Pesticide Residues Recovery studies by QuEChERS Sample Technique Using Gas Chromatography

The quantification of pesticides from oil matrices is a challenging analytical task because of the high triglyceride content of the samples (100%) and the possible presence of lipophilic analytes. The most often used techniques for extraction and clean-up are liquid–liquid partitioning with acetonitrile or an acetonitrile-hexane mixture, followed by gel-permeation chromatography or solid-phase extraction (SPE) (Li et al., 2009; Muccio et al., 2006). However, in recent times, pesticide analysis has seen an increasing need for multi-residue methods, leading to the development of versatile multi-analyte sample preparation methodologies.

Several multi-residue methods have been reported for the measurement of pesticides, particularly in fruits and vegetables (Soler et al., 2005; Kmell et al., 2008; Zhao et al., 2004). The QuEChERS method, which stands for Quick, Easy, Cheap, Effective, Rugged, and Safe, was introduced in 2003 by Anastassiades et al. (2003) for the determination of pesticide residues in various food products. The method was mainly based on a salt-out acetonitrile extraction followed by a dispersive solid-phase extraction (d-SPE) clean-up procedure using a primary-secondary amine (PSA) sorbent and anhydrous MgSO₄.

The European Union Standards (EU), Food and Agriculture Organization of the United Nations (FAO), and the World Health Organization (WHO) have defined Maximum Residue Limits (MRL) in edible oils for various pesticide residues in the range of 0.002–2.0 mg/kg. However, MRL values for pesticides in edible oils have not yet been formulated in several countries, including India (Pragney et al., 2014).

According to the International Union of Pure and Applied Chemistry (IUPAC, 2009), recovery is defined as the proportion of the amount of analyte present in or added to the analytic portion of the test material that is extracted and presented for measurement. In pesticide analysis, recovery values are considered acceptable between 70-120% for routine analysis, as recommended by the Codex Alimentarius guidelines (Khay et al., 2009) and EU Commission guidelines. Poor recovery and high recovery values have been reported in some studies due to the interference of the food matrix and the high-fat content of samples (Argauer et al., 1997; Przybylski and Segard, 2009).

Some authors (Doong and Lee, 1999; Serrano R et al., 2003) have reported that matrix interference can be reduced through clean-up procedures, leading to good recoveries even from high-fat foods. Subsequently, the QuEChERS method was modified for the analysis of high oil-content matrices, particularly avocado or olive oils (Cunha et al., 2007). However, a literature survey reveals that vegetable oils (with the exception of olive oil) have not been thoroughly investigated using the QuEChERS approach (Ferreret et al., 2005; Cunha et al., 2007).

The evaluation of different combinations of sorbents, such as PSA, C18, and GCB, for the dispersive SPE (d-SPE) step of the QuEChERS procedure has been reported (Rajski et al., 2013). The aim of the present study is to evaluate the analytical performance and applicability of the proposed modified QuEChERS method on sunflower oil matrices using dispersive-SPE (d-SPE) clean-up sorbent combinations and their effects on method performance parameters such as recovery studies and limit of detection (LOD).

However, for sunflower oil matrices containing large amounts of pigment and fats, which can interfere with the analysis, activated charcoal could play a significant role in eliminating pigments in the extract. In the present work, the QuEChERS method was further modified in the d-SPE clean-up step by using activated charcoal to improve recovery values.

Chemicals and Reagents

All the chemicals used in the present study are of Analar grade, and solvents were used under LC–MS grade & LC grade, procured from M/S Thermo Fisher Scientific (Fair Lawn, NJ). All the pesticide standards such as chlordane, dicofol, and dieldrin were supplied by M/S Sigma Aldrich, Bangalore. The QuEChERS extraction kit was procured from M/S Phenomenx Company, Hyderabad.

Instrumentation

The experiments were performed using the Agilent Technologies 7890 model Gas Chromatography, equipped with an ECD. A HP-5 capillary column (0.25 mm i.d., 30 m, and 0.25 μm phase film diameter) was used. The total run time for the instrumentation was 40 minutes. The carrier gas (Nitrogen) was maintained at a flow rate of 1.0 mL min⁻¹ for the experimentation.

