Analysis of Multi-Residue Pesticides in Vegetable Samples Using Solid-Phase Micro-Extraction (SPME) Coupled to Gas Chromatography-Electron-Capture Detector (GC-ECD)

Vegetables are important for the maintenance of health. They provide vitamins, antioxidants, enzymes, minerals and fiber. Daily consumption of at least 200-300 grams of fruits and 250 grams of vegetables is recommended for all age groups. India is the second largest user of pesticides in Asia. However, pesticide consumption in India is relatively low i.e. 0.33 kg/ hectare. When compared to the pesticide consumption of 3.07 kg /hectare in France, 4.17 kg /hectare in Italy and 13.1 kg/ hectare in Japan and more than 15 kg/ hectare in United Kingdom, Canada and United States (Associated Chambers of Commerce and Industry of India (ASSOCHAM). According to Indian Pesticide Report it is estimated that India loses approximately 18% of its crop yield anually worth Rs.900 billion due to pest attack. As a result, farmers began to rely on using more and more pesticides to protect crops from pests, weeds, rats, mice, flies and other insects from consuming and contaminating crops in agricultural field as well as for protecting fruits, vegetables, legumes and cereals etc. when they are being stored. Many researchers have reported the residues of OCs, OPs and pyrethroids, along with fungicide and herbicides


Introduction
Vegetables are important for the maintenance of health. They provide vitamins, antioxidants, enzymes, minerals and fiber. Daily consumption of at least 200-300 grams of fruits and 250 grams of vegetables is recommended for all age groups. India is the second largest user of pesticides in Asia. However, pesticide consumption in India is relatively low i.e. 0.33 kg/ hectare. When compared to the pesticide consumption of 3.07 kg /hectare in France, 4.17 kg /hectare in Italy and 13.1 kg/ hectare in Japan and more than 15 kg/ hectare in United Kingdom, Canada and United States (Associated Chambers of Commerce and Industry of India (ASSOCHAM). According to Indian Pesticide Report it is estimated that India loses approximately 18% of its crop yield anually worth Rs.900 billion due to pest attack. As a result, farmers began to rely on using more and more pesticides to protect crops from pests, weeds, rats, mice, flies and other insects from consuming and contaminating crops in agricultural field as well as for protecting fruits, vegetables, legumes and cereals etc. when they are being stored. Many researchers have reported the residues of OCs, OPs and pyrethroids, along with fungicide and herbicides in fruit and vegetables of indian origin [1][2][3][4][5][6][7][8][9][10][11][12][13] .
Several methodologies have been developed for sample preparation prior to the analysis of pesticides from biological, food or environmental samples [14,15] . SPME was first developed in 1989 by Pawliszyn and coworkers and has been marketed by Supelco since 1993. Subsequently, the technique has grown enormously [16][17][18] . This technique integrates sampling, extraction, pre concentration and sample introduction in a simple single step procedure. Although SPME was initially introduced for the extraction of organic compounds from water samples [19] , but, soon after, SPME have been successfully applied for the determination of pesticides in various complex sample matrices [20][21][22][23][24][25][26][27][28] . Application of SPME was described by many researchers for the determination of pesticide residues determination in fruits or for the determination of volatile organic compounds in food commodities. SPME is the advanced micro extraction technique that is being increasingly used in the isolation and extraction of organic contaminants from environmental/food/biological samples. In this study, an easy and solventless SPME method has been developed and validated followed by GC-ECD analysis for the simultaneous determination of multi-residue pesticides (24 Nos.) from raw vegetable samples.

Instrumentation
Perkin Elmer Clarus 500 Gas chromatograph equipped with Ni 63 electron capture detector (GC-ECD) was used with a split less injector (0.75 mm I.D. glass liner, split less time) for thermal desorption. All the separations were carried out on Elite -5 (30 m X 0.25 mm, 0.25 µm) capillary column (Perkin Elmer). The oven temperature was started at 200°C with 2min hold and then increased to 280°C at a rate of 2°C /min. The fiber depth in the injector was set to 2.5 cm and the thermal desorption time in the split less injector was 10 min at 280°C. The temperatures of injector port and detector were set at 280°C and 350°C, respectively. Ultrapure Nitrogen (99.999%) was used as carrier gas with a flow rate of 0.79 ml /min.

