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Cloud point extraction coupled with ultrasonic-assisted back-extraction for the determination of organophosphorus pesticides in concentrated fruit juice by gas chromatography with flame photometric detection
Food Chemistry 127 (2011) 683–688
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Analytical Methods
Cloud point extraction coupled with ultrasonic-assisted back-extraction for the determination of organophosphorus pesticides in concentrated fruit juice by gas chromatography with flame photometric detection Wei-jun Zhao a, Xiao-ke Sun a, Xiao-ni Deng b, Lin Huang c, Ming-min Yang d, Zhi-ming Zhou d,⇑ a
Department of Quality Assurance, Shaanxi Haisheng Fresh Fruti Juice Co., Ltd., Qianxian, Shaanxi Province 713300, China Ministry of Agriculture Supervision & Center for Fisheries Environment and Quality of Fishery Products (Xi’an), Xi’an, Shaanxi Province 710086, China The Nineth Department of Xi’an Modern Chemistry Research Institute, Xi’an, Shaanxi Province 710065, China d College of Science, Nanjing Agricultural University, Nanjing, Jiangsu Province 210095, China b c
a r t i c l e
i n f o
Article history: Received 23 December 2009 Received in revised form 27 October 2010 Accepted 29 December 2010 Available online 13 January 2011 Keywords: Cloud point extraction (CPE) Organophosphorus pesticides (OPPs) Ultrasonic-assisted back-extraction Gas chromatography with flame photometric detection (GC-FPD) Concentrated fruit juice
a b s t r a c t A new method for the determination of nine organophosphorus pesticides (OPPs): Dichlorvos, methamidophos, acephate, diazinon, dimethoate, chlorpyrifos, parathion-methyl, malathion and parathion-ethyl in concentrated fruit juice was developed using the cloud point extraction coupled with ultrasonicassisted back-extraction prior to gas chromatography with flame photometric detection (GC-FPD) analysis. The parameters and variables that affect the extraction were investigated. Under optimum conditions: a solution containing 6% (W/V) polyethylene glycol 6000 (PEG 6000) and 20% (W/V) Na2SO4 for the extraction of the OPPs. The coacervation phase obtained was back extracted with ethyl acetate. The upper ethyl acetate solution was centrifugated simply for further cleanup for the sake of automatic injection. A preconcentration factor of 50 was obtained for these nine pesticides. Using this method, the limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.5–3.0 and 1.5–9.0 lg kg1 in concentrated fruit juice, respectively; the relative standard deviations (RSD) were <9%. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Organophosphorus pesticides are an important source of environmental contamination owning to their widespread use in agriculture; this involves their later passage to the fruit with the ensuing risk for the population. Studies showed that mixture of cholinesterase-inhibiting OPPs formation adversely affected embryogenesis in mice which could have implications for susceptible population from occupational and environmental exposures (Gomes, Anilal, & Lloyd,1999). In addition, OPPs were known to reduce the activity of neurotransmitters and hence to cause irreversible effects on the nervous system. Therefore, accomplishment of sensitive, rapid and simple analytical methods of the OPPs in concentrated fruit juice are paramount. Several studies have been reported on the analysis of organophosphorus pesticides (Ahmadi, Assadi, Milani Hosseini, & Rezaee, 2006; Jin et al., 2004; Lambropolou & Albanis, 2003; Lin, Huang, & Liu, 2006; Pinto, pavón, & Cordero, 1995; Rastrelli, Totaro, & Simone, 2002). Pesticide samples are usually enriched by liquid–liquid extraction (Barcelo, Porte, Cid, & Albaines, 1990; Hyötyläinen, Lüthje, Rautiainen-Rämä, &
⇑ Corresponding author. Fax: +86 25 84395255. E-mail address: [email protected] (Z.-m. Zhou). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.122
Riekkola, 2004) or solid-phase extraction (Liu & Lee, 1998; Martinez, Gonzalo, Moran, & Mendez, 1992; Zhu et al., 2009); these conventional methods have been involved in an extraction technique which suffers from some disadvantages such as a great expense and toxicity of solvents used. The cloud point extraction (CPE) was introduced for the first time by Watanabe and co-workers in 1976 (Miura, Ishii, & Watanabe, 1976): Cloud point of aqueous solutions of the surfactant micellar systems is a temperature at which the solution becomes turbid before separating into two phases, i.e. a surfactant-rich phase and an aqueous phase. The surfactant-rich phase is able to extract and pre-concentrate analytes. The CPE methodology based on surfactant-mediated phase separation has been successfully used for the preconcentration of species of widely differing character as a previous step to their later determination by high-performance liquid chromatography (HPLC) and/or gas chromatography (GC) (Sanz, Halko, Ferrera, & Rodriguez, 2004; Shen & Shao, 2006). Cloud point extraction methodology can offer an interesting alternative to the extraction systems due to many advantages. Surfactants are less toxic and cheaper than the organic solvents. CPE method with surfactant provides possibility of extracting and preconcentrating analysis in a single step using a simple procedure, and also the CPE technique can achieve results similar to those obtained by traditional extraction techniques. The surfactant-rich
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phase is compatible with hydroorganic mobile phases, so CPE has been exploited to combine with HPLC. On the other hand, the application of CPE as a preconcentration followed by GC is very rare; the reason is mainly because the viscous nature of the surfactant endangers the blocking of the capillary column. Methods to avoid these disadvantages were reported (Froschl, Stangl, Niessner, & Fresenius, 1997). In this work, we described a competitive method of CPE for the rapid and effective extraction and preconcentration of nine OPPs from concentrated fruit juice coupled with GC-FPD by automated injection. The preconcentrated analytes were back-extracted from the obtained surfactant-rich phase into ethyl acetate by the assisted of ultrasonic. The ethyl acetate solution obtained from back-extraction was centrifuged for further cleanup and then directly injected into the GC for analysis. The aim of this paper was to investigate the applicability of cloud point extraction coupled with ultrasonic-assisted back-extraction to extract and preconcentrate organophosphorous pesticides from concentrated fruit juice (apple, pear) prior to gas chromatography.
