Determination of Carbendazim and Chlorpyrifos in Selected Fruits and Vegetables Samples Using QuEChERS- HPLC-FD

A new method was developed to determine pesticide residues in fruit and vegetable extracts with short time analysis. An optimum results were achieved using binary mobile phase consisting of (methanol: water; 95:5, v/v) and fluorescence detection (λEx and λEm set at 280 and 340 nm, respectively) was used. The dynamic range was between 0.100 to 10 mg L with relative standard deviation less than 0.45%, (n=4). Limits of detection and recoveries for carbendazim and chlorpyrifos were 0.073 mg L (84.2-106.5%) and 0.062 mg L (85.7-90.3%), respectively. The results revealed that the concentrations of carbendazim and chlorpyrifos residue in all collected samples were below than the EU legal limit.


INTRODUCTION
Pesticides include herbicides, insecticides, fungicides, and nematicides which are described as chemicals that kill or slow down the growth of undesirable organisms [1].Pesticides constitute a very important group of chemical compounds that have to be controlled for their high toxicity and their widespread use in agricultural practice in field and post-harvest protection [2,[3][4][5].Although the use of pesticides provides unquestionable benefits in providing a plentiful, low cost supply of high quality fruits and vegetables, their incorrect application may leave harmful residues, which involve possible health risk [3,6].The toxic chemicals taken up by plants during cultivation or contaminated during preservation are passed on the food chain causing serious health effect in human beings [3,7].In recent years, issues of pesticide residue in the environment, vegetables and fruits are arousing more and more public attention [2,8,9].Most of recently studies find that the pesticide has a direct related with carcinogenicity and neurotoxicity on human health [9,10,11].The riskiness of pesticide is ability to migrate from the intended application area is influenced by the particular chemicals properties, its propensity for binding to soil, its vapor pressure, its water solubility, and its resistance to being broken down over time [3].A maximum acceptable daily intake of pesticide is 0.5 µg kg −1 body weight [12].Carbendazim and chlorpyrifos have found a wide applications in Turkey on citrus fruits, cotton, corn, almond and apple trees (see Figure 1) [4,13,14].
Chlorpyrifos, has been widely employed in agriculture to kill pests which has a wide environmental endocrine disruptor and its residual mainly exists in crops, livestock, poultry products and also led to their migration into aquifers which is potentially hazardous to health [1,2,5,8,15].Unfortunately, Chlorpyrifos with a thiophosphoryl (P=S) functional group is important causes of morbidity and mortality following intentional self-poisoning or in cases of occupational or environmental exposure [1,5,16,17].The American Department of Pesticide Regulation (ADPR) gave chlorpyrifos a high priority for risk assessment for its acute toxicity [18].Recently studies have found that carbendazim cause infertility and destroy the testicles of human males [19,20], and Chlorpyrifos cause lung cancer [15,21,22] and acts by interfering with cholinesterase, an enzyme that is essential for the proper working of the nervous system of both humans and insects [1,5,15,17,18,23].Effects of carbendazim and chlorpyrifos were seen in liver, thyroid, thymus and blood in the combination groups [23]. 2 mg kg -1 of chlorpyrifos as a dose it showed significant inhibition of DNA synthesis in all brain regions of old rats within 4 hours of treatment [15].The risk assessment of pesticides mixtures (carbendazim and chlorpyrifos) is may be more dangerous than approach [12].Thus the analytical methodologies employed must be capable of residue measurement at very low levels and must also provide clear-cut evidence to confirm both identity and quantity of any residues detected [7,22,24].
Fluorescence detection in HPLC pesticide analysis is one of the selective and sensitive detection systems but it has been classically limited by the fact that a few pesticides are fluorescent such as carbendazim (benzimidazole) set at (λex: 280nm and λem: 300nm) and triazophos (organophosphorus) set at (λex: 250nm and λem: 305nm) [24].The fluorescence spectra revealed that chlorpyrifos causes the quenching of the fluorescence emission of serum albumin which determined at (λex: 275nm and λem: 350nm) as reported by Han et al. [17].A numerous publications and research papers focus on separation methods to detect carbendazim and chlorpyrifos for simultaneous determination using RP-HPLC-UV/Vis [23,25] and LC-MS-MS [7].The aim of this study was to develop a simple, a reliable, a sensitive, a selective and an affordable technique to test the presence of carbendazim and chlorpyrifos in selected fresh samples of (appel, grapefruit, tangerine, spinach and parsley) which grown in Turkey by QuEChERS-HPLC-FD analysis without any derivatization.Most of recently studies for simultaneous determination of carbendazim and chlorpyrifos has been reported by HPLC using UV/Vis or PDA (photo diode array) or MS detectors.As far as we know from recently studies, this is the first time that carbendazim and chlorpyrifos were simultaneously analyzed by QuEChERS-HPLC-FD.