Sample collection site
Total 110 (10 samples of each variety) vegetable samples of Cabbage, Tomato, Cauliflower, Chilli, Okra, Brinjal, Bottle gourd, Cucumber, Beetroot, Spinach and Radish were collected from different market locations within Lucknow city, India (26° 55' N, 80° 59' E) in September-October 2011. Samples were procured and kept in refrigerator and analyzed next day of the collection. SPME procedure 10 gm of vegetable sample was homogenized with 10 ml of Milli-Q (Millipore, Milford, MA, USA) water using a high speed blender. Further 2.0 ml of homogenate, 0.6 g of NaCl and magnetic flea were added in 4.0 ml amber glass vial capped with PT-FE-faced silicon septa. PDMS (100 µm) fiber was then exposed in glass vial in headspace for 20 min at 80°C. At stirring speed 400 rpm. After adsorption the syringe was removed from the vial and thermal desorption of the analytes was carried out by inserting the fiber into the GC injection port (at 280°C) for 10 minutes. Magnetic fleas were cleaned with detergent and water and finally rinsed with acetone for next set of SPME extraction.
The values of linearity range, coefficients of determination (r 2 ) limit of detection (LOD), and limit of quantification (LOQ) corresponding to individual pesticide have been depicted in Table 1.
Recovery study was conducted by performing HS-SPME on the homogenate vegetable sample being spiked with mix pesticide standard solution at five-level concentrations as depicted in Table 1. Three replicate samples were used at each concentration. Control samples were processed along with spiked ones. GC-ECD chromatogram of targeted analytes recovered using HS-SPME from homogenate vegetable sample being spiked with mix pesticides standard solution at 0.02 mg/ kg as shown in Fig.1. Recovery percentages corresponding to each targeted analytes were found in the range of 86.1 -96.4 and linearity curve with R 2 ranging between 0.9847 and 0.9986. were also depicted in Table 1.

Optimization of extraction conditions
Different parameters influencing HS-SPME were optimized such as extraction temperature, extraction time, sodium chloride amounts, desorption temperature, desorption time of the fiber in the GC injector port. All the conditions of SPME coupled to GC-ECD were systematically optimized using such an approach that the finally optimized conditions could support reasonable extraction of all the analytes of dissimilar volatilities.
In order to study the temperature effect on extraction of targeted analytes from spiked vegetable sample, HS-SPME was performed at 60, 70, 80 and 90°C and the peak areas of target analytes were observed. Maximum area of analytes was achieved at 80 o C because when the sample temperature is increased, the vapor pressure of the analyte and consequently the concentration of analyte in the headspace were increased. From a kinetic view, higher temperatures increase the transfer rate of the analytes from the matrix (e.g. water) towards the headspace, while thermodynamically higher temperatures decrease the analytes partition coefficients between the headspace and SPME fiber. Peak areas were decreased when temperature exceeded 80°C because adsorption is an exothermic process and disfavored at high temperature therefore 80°C was selected as working temperature for the analysis. (Figure 2)  Spiked vegetable sample were treated with NaCl (w/v) 0, 10, 20, 30, 40%. The 30% (w/v) NaCl concentration was selected for extraction because peak areas of pesticides were declined when more than 30% NaCl (w/v) was added. Addition of sodium chloride reduced the solubility of target compounds in water by increasing distribution of analytes in the headspace and onto the SPME fiber. The presence of higher concentrations of sodium chloride within the matrix may reduce the molecular movement of targeted analytes, resulting in decrease in extraction efficiency because excess concentration of NaCl deposited on SPME fiber. (Figure 4)

Recovery of Proposed Method
To evaluate extraction recoveries, extractions were performed in triplicates on spiked vegetable sample at finally optimized extraction conditions Recoveries were found in the range of 86.1-96.4% as shown in Table 1.

Result and Discussion
Residues of HCH, DDT, methylparathion, malathion, chlorpyrifos, monocrotophos, endosulfan, cypermethrin, fenvalerate pesticides were detected in vegetable samples ( Table 2)   -HCH was detected in 28 out of the 110 samples. The average lower concentration was 0.11 mg/kg in Brinjal samples while higher mean concentration was 0.37 mg/kg found in Tomato samples. The residues of -HCH were above MRL in 09 samples as per PFA Act.
∑DDT was detected in 21 out of the 110 samples. The average lower concentration was 0.35 mg/kg in Okra samples while average higher concentration was 0.51 mg/kg found in Tomato samples. The residues of -HCH were above MRL in 07 samples as per PFA Act.
Methyparathion was detected in 38 out of the 110 samples. The average lower concentration was 0.21 mg/kg in Okra samples while higher concentration was 0.85 mg/kg found in Cauliflower samples. The residues of methyparathion were above MRL in 12 samples.
Malathion residues were found in 47 samples out of the 110. Minimum average concentration was found 0.41 in case of Brinjal samples and maximum concentration 0.62 mg/kg was found in cabbage samples. The residues of Malathion were below maximum residue level.
Chlorpyrifos residues were detected in 49 out of the 110 samples. The average lower concentration was 0.01 mg/kg in cabbage samples and high 0.153 mg/kg was found in okra samples. The residues of chlorpyrifos were above MRL in 08 samples.
Total Endosulfan (α-Endosulfan, β-Endosulfan and Endosulfan sulfate) was found in 64 vegetable samples. Lowest average concentration was 0.29 mg/kg in Okra samples while highest concentration was 0.57 mg/kg in Beetroot samples. The residues of Endosulfan were above MRL in 17 samples.
Cypermethrin was detected in 48 out of the 110 samples. The average lower concentration was 0.08 mg/kg in Okra samples while higher concentration was 0.28 mg/kg found in