Optimization and Efficiency Comparison of Dispersive and Cartridge Solid Phase Extraction Cleanup Techniques in the Analysis of Pesticide Residues in Some Vegetables Using Gas Chromatography-Mass SpectrometrySpectrometry

Author's: Anass A. Alnedhary1, Mahfoudh M. AL-Hammadi2, Abdualqawi A. Numan3, Fatima A. Murshed2*
Authors' Affiliations
1Department of Chemistry, Faculty of Education, Khawlan Branch, 2Department of Chemistry, Faculty of Science, 3Science Curricula Department, Faculty of Education, Sana’a University, Yemen.*Corresponding Author: Fatima A. Murshed Email:fatima.murshed2018@gmail.com
Article Type: Research Articles     Published: Jan. 31, 2020 Pages: 40-54
DOI:        Views 894       Downloads 0


This work was conducted to demonstrate the optimization procedures and results for a sample of preparation method combining Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction with cartridge solid phase extraction (c-SPE) cleanup utilized for the analysis of pesticides residues in some vegetables using gas chromatography-mass spectrometry (GC-MS). The method applied for the analysis of four pesticides of different classes; dimethoate (Organophosphorus), fenvalerate (Pyrethroid), difenoconazole (Triazole) and deltamethrin (Pyrethroid) on four types of vegetables (i.e. tomato, potato, cucumber, and carrot). The procedures simply involve the use of acetonitrile containing 1% acetic acid for the extraction, and for cleanup; a manually prepared solid-phase extraction cartridge containing primary secondary amine (PSA) and normal charcoal were used. The validated GC-MS analysis method for the pesticide residues in the selected vegetables has high linearity with R2 ranged from 0.9965 to 0.9999. The precision of the method estimated as relative standard deviation (%RSD) was ≤ 9.4% for all target pesticides which were indicative of the high repeatability of the optimized method. The accuracy calculated as average recoveries (%R) was between 80.52% and 99.63%. LODs for target pesticides in spiked cucumber, tomato, carrot, and potato samples ranged between 0.0950 and 0.5590 ng/g. The combined sample preparation method is cost-effective and has shown good simplification, recovery and cleanup capacity and proved to be efficient and suitable for the proposed application.

Keywords: QuEChERS, d-SPE, c-SPE, Cleanup, Pesticides, GC-MS.

Citation: Alnedhary, A.A., AL-Hammadi, M.M., Numan, A.A., Murshed, F.A., 2020. Optimization and Efficiency Comparison of Dispersive and Cartridge Solid Phase Extraction Cleanup Techniques in the Analysis of Pesticide Residues in Some Vegetables Using Gas Chromatography-Mass Spectrometry. PSM Biol. Res., 5(1): 40-54.

Copyright: ©2020 PSM. This work is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial 4.0 International License.


Pesticides are artificially synthesized compounds produced to fight pests and diseases of plants to increase and improve agricultural products. Although their use has tremendously increased agricultural production in many parts of the world (Galani et al., 2018; Osadebe et al., 2018), their uses have been of concern due to their toxicity and adverse effects on human health. Thus, efforts have to be made to ensure that pesticide contaminations were kept at levels below the maximum residue levels (MRLs). Pesticides are classes of chemical substances either naturally or synthetically made to fight diseases affecting crops (Cabras, 2003; Mahmood et al., 2016; Akhtar et al., 2018). Pesticides are classified into different categories including target organisms, chemical structures, mode of action, and their environmental persistence and pathway of movement into the target organisms. WHO classified them into four classes: extremely dangerous, highly dangerous, moderately dangerous and slightly dangerous (Rajveer et al., 2019). The detection and quantitation of the presence of different classes of pesticides particularly in trace levels in complex matrixes such as vegetables presenting a challenge for scientists (Kataoka et al., 2000; Lambropoulou and Albanis, 2007).