Extraction Procedure

The sunflower oil samples were collected from department stores in the local area around Kadapa District, Andhra Pradesh, India. The modified QuEChERS procedure was adopted for the extraction of the target analyte from the sample.

A 3 g oil sample was placed in a 50-mL falcon tube. Subsequently, 7 mL of distilled water and 10 mL of acetonitrile solution (MeCN) were added to the tube, and it was shaken by a mechanical shaker for 10 minutes. QuEChERS extraction salts (4 g anhydrous MgSO₄, 1 g NaCl) were added to the tube, which was then shaken again for another 10 minutes and subjected to centrifugation at 5000 rpm for 5 minutes.

Then, 3 mL of the supernatant was transferred to a dispersive centrifuge tube (15 mL) containing 150 mg of PSA and 150 mg of anhydrous MgSO₄. Different amounts of activated charcoal (20 mg, 30 mg, 40 mg, and 50 mg) were added, stirred in a vortex for 2 minutes, and centrifuged again at 5000 rpm for 5 minutes.

An aliquot of 2 mL of the MeCN extract was transferred to a vial, dried under a gentle nitrogen stream, and the resultant residue was re-dissolved with acetonitrile solvent.

Application of QuEChERS Method for Pesticide Estimation

The established QuEChERS method was employed for the estimation of dicofol, chlordane, and dieldrin in sunflower oil. The low volatility is also one of the factors that do not affect quantitation. However, fat and pigments can thermally degrade in the injection liner or remain in the columns, which can lead to increased maintenance of the instrument frequently.

As a result, the pesticide residue analysis in oil samples such as sunflower oil, groundnut oil, olive oil, and other edible oils could be a difficult task. In an analytical method, various types of extraction methods and clean-up steps must be followed to obtain maximum analyte recovery with minimum matrix interference at the final measurement step (Bennett et al., 1997).

Several permutations of QuEChERS sample preparation approaches are useful to improve results or practical efficiency of recovery for the targeted analyte. In this study, the modified QuEChERS method has been adopted by employing activated charcoal, which effectively extracted the title compound from samples. The accurate quantification of pesticide residues is normally expressed in terms of the recovery value of the analyte.

The different concentrations of extractants of analytes were subjected to gas chromatography, and the evaluated analytical data is depicted in Table 1.

Table 1. Recovery values (%) with applied different concentrations of AC

Method Validation

The analytical method validation was carried out on the basis of SANCO guidelines (E.C.2009). The LOD of the method for each pesticide was measured at a signal-to-noise (S/N) ratio of 3:1. The LOQ values were calculated at a signal-to-noise (S/N) ratio of 10:1. The limit of detection (LOD) and limit of quantification (LOQ) for target analytes were in the range of 0.001–0.011 mg/kg and 0.002–0.030 mg/kg, respectively.

The linearity of the developed method was tested over the concentration range of 5 to 1000 ng/mL by spiking each pesticide standard sample. The regression coefficient (R²) values calculated for Dicofol, Chlordane, and Dieldrin were found to be 0.992, 0.995, and 0.998, respectively.

The calculated precision values, evaluated in terms of relative standard deviations (RSDs, %), for each pesticide residue were as follows:

• Dicofol: 1.6%
• Chlordane: 3.8%
• Dieldrin: 2.4%

The retention times observed for these three pesticides were 16, 22.5, and 26.5 minutes, respectively. The validated procedure was employed for recovery studies of spiked samples using different concentrations of AC and was found to be in the range of 79%–105%. The results are shown in Table 1 and Fig. 1

Conclusion

The modified and established QuEChERS method for the extraction of organochlorine pesticides in samples of sunflower oil was employed, and subsequent quantification by GC-ECD showed good precision, accuracy, linearity, and sensitivity. Hence, it is recommended for the routine analysis of various pesticide samples.

The extracted analytes were clearly detected without interference from the matrix in the proportional addition of AC to PSA sorbent. The clean-up efficiency also improved with the proposed method. The validated method proved to be fast and effective.

In the modified QuEChERS method, the combination of PSA + AC is economically cheaper compared to PSA and GCB sorbent combinations. The optimum quantity of AC was proposed for good analyte recoveries, but a higher quantity of AC caused lower recoveries.