Reagent and Materials
Standard samples of carbendazim (2000 mg L -1 ) and chlorpyrifos (1000 mg L -1 ) were HPLC grad and obtained from (Agilent, USA) which keep it in freezer away from light (stock solutions).Standard working solutions was prepared by diluting appropriate amounts of the standard samples (stock solutions) in acetonitrile.All chemical reagents used for analysis carbendazim and chlorpyrifos by QuEChERS-HPLC-FD were analytical Grade (99.99%) of BDH Prolabo (Australia).The reagents include acetonitrile and methanol.While acetic acid obtained from Merck (Darmstadt, Germany).Anhydrous magnesium sulfate and anhydrous sodium acetate were analytical grad of Carlo Erba (Milano, Italy).Bulk sorbents (50 µm particle size) for dispersive-SPE including primary secondary amine (PSA) and graphitized carbon black (GCB) were obtained from Sigma-Aldrich (USA).Reverse-osmosis ultrapure type quality water used was obtained from Millipore water purification system (DIRECT-Q 8UV, USA).

Collection of Fruit and Vegetable Samples
In the month of December 2015, seven different types of fruit and vegetable were obtained from different agricultural area in Yozgat and Kayseri include apples (3 different agricultural area), grapefruit, tangerine, spinach and parsley.Approximately, 1 kg of each type of fruit or vegetable were obtained and stored in plastic bags at 4°C in a refrigerator prior to analysis.

QuEChERS Extraction Method
A quick, easy, cheap, effective, rugged, and safe (QuEChERS).One of three official QuEChERS extraction procedures is AOAC which was adopted for this study [9,26].All the samples were cut and ground to fine pieces.A 10 g of freezing sample is weighed into a 50 mL centrifuge tube (freezing the sample is recommended to minimize pesticide degradation).Ten millilitres of acidic acetonitrile (1% acetic acid) are added and the tube is shaken for one minute.Next, 5 g of anhydrous (MgSO4:NaOAc, 4:1, w/w) are added and the tube is shaken for an additional minute followed by centrifugation for 10 minutes at 4000 rpm.After centrifugation, three layers are observed.The top layer is the acetonitrile extract that will be transferred to the clean up step.The middle layer is the solid plant material and the bottom layer is the aqueous magnesium solution.
The dispersive solid phase extraction (d-SPE) clean up centrifuge tubes (15 mL size) contain anhydrous MgSO4 (1250 mg), PSA primary secondary amine sorbent (PSA, 210 mg) and graphitized carbon black (GCB, 40 mg).After the acetonitrile extract layer is transferred to the 15 mL d-SPE tube, it is manually shaken for 30 seconds and then centrifuged for 10 minutes at 4000 rpm.The final extract is typically analysed by HPLC-FD after filtered through a 0.45 μm syringe filter.