The first step in pesticide analysis of vegetables is the preparation of the sample for analysis, which involves cutting, grinding and blending to form a homogeneous sample structure. Subsampling is then taken for further treatment. This step is important because the success of subsequent steps depends on obtaining a homogeneous sample. Extraction and cleanup are the two steps used to extract the pesticide residue of interest from the matrix and to remove interferences that could compromise pesticide detection and quantitation. As the matrix gets complicated, the cleanup procedure gets more involved to ensure that the instrument performance is not compromised (Huertas-Pérez et at., 2019; Vaclavik et al., 2018). Among sample preparation techniques that have been used in the pesticide cleanup step, some stand out, which include solid-phase extraction (SPE), matrix solid-phase dispersion (MSPD) and solid-phase micro extraction (SPME) which were developed with the aim of simplifying steps. Furthermore, stir bar sorptive extraction (SBSE) has been found to provide low detection limits, especially for hydrophobic analytes. Supercritical fluid extraction (SFE), accelerated solvent extraction (ASE) and microwave-assisted extraction (MAE) (Wilkowska and Biziuk, 2011; Lambropoulou and Albanis, 2007) are also of great value as tools for pesticides sample preparation. The ability of the QuEChERS method to extract various compounds of different chemical classes is a major advantage over traditional methods that are typically capable to extract only one analyte or multiple analytes of the same chemical class (Wilkowska and Biziuk, 2011). Furthermore, proficiency testing employing the QuEChERS method demonstrates that the method is highly robust, and successfully transferred between the participating laboratories (Kaczyński and Łozowicka 2017; Lee et al., 2016). The first and the most significant modifications were developed to expand the method applicability to some pesticides that are ionized and/or degraded during the extraction, depending on the pH of the matrix (Gonzalez-Curbelo et al., 2015). Thus, the first modification proposed for the QuEChERS method was the addition of a buffering step, where the buffering effect (pH 4.8) promoted the addition of sodium acetate and 1% acetic acid in acetonitrile MeCN. This method was adopted in 2007 by the Association of Official Analytical Chemists (AOAC) as an official method for the determination of pesticide residues (Gonzalez-Curbelo et al., 2015; Lehotay et al., 2005; Schenck and Hobbs, 2004; Wilkowska and Biziuk, 2011; Lehotay et al., 2007; Lehotay et al., 2010). To remove matrix components in the clean-up step, modifications of the original d-SPE step by used graphitized carbon black (GCB) and C18 sorbent. QuEChERS offers several advantages over most conventional techniques because it does not require glassware or auxiliary equipment (e.g. vacuum manifolds), uses low volumes of solvent, generates little solvent waste and provides high recovery of analytes (Seccia et al., 2011; Acebal et al., 2016; Ma et al., 2016).

The main disadvantage of QuEChERS (d-SPE cleanup) is that for 1g sample per milliliter of final extract, the obtained concentration of the extract is usually lower than the concentration that could be obtained by the use of most traditional procedures such as (LLE). Thus, the final extract must be concentrated to a greater extent to furnish the necessary sensitivity and to achieve the desired limits of quantification (LOQ). Despite this drawback, the quantitative results obtained from a large number of pesticides indicate that combination of QuEChERS as a (d-SPE) with hyphenated methods of detection (GC-MS, GC-MS/MS, LC-MS) provides scientists with the capability to achieve efficient and effective monitoring of pesticide residues in food (Lambropoulou and Albanis, 2007).

SPE could be used for different purposes including a sample cleaning and enrichment where the sample passes through adsorbent loaded in a cartridge. The analyte of interest could be adsorbed on the surface of the adsorbent (and is eluted later on) and interferences pass through or vise visa. Various adsorbing materials are commonly available including C18, normal-phase aminopropyl (-NH2) and primary secondary amine (PSA), anion-exchanger three-methyl ammonium (SAX) and adsorbents such as graphitized carbon black (GCB). The efficiency and selectivity of these adsorbents vary depending on their nature and thus, the physicochemical properties of the analyte under investigation will give guidance to select the appropriate adsorbent (Lambropoulou and Albanis, 2007). In some cases, a user prefers using her/his own adsorbent to suit the intended application. SPE has some attractive features such as the cost-effectiveness in which only a small amount of solvent is needed, and it is easy to use and to authorize. Numerous methods have been published on the analysis of several hundreds of pesticide residues of different types of food and environmental samples using various analytical systems. Each of the published methods has some advantages and limitations which make it successful with few types of commodities and fail with others. This reason drives the continuous development of the sample preparation procedures as the core and the most important step in any analysis method. The aim of this work was to optimize, validate the sample preparation and apply the gas chromatographic method for the detection and quantitation of pesticide residues in some vegetables.


All pesticide standard: Dimethoate, 99.6%, Fenvalerate, 98.3%, Difenoconazole, 99.3%, Deltamethrin, 98%) were from Sigma-Aldrich and Fluka/ (Zwijndrecht, The Netherlands). Individual pesticides standard solutions (1000 µg/mL) for all target pesticides were prepared in hexane-acetone (9:1) and kept at (-4 °C) until use (Bozena et al., 2015; Bozena et al., 2016)

All used solvents were HPLC grade. Primary Secondary Amin (PSA 40 mm particle size, Agilent, USA), activated charcoal 15-30 mesh size (Merck, Germany) and C18 (Supelco, USA) were also used as adsorbents.

