Gas Chromatography-Mass Spectrometer Electron Ionization (GC-MS-EI) method for the Analysis of MalathionResidue in Tomato

In this a study tomato sampleswere collected from Khartoum, extracted with acetone, dichloromethane and petroleum ether (1:1:1) and cleaned up by florisil column. Malathion quantitative determination is carried out by gas chromatographmass spectrometerusing the optimum ionization mode electron ionization (EI). The detection ofmalathionis confirmed by retention time and comparison of primary and secondary ions.Recovery studies were performed at two spikes (0.5,0.25mg kg -1 ) fortification levels of malathion and the recovery obtained ranged from 81% to 97%. The method showed good linearity(R2> 0.995) over the range assayed (from 0.05 to 7.0mg L -1 ) and the calculated limits of detection (LOD) and quantification(LOQ) were0.03 mg kg -1 and 0.11 mg kg -1 , respectively. These limits werelower than the maximum residue levels(MRL) established by European legislations (0.5mg kg -1 ).


INTRODUCTION
During the last two decades there have been growing social concerns over issues related to public health, environmental quality, and food safety. One of the major controversies inciting these concerns involves the production and consumption of fresh fruit and vegetables. Research has shown that diets with greater proportions of fruit and vegetables can prevent or delay a number of life threatening diseases. At the same time, public acceptance and adoption of these findings is being discouraged by ongoing re-evaluations of the possible health risks associated with minute amounts of pesticide residues sometimes found in or on these foods.The application of pesticides is essential in modern agricultural practices to control pest and diseases that damage fruit and vegetables. However, it has the drawback of pesticide residues which remain on fruit and vegetables, constituting a possible risk to consumers [1] Therefore, governments and international organizations FAO, WHO) have established maximum residue levels (MRLs), limiting the amount of pesticides in foods. Currently organophosphates, carbamates and pyrethroids are mostly used while someorganochlorine insecticides have been banned because of their toxicity, persistent and bioaccumulation in the environment [2].
The most frequently used technique for analysis of pesticide residues in fruit and vegetables is gas chromatography with different selective detectors. Such as flame photometric (FPD) [8], nitrogen-phosphorus (NPD) [9], and electron-capture detectors(ECD) [10,11]. Numerous methods use gas chromatography coupled with mass spectrometry (GC-MS) due to the possibility of confirming pesticide identity in these matrices [12,13].Liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) [14,15]has lately become a powerful analytical technique for the identification and quantification of residues in fruit and vegetable. A critical review of literature showed that different solvents such as nhexane, petroleum ether,methylene chloride and acetone or ethyl acetate have been used for extraction of pesticide residue from fruit and vegetables [16]. As more polar pesticides, such as organophosphate and phenoxyaceticacid, polar solvents such as chloroform, acetone, acetonitrile and methanol were found to be good [17].Ethyl acetate isfound to be a good solvent as compared to other solvents forthe extraction of residues of several pesticides from fruit and vegetables because its polarity is high and it is a less volatile and thermally labile compound [18].
Malathionis a broad spectrum, non-systemic Organophosphorous insecticide that is used on a wide variety of crop sites and on various non-crop sites, including greenhouses, nurseries, home and garden, and public health. The chemicalstructure ofmalathion is shown in Figure 1. It is very highly toxic to fish and aquatic invertebrates but does not appear to be toxic to plants. Some residential and agricultural uses can have rather high application rates and resulting exposure.

Fig. 1.Malathion or 2-(dimethoxyphosphinothioylthio) butanedioic acid diethyl ester
The present work is designed to study the residues of malathion pesticides in tomato, a sample is extracted with simple and effective procedure using low volume of organic solvent , cleanup is carried by florisil columns and residue levels were determined by gas chromatography (GC) with mass spectrometer detector (GC-MSD.

