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Eurasian Journal of Analytical Chemistry ISSN: 1306-3057 2017 12(2):17-30

DOI 10.12973/ejac.2017.00151a

© Authors. Terms and conditions of Creative Commons Attribution 4.0 International (CC BY 4.0) apply.

Correspondence: Mohammed Akkbik, Bozok University, Science and Technology Application and Research Center,

TR-66100, Yozgat, Turkey.

mohammed.akkbik@bozok.edu.tr

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

HPLC-FD

Orhan Hazer Bozok University, TURKEY

Mohammed Akkbik Bozok University, TURKEY

Dilara Demir Bozok University, TURKEY

Yasemin Turhan

Bozok University, TURKEY

Received 11 April 2016 ▪ Revised 9 September 2016 ▪ Accepted 12 September 2016

ABSTRACT

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-1 with relative standard deviation less than 0.45%, (n=4). Limits of detection and

recoveries for carbendazim and chlorpyrifos were 0.073 mg L-1 (84.2-106.5%) and 0.062 mg

L-1 (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.

Keywords: carbendazim, chlorpyrifos, fruit & vegetable extracts, QuEChERS -HPLC-FD

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, 3-5]. 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,

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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

Figure 1. Structures of carbendazim and chlorpyrifos

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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.

MATERIALS AND METHODS

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

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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

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(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]:

𝐿𝑂𝐷 𝑜𝑟 𝐿𝑂𝑄 = 𝑘(𝐵/𝑆) (1)

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.

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Specificity

Specificity of the proposed method was evaluated by peak purity curves through

resolution factors (𝑅𝑠), peak asymmetry factor (As) and number of theoretical plates (N). The

resolution factor 𝑅𝑠 was calculated based on equation 2 [28]:

𝑅𝑠 = (𝑡2 − 𝑡1) + (𝑊2/2 + 𝑊1/2) (2)

Where 𝑡1 and 𝑡2 are the retention times of the two components, 𝑊1 and 𝑊2 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]:

𝐴𝑠 = 𝑏/𝑎 (3)

Where 𝐴𝑠 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]:

𝑁 = 16(𝑡𝑅/𝑊1)2 (4)

Where, N is the number of theoretical plates, 𝑡𝑅 is retention time and 𝑊1is width at the

bases of the peak.

RESULTS AND DISCUSSION

Optimization of analytical conditions

Effect of mobile phase composition

In this study, the optimum mobile phase combinations was achieved by testing the

following binary mixtures of (methanol:water).

1. (75:25 v/v) adopted by Parveen et al. [30] for analysis chlorpyrifos in apple and

citrus fruits using HPLC-DAD.

2. (70:30 v/v) adopted by Saad et al. [22] for analysis fungicides in oranges using

HPL-FD.

3. (90:10 v/v) adopted by Venkateswarlu et al. [7] for analysis carbendazim and

chlorpyrifos in Indian grapes using LC-ETMS.

4. (65:35 v/v) adopted by Liu et al. [31] for analysis carbendazim in apple juice

using HPLC-FD.

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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.

When the excitation wavelength was set at 280 nm, strong fluorescence signals were obtained for carbendazim and chlorpyrifos. While, at 250 and 275 nm were obtaining the lowest fluorescence signals for carbendazim and chlorpyrifos. On other hand, setting the emission wavelength at different values (300, 305, 315, 340 and 350) nm produced different fluorescence signal strengths for carbendazim and chlorpyrifos. Therefore, the optimum combination of excitation wavelength (λEx: 280 nm) and emission wavelength (λEm: 340 nm)

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

asymmetry factor (As) of carbendazim and chlorpyrifos

Carbendazim Chlorpyrifos

(Methanol:Water) tR N As tR N As

(65:35) 0 0 0 0 0 0

(70:30) 0 0 0 0 0 0

(75:25) 8.41 1342.5 1.45 0 0 0

(90:10) 5.50 1958.6 1.20 7.62 5827.8 1.60

(95:5) 3.99 2148.7 1.10 5.56 12855.0 1.15

(100:0) 0 0 0 0 0 0

Table 2. Effect of emission (𝜆𝐸𝑥) wavelength and excitation (𝜆𝐸𝑚) wavelength on retention time (𝑡𝑅),

number of theoretical plates (N) and asymmetry factor (𝐴𝑠) of carbendazim and chlorpyrifos

Carbendazim Chlorpyrifos

References (λEx- λEm)nm tR N As tR N As

Han et al. [17] (275-350) 0 0 0 0 0 0

Asensio-Ramos et al. [24] (250-305) 0 0 0 0 0 0

Asensio-Ramos et al. [24] (280-300) 4.82 580.8 1.50 0 0 0

Liu et al [31] (280-315) 4.17 1373.9 1.25 0 0 0

This study (280-340) 3.98 2068.9 1.10 5.57 12803.3 1.15

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was selected as the most suitable for carbendazim and chlorpyrifos, and which gave reasonable fluorescence signals (Rs>2.0, As ≤ 1.15 and N∼ 12800).

