Determination of organophosphorus pesticide residues in vegetables using solid phase micro-extraction coupled with gas chromatography–flame photometric detector

2.1 Reagents and chemicals

Certified individual pesticide standards, namely ethoprophos (93.1%), sulfotep (97.4%), tolclofos-methyl (99.4%), diazinon (98.3%), fenitrothion (95.2%), chlorpyrifos (99.9%), isofenphos (95.6%), methidathion (95.8%), ethion (97.8%), triazophos (96.5%) and leptophos (96.7%) were obtained from Fluka (Buchs, Switzerland). Individual stock standard solutions (500 mg/L) were prepared by dissolving an accurate weight of each pesticide in 100 mL of acetone. Working standard solutions (10 mg/L) for the eleven OPs were prepared by serial dilution of the individual stock with acetone. All standards were stored under refrigeration at 4 °C. Acetone and methanol (analytical grade) were supplied by QRec (>99%, Chonburi, Thailand) and sodium chloride (>99.5%) was obtained from Merck (Darmstadt, Germany). The properties and the chemical structures of the studied OPs are shown in Table 1 and Fig. 1, respectively.

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Figure 1 Chemical structures of the studied organophosphorus pesticides.

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2.2 Instrumentation and apparatus

A HP5890 series II plus GC unit (Hewlett–Packard, PA, USA) with split/splitless injector coupled with flame photometric detector (FPD) was used. The data were analysed using Chemstation software (Ver. A.10.02). A capillary column, DB-5 (5% phenyl methylpolysiloxane, 30 m × 0.25 mm id × 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA) was used to separate the pesticides using the following temperature program: Initial temperature, 100 °C; held for 1 min; heated to 200 °C at 20 °C/min and held for 8 min; heated to a final temperature of 250 °C at 10 °C/min and held for 7 min. The injector temperature was maintained at 260 °C for effective fibre desorption and detector temperature was at 275 °C. Nitrogen (CP grade, 99.999%) was used as carrier gas and supplied by SGS (Malaysia). The flow of carrier gas was 1.0 mL/min and the injection was performed using splitless mode.

Detected OPs were confirmed using a GC–MS system (HP7890/MSD 5975C, Agilent, CA, USA) operated in the splitless mode. A capillary column DB-5MS (5% phenyl methylpolysiloxane, 30 m × 0.25 mm id × 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA) was used with helium carrier gas at 1.5 mL/min. The oven temperature program was set as previously mentioned for GC–FPD system. The mass spectrometer was operated in the electron impact ion (EI) mode with a source temperature of 230 °C. The chromatographic resolution was performed in scan mode from m/z 50 to 400. The confirmation of pesticides peaks has been done by comparing the fragment ions with NIST library and the standard reference with similarity index more than 70%. All prepared reagents were kept in a dispenser (BRAND, Germany) with capacity of 1–10 mL.

2.3 Sample preparation

Pesticide-free samples (cabbage, kale and mustard) were collected from Agro Technology Park, Malaysian Agricultural Research Development Institute (MARDI), Cameron Highlands, Malaysia. A representative sample (0.5 kg) was finely chopped into small pieces, combined and homogenised using a Robot coupe food processor R3 (Jackson, MS, USA). After the sample was homogenised, 0.5 g of the sample was accurately weighed in 20 mL vial followed by the addition of 2 mL of methanol/acetone (1:1, v/v) as extraction solvent and 1 mL of NaCl solution (10%, w/v). The sample was then diluted to 10 mL with water and sealed with septum cap for the SPME analysis. Pesticides-free vegetables were obtained from Malaysian Agriculture and Development Institute (MARDI) and used for recovery studies. Vegetable samples (eight mustard, seven kale and five cabbage samples) were purchased from six local supermarkets in Ipoh, Perak. All the samples are products of Cameron Highland, Malaysia.

2.4 SPME procedure

SPME sampling stands, fibre holder and commercial fibre assembly coated with 100 μm polydimethylsiloxane (PDMS), 65 μm polydimethylsiloxane/divinylbenzene (PDMS/DVB), 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) and 85 μm polyacrylate (PA) were obtained from Supelco (Bellefonte, PA, USA). The fibre was attached to the holder for manual sampling and was pre-conditioned before use according to the manual given by the manufacturer. The fibre inside the holder was attached to the SPME sampling stand and was pushed out from the needle and immersed into the sample solutions (prepared as described in Section 2.3) for 30 min at ambient temperature. The solution was stirred at a constant speed (1275 rpm) during the extraction using a magnetic stirrer. After the extraction, the fibre was immediately transferred to the GC injector and desorbed for 11 min at 260 °C. The extraction efficiency is based on total peak areas of the OPs.

