples. According to regulation (EC) No 396/2005, the LOQ for fluopyram in plant matrices is 0.01 mg kg-1. The MRL of fluopyram is 0.9 mg kg-1 for tomato, 1.5 mg kg-1 for grape, 0.6 mg kg-1 for apple, 0.4 mg kg-1 for waternekibin EU and 0.3mg kg-1 in apple, 2.0 mg kg-1 in grape in Canada. These developed LOQs are below the MRL established by the EU and Japan, indicating that the proposed method is suitable for the quantitation of fluopyram and TFS in the studied matrices.

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Table 2: Comparison of matrix-matched calibration and solvent calibration at 10-1000 µg/L. Compound

TFS

FLU

TFA

Matrix

2

Matrix effect (%)

LOQ -1 (μg/kg )

LOD -1 (μg/kg )

Regression equation

R

Acetonitrile

Y= 8.2464x + 137.6

0.9988

-

-

-

Pear Apple Grape Cowpea Tomato Watermelon Watermelon (peel)

Y = 8.8804x + 180.91 y = 10.014x - 63.515 y = 9.5928x + 115.91 y = 5.8778x - 113.62 y = 6.6477x - 182.73 y = 9.1166x - 351.61

0.9882 0.9993 0.9994 0.9878 0.9864 0.9666

7.7 21.3 16.3 -28.7 -19.3 10.6

3 1 3.6 4 5.1 1.1

1 0.3 1.2 1 1.7 0.4

y = 8.6611x - 45.193

0.9996

5.0

2

0.7

Acetonitrile

Y= 8.2137x +178.6

0.9990

-

-

-

Pear Apple Grape Cowpea Tomato Watermelon Watermelon (peel)

Y= 7.127x + 247.44 Y= 8.8187x - 21.902 y = 9.3452x + 257.61 y = 7.8165x - 95.889 y = 9.2926x - 316.04 y = 9.1823x - 132.21

0.9711 0.9994 0.9973 0.9851 0.9761 0.9964

-13.2 7.4 13.8 -4.8 13.1 11.8

5 2 2 4 2.9 3

1.7 0.7 0.7 1.3 1 1

y = 9.0019x - 79.417

0.9958

9.6

7

1

Acetonitrile

Y= 4.0375x – 21.055

0.9991

Pear Apple Grape Cowpea Tomato Watermelon Watermelon (peel) Watermelon Watermelon (peel)

Y= 7.127x + 247.44 Y= 5.0076x - 60.314 y = 7.1293x + 224.38 y = 3.7848x - 80.967 y = 4.0133x - 80.618 y = 6.0188x - 202.23

0.9964 0.9976 0.9772 0.9954 0.9936 0.9837

76.5 23.8 76.6 -6.2 -0.6 49.1

2 3 2 6 6 3

0.7 1 0.7 2 2 1

y = 6.9538x - 59.468

0.999

72.2

2

0.7

y = 34.1x - 547.43

0.9938

58.4

0.8

0.3

y = 33.17x - 162.1

0.998

54.1

1

1

3.5: Dissipation of fluopyram in tomato The data in table 4 showed the dissipation of fluopyram in tomatoes under field condition and half-lives. The decrease of fluopyram was gradual and continuous after application. The average residue of fluopyram in Henan and Beijing tomatoes samples at 1.5 times were 0.125 mg/kg and 0.160 mg/kg, with the half-lives of 2.8 d and 12.0 d in Henan and Beijing, respectively. And the

initial concentrations in tomatoes samples were 0.117 mg/kg and 0.136 mg/kg after 2 day with dissipation rate 19.4% and 14.9% for Henan and Beijing respectively, the concentration were reduced dissipation rate 86.1 % and 47.5 % by the 10 days after application and treatment for Henan and Beijing respectively.

Table 4: Data for Regression equation, Correlation Coefficient (r) and Half-life (days)under field condition Matrix Tomato

Field location Henan Beijing

Regression equation y=0.1182 e-0.2496x y=0.1463 e-0.0578x

3.6 Terminal residue of fluopyram in tomato The terminal residue of fluopyram in tomato from treated plots are summarized in table 5. In Henan and Beijing test, samples after the treatment of 3 daysat two times and three times at levels of 250 g a.i/ha were lower than 0.399 mg/kg and 0.197 mg/kg, respectively. The final residue of fluopyram

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Correlation Coefficient (r)

Half-life (day)

0.833 0.939

2.8 12.0

in the tomato samples after the treatment of 3 daysat two times and three times at levels of 375 g a.i/ha were lower than 0.294 mg/kg and 0.500 mg/kg, respectively. The terminal residue results indicated that the residue of fluopyram in tomato in Henan and Beijing was lower than the MRLs set by EU (0.9 mg/kg) on 3 days after last application at

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the tested dosages. Hence, a safe pre-harvest interval (PHI) of 3 days is suggested before Table 5: Terminal residue of fluopyram in tomatoes Dosage Spray Interval Mean ± SD (mg/kg) (g a.i./ha) times (days) 2012 Henan 2 7 0.01 5 < 0.01 3 < 0.01 1 < 0.01 250 3 7 < 0.01 5 0.021±0.0025 3 0.019±0.00125 1 < 0.01 2 7 < 0.01 5 0.011 3 < 0.01 1 0.019±0.0040 375 3 7 < 0.01 5 0.011 3 0.035±0.0052 1 0.017±0.0040

