Dynamic microwave-assisted extraction combined with liquid phase microextraction based on the solidification of a floating drop for the analysis of organochlorine pesticides in grains followed by GC

Guijie Li, Xu Zhng, Tingting Liu, Hongxiu Fn, Hongcheng Liu,Shngyu Li, Dwei Wng,*, Ln Ding*

a College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China

b Jilin Province Product Quality Supervision and Inspection Institute, Changchun 130103, China

c College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China

Keywords:

Dynamic microwave-assisted extraction

Liquid phase microextraction

Gas chromatography

Organochlorine pesticides

Grain

ABSTRACT

A convenient, cost-effective and fast method using dynamic microwave-assisted extraction and liquid phase microextraction based on the solidification of a floating drop was proposed to analyze organochlorine pesticides in grains including rice, maize and millet. Twelve samples can be processed simultaneously in the method. During the extraction process, 10% acetonitrile-water solutions containing 110 μL of n-hexadecane were used to extract organochlorine pesticides. Subsequently, 1.0 g sodium chloride was placed in the extract, and then centrifuged and cooled. The n-hexadecane drops containing the analytes were solidified and transferred for determination by gas chromatography-electron capture detector without any further filtration or cleaning process. Limits of detection for organochlorine pesticides were 0.97-1.01 μg/kg and the RSDs were in the range of 2.6%-8.5%. The developed technology has succeeded in analyzing six real grains samples and the recoveries of the organochlorine pesticides were 72.2%-94.3%. Compared with the published extraction methods, the developed method was used to analyze organochlorine pesticides in grains, being more environmentally friendly, which is suitable for the daily determination of organochlorine pesticides.

1. Introduction

Organochlorine pesticides (OCPs) were widely used to control pests during the growth of grains due to their wide range of control, better insecticidal and lower toxicity effect than other pesticides since the 1940s [1,2]. The use of pesticides in agriculture can improve agricultural productivity [3]. However,the use of pesticides has caused many adverse effects on the environment and human health [4,5].

China has banned the production and use of OCPs in agriculture since 1983. Although OCPs have been banned for many years, due to their high chemistry stability, this kind of pesticides does not disappear completely in the environment, they are still present in different samples [6-11]. Moreover, the residues of OCPs still can pose a potential threat or health problems to humans [12]. Considering the environment and food safety, it is necessary to develop a convenient, fast and efficient analytical approach to detect OCPs from grains.

Sample preparation is the most crucial step in analysis of complex samples [13]. The common pretreatment methods for OCPs in grain samples included ultrasonic extraction (UE) [14], solid phase extraction (SPE) [15], solid phase microextraction (SPME)[16], accelerated solvent extraction (ASE) [17]. However, tedious steps are also required with long time and complicated operation in the above methods. A large amount of organic solvents are needed for UE, and the active substances are easy to be destroyed in the extraction process. ASE needs not only a large number of samples,but also toxic solvents. SPE also needs large amount of organic solvents and complicated purification steps [18]. SPME is mainly combined with GC-MS to analyze volatile and semi volatile pesticide residues in pharmaceutical, environmental, food, animal and plant samples. Its advantages include less sample quantity, fast analysis speed, no solvent, high sensitivity and selectivity, ability to analyze complex samples and automation [19]. However, this method has its limitations, because it is only suitable for direct analysis of homogeneous samples (such as water samples).

In 2007, Zanjani et al. [20] first proposed liquid phase microextraction based on the solidification of a floating drop (LPMESFO), the technology overcomes some shortcomings of traditional liquid-phase microextraction technology, such as poor selectivity and the use of high toxic solvents. LPME-SFO, as an emerging sample pretreatment process, has been widely applied to extract trace poisonous substances in water, biological samples [21-28] and herbal plant samples [29]. In this technology, the target analytes in aqueous solution are enriched by a small amount of organic solvent, and then the organic solvent is solidified together in an ice water bath, which can be transferred to analyze. Therefore, sample extraction, separation and preconcentration can be completed in one step in this method.

Dynamic microwave-assisted extraction (DMAE), which has emerged in recent years, can effectively extract organic pollutants from food and environmental samples, and has lots of advantages such as short extraction time, simple equipment, low cost, solvent saving, convenience and high extraction efficiency [30-32]. DMAE is a dynamic extraction process, which can continuously provide fresh extraction solvent to the extraction vessel, and can be combined with other sample pretreatment technologies.