2. Experimental 2.1. Reagents and solutions Nine organophosphorus pesticides (dichlorvos, methamidophos, acephate, diazinon, dimethoate, chlorpyrifos, parathionmethyl, malathion and parathion-ethyl) were purchased from Dr. Ehrenstorfer (Augsburg, Germany); purity P96%. Fig. 1 shows the structures of nine organophosphorus pesticides. 0.01000 g of each pesticide was dissolved in 10.00 mL ethyl acetate (Damstadt, Germany) to obtain a standard stock solution with a concentration of 1000 mg L1, A fresh 20.00 mg L1 standard solution containing the nine pesticides was prepared in ethyl acetate every four days and stored at 4 °C. Working mixed standard solutions (0.4 and 2.0 mg L1) were obtained everyday by diluting intermediary mixed standard solution. Anhydrous sodium sulfate and sodium chloride were purchased from Bodi Chemical Reagent Co. (Tianjin, China). Polyethylene glycol (PEG) 4000, 6000, 10,000 were O O O P O
Cl Cl
C 4H 7Cl 2O 4P Mol. Wt.: 220.98 Dichlorvos
O
N N
O O P S
O P O N H S C 4H 10NO 3PS Mol. Wt.: 183.17 Acephate O
O P O H 2N S C 2H 8NO 2PS Mol. Wt.: 141.13 Methamidophos
Cl S O P O O
S P O S O
H N
Cl N
Cl
O C12H21N2O3PS Mol. Wt.: 304.35 Diazinon ON+ O S O P O O C8H10NO 5PS Mol. Wt.: 263.21 Parathion-methyl
C9H11Cl 3NO 3PS Mol. Wt.: 350.59 Chlorpyrifos
C5H12NO 3PS2 Mol. Wt.: 229.26 Dimethoate
O O O
O O
S S P O O C10H19O6PS 2 Mol. Wt.: 330.36 Malathion
ON+ S O P O O C10H14NO 5PS Mol. Wt.: 291.26 Parathion-ethyl
Fig. 1. The structures of nine organophosphorus pesticides.
obtained from Guangdong Guanghua Chemical Factory (Guangdong, China) and dissolved with distilled water to obtain stock solution of 20% (W/V). 2.2. Apparatus A gas chromatograph (GC-17A) (Shimadzu, Japan) with a split/ splitless injector system, and a flame photometric detection with phosphorus filter were used. Ultra pure nitrogen (99.9999%, China) was passed through a molecular sieve trap and an oxygen trap (Beijing, China) was used as the carrier gas at a constant linear velocity of 44.6 cm/s. The injection port was held at 250 °C and used in the splitless mode with splitless time of 1.2 min. For reduction of degradation products deactivated glass liner was used. Long life septum (Chromseal) was used in the injection port. Separation was carried out on a DB-1701, 30 m 0.32 mm capillary column with a 0.25 lm stationary film thickness (Agilent Technologies). Hydrogen gas was generated with hydrogen generator of GH500B from ZhongXingHuiLi (Beijing, China) for FPD at a flow of 130 mL/min. The flow of zero air (99.999%) from HuiLong (Berjing, China) for FPD was 100 mL/min. An ultrasonic from Kunshan ultrasonic instrument plant (Jiangsu, China) was used as back-extraction of samples. A centrifuge from Feige instrument and metre Co. (Shanghai, China) was used to accelerate phase separation. 2.3. GC analytical conditions In order to acquire the optimum conditions for OPPs determination, the oven temperature was programmed as follows: initial 150 °C, from 150 °C (held for 0.5 min) to 185 °C at the rate of 20 °C/min; from 185 °C (held 2.0 min) to 200 °C at the rate of 20 °C/min; from 200 °C (held 5 min) to 230 °C at the rate of 30 °C/min and held at 230 °C for 3 min. The total time for one GC run was 14 min. The FPD temperature was maintained at 250 °C. 2.4. Concentrated fruit juice storage The concentrated fruit juice (apple, pear) specimen was collected from Shaanxi Haisheng Fresh Fruit Juice Co. Ltd of Qianxian plant (China). The physical/chemical characteristic of concentrated fresh fruit juice as follow: Brix (deg.) 70.0–70.5. Acid (Titratable) % as malic acid (wt/wt) @ 11.5°Brix: 1.5–2.5. pH (@ 11.5°Brix) 3.4– 4.2. Colour (% transmittance) @ 440 nm @ 11.5°Brix P70%. Clarity (% transmittance) @ 625 nm @11.5°Brix P97%. Absorbance (@ 420 nm @ 11.5°Brix) < 0.300. Turbidity (NTU @ 11.5°Brix) < 2.0. Before use, it would be stored in plastic containers at 4 °C. 2.5. Extraction and analysis In a typical extraction experiment, 2.0 g of concentrated fruit juice was placed in a 15 mL screw-capped centrifuge tube, 50 lL