Conclusion In this paper, a simple UHPLC-MS/MS method was developed and validated for the simultaneous determination of residues of three compounds (fluopyram, trifloxystrobin and its metabolite) in agricultural products (pear, apple, grape, tomato, cowpea and watermelon). Extracts containing the target compounds were analyzed and validated by UHPLC–MS/MS. This method allowed separation of the three target pesticides in less than 3.0 min with good specificity. The recovery results were from 71.7% to 116.4%with RSD in the range of1.4% to 20% and the correlation coefficient (r2) was higher than 0.966 for all analytes in six matrices. In addition, the specificity, calibration curves, precision, and reproducibility were tested successfully. The proposed method is recommended and easy and useful in analysis all these three pesticides to ensure food safety. The trial results showed that the half-lives in tomato were2.8 d in Henan and 12.0 d in and Beijing respectively. The terminal residue results would be helpful for the Chinese government to establish the MRL of fluopyram in tomato and to provide guidance on the proper and safe use in China. Acknowledgments This work was supported by National Natural Science Foundation of China (31272070 and 31371970).

harvesting of tomato.

Beijing 0.061±0.0169 0.179±0.0270 0.278± 0.1049 0.223±0.0048 0.059±0.0175 0.074±0.0121 0.032± 0.0111 0.313±0.2730 0.087± 0.198 0.175± 0.0048 0.099± 0.0300 0.228± 0.1517 0.073± 0.02358 0.145±0.0728 0.365± 0.2255 0.242± 0.0706 solid-phase microextraction and gas chromatography coupled with electron-capture and mass spectrometric detection. Journal of Chromatography A, 893, 143–156. Hervé F.A., Themis J.M.,2010. Progress in understanding molecular mechanisms and evolution of resistance to succinate dehydrogenase inhibiting (SDHI) fungicides in phytopathogenic fungi. Crop Protection, 29, 643-651 Xingang Liu, Xu Wang, Jun Xu, Fengshou Dong, Wencheng Song and YongquanZhenga, 2011. Determination of tebuconazole, trifloxystrobin and its metabolite in fruit and vegetables by a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method using gas chromatography with a nitrogen–phosphorus detector and ion trap mass spectrometry, Biomedical Chromatography, 25, 1081–1090 Abreu S.D, Caboni P., Cabras P., Garau V.L.,Alves A. 2006. Validation and global uncertainty of a liquid chromatographic with diode array detection method for the screening of azoxystrobin, kresoxim-methyl, trifloxystrobin, famoxadone, pyraclostrobin and fenamidone in grapes and wine.AnalyticaChimicaActa 573:291–297 Likas D.T., Tsiropoulos N.G.,Miliadis G.E., 2007. Rapid gas chromatographic method for the determination of famoxadone, trifloxystrobin and fenhexamid residues in tomato, grapeand wine samples. Journal of Chromatography A, 1150, 208–214.

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Grape Leaves, Grape Berries and Soil. Bulletin of Environmental Contamination and Toxicology 84, 305– 310. Lilia Araujo, Carlos Rojas, Dalia Cubillan, Noreiva Villa, Jair Mercado, AvismelsiPrieto, 2010. Determination of Trifloxystrobin, Tebufenozide, and Halofenozide in Foods by MicellarElectrokinetic Capillary Chromatography. Journal Analytical Letters, 43, 2340-2348 SoudaminiMohapatra,RekhaAjithakumar, 2014. Persistence of trifloxystrobin and tebuconazole in banana tissues and soil under the semi-arid climatic conditions of Karnataka, India International Journal of Environmental Analytical Chemistry, 94, 506–518. JiayingXue, Huichen Li, Fengmaoliu, Wenqing Jiang, Xiaochu Chen, 2014. Determination of strobilurin fungicides in cotton seed by combination of acetonitrile extraction and dispersive liquid−liquid microextraction coupled with gas chromatography, Journal of Separation Science. Journal of Separation Science, 37 .845-852. S.K. Patyal, I.D Sharma, R.S. Chandel, K.J. Dubey, 2013. Dissipation kinetics of trifloxystrobin and tebuconazole on apple (Malusdomestica) and soil–A multi location study from north western Himalayan region.Chemosphere, 92.949 -954. StanisławSadło , Magdalena Duda , BartoszPiechowicz&AgnieszkaJaźwa, 2013. Comparative study on disappearance trends of captan and trifloxystrobin residues on fruit and apple tree leaves using internal normalisation method. Food Additives &ContaminantsA.30, 826-832. SarabjitKaur, ReenuTakkar, UravashiBhardwaj, Rajinder Kumar, R.S. Battu&Balwinder Singh, 2012. Dissipation Kinetics of Trifloxystrobin and Tebuconazole on Wheat Leaves and Their Harvest Time Residues in Wheat Grains and Soil. Bulletin of Environmental Contamination and Toxicology, 89, 606-610. ArmindoMelo, Sara C. Cunha, CatarinaMansilha, Ana Aguiar, OlíviaPinho, Isabel M.P.L.V.O. Ferreira, 2012. Monitoring pesticide residues in greenhouse tomato by combining acetonitrile-based extraction with dispersive liquid–liquid microextraction followed by gaschromatography–mass spectrometry. Food Chemistry, 135, 1071- 1077. Dong Bizhang, Jiye Hu, 2014. Dissipation and residue determination of fluopyram and tebuconazole residues and watermelonand soil by GC-MS. International Journal of Environmental Analytical Chemistry, 94, 493-505.

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