On the basis of the previous studies, this paper established a fast analytical method based on DMAE-LPME-SFO for the analysis of OCPs from grains, using GC-electron capture detector (ECD) for determination of the targets. Fresh solvent continuously pumped into the extraction tube, the amount of organic solvent used in the process was very small, multiple samples can be processed at the same time,and the experimental conditions affecting the extraction efficiency of target analytes were optimized.

2. Materials and methods

2.1 Instruments and installations

The equipment system used in this method was shown in Fig. 1,which consisted of a vacuum pump (HPD-25, Shanghai, China), a vacuum SPE manifold (Chromatography, America), and a microwave oven (NN-GF599M, Panasonic, Japan). Glass centrifuge tubes in the vacuum SPE manifold were used as collection tubes. The sample extractors were made of polyethylene, which were put in the microwave oven. The upper ends and the bottoms of the sample extractors were connected with the solvent container and the vacuum SPE manifold by T1 and T2 tubes (26 cm long, 11.0 mm id). An Agilent 7890B gas chromatograph system, which equipped with ECD was used.

2.2 Chemicals and samples

OCPs standards including α-hexachlorocyclohexane (HCH),β-HCH, γ-HCH, δ-HCH, P,P′-DDT, O,P′-DDT, P,P′-DDE and P,P′-DDD at concentrations of 100 μg/mL were bought from the National Institute of Metrology of China. Analytical grade sodium chloride, acetone, acetonitrile, ethyl acetate were bought from Beijing Chemical Plant, and quartz sand (25-50 mesh) was purchased from Shanghai National Pharmaceutical Company. Chromatographic grade undecyl alcohol, dodecyl alcohol and n-hexadecane were purchased from Fisher Corporation. Ultrapure water was acquired by milli-Q treatment equipment.

2.3 Sample pretreatment

Fig. 1 Schematic diagram of DMAE-LPME-SFO.

The samples included 2 kinds of maize, 2 kinds of rice, and 2 kinds of millet, which were obtained from the local farm product market. The grains were crushed by a high speed crusher and then passed through a 40-mesh sieve. The grain sample powder were stored under dried and sheltered conditions before experimental use. All the samples were tested using the national standard method and the results showed that they did not contain the 8 target OCPs.The sample 1 (maize) was made into spiked sample to optimize the experimental conditions. For recovery studies, spiked grain samples were prepared by adding the different concentrations of the OCPs standard solution into the samples. After shaking for 15 min, the mixture was placed to stand at room temperature in the dark for 12 h,and then analyzed.

2.4 Procedure of DMAE-LPME-SFO

Firstly, 0.50 g grain sample and 3.0 g of quartz were accurately weighed and mixed thoroughly. The mixture were transferred to the sample extractor and then compacted gently. In the same way, 12 samples were prepared and placed in the microwave system. The upper ends and the bottoms of the sample extractors were connected with the solvent container and the vacuum SPE manifold by T1 and T2 tubes. Twelve collection tubes were put into the vacuum SPE manifold, the microwave power was turned on and adjusted to 400 W. At the same time, 10% acetonitrile-water solutions containing 110 μL of n-hexadecane was driven by the vacuum pump through the sample extractor at the flow rate of 2.0 mL/min.When the volume of the extract in the collection tube was 12 mL,the vacuum pump and microwave oven were turned off, and the extraction process was finished.

Finally, sodium chloride (1.0 g) was added to the collection tube. After centrifugation (2 min, 4 500 r/min), the n-hexadecane containing target analytes floated on the top of the extract.After the collection tube was put into an ice water bath for 2 min, the solidified n-hexadecane was take out and placed in a gas chromatograph sample bottle, and then melted at room temperature, took 1 μL for GC analysis.

2.5 GC analysis conditions

The target analytes were detected by an Agilent 7890B gas chromatograph, which was equipped with ECD; A DB-1701 capillary column (30 m × 0.320 mm, 0.25 μm) was conducted as chromatographic separation column, the temperature of column temperature was 185 °C, the temperature of ECD detector was 225 °C, the inlet temperature was 195 °C. Heating program was set as follows: 120 °C kept for 1 min, rose to 200 °C within 4 min at a speed of 20 °C/min, rose to 230 °C within 3 min at a speed of 10 °C/min, then rose to 250 °C within 4 min, at a speed of 5 °C/min, kept 2 min, finally rose to 280 °C within 3 min, at a speed of 10 °C/min, kept 3 min. The flow rate of carrier gas (N2) was 1.0 mL/min. The injection volume was 1 μL.