2.5 Method validation

Method validation (linearity, limits of detection (LOD) and quantitation (LOQ), repeatability and recovery) was performed using the proposed SPME–GC–FPD method according to ICH and SANCO 2013 guidelines. Linearity was investigated over the range of 0.1–100 μg/L. The LOD and LOQ values were calculated based on the standard deviation of the response of the standard blank and the slope of calibration curve at signal-to-noise ratio of 3 and 10, respectively. Repeatability, expressed as relative standard deviation (% RSD), was done by extracting six replicates at three concentration levels (5, 50 and 100 μg/L). Recovery studies were carried out using three vegetable matrices (cabbage, kale and mustard) spiked at three fortification levels (5, 50 and 100 μg/L). All spiked samples were extracted in triplicates.

3.1 Gas chromatography (GC) separation

Preliminary separation conditions were based on the work of Yao et al. (2001). Injector and detector temperatures used were 230 and 250 °C, respectively. The oven temperature was programmed as follows: initial temperature 60 °C held for 1 min, heated to 150 °C at 20 °C/min and held for 1 min, heated to a final temperature of 230 °C at 10 °C/min. Under these conditions, only six peaks were obtained with a total run time of 14.50 min. In order to achieve separation of all analytes that were injected, the oven temperature program was modified. The holding time was increased to obtain adequate retention times for the eleven OPs. The initial temperature (60, 80, 100 and 120 °C) was investigated, with 100 °C giving the best results. Two different temperature ramps were also optimised to achieve full separation of the analytes in short analysis time. The best ramps were 100 to 200 °C at 20 °C/min and held for 8 min; heated to 250 °C at 10 °C/min (hold for 7 min). Under the optimum chromatographic conditions, all the peaks were separated in 26 min.

3.2 Extraction mode and fibre coating

The extraction efficiency for each fibre is related to the logarithm octanol–water partition coefficient (log K_ow) and volatility (PV) (Table 1). The log K_ow indicates the polarity strength and their tendency towards the hydrophilic and lipophilic layers. Log K_ow of the OPs was between 2.57 (methidathion) and 6.31 (leptophos) which reflect their tendency towards lipophilic layers. Furthermore, these compounds are volatile with vapour pressures between 0.20 mPa (ethion) and 78 mPa (ethoprophos).

Three extraction modes, i.e., headspace (HS), direct immersion (DI) at 60 °C and ambient temperature were investigated by comparing the sum of the peak areas of the extracted OPs. DI mode at ambient temperature exhibited the highest extraction efficiency (the highest total peak areas) for all compounds studied (Fig. 2). It is anticipated that the DI–SPME will provide more sensitive results compared to the HS–SPME due to the direct contact between the coating materials and the analytes. The HS mode was able to extract all the pesticides except methidathion and triazophos. The least satisfactory is using direct immersion mode at 60 °C, which is unable to extract three pesticides namely fenitrothion, methidathion and triazophos. This is due to the possible degradation of these compounds under heating condition (Kin and Huat, 2010). Thus, DI mode without heating was selected for further experiments.

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Figure 2 Extraction efficiency by three modes of SPME: headspace (HS), direct immersion (DI) at ambient temperature and direct immersion (DI) at 60 °C.

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Four types of commercial SPME fibre coatings: 100 μm PDMS (85 μm PA, 65 μm PDMS–DVB and 50/30 μm DVB/CAR/PDMS (tripolar phase) were used to extract eleven OPs (50 μg/L) in aqueous solution. 85 μm PA fibre exhibited the higher extraction efficiency for the eleven OPs used, especially for chlorpyrifos and ethion (Fig. 3). This fibre was used for further work. The good ability of PA fibre in extracting pesticide residues was previously reported by Tankiewicz et al. (2013).

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Figure 3 Comparison of different SPME fibres in extracting OPs at concentration of 50 μg/L. Extraction time, 30 min; stirring speed, 800 rpm; desorption time, 10 min and desorption temperature 240 °C in direct immersion mode.

