Trace level determination of insecticide using gas chromatography, and the application for residual monitoring in local Syrian vegetables

2.1 Apparatus

A Shimadzu GC version 2010 gas chromatography with an electron capture detector (ECD) was used. A trb-1, 30 m × 0.32 mm i.d. capillary column with a 0.25 μm film thickness was used. The temperature program: initial temperature of 120 °C held for 1 min, 8 °C/min ramp to the final temperature at 250 °C, held for 2.5 min. The injector temperature was at 250 °C and the detector temperature was maintained at 300 °C. Nitrogen gas (99.999%) was used as the carrier gas with a flow rate of 6 ml/min at a pressure of 105 kPa., the injection volume of 1 μl was used in a splitless mode.

2.2 Reagents and chemicals

Reagent-grade chemicals were of the highest purity available from their sources. Methanol, acetone, ethyl acetate, acetonitrile, hexane, dichloromethane and diethyl ether were purchased from the Merck Company. Anhydrous sodium sulfate and sodium chloride were purchased from J. T. Baker. The pesticide standards of Dimethoate (Di), Chlorpyrifos-ethyl (CPE), Cypermethrin (Cyp), and Deltamethrin (Del), were of 90.0–99.5% purity and purchased from Accu Standard Inc. New Haven, CT, USA.

A stock solution pesticide of 1000 μg mL⁻¹ was prepared in acetone. Working solutions of each pesticide were prepared daily by diluting the stock solution of corresponding pesticide to give a concentration within the range of 0.01−0.5 μg mL⁻¹. An internal standard of 1-Chloro-4-fluorobenzene (1 mg/kg) was used.

2.3 Pesticide extraction from vegetable sample

A preliminary study was carried out to optimize the extraction procedures, by trying three different solvents: Acetone, acetonitrile, and ethyl acetate (Seddik et al., 2010, 2012).

Vegetables of cucumber, tomato, squash, and eggplant were collected from Maraet Alnouman areas (Edlab, Syria), that are known to be contaminated with the studied pesticides. The same vegetables were provided from local market for analytical comparison. Vegetable sample of 1 kg weight was cut into pieces and mixed properly for solvent extraction. The extraction process was made on 10 g of sample with 100 mL extracting solvent. A proper quantity of standard solutions of indicated pesticide was spiked to give 0.0 (blank), 0.05, 0.10 and 0.50 mg/kg for each compound, the mixture was then stirred to homogenize for 5 min. The resulting mixture was then filtrated using 5A (541) filter paper. The extraction and filtration processes were repeated on the residue with 50 mL of extraction solvent. The resulting filtrates were then mixed and evaporated using rotary evaporator with a temperature less than 40 °C to 50 mL volume. The resulting mixture was then mixed with 100 mL NaCl 10% (w/v) and subjected to purification extraction process with 20 mL × 5 batches (100 mL the final volume) of various Ethylacetate:Hexane ratio. The organic extracted phase was then filtered on 5A (541) filter paper after moisture absorption using 10 g of anhydrous sodium sulfate. The filtrate was then evaporated to dryness and the residue dissolved again with acetone using Pasteur pipe and the volume was made to 10 mL using the same solvent to give the Pesticide Extract (PE).

2.4 Solid-phase extraction (SPE) and cleanup

Solid-phase extraction (SPE) was performed using an LC-Silica Gel (100–200 mesh), or LC-Florisil (magnesium silicate, 100–200 mesh) provided from Supelco. The extraction columns were washed with 6 ml of ethyl acetate and conditioned by passing 2 ml of methanol, followed by 2 ml of deionised water. The cartage sorbent was never allowed to dry during the conditioning and sample loading steps. Sample loading was 5 mL of the PE volume, and performed under a vacuum in a flow rate of 5 ml/min. The pesticides were eluted from cartage with three × 2 ml portions of the eluting solvent. The eluates were collected in a 15 ml tube under gravity flow. The eluate evaporated to dryness, the residue dissolved with acetone, spiked with 100 μL internal standard, the final volume was made to 5 mL, and 1 μL of this solution was injected into the GC–ECD for analysis.

2.5 GC calibration curve

Proper volumes of the target pesticides’ working solution were transferred to the volumetric flask to cover a concentration range of 0.01–0.50 mg L⁻¹. Three standard extractions were made for each concentration levels of the mixture solution. The calibration graph was plotted by the ratio (S/Sa) of the peak area of the analyte (S) to the peak area of the internal standard (Sa); versus the concentration of the analyte. Calibration lines were used for quantification in subsequent experiments.