of working standard solution containing nine organophosphorus pesticides was added in it. And then, 3.0 mL of PEG 6000 stock solution (20%, W/V) was added in sequence. The mixture was diluted to the 10.0 mL with distilled water. Then 2.0 g of anhydrous sodium sulfate (Na2SO4) was subsequently added. After Na2SO4 completely dissolved, the mixture solution was stirred for 15 min on the vortex and then left to centrifugate for 5 min at 4000 rpm. The surfactant-rich phase and aqueous phase were separated; the aqueous phase was sucked out by the aid of a syringe and surfactant rich phase was left in the vial and its volume was a little less than 0.5 mL, added 0.2 mL of ethyl acetate in centrifuge tube and the preconcentrated analytes were extracted into the ethyl acetate phase by applying ultrasonic waves for 20 min. This process was controlled in a hermetical condition. Two distinct layers were formed, and the upper phase was carefully transferred to a
W.-j. Zhao et al. / Food Chemistry 127 (2011) 683–688
2.0 mL EP vial with a 0.2 mL dragon MED pipettor, and then centrifugated for 2 min at 4000 rpm for further cleanup. The clean ethyl acetate solution was then transferred to an autosample vial and analysed by GC-FPD through automatic injection. Because of using automatic injection, the study adopted the external standard method. 3. Results and discussion 3.1. Optimisation of the CPE conditions 3.1.1. Effect of surfactant concentration During CPE, it is necessary to optimise the surfactant concentration for sufficient extraction of target analytes. The effect of the surfactant concentration was determined by measuring a series of aqueous solutions containing organophosphorus pesticides, Na2SO4 (20%, W/V) and different concentrations of PEG 6000. In order to obtain the optimised extraction condition and best extraction efficiency, we used the recovery of analytes to evaluate the extraction efficiency under different conditions. We selected 5 compounds as representatives of the OPPs, and showed their behaviour under these extraction conditions. Fig. 2 shows the effect of surfactant concentration on the extraction efficiencies of the OPPs. It can be found that the extraction efficiency of five OPPs increased with concentration of the surfactants. At PEG 6000 concentration of 6% (W/V), the extraction efficiency of acephate and methamidophos was about 72%, 74%, respectively. The three others were all above 77%, and when the concentration of surfactant was beyond 6%, the efficiency remained constant. However, the increase of surfactant concentration would increase the volume and the viscidity of surfactant-rich phase, which can affect the volume of ethyl acetate in ultrasonic waves-assisted back-extraction. In this paper, in order to achieve a high preconcentration factor, 6% (W/V) of PEG 6000 was chosen as extractant. PEG 4000 and PEG 10,000 were also used as extracted reagents, through experiment, it can be found out that in the same extract conditions, the recovery of analytes was poor when using PEG 4000 and viscidity of PEG 10,000 was so high that infection of back-extraction process. On the other hand, there were many advantages of using PEG as extracted reagents. For the first; the phase balance temperature was lower while PEG was used as extracted reagents. After electrolyte addition, the two-phases were separated at room temperature. Second; the abilities of the PEGs for two-phase formation are increasing with the increased of the molecular weight (Ganong & Delmore, 1991). So it was easy for us to select extracted reagents. Third; it can achieve results similar to those obtained by traditional extraction techniques. 3.1.2. Effect of pH and temperature The physical/chemical characteristic of pH (@ 11.5°Brix) for concentrated fruit juice is 3.4–4.2. The phase behaviour was also
Fig. 2. Effect of surfactant concentration on the extraction efficiency CPE conditions: Na2SO4 20% (W/V), stir for 15 min on the vortex Back-extraction conditions: ethyl acetate 0.2 mL, ultrasonic time 20 min.