Chromatographic analysis
Reversed phase HPLC with fluorescence detector (RP-HPLC-FD) has an important analytical technique for analysis a traces of carbendazim and chlorpyrifos because a fluorescence detector is probably sensitive among the existing modern of HPLC detectors.The quantitative and qualitative analysis of carbendazim and chlorpyrifos were carried out with a Shimadzu HPLC autosampler system model LC-20AT (Kyoto, Japan) consisting of degasser, tertiary pump, auto sampler, column oven and Shimadzu RF-20A fluorescence detector.A 20 μL sample was injected and the chromatographic separation was performed on a RP-C18 Inertsil ODS-3 (5μm) column, 4.6 mm×250 mm (Japan).The HPLC optimum analytical conditions was based on recent study reported by Saad et al. [22] with slight modification using 280 nm as excitation wave length (λEx) and 340 nm as emission wave length (λEm).The fruit and vegetable extracts were analyzed isocratically using (methanol: water; 95: 5, v/v) as mobile phase at 40 °C of column temperature with flow rate at 0.8 mL min -1 to achieve the optimum resolution of carbendazim and chlorpyri.

Optimization of chromatographic conditions
The effects of different chromatographic conditions on the instrumental responses create a situation where one has to compromise between different experimental variables in order to achieve the best chromatographic separation.In order to achieve the optimum separation for simultaneous determination of carbendazim and chlorpyrifos, following conditions were studied: (I) six different combinations of the most common binary mixture of methanol: water (65:35, 70:30, 75:25, 90:10, 95:5 and 100:0, v/v),with excitation and emission wavelengths, column temperature and flow rate kept constant at (λEx-λEm, 280-340) nm, 40°C and 0.8 mL min -1 , respectively.(II) the excitation and emission wavelengths (λEx-λEm) were tested at (250-305, 275-350, 280-300, 280-315 and 280-340) nm with binary mixture of (methanol:water, 95:5, v/v) as mobile phase with flow rate and column temperature maintained at 0.8 mL min -1 and 40°C, respectively.(III) Flow rate was varied from 0.2 to 1.2 mL min -1 with binary mixture of (methanol:water, 95:5, v/v) as mobile phase, excitation and emission wavelengths and column temperature maintained at (λEx-λEm, 280-340) nm, 40°C.Moreover, the effects of different factors such as resolution factor (Rs), theoretical plates (N), and asymmetry factor (As) were systematically addressed on system suitability parameters.

Validation of the developed method
After the chromatographic method had been developed and optimized, it must be validated.The validation of an analytical method verifies that the characteristics of the method satisfy the requirements of the application domain.The proposed method was validated in the light of (International Conference on Harmonization) ICH Guidelines [27,28] for linearity, accuracy, sensitivity, specificity and robustness.Consequently, the following were performed.

Linearity
Linearity of an analytical method was established by automatic injections of the standard mixture solutions in the investigated ranges from low to high concentrations, each concentration was repeated four times.Five different concentrations of carbendazim and chlorpyrifos (0.1, 1, 2.5, 5 and 10) mg L -1 were constructed in the specified concentration range.The calibration plot (peak area ratio of carbendazim and chlorpyrifos versus its concentration) was generated by replicate analysis (n = 4) at all concentration levels and the linear relationship was evaluated using the least square method within Microsoft Excel program.

Sensitivity
The instrumental response sensitivity is the slope of the calibration line because a method with a large slope is better able to discriminate between small differences in analyte content.Limit of detection (LOD) and limit of quantitation (LOQ) were determined according to following equation [27]: where k is a constant (3 for LOD and 10 for LOQ), B is the standard deviation of the analytical signal, and S is the slope.

Accuracy
The accuracy of the method (recovery) was assessed by adding two know amount of carbendazim and chlorpyrifos at two different fortification levels (0.100 and 1.00 mg L -1 ) was evaluated in order to assess the extraction efficiency of the proposed two methods.For this, 25 g of blank sample (apples grown without application of any pesticide) were spiked with 0.10 mg kg -1 and 1.00 mg kg -1 of carbendazim and chlorpyrifos.Resulting samples were mixed and allowed to stand for 15 minutes before extractions.Six replicates at each fortification level were prepared.