2.6 Statistical analysis

The presented data were shown in the mean values of three replicates ± standard deviation (SD). Statistical analysis of all the results were done using one-way ANOVA, and significant differences were evaluated based on Duncan’s multiple comparison test.Differences with P ＜ 0.05 were regarded as statistically significant.

3. Results

3.1 Optimization of DMAE conditions

3.1.1 Microwave power effect

The microwave power on the recoveries of OCPs in the range of 0-1 000 W was investigated. As shown in Fig. 2, with the microwave radiation power increased ranging from 0 to 400 W, the recoveries of the target analytes increased, and gradually decreased thereafter. High microwave power may lead to high temperature in the microwave oven, which may lead to the volatilization or degradation of target OCPs. Therefore, 400 W was selected in the subsequent experiments.

Fig. 2 influence of microwave power on the recoveries of OCPs (n = 3).

3.1.2 influence of extraction solvent

In the preliminary experiment, we investigated the effects of acetone-water (20%, V/V), acetonitrile-water (20%, V/V), and ethyl acetate-water (20%, V/V) on the recoveries of OCPs in grains.The results showed that 20% acetonitrile-water solution had the best extraction effect. Compared with acetone and ethyl acetate,acetonitrile has a higher boiling point, so it is not easy to boil in the process of microwave-assisted extraction. Therefore, acetonitrilewater solution was selected for further experiments.

Subsequently, we investigated the effect of acetonitrile concentration on the recoveries of OCPs. Acetonitrile-water solutions(10 mL, 0-25%, V/V) were used in the experiments, the results were demonstrated in Fig. 3, acetonitrile concentration within 0-10%, the recoveries of OCPs increased, and then gradually decreased. It may be that too much acetonitrile leads to poor phase separation, incomplete collection of n-hexadecane. So we chose 10% acetonitrile-water solutions as extraction solvent.

Fig. 3 influence of acetonitrile solution concentration on the recoveries of OCPs (n = 3).

So as to obtain optimum extraction conditions of the targets analytes, we investigated the influence of different volumes of acetonitrile-water solutions on the recoveries of OCPs.As shown in Fig. 4, when the volume of acetonitrile-water solutions increased from 6 mL to 12 mL, the recoveries of OCPs increased significantly (P ＜ 0.05). When the volume of acetonitrile-water solutions was above 12 mL, the recoveries of OCPs decreased slightly. It may be that with the increase of the volume of acetonitrile-water solutions, subsequent phase separation is difficult, leading to the decrease of n-hexadecane collection and the increase of dissolution of OCPs in water, so the recoveries of OCPs slightly decreased. In addition, with the increase of volume of acetonitrile-water solutions, the extraction time is also prolonged, which is not conducive to rapid analysis. Therefore, in the experimental process, 12 mL was selected.

Fig. 4 influence of extraction solution volume on the recoveries of OCPs (n = 3).

3.1.3 Extraction solvent flow rate

We studied the effect of the extraction solvent flow rate(0.5-2.5 mL/min) on the recoveries of OCPs. The experimental results were shown in Fig. 5. When the flow rate was in the range of 0.5-2.0 mL/min, the recoveries of OCPs increased, and then continued to increase to 2.5 mL/min, slowly decreased. This was because when the collected extract volume remained the same, the larger flow rate of the solvent, the extraction was inadequate, resulting in a rapid reduction in the recoveries of OCPs. However, when the flow rate of the solvent was too small, the corresponding extraction time would be extended. Thus, 2.0 mL/min of the extraction solvent flow rate was chosen in further studies.

Fig. 5 influence of flow rate of extraction solution on the recoveries of OCPs (n = 3).

3.2 The optimization of LPME-SFO conditions

3.2.1 Extraction solvent used for LPME-SFO

The 3 common LPME-SFO extraction solvents, 1-undecanol,1-dodecanol, and n-hexadecane were examined in the study. The experimental results showed that n-hexadecane can provide best effect on the target OCPs, so n-hexadecane was chosen as extraction solvent used for the LPME-SFO in the study.

3.2.2 Effect of volume of n-hexadecane

During the experiment, we investigated different volumes of n-hexadecane ranging from 50 μL to 130 μL on the recoveries of the target analytes. The experimental results were shown in Fig. 6. When the n-hexadecane was 110 μL, the recoveries of OCPs reached the maximum value. Therefore, 110 μL of n-hexadecane was chosen in subsequent study.