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investigated as a function of pH. The effect of pH on the extraction efficiency of nine OPPs was studied over the pH range 3.5–8.0. The experimental results indicated that exclusive of parathion-methyl and diazinon, the extraction efficiencies of all other pesticides were reduced. The Effect of pH on the extraction efficiency was shown in Fig. 3. The main reason of reduction was these pesticides can be hydrolysed in alkalescent solution. Jia et al. (2008) used pH 6.0 phosphate buffer to adjust urine specimen, we adopt this method, but the result indicated that the recovery of all pesticides had not obviously changed when compared with non-addition of phosphate buffer solution. So pH was not adjusted when using CPE. The temperature was also investigated. As a universal observation, an elevated temperature leads to dehydration of the micelle and increase of the phase-volume ratio, therefore, the pre-concentration efficiency can be enhanced. On the other hand, it was proved by experiments that the stability of most organophosphorus pesticides reduced with the rise of temperature. The surfactant of PEG 6000 can be dehydration at room temperature. Therefore, room temperature was adequate for the CPE procedure. 3.1.3. Effect of salt type and concentration Addition of a salt can increase the incompatibility between the water structures in hydration shells of ions and surfactant macromolecules which can reduce the concentration of ‘‘free water’’ in surfactant-rich phase and consequently reduce the volume of the phase. Fig. 4 shows that the salt (Na2SO4) impacts on the extraction efficiencies of pesticides. The extraction recovery varied from 15% to 93% when concentration of salt increased from 5% to 25%. Addition of salt can accelerate phase separation by enhancing the micellar concentration in the surfactant rich phase, and high concentration of salt can induce a fall of temperature of cloud point in CPE procedure. The volume of surfactant rich phase can be decreased by adding of salt, which can allow increasing pre-concentration factor, but the phase would become too sticky to be dealt with. Taking the above into account, 20% (W/V) Na2SO4 was chosen for cloud point extraction. CPE-NaCl system was also investigated, between above two CPE systems, the extraction efficiency of CPENa2SO4 system is the highest; on the other hand, when Na2SO4 was added in surfactant solution, the time achieved phase separation was the shortest. 3.1.4. Ultrasonic-assisted back-extraction In this paper, ethyl acetate was selected as back-extraction reagent. This process was controlled in a hermetical condition, which can reduce the volatilisation of ethyl acetate. Then ultrasonic was applied to back-extraction the analytes from the surfactant-rich phase into ethyl acetate. Through experiment, it can be found that ethyl acetate can provide good extraction efficiency and reproducibility. When the volume of ethyl acetate was 0.2 mL, the pesticides were quantitatively extracted into ethyl acetate within 20 min. Further application of ultrasonic up to 30 min gave no significant enhancement in the analytical response. And the two phases (ethyl
Fig. 3. Effect of pH on the extraction efficiency CPE conditions: PEG 6000 6% (W/V), Na2SO4 20% (W/V), stir for 15 min on the vortex. Back-extraction conditions: ethyl acetate 0.2 mL, ultrasonic time 20 min.
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Fig. 4. Effect of salt on the extraction efficiency CPE conditions: PEG 6000 6% (W/V), stir for 15 min on the vortex. Back-extraction conditions: ethyl acetate 0.2 mL, ultrasonic time 20 min.
acetate and surfactant-rich phase) were separated completely. Therefore, a 20 min application of ultrasonic was selected for further study. There are two important factors to be taken into account in optimising the volume of ethyl acetate: the preconcentration factor and the required volume of the automated injection. Pre-concentration factor calculated as the ratio of the volume of ethyl acetate (Vu) for back-extraction of surfactant-rich phase to the total volume of the solution (VL) used for CPE. Regarding VL/Vu, the general tendency expected would be a decrease of volume of rich phase. It can be seen from Fig. 5, the extraction recovery was relatively high when 0.2 mL of ethyl acetate was used as back-extraction, beyond 0.2 mL, the recovery of five OPPs become constant. On the other hand, the preconcentration factor reduced with increased the volume of the ethyl acetate. Taking in above two aspects, a volume of 0.2 mL was finally selected as the optimum and the preconcentration factor calculated theoretically was 50 for 10 mL of a sample. 3.2. Centrifugation cleanup During the experiment, it was found that the upper ethyl acetate phase was not clear after ultrasonic-assisted back-extraction. In order to avoid blocking of the capillary column and achieving automated injection successfully, centrifugation was adopted for further cleanup. After back-extraction, the upper ethyl acetate phase was centrifuged for 5 min at 4000 rpm, and then immediately for analysis.