Gas Chromatography-Mass Spectrometry (GC-MS): GCMS-QP2010 (Shimadzu, Kyoto, Japan) was used in electron ionization (EI) mode. Analytes were separated in a fused silica capillary column DB 5MS (5% phenyl polysiloxane as polar stationary phase), (0.25 mm x 30 m, 0.25 µm film thickness, supplied by Agilent, Palo Alto, CA, USA). GC–MS was equipped with a split/splitless injector and the splitless mode at 250 °C was used. The oven temperature was set initially at 85 °C (2 minutes), and raised to 280 °C at 15 °C min−1 and hold at 280 °C for 10 minutes. The total run time was 25 minutes. The temperatures of the mass detector interface and ion source were set at 280 °C and 200 °C, respectively. Helium gas (99.999%) was used as the carrier gas with a flow rate of 1.29 mL. min-1. The solvent cut of time was set at 4 minutes. Selected ion monitoring (SIM) mode was used in the quantitation step. The optimization of the retention times and chromatographic resolution were done in the scan mode from m/z 50 to 550 at 0.5 sec. per scan.

Blank and Spiked Samples:

Blank vegetable samples of cucumber, tomato, carrot, and potato were collected from organic cultivation sources and used for method development, calibration, and recovery studies. They were first analyzed to ensure the absence of the target pesticide residues. Vegetable samples were chopped into small pieces before mixer blending then homogenized and spiked with suitable amounts of pesticide mixture to levels of 0.01, 0.05, 0.1, 0.5, 1.5 and 2 µg/g and used for calibration and validations study. The spiked samples were properly homogenized and kept overnight before the extraction and cleanup procedures.

Preparation of Solid Phase Extraction Cartridges (c-SPE):

10 mL medical syringes were packed with suitable weights of PSA, activated charcoal and 1 g of anhydrous sodium sulfate was added to the top of each cartridge. Thin discs made up of pre-cleaned medical cotton were inserted at the bottom, top, and between the sorbents layers.

QuEChERS Extraction:

(AOAC Official Method 2007.01) The QuEChERS method was used for sample extraction (AOAC, 2011). 5 g of grounded vegetable and 5mL of H2O for carrot and potato, and for cucumber and tomato, 10 g of a grounded sample was taken to 50 mL extraction tube. 10 mL of acetonitrile containing 1% acetic acid was added to each wet sample. After a one-minute shake, buffering extraction salts 4 g anhydrous magnesium sulfate, 1 g anhydrous sodium acetate was added. Following another two-minute shake, the sample was centrifuged for 5 minutes at 5000 rpm. Finally, the acetonitrile layer was separated and used for the cleanup procedures.

Cleanup of d-SPE and c-SPE

Dispersive Solid Phase Extraction Cleanup (d-SPE)

8 ml of the supernatant (acetonitrile layer) was transferred to a 15 mL PTFE tube, 500 mg anhydrous magnesium sulfate, suitable amounts of PSA and activated charcoal was added. The extract was shaken for 2 minutes and then centrifuged at 5000 rpm again for 5 minutes. The supernatant layer was filtered through a 0.45 µm syringe filter before analysis.

Cartridge Solid Phase Extraction Cleanup (c-SPE):  8.0 mL of the acetonitrile layer was transferred into an SPE cartridge packed with PSA in the bottom, activated charcoal as a middle layer and anhydrous sodium sulfate on the top, which was formerly conditioned with 5 mL of acetonitrile: toluene (3:1), the conditioned solvent mixture was discarded. After elution with 20 mL of acetonitrile: toluene (3:1), the collected eluents were then evaporated using a rotary evaporator near to dryness before reconstituted to 2 mL using acetone: hexane (1:9).

The resulting final extracts for all matrixes with cleanup by either a d-SPE or c-SPE procedures were analyzed by GC-MS.

Optimization and Efficiency Comparison of the Cleanup Methods

Optimization of Dispersive Solid Phase Cleanup (d-SPE) and (c-SPE) Procedures

For (d-SPE) and (c-SPE) cleanup optimization, the amount of each sorbent was studied to find the optimum amounts of PSA, charcoal, and C18 for the cleanup of vegetable extracts.

Comparison of Sample Preparation Efficiency Using d-SPE and c-SPE in Vegetable Samples After the optimization of the d-SPE and c-SPE cleanup procedures for selected vegetables, the method of efficiency for the analysis of pesticides residues in the selected types of vegetables using d-SPE and c-SPE was comprised (Maciej, 2019; Michelle et al., 2013; Tomás et al., 2018).



Pesticides were identified according to the retention times, the quantification and three confirmation ions with the assistance of the National Institute for Standards and Testing (NIST,s) and Wily,s libraries (El Shoubaky and Salem, 2014; Lincy et al., 2015). The quantitation was based on the Total Ion Chromatogram (TIC) of peak areas of pesticides. Table 1 summarized the selected pesticides with their quantification and confirmation ions used in SIM mode to analyze dimethoate, fenvalerate, difenoconazole and deltamethrin in cucumber, tomato, carrot, and potato.