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.

Method Validation 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 (R2). The

chromatographic responses were found to be linear over an analytical range of 0.10–10.00 mg

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.

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

asymmetry (𝐴𝑠) of carbendazim and chlorpyrifos

Carbendazim Chlorpyrifos

mL min-1 tR N As tR N As

0.2 18.77 2505.3 1.70 28.06 3888.2 1.85

0.4 9.59 4088.3 1.50 14.65 4239.4 1.65

0.6 6.49 4213.3 1.25 10.08 8028.2 1.40

0.8 3.99 6432.0 1.10 5.55 12814.2 1.15

1.0 3.65 5340.7 1.15 4.93 9721.9 1.20

1.2 3.34 4483.6 1.15 4.53 8208.4 1.20

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

Analyte Rt

(minutes) Calibration equation R2

RSD

(%)

LOD

(mg kg-1)

LOQ

(mg kg-1)

Carbendazim 3.98 ± 0.01 y = 1051.4x+330025 0.9999 0.45 0.07 0.22

Chlorpyrifos 5.56 ± 0.01 y = 887.86x+288484 0.9999 0.43 0.06 0.19

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The calibration curve of carbendazim and chlorpyrifos showed a good regressions

(correlation coefficients, R2≥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.

Figure 2. Typical chromatogram of carbendazim (1 mg L-1) and chlorpyrifos (1 mg L-1), B. Chromatogram

of spiked spinach with carbendazim and chlorpyrifos at 0.1 mg L-1, C. Chromatogram of spiked parsley

with carbendazim and chlorpyrifos at 0.1 mg L-1 using (Methanol: water; 95: 5, v/v) as mobile phase with

flow rate 0.8 mL min-1 at (𝜆𝐸𝑥 : 280 nm and 𝜆𝐸𝑚: 340 nm) by HPLC-FD after QuEChERS extraction

procedure

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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

Table 5. Recoveries of carbendazim and chlorpyrifos fortified to apples (mg kg-1, n=6)

Spiked

level

Recovery of carbendazim (%) Recovery of chlorpyrifos (%)

R1 RSD1 R2 RSD2 Average R1 RSD1 R2 RSD2 Average

0.100 83.9 8.14 84. 6.82 84.2 92.5 10.61 88.1 9.96 90.3

1.000 104.6 4.39 108.4 3.97 106.5 84.8 7.14 86.6 5.93 85.7

R1,2: Recoveries 1 and 2

Table 6. Maximum residue limits of the European Union (EU) for carbendazim and chlorpyrifos (mg

kg−1) in apple, citrus, parsley and spinach

Name Apple Citrus Parsley Spinach

Carbendazim 2.00 5.00 0.10 0.10

Chlorpyrifos 0.50 0.50 0.05 0.05

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

Analyte

(µg kg-1)

Apple1

(Kayserig)

Apple2

(Yozgatm)

Apple3

(Yozgatm)

Grapefruit

(Yozgatm)

Tangerine

(Yozgatm)

Spinach

((Yozgatm)

Parsley

(Yozgatm)

Carbendazim 423±7.7 n.d n.d n.d 964 ± 14 n.d 71 ± 9

Chlorpyrifos 301±11 203 ± 9 306 ± 10 188 ± 6 159 ± 5 38 ± 3 44 ± 4

d: not detected. g: Garden, m: Market

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treatment with chlorpyrifos which means increase a contamination level of chlorpyrifos in

parsley.

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).

ACKNOWLEDGEMENT

The authors would like to acknowledge Bozok University, science and technology application

and research center for providing HPLC-FD instrument for this study.

Figure 3. A. Typical chromatogram of carbendazim (1 mg L-1) and chlorpyrifos (1 mg L-1), B.

Chromatogram of Apple1 using (Methanol: water; 95: 5, v/v) as mobile phase with flow rate 0.8 mL min-

1 at (𝜆𝐸𝑥 : 280 nm and 𝜆𝐸𝑚: 340 nm) by HPLC-FD after QuEChERS extraction procedure

O. Hazer et al.

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