Sakkas, Albanis, & Lampropoulou, 2005; Mo, Zheng, Chen, Zhao, & Yang, 2009; Sikalos & Paleologos, 2005; Zygoura, Paleologos, Riganakos, & Kontominas, 2005). In our work, we apply this technique to GC-FPD with automatic injection. According to the optimal extraction procedure, a blank concentrated fruit juice and a concentrated fruit juice spiked with 50 lg kg1 of each organophosphorous pesticides were subjected to the CPE-back extraction produce. The chromatograms were shown in Fig. 6. The blank chromatogram (Fig. 6a) presented a smooth baseline. As can be seen from the Fig. 6b, nine pesticides were separated under 14 min, and their peaks appeared free from any interference. Fig. 6c indicated in the case of the extract obtained from concentrated fruit juice spiked with 50 lg kg1 of each organophosphorus pesticide. There were nine peaks corresponding with the peaks of pesticides in Fig. 6b.
3.3.2. Linear range, detection limit and repeatability of this method Based on the method development described above, the linear range of the method was tested by varying the different concentration of the mixed nine standard solutions. The peak areas for the OPPs were proportional to their concentrations (lg kg1) in good linear relationships. The relative standard deviations (RSD) ranged between 1.03% and 8.41%. The limit of detection (LOD) of each OPPs was calculated based on a signal-to-noise ratio 3:1. The limit of quantification (LOQ) was defined as three times the LOD, the results were shown in Table 1.
3.3.3. Comparison of this method with others In order to estimate the GC-FPD method of determination of nine OPPs involving CPE-ultrasonic back extraction, the determination results in concentrated fruit juice sample were compared with this obtained by the method of S/V 0334-1995 (SN 0334-1995), which was the official method about import and export for concentrated fruit juice of determination of OPPs in China. The comparison results were shown in Table 2. Through comparison, it verified the applicability of the proposed method. To further confirm the reliability of the method, we prove the method in three different laboratories. They all verify this proposed
3.3. Analytical characteristic 3.3.1. GC-FPD analysis Cloud point extraction coupled with the assisted back extraction has been combined with GC analysis successfully applied in many studies concerning the extraction of analytes (Giokas,
Fig. 5. Effect of the volume of ethyl acetate on the extraction efficiency CPE conditions: PEG 6000 6% (W/V), Na2SO4 20% (W/V), stir for 15 min on the vortex. Back-extraction conditions: Ultrasonic time 20 min.
Fig. 6. GC-FPD chromatograms: (a) blank concentrated fruit juice, (b) standards, (c) concentrated fruit juice spiked with working mixed standard solutions (50 lg kg1). Peak No: (1) Dichlorvos; (2) Methamidophos; (3) Acephate; (4) Diazinon; (5) Dimethoate; (6) Chlorpyrifos; (7) Parathion-methyl; (8) Malathion; (9) Parathion-ethyl.
W.-j. Zhao et al. / Food Chemistry 127 (2011) 683–688 Table 1 Analytical parametres of the cloud point extraction (CPE) – back extraction method.
a b c
Organophosphorus pesticides (OPPs)
R2
RSDa (%)
Linear range (lg kg1)
(LOD)b (lg kg1)
(LOQ)c (lg kg1)
Dichlorvos Methamidophos Acephate Diazinon Dimethoate Chlorpyrifos Parathion-methyl Malathion Parathion-ethyl
0.9995 0.9994 0.9985 0.9995 0.9984 0.9999 0.9996 0.9983 0.9997
4.56 2.98 8.41 1.51 6.57 2.27 1.03 1.88 4.39
5.0–200 5.5–200 8.0–200 4.0–200 5.5–200 4.0–200 4.0–200 4.0–200 5.0–200
1.5 2.0 3.0 0.5 2.0 1.0 1.0 1.0 1.5
4.5 5.0 7.0 1.5 5.5 3.0 3.0 3.0 4.5
Relative standard deviation, n = 5. Represents of limit of detection. Represents of limit of quantification.
Table 2 Comparing the performance of this method with that of the official authority method about import and export of China. Organophosphorus pesticides (OPPs)
Added (lg kg1)
Found, lg kg1/ RSDa, % CPE-GC method
Found, lg kg1/RSDa, % SN 0334-1995 method
Dichlorvos
10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0
7.65/3.56 36.4/4.44 7.45/3.53 36.8/3.01 7.31/5.49 36.2/3.97 9.15/0.53 46.2/1.47 7.15/3.97 36.2/3.71 8.54/2.03 41.5/2.55 9.00/2.62 45.4/2.52 9.36/2.87 47.1/1.09 8.97/1.88 45.1/1.13
8.31/4.44 41.4/3.87 7.65/2.59 37.4/2.16 7.24/3.10 35.4/2.01 8.87/0.56 45.8/1.07 8.47/2.31 42.5/1.97 9.71/4.79 47.5/5.19 9.06/6.55 44.2/4.39 9.11/2.33 44.7/1.06 9.28/1.68 46.6/1.47
Methamidophos Acephate Diazinon Dimethoate Chlorpyrifos Parathion-methyl Malathion Parathion-ethyl a
Relative standard deviation, n = 5.