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3.3 Optimisation of DI–SPME method

Factors affecting the SPME process such as absorption time, stirring speed, salting out effect, desorption temperature and desorption time were evaluated. An extraction procedure was established with the initial conditions: extraction time, 30 min; stirring rate, 800 rpm, 10% NaCl solution, in accordance with data obtained from literature (Kin and Huat, 2009).

The influence of extraction time (20–40 min) was studied using 85 μm PA fibre directly immersed in deionised water containing 50 μg/L OPs with the addition of NaCl solution (Fig. 4(A)). It was observed that the signal for all of the OPs increased as the extraction time increased up to 30 min and dropped thereafter, especially for triazophos and fenitrothion. Thus 30 min exposure was therefore selected for the following experiments.

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Figure 4 Optimisation parameters for extraction of OPs (50 μg/L) with 85 μm PA coating: (A) Absorption time, (B) stirring speed, (C) effect of NaCl, (D) desorption time, and (E) desorption temperature.

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In order to achieve faster extraction equilibrium and improve the mass transfer of the analytes from solution to the fibre, different stirring speeds (1050, 1200, 1275, 1350 and 1500 rpm) were studied. The signal for most of the pesticides decreased as the stirring velocity was increased, starting at 1350 rpm (Fig. 4(B)). Thus, 1275 rpm was used for the rest of the studies.

The effect of ionic strength of the solution on the SPME procedure was studied using different concentrations (5, 10, 20 and 30% (w/v)) of sodium chloride. Generally, the addition of suitable amount of salt will decrease the solubility of the compounds in the liquid phase, thus enhancing the extraction of the compounds to the fibre coating. It was observed that the peak areas increase with the addition of salt up to 10% NaCl especially for chlorpyrifos, ethion, tolclofos-methyl and isofenphos (Fig. 4(C)). The use of higher salt content (more than 10%) will decrease the instrumental response due to the deposition of salt. Further experiments were conducted using 10% (w/v) NaCl.

Duration of desorption (9, 10, 11, 12 and 13 min) was investigated, high sensitivity was obtained when desorbed for 11 min (Fig. 4(D)). Five desorption temperatures (230–270 °C) were also studied. The best result was obtained at 260 °C (Fig. 4(E)). Long exposure periods of the PA fibre at high temperature can cause thermal degradations, resulting in lower efficiency.

3.4 Adopted extraction conditions

The adopted extraction conditions were: absorption time, 30 min; stirring speed, 1275 rpm; ionic strength, 10% NaCl; desorption temperature, 260 °C; desorption time, 11 min. The proposed method shows some advantages over the previous reported methods in terms of heating requirement and types of analytes. The proposed method was performed at ambient temperature, compared to the previously DI–SPME methods, where the sample was heated in the range 40–75 °C (Filho et al., 2010; Yao et al., 2001; Campillo et al., 2006). The procedure (ambient temperature) was purposely used to provide good pre-concentration for the extracts and prevent any degradation of the analytes. Two of these OPs, tolclofos-methyl and leptophos, are for the first time reported using SPME. The proposed method showed considerable advantages over the previously reported methods such as LLE, SPE, DLLME and MAE in terms of solvent consumption and waste generated (Fenoll et al., 2007; Bidari et al., 2011; Wang et al., 2009; Zhao et al., 2012).

3.5 Method validation

The linearity range, limits of detection (LOD) and quantitation (LOQ), repeatability and recovery of the DI–SPME method were investigated under the optimised conditions. The linearity range was studied by varying the concentration of the standard solution from 0.1 to 100 μg/L. Calibration study was tested by spiking the working standard solutions into water containing NaCl solution. The calibration curves were illustrated by plotting the peak area versus concentration of each pesticide. Over the studied range (0.1–100 μg/L), the calibrations were linear only for sulfotep, tolclofos-methyl, chlorpyrifos, ethion and triazophos. Diazinon, isofenphos and methidathion were linear over 0.3–100 μg/L, while the linearity range for ethoprophos, fenitrothion and leptophos was 0.5–100 μg/L (Table 2). All calibrations were well correlated with determination coefficients, r² > 0.99.

The LOD values obtained were in the range from 0.01 (triazophos) to 0.14 μg/L (ethoprophos, fenitrothion, leptophos), meanwhile the LOQs ranged from 0.03 to 0.42 μg/L, which is lower than the MRL (1 mg/kg). The values obtained were found to be comparable to the previously reported method using GC–FPD (0.05–0.30 μg/L) as reported by Yao et al. (2001) but lower than the other reported methods using GC–MS (2–109 μg/L) (Zambonin et al., 2004), (1–33 μg/L) (Filho et al., 2010), and (0.01–50 μg/L) (Cortés-Aguado et al., 2008).