3.1 The choice of extraction solvent

Solvents such as Acetone, Acetonitrile, and Ethylacetate were used to prepare pesticide extract (PE) directly from vegetables. Tables 2–4 summarize the average recovery of four pesticides studied (Di, CPE, Cyp, and Del) from Acetone, Acetonitrile, and Ethylacetate, respectively. The results indicate that the average recovery using acetonitrile was not exceeding 72 ± 3 with Cyp from tomato. Better recovery was shown with Ethylacetate with ratio not more than 85 ± 6 with Cyp from Cucumber, but some recovery rate was still less than 80%. Whereas, using acetone the recovery was within the range of 80–97%, that indicating the best solvent used in this work for PE process was Acetone.

3.2 Purification extraction process

It was found, Fig. 1, that using hexane alone as a solvent for the extraction process resulted in low recovery with less than 80% for Di, Cyp, and Del. On the other side, using Ethylacetate alone shows increases with recovery to values exceeding 80% for Di, CPE, and Cyp. Varying the mixing ratio of Ethylacetate:Hexane, indicates that the ratio of 95:5 shows recovery exceeding 80% for the four pesticides from tomato.

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Figure 1

Effect of the mixture ratio (Ethylacetate:Hexane) used at the LLE process at the recovery ratio of the pesticides (Di, CPE, Cyp, Del) in Tomato samples extraction by acetone.

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The solvent mixture Ethylacetate:Hexane ratio of 95:5 (v:v) was used as the extraction solvent for further finding in this work.

3.3 Solid-phase extraction (SPE) and cleanup

The cleanup process has been done by using the solid phase by cartridges filled with either Florisil or silicagel, and determining the recovery average of each pesticide (Di, CPE, Del, Cyp) from Tomato samples. Table 5 shows that the cleanup process may result in a decrease of the recovery average for all of the studied pesticides. Despite that, the cleanup procedure was employed using, preferably, the Florisil cartage, only if required to inhibit the destruction of GC column.

Varying the eluting solvent mixture using Hexane:Acetone, Fig. 2, resulted in higher recovery values at 95:5 (v:v). As a try, replacing the acetone with more polar solvents, using Hexane:Methanol mixture resulted in high GC peaks interference (chromatographic resolution between peaks less than 1.2). Whereas, decreasing the polarity using Hexane:Ether, resulted in a less recovery ratio, that was not exceeding 80% with the four pesticides.

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Figure 2

Effect of acetone ratio in hexane as an eluting solvent in SPE process for the recovery average of the pesticides (Di, CPE, Cyp, Del) from Tomato samples.

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3.4 Method validation

The method shows linearity for the four pesticides in the same mixture, with no interference. Linear equations are presented in Fig. 3, showing high correlation coefficient with values more than 0.998 (n = 3). The limit of detection for Di, CPE, Cyp, and Del, were 1, 0.96, 1, 3, and 1.9 ng mL⁻¹, respectively. Calculating the quantification limit for the pesticides, that was calculated as three times the standard deviation of the blank divided by the slope of the respective calibration graph (Miller and Miller, 1993), indicating values not exceeding 3 ng mL⁻¹.

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Figure 3

The constructed calibration curves for (a) Dimethoate, (b) Chlorpyrifos-ethyl, (c) Deltamethrin, and (d) Cypermethrin in tomato using 1-Chloro-4-fluorobenzene as an internal standard.

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The precision was assessed according to the IUPAC recommendations by analyzing 0.01, 0.025, 0.05, 0.1, 0.25, and 0.5 μg mL⁻¹, the recovery of the pesticides from vegetables was within the range (80–112%) with RSD% ⩽ 5% (n = 3). The Student’s t-test values (the tabulated t-value for the 95% confidence level and n = 3 is 4.303 (Miller and Miller, 1993)). The t-test could not detect any systematic error and proved accuracy of the proposed method.

The chromatogram on the recovery of tomato spiked at 0.05 mg/kg is indicated in Fig. 4.

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Figure 4

Chromatogram on the recovery of tomato spiked at 0.05 mg/kg of (1) internal standard, (2) Di, (3) CPE, (4) Cyp, and (5) Del.

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4.1 The study of residual effect to the pesticides

The analytical results for the determination of the trace level of Di, CPE, Del, and Cyp pesticide residuals in 110 various vegetable samples that were obtained from bazaars, or known farms of the Maraet Al noman area (Idlb, Syria) are summarized in Table 6. The indicated results show that high percentage (29%) of total vegetable samples was polluted with one or two of the studied pesticides. From these polluted samples there were thirty samples contaminated above the maximum regulated level (MRL), of which three samples contain CPE, four samples contain Di, two samples contain Cyp, and four samples contain Del. The contaminated samples indicate high levels of various pesticides as pollutants, which demand more attention in the region.