Table 3 Recovery of the cloud point extraction (CPE) – back extraction method.
a
Organophosphorus pesticides (OPPs)
Added (lg kg1)
Found, lg kg1/ RSDa, (%)
Average recovery (%)
Dichlorvos Methamidophos Acephate Diazinon Dimethoate Chlorpyrifos Parathion-methyl Malathion Parathion-ethyl
50 50 50 50 50 50 50 50 50
35.8/3.98 38.7/2.07 36.0/5.14 46.8/3.55 38.2/1.86 40.7/2.89 44.6/2.04 47.3/0.98 46.5/1.90
71.6 77.4 72.0 93.6 76.4 81.4 89.2 94.6 93.0
Relative standard deviation, n = 5.
method for determination of nine organophosphorus pesticides in concentrated fruit juice. 3.3.4. Interference test For the above mentioned experiment, concerning interference of foreign chlororganic pesticides with OPPs was necessary. 20.00 mg L1 mixed standard solution containing the a-HCH, bHCH, c-HCH, d-HCH, heptachlor, aldrin, dieldrin, O,P0 -DDT, P,P0 DDE, P,P0 -DDD and P,P0 -DDT, were prepared in petroleum ether. Working mixed standard solutions 2.0 mg L1 were obtained by diluting mixed standard solution with petroleum ether. Before extraction experiment, 50 lL of working mixed standard solutions
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contained chlororganic pesticides was added in 2.0 g of concentrated fruit juice, then the pre-processing and analytical process were done according to the conditions mentioned above. The recovery results of OPPs were shown in Table 3. It can be seen that the recovery of OPPs had not changed when compared with nonaddition of chlororganic pesticides.
4. Conclusions In order to overcome the problems arising from the use of organic solvents in an extraction procedure, CPE coupled with ultrasonic-assisted back-extraction for determination of nine OPPs in concentrated fruit juice by GC-FPD with automated injection was successfully developed. The upper ethyl acetate phase obtained from back-extraction was centrifugated for further cleanup to achieve automatic injection. The performance of the experiment indicates that for determination of nine OPPs in concentrated fruit juice, it can be used as a rapid, easy, sensitive, convenient and practical method.
Acknowledgements The authors acknowledge the financial support from the Shannxi Haisheng Fresh Fruit Juice Co. Ltd of Qianxian Plant in China. The authors also acknowledge the support of Qianxian Plant for providing the sample of concentrated fruit (apple, pear) juice. The author thanks three other laboratories for further confirmation of this method.
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Sanz, G. P., Halko, R., Ferrera, Z. S., & Rodriguez, J. J. S. (2004). Micellar extraction of organophosphorus pesticides and their determination by liquid chromatography. Analytica Chimica Acta, 524, 265–270. Shen, J. C., & Shao, X. G. (2006). Determination of tobacco alkaloids by gas chromatography–mass spectrometry using cloud point extraction as a preconcentration step. Analytica Chimica Acta, 561, 83–87. Sikalos, T. I., & Paleologos, E. K. (2005). Cloud point extraction coupled with microwave or ultrasonic assisted back extraction as a preconcentration step prior to gas chromatography. Analytical Chemistry, 77, 2544–2549. SN 0334-1995, Method for the determination of 22 organophosphorus pesticides multi-residues in fruits and vegetable for export. Zhu, F., Ruan, W. H., He, M. H., Zeng, F., Luan, T. G., & Tong, Y. X. (2009). Application of solid-phase microextraction for the determination of organophosphorus pesticides in textiles by gas chromatography with mass spectrometry. Analytica Chimica Acta, 650, 202–206. Zygoura, P. D., Paleologos, E. K., Riganakos, K. A., & Kontominas, M. G. (2005). Determination of diethylhexyladipate and acetyltributylcitrate in aqueous extracts after cloud point extraction coupled with microwave assisted back extraction and gas chromatographic separation. Journal of Chromatography A, 1093, 29–35.