Reagents and Chemicals
Acetone, dichloromethane, petroleum ether and n-hexane, of special gradingfor the pesticide residue analysis, were obtained fromScharlau Company

Chromatographic conditions
Heliumwas used as carrier gas at a constant flow-rate of 0.9 mL min -1 . The column temperature was programmed as follows: 90 °C for.6 min, 20°C /min to 200°C (6min.) and 20°C /min to 260°C (5min) .The solvent delay was 2.5 min. The total analysis time was 2 min. The injection port was maintained at 200 °C and 1 µL, sample volumes were injected in splitless mode. The data were acquired and processed using Shimadzu GC Solution software. The eluent from the GC column was transferred via a transfer line heated at 280 °C and fed into a 70 eV electron impact ionization source, also maintained at 280 °C. Table 1lists the pesticides along with their retention times, molecularmass, the target and qualifier ions, and their qualifierto target abundance ratios. The target abundances were determined by injection of pesticide standards under the same chromatographic conditions using full scan mode with the mass/charge ratio ranging of the m/z 10 to 400. In these evaluations, the characteristic ions were chosen, and the MS system was then programmed in selective ion monitoring (SIM) mode for quantification of pesticide. The choice of the ions for SIM acquisition was based on the best S/N ratioswasm/z100, 125 and 127.Values of m/z in bold type correspond to the quantification ion for analyte.

Sample preparation
Two different weights (49.70 g, 52.69 g) of sample was sprayed by formulation (10 mg L -1 ) by different volumes (1.25mL,2.5mL ), respectively, then was left until they were dry , extracted , cleaned up and determined .Real sample was carried out by taking sample without spraying and then was extracted, cleaned up and determined.

Extraction
Each sprayed sample was cut and putin blender and homogenized for (30 sec) with 30 mL of acetone 60 mL of dichloromethane and petroleumether (1:1) were added and the mixture was homogenized for (1min) then centrifuged at 4000 rpm for (5 min), the volume of extract was concentrated in rotary evaporator with water bath at 35˚C and then was cleaned up by florisil column and determined [ 19] .

Cleaned up
All samples were cleaned up by florisil column before analysis by GC-MS. Florisil (20g) in hexane was allowed to settle in a chromatographic column (45cm×20mm) by tapping the column. To the tap of florisil, a layer of 1 to 2cm deep anhydrous sodiumsulphate was added. Then the column was eluted with 200 mL of hexane and the liquid was discarded. Concentrated sample of tomato extract (1 mL) in hexane was transferred to the column,and then the column was eluted with 200 mLof 15% diethyl ether in hexane followed by 200 mL of 50% diethyl ether in hexane. The solution was evaporated to 5 mLand injected in GC-MS.

Gas chromatographic determination
Pesticides residue levels were determined by GC-MS. Representative mass spectrum andchromatograms of a standard pesticideare shown in (Figure 2 (a), (b), and that for a tomato sample spiked with the formulation of the malathionsolution and real sample are shownin ( Figure 3 (a), (b), respectively.

Method validation
The MS response for pesticides was linear in the concentration assayed (0.05-7.0mg L -1 ) with determination coefficients >0.995for pesticides. The results are shown inTable 2.  Limit of Detection and Quantitation The limit ofdetection (LOD) of the method was determined at a signal-to-signal ratio of 3 for the pesticides in tomato by GC-MS, whereas the limit of quantification was obtained at a signal-to-signal ratio of 10.The LOD is 0.03 mg kg -1 and the LOQ is 0.11 mg kg -1 .

Recovery
A study of recoveries for pesticide at two different fortification levels was carried out in order to assess the extraction efficiency of the method. For that, two tomato samples were spikedwith (0.5,0.25 mg kg -1 )of pesticide and processedas described. A recovery data obtained are shown in Table 3. Analysis of real samples Tomato samples were analyzed following the extraction methods described above. Pesticide concentration levels in the real samplewere found to be 0.3mg kg -1 . Analysis of samples showed the validity of method used, which allowed the determination and identificationof pesticides present in the samples.

CONCLUSION
The results of this study show that the method to determine residues of pesticides in tomatois rapid, simple, sensitive and uses small volumesof solvents for sample extraction , reducing the risk for human health andthe environment.Good recovery and low detection throughmethod were obtained for the pesticides studied,including new generations of pesticides, since their decompositions quicker and has a less damaging effect on theenvironment. The method shows advantages comparedwith other conventional methods in that, the use of a lowvolume of organic solvent in the sample extraction,it avoids the use of a chlorinated hydrocarbon, and the time of extraction is short. J u l y 1 5 , 2 0 1 3 .