Specificity
Specificity of the proposed method was evaluated by peak purity curves through resolution factors (  ), peak asymmetry factor (As) and number of theoretical plates (N).The resolution factor   was calculated based on equation 2 [28]: Where  1 and  2 are the retention times of the two components,  1 and  2 are the corresponding widths at the bases of the peaks obtained by extrapolating the relatively straight sides of the peaks to the baseline.The asymmetry factor is a measure of peak tailing and was calculated based on equation 3 [29]: Where   is peak asymmetry factor, b is the distance from the point at peak midpoint to the trailing edge and a is the distance from the leading edge of peak to the midpoint (a and b were measured at 10% of peak height).The number of theoretical plates (N) were calculated using equation 4 [28]: Where, N is the number of theoretical plates,   is retention time and  1 is width at the bases of the peak.

Effect of mobile phase composition
In this study, the optimum mobile phase combinations was achieved by testing the following binary mixtures of (methanol:water).
Table 1 summarizes the effect of mobile phase compositions on retention time (tR), an asymmetry factor (As) and a number of theoretical plates of carbendazim and chlorpyrifos.Minimum retention times of carbendazim (3.99 min) and chlorpyrifos (5.56 min) were obtained at (methanol:water, 95:5, v/v) level as mobile phase, which makes the method rapid, a one of the most desirable criteria.Though retention time was shorter, satisfactory resolution (Rs>2.0)and asymmetry values were achieved (As ≤ 1.15).An adequate theoretical plates (∼ 12800) is indicative of a good column performance.On the other hand, carbendazim was separated using (methanol:water, 75:25, v/v) as mobile phase, while chlorpyrifos were not separated by other binary mixtures of methanol:water (65:35, 70:30 and 100:0).

Effect of excitation and emission wavelengths
For the fluorescence detection, a spectrum of carbendazim and chlorpyrifos standard solutions was tested to obtain the best fluorescence signals.Various (emission/excitation) wavelengths were applied to obtain the best values in order to enhance the detection for carbendazim and chlorpyrifos, as shown in Table 2.

Effect of mobile phase flow rate
Mobile phase flow rate was studied at varied values (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2) and was enumerated in Table 3.
From Table 3, it can be observed that theoretical plates were highest at flow rate of 0.8 mL min -1 with asymmetry factors less than 1.20.The change in flow rate had slight significant effect on resolution factor while retention time decreased as the flow rate increased.
In the present study, we found that use of (methanol: water; 95: 5, v/v) as mobile phase with the detector set at (λex: 280 nm and λem: 340 nm) and flow rate is 0.8mL min -1 , yielded the most satisfactory separation of carbendazim and chlorpyrifos can be achieved within 7 minutes.

Linearity
The linearity of analytical procedure of carbendazim and chlorpyrifos was constructed by spiking six different concentrations (0.1, 1.0, 2.5, 5.0, and 10.0 mg L -1 ) with four replicates and evaluated by plotting detector response (peak area) versus carbendazim and chlorpyrifos concentration (mg L -1 ) to obtain the calibration curve and correlation coefficient (R 2 ).The chromatographic responses were found to be linear over an analytical range of 0.10-10.00mg L -1 and found to be quite satisfactory and reproducible with time.The linear regression equation was calculated by the least squares method using Microsoft Excel program and summarized in Table 4.The calibration curve of carbendazim and chlorpyrifos showed a good regressions (correlation coefficients, R 2 ≥0.9999) with R.S.D ≤ 0.45 and Rs > 2.0, indicating a strong linear relationship between the variables and suggesting that the developed HPLC-FD method had an excellent linearity.