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Analytical Methods
Cloud point extraction coupled with ultrasonic-assisted back-extraction for the determination of organophosphorus pesticides in concentrated fruit juice by gas chromatography with flame photometric detection Wei-jun Zhao a, Xiao-ke Sun a, Xiao-ni Deng b, Lin Huang c, Ming-min Yang d, Zhi-ming Zhou d,⇑ a
Department of Quality Assurance, Shaanxi Haisheng Fresh Fruti Juice Co., Ltd., Qianxian, Shaanxi Province 713300, China Ministry of Agriculture Supervision & Center for Fisheries Environment and Quality of Fishery Products (Xi’an), Xi’an, Shaanxi Province 710086, China The Nineth Department of Xi’an Modern Chemistry Research Institute, Xi’an, Shaanxi Province 710065, China d College of Science, Nanjing Agricultural University, Nanjing, Jiangsu Province 210095, China b c
a r t i c l e
i n f o
Article history: Received 23 December 2009 Received in revised form 27 October 2010 Accepted 29 December 2010 Available online 13 January 2011 Keywords: Cloud point extraction (CPE) Organophosphorus pesticides (OPPs) Ultrasonic-assisted back-extraction Gas chromatography with flame photometric detection (GC-FPD) Concentrated fruit juice
a b s t r a c t A new method for the determination of nine organophosphorus pesticides (OPPs): Dichlorvos, methamidophos, acephate, diazinon, dimethoate, chlorpyrifos, parathion-methyl, malathion and parathion-ethyl in concentrated fruit juice was developed using the cloud point extraction coupled with ultrasonicassisted back-extraction prior to gas chromatography with flame photometric detection (GC-FPD) analysis. The parameters and variables that affect the extraction were investigated. Under optimum conditions: a solution containing 6% (W/V) polyethylene glycol 6000 (PEG 6000) and 20% (W/V) Na2SO4 for the extraction of the OPPs. The coacervation phase obtained was back extracted with ethyl acetate. The upper ethyl acetate solution was centrifugated simply for further cleanup for the sake of automatic injection. A preconcentration factor of 50 was obtained for these nine pesticides. Using this method, the limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.5–3.0 and 1.5–9.0 lg kg1 in concentrated fruit juice, respectively; the relative standard deviations (RSD) were <9%. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Organophosphorus pesticides are an important source of environmental contamination owning to their widespread use in agriculture; this involves their later passage to the fruit with the ensuing risk for the population. Studies showed that mixture of cholinesterase-inhibiting OPPs formation adversely affected embryogenesis in mice which could have implications for susceptible population from occupational and environmental exposures (Gomes, Anilal, & Lloyd,1999). In addition, OPPs were known to reduce the activity of neurotransmitters and hence to cause irreversible effects on the nervous system. Therefore, accomplishment of sensitive, rapid and simple analytical methods of the OPPs in concentrated fruit juice are paramount. Several studies have been reported on the analysis of organophosphorus pesticides (Ahmadi, Assadi, Milani Hosseini, & Rezaee, 2006; Jin et al., 2004; Lambropolou & Albanis, 2003; Lin, Huang, & Liu, 2006; Pinto, pavón, & Cordero, 1995; Rastrelli, Totaro, & Simone, 2002). Pesticide samples are usually enriched by liquid–liquid extraction (Barcelo, Porte, Cid, & Albaines, 1990; Hyötyläinen, Lüthje, Rautiainen-Rämä, &
⇑ Corresponding author. Fax: +86 25 84395255. E-mail address: [email protected] (Z.-m. Zhou). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.122
Riekkola, 2004) or solid-phase extraction (Liu & Lee, 1998; Martinez, Gonzalo, Moran, & Mendez, 1992; Zhu et al., 2009); these conventional methods have been involved in an extraction technique which suffers from some disadvantages such as a great expense and toxicity of solvents used. The cloud point extraction (CPE) was introduced for the first time by Watanabe and co-workers in 1976 (Miura, Ishii, & Watanabe, 1976): Cloud point of aqueous solutions of the surfactant micellar systems is a temperature at which the solution becomes turbid before separating into two phases, i.e. a surfactant-rich phase and an aqueous phase. The surfactant-rich phase is able to extract and pre-concentrate analytes. The CPE methodology based on surfactant-mediated phase separation has been successfully used for the preconcentration of species of widely differing character as a previous step to their later determination by high-performance liquid chromatography (HPLC) and/or gas chromatography (GC) (Sanz, Halko, Ferrera, & Rodriguez, 2004; Shen & Shao, 2006). Cloud point extraction methodology can offer an interesting alternative to the extraction systems due to many advantages. Surfactants are less toxic and cheaper than the organic solvents. CPE method with surfactant provides possibility of extracting and preconcentrating analysis in a single step using a simple procedure, and also the CPE technique can achieve results similar to those obtained by traditional extraction techniques. The surfactant-rich
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phase is compatible with hydroorganic mobile phases, so CPE has been exploited to combine with HPLC. On the other hand, the application of CPE as a preconcentration followed by GC is very rare; the reason is mainly because the viscous nature of the surfactant endangers the blocking of the capillary column. Methods to avoid these disadvantages were reported (Froschl, Stangl, Niessner, & Fresenius, 1997). In this work, we described a competitive method of CPE for the rapid and effective extraction and preconcentration of nine OPPs from concentrated fruit juice coupled with GC-FPD by automated injection. The preconcentrated analytes were back-extracted from the obtained surfactant-rich phase into ethyl acetate by the assisted of ultrasonic. The ethyl acetate solution obtained from back-extraction was centrifuged for further cleanup and then directly injected into the GC for analysis. The aim of this paper was to investigate the applicability of cloud point extraction coupled with ultrasonic-assisted back-extraction to extract and preconcentrate organophosphorous pesticides from concentrated fruit juice (apple, pear) prior to gas chromatography.