To evaluate the precision of the developed method, intra-day repeatability (n = 6) was performed using three concentration levels (5, 50 and 100 μg/L) of the studied pesticides. % RSD values varied between 2.44% (ethoprophos) and 17.9% (leptophos) (Table 3). Overall, the repeatabilities obtained for each pesticide in all concentration levels indicated satisfactory extraction.

The effect of different sample weight (0.5, 1.0, 2.0 and 3.0 g) on the recoveries was also investigated. The recoveries were found to increase as the mass of the sample decreased. The best recoveries were obtained when 0.5 g sample was used. Centrifugation step was not applied due to the lower recoveries. Table 3 shows recovery results for samples that were spiked with different levels of pesticides. Satisfactory recoveries (74.6–117%) for most of the studied pesticides were obtained except leptophos (16.7–48.7%), probably due to its high molecular weight (412.7) and low polarity (log K_ow = 6.31), making it difficult to extract when spiked into real samples. Despite leptophos, the obtained recoveries were within the acceptable range (70–120%) and comparable with the previous reported works using DI–SPME (72–117%) (Filho et al., 2010), HS–SPME (71–98%) (Kin and Huat, 2009), matrix solid phase dispersion (62–102%) (Navarro et al., 2002), accelerated solvent extraction (62–107%) (Wu et al., 2011), vortex-assisted liquid–liquid microextraction (72–106%) (Jia et al., 2010), microwave-assisted extraction (76.5–109%) (Zhao et al., 2012) and stir bar sorptive-extraction (77–119%) (Hu et al., 2013) and better than the obtained recoveries using solid phase extraction (63–137%) (Yang et al., 2011). Typical chromatogram for the spiked cabbage sample is shown in Fig. 5(A).

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Figure 5 Typical chromatograms obtained for: (A) spiked cabbage with OPs (5 μg/L), (B) mustard, (C) kale and (D) cabbage samples using DI–SPME–GC–FPD with 85 μm PA in three different locations. Peak label: (1) ethoprophos, (2) sulfotep, (3) diazinon, (4) tolclofos-methyl, (5) fenitrothion, (6) chlorpyrifos, (7) isofenphos, (8) methidathion, (9) ethion, (10) triazophos, (11) leptophos, (i) trichlorfon, and (ii) Demephion.

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3.6 Application to commercial samples

The developed DI–SPME–GC–FPD method using 85 μm PA fibre was applied for the determination of OPs in twenty vegetable samples. The samples were immediately processed according to the sample preparation procedure as mentioned before. The analysis was performed in triplicate and between each run of the sample, a blank was carried out to avoid carryover and possible contamination from the extraction process.

The results obtained for the positive samples are summarised in Table 4. Of the twenty samples that were analysed, six samples were found to contain pesticides residues. All positive samples contained chlorpyrifos (0.22–1.83 μg/kg) and were mostly detected in mustard samples. The residue levels of the pesticides determined were lower than the MRLs (1 mg/kg) specified by the International Food Act & Regulation 1985 and Codex Alimentarius Commission. Chlorpyrifos is one of the most widely used pesticides and commonly found in vegetables sold by retailers in the Ipoh area. The results are in agreement with the data compiled from 2004 to 2010 by the Pesticide Analytical Centre (Department of Chemistry Malaysia) which found chlorpyrifos to be the highest OPs detected in vegetables for nine districts in Ipoh.

The presence of chlorpyrifos, ethion and triazophos was confirmed by GC–MS analysis on the suspected sample. Under the selected extraction conditions, co-extraction for some compounds was observed. These compounds were also identified using GC–MS. Two of OPs that were outside the scope of the studies (demephion and trichlorfon) were successfully identified in mustard samples. These findings suggest that the SPME method can be applied to a wide range of compounds in combination with various types of detectors. Typical chromatograms for mustard, cabbage and kale samples subjected to DI–SPME–GC–FPD method are shown in Fig. 5(B–D).

In the current study, the standard deviations (SDs) obtained from the samples show the possibility of using only a small mass (0.5 g) to represent the sample. This is considered advantageous when compared to the previous reported methods where large amounts of sample are required. This is made possible by the reproducibility of the homogenisation and extraction processes.