Sensitivity
The sensitivity was estimated by the limit of detection (LOD) and limits of quantification (LOQ).The LODs values obtained (see Table 4) are 0.07 and 0.06 mg kg -1 for carbendazim and chlorpyrifos respectively, while the LOQs for carbendazim and chlorpyrifos are 0.22 and 0.19 mg kg -1 , respectively.The detection limits of presently developed method is lower than that reported earlier by Bedendo et al. [3] for carbendazim (0.35 mg L -1 ) and chlorpyrifos (0.075 mg kg -1 ) in (regular carton orange juice, light carton orange juice, fresh orange juice) using LC-MS/MS and by Martindale, R.W. [32] for carbendazim (0.100 mg kg -1 using HPLC-FD) in potatoes.The new method offers a good reproducibility and acceptable accuracyy.

Accuracy
The accuracy was tested by the determination of the average recoveries of carbendazim and chlorpyrifos by spiking two know amount of carbendazim and chlorpyrifos at two different fortification levels (0.10 and 1.0 mg L -1 ) was evaluated in order to assess the extraction efficiency of the proposed method (see Fig. 2).The spiked samples were extracted according to the procedures described above.The results are summarized in Table 5.The average of carbendazim and chlorpyrifos recoveries at 0.1 mg kg -1 exceeded 84.1% while at 1.0 mg kg -1 , exceeded 85.6%.Good repeatability at all spiked levels for all samples was achieved (n=6) since all the RSD values were below 10% (see Table 5).

Sample analysis
The developed QuEChERS-HPLC-FD method was investigated to determine of carbendazim and chlorpyrifos residues in selected fresh fruits & vegetables samples.Seven fresh samples were extracted by QuEChERS, then the developed method using HPLC-FD was applied for the determination of carbendazim and chlorpyrifos residues (Tables 6 & 7 and Figure 3).
The present results are anticipated since QuEChERS is selective towards carbendazim and chlorpyrifos [33,34,35], and showed that the level of carbendazim and chlorpyrifos residues in all selected samples are below than the EU legal limit.There was a permissible occurrence of carbendazim and chlorpyrifos residues in parsley which grown without application of any pesticide.It confirms that the pesticide residues (carbendazim and chlorpyrifos) were transfered by a surface water to soil agricultural of parsley [8,14,36].Crum et al. [37] had found in his study that the highest sorption of nine common pesticides (include chlopyrifos and carbendazim) to aquatic macrophytes (its structure is very similar to parsley) was chlorpyrifos.El-Shahawi, [38] reported that increased of total elements in parsley after

CONCLUSION
The developed method using QuEChERS-HPLC-FD is quick, accurate, sensitive, good recoveries, convenient and effective for monitoring of carbendazim and chlorpyrifos residues in fresh fruits & vegetables samples which were collected from a different agricultural area (Yozgat and Kayseri, Turkey).It combines the advantage of fluorescence detection and allowed discrimination between two target fluorescent pesticides that were marginally separated by liquid chromatography.All the selected samples contained residue of carbendazim chlorpyrifos lower than the maximum residue limits (MRLs) of the European Union (EU).

Figure 1 .
Figure 1.Structures of carbendazim and chlorpyrifos

Table 1 .
Effect of mobile phase composition on retention time (tR), number of theoretical plates (N) and asymmetry factor (As) of carbendazim and chlorpyrifos

Table 2 .
Effect of emission (  ) wavelength and excitation (  ) wavelength on retention time (  ), number of theoretical plates (N) and asymmetry factor (  ) of carbendazim and chlorpyrifos

Table 3 .
Effect of mobile phase flow rate on retention time (  ), number of theoretical plates (N) and asymmetry (  ) of carbendazim and chlorpyrifos

Table 4 .
Results of the validation study using HPLC-FD (n=4)

Table 6 .
Maximum residue limits of the European Union (EU) for carbendazim and chlorpyrifos (mg kg −1 ) in apple, citrus, parsley and spinach

Table 7 .
Concentration of carbendazim and chlorpyrifos residues in fresh fruits & vegetables samples (µg kg −1 ) collected from different agricultural area, Yozgat and Kayseri, Turkey treatment with chlorpyrifos which means increase a contamination level of chlorpyrifos in parsley.