Determination of Carbendazim and Chlorpyrifos in … Selected Fruits and Vegetables Samples ... Pesticides include herbicides, insecticides, ... a selective and an affordable technique
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Eurasian Journal of Analytical Chemistry ISSN: 1306-3057 2017 12(2):17-30
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.
O. Hazer et al.
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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
This study (280-340) 3.98 2068.9 1.10 5.57 12803.3 1.15
O. Hazer et al.
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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)
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.
28
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