Fig. 6 influence of volume of the n-hexadecane on the recoveries of OCPs (n = 3).

3.2.3 Effect of amount of NaCl

Adding a certain amount of salt can improve the ionic strength in the water phase, so it is beneficial to enrich the target substances[33]. We studied the amount of NaCl on the recoveries of the target analytes ranging from 0-2.0 g (Fig. 7). The experimental results showed when the amount of NaCl added was 1.0 g, the recoveries of OCPs were the largest, so we chose to add 1.0 g NaCl in the study.

Fig. 7 influence of amount of NaCl on the recoveries of OCPs (n = 3).

3.3 Method validation

3.3.1 Linear range and detection limit

The 8 OCPs were added to the solvent and the extraction solution of the blank grain samples respectively to establish the solvent standard curves and matrix standard curves, respectively. The slopes of the solvent standard curves were slightly lower than that of the matrix standard curves, which indicated that there was the matrix enhancement effect for the determination of the OCPs in the study.However, the slopes were not obvious different in the grain samples,as shown in Table 1. But to make the results more accurate, the standard curves of maize matrix was used for quantitative analysis.The linear range of the standard curves was 5-150 μg/kg, and the linear determination correlation coefficients were 0.996 3-0.999 2.The detection limits (LODs) and quantitative limits (LOQs) of the method were calculated as the OCPs concentrations producing signal/noise ratio of 3 and 10, respectively. The linear range,correlation coefficients, LODs and LOQs values of the established method were listed in Table 1. The GC chromatograms of spiked grain samples (20 μg/kg) were shown in Fig. 8.

Fig. 8 Gas chromatograms of organochlorines pesticides in maize (a), rice (b), millet (c) spiked (20 μg/kg). 1. α-HCH, 2. γ-HCH, 3. β-HCH, 4. δ-HCH,5. P,P’-DDE, 6. O,P’-DDT, 7. P,P’-DDD, 8. P,P’-DDD.

Table 1Analytical parameters.

Table 2Intra-, inter-day precision and recoveries of the proposed method (n = 6).

3.3.2 Precision and accuracy

The precision of OCPs with different concentrations (5, 50, 150 μg/kg)were determined 6 times in a day, and the same experiment process was carried out in 6 consecutive days. The experimental results were shown in Table 2. The recoveries of OCPs in this experiment were in the range of 70.6%-90.1%. The intra-day relative standard deviation(RSDs) were 2.6%-8.5%, and the inter-day RSDs were 3.1%-8.5%.The results showed that the reproducibility of this method was good.

3.3.3 Application of methods

So as to examine the practical application of the developed method, it was applied to detect OCPs in six grains samples. No target analyte was detected in the above samples. Subsequently, we examined the recoveries of OCPs at different spiked concentrations(10, 50, 150 μg/kg) in the above grains samples and the recoveries and RSDs of OCPs were 72.2%-94.3% and 2.5%-8.6%, respectively.The detailed experimental results were summarized in Table 3.

3.3.4 Comparison of methods

In this study, the analytical performances of the established method were compared with some published studies for the detection of OCPs in grains, such as dynamic microwave-assisted extraction coupled with solid phase extraction (DMAE-SPE) [34], matrix dispersive solid phase extraction (MSPD) [35], microwave-assisted extraction [36], QuEChERS [37], and the details were summarized in Table 4. It can be seen that, the proposed method was highly competitive, less organic reagents were used in the experimental process, which was more conducive to environmental protection.The sample treatment time was shorter, which effectively reduced the experimental operation process and the method has achieved satisfactory experimental results.

Table 3Analytical results of real samples (n = 3).

Table 4Comparison of the developed method with other methods used in the literatures.

4. Conclusion

A fast and simple method based on DMAE-LPME-SFO followed by GC-ECD was established for the analysis of OCPs in grains in the study.The main advantages of this method were that the amount of organic solvent was small, the sample pretreatment was simple, and multiple samples can be processed at the same time. The operation process of the method was carried out in an airtight reactor, which can greatly reduce the exposure of poisonous solvents to operators. The results showed that the method was an environmentally friendly technology with great potential for the daily detection of OCPs in different samples.

conflict of interest

The authors declared that they have no conflicts of interest to this work.

Acknowledgement

The study was financially supported by the National Science and Technology Support Program of China (Grant No. 2013BAD16B08).The authors have declared no conflict of interest.