2. Experimental 2.1. Reagents and solutions Nine organophosphorus pesticides (dichlorvos, methamidophos, acephate, diazinon, dimethoate, chlorpyrifos, parathionmethyl, malathion and parathion-ethyl) were purchased from Dr. Ehrenstorfer (Augsburg, Germany); purity P96%. Fig. 1 shows the structures of nine organophosphorus pesticides. 0.01000 g of each pesticide was dissolved in 10.00 mL ethyl acetate (Damstadt, Germany) to obtain a standard stock solution with a concentration of 1000 mg L1, A fresh 20.00 mg L1 standard solution containing the nine pesticides was prepared in ethyl acetate every four days and stored at 4 °C. Working mixed standard solutions (0.4 and 2.0 mg L1) were obtained everyday by diluting intermediary mixed standard solution. Anhydrous sodium sulfate and sodium chloride were purchased from Bodi Chemical Reagent Co. (Tianjin, China). Polyethylene glycol (PEG) 4000, 6000, 10,000 were O O O P O
Cl Cl
C 4H 7Cl 2O 4P Mol. Wt.: 220.98 Dichlorvos
O
N N
O O P S
O P O N H S C 4H 10NO 3PS Mol. Wt.: 183.17 Acephate O
O P O H 2N S C 2H 8NO 2PS Mol. Wt.: 141.13 Methamidophos
Cl S O P O O
S P O S O
H N
Cl N
Cl
O C12H21N2O3PS Mol. Wt.: 304.35 Diazinon ON+ O S O P O O C8H10NO 5PS Mol. Wt.: 263.21 Parathion-methyl
C9H11Cl 3NO 3PS Mol. Wt.: 350.59 Chlorpyrifos
C5H12NO 3PS2 Mol. Wt.: 229.26 Dimethoate
O O O
O O
S S P O O C10H19O6PS 2 Mol. Wt.: 330.36 Malathion
ON+ S O P O O C10H14NO 5PS Mol. Wt.: 291.26 Parathion-ethyl
Fig. 1. The structures of nine organophosphorus pesticides.
obtained from Guangdong Guanghua Chemical Factory (Guangdong, China) and dissolved with distilled water to obtain stock solution of 20% (W/V). 2.2. Apparatus A gas chromatograph (GC-17A) (Shimadzu, Japan) with a split/ splitless injector system, and a flame photometric detection with phosphorus filter were used. Ultra pure nitrogen (99.9999%, China) was passed through a molecular sieve trap and an oxygen trap (Beijing, China) was used as the carrier gas at a constant linear velocity of 44.6 cm/s. The injection port was held at 250 °C and used in the splitless mode with splitless time of 1.2 min. For reduction of degradation products deactivated glass liner was used. Long life septum (Chromseal) was used in the injection port. Separation was carried out on a DB-1701, 30 m 0.32 mm capillary column with a 0.25 lm stationary film thickness (Agilent Technologies). Hydrogen gas was generated with hydrogen generator of GH500B from ZhongXingHuiLi (Beijing, China) for FPD at a flow of 130 mL/min. The flow of zero air (99.999%) from HuiLong (Berjing, China) for FPD was 100 mL/min. An ultrasonic from Kunshan ultrasonic instrument plant (Jiangsu, China) was used as back-extraction of samples. A centrifuge from Feige instrument and metre Co. (Shanghai, China) was used to accelerate phase separation. 2.3. GC analytical conditions In order to acquire the optimum conditions for OPPs determination, the oven temperature was programmed as follows: initial 150 °C, from 150 °C (held for 0.5 min) to 185 °C at the rate of 20 °C/min; from 185 °C (held 2.0 min) to 200 °C at the rate of 20 °C/min; from 200 °C (held 5 min) to 230 °C at the rate of 30 °C/min and held at 230 °C for 3 min. The total time for one GC run was 14 min. The FPD temperature was maintained at 250 °C. 2.4. Concentrated fruit juice storage The concentrated fruit juice (apple, pear) specimen was collected from Shaanxi Haisheng Fresh Fruit Juice Co. Ltd of Qianxian plant (China). The physical/chemical characteristic of concentrated fresh fruit juice as follow: Brix (deg.) 70.0–70.5. Acid (Titratable) % as malic acid (wt/wt) @ 11.5°Brix: 1.5–2.5. pH (@ 11.5°Brix) 3.4– 4.2. Colour (% transmittance) @ 440 nm @ 11.5°Brix P70%. Clarity (% transmittance) @ 625 nm @11.5°Brix P97%. Absorbance (@ 420 nm @ 11.5°Brix) < 0.300. Turbidity (NTU @ 11.5°Brix) < 2.0. Before use, it would be stored in plastic containers at 4 °C. 2.5. Extraction and analysis In a typical extraction experiment, 2.0 g of concentrated fruit juice was placed in a 15 mL screw-capped centrifuge tube, 50 lL of working standard solution containing nine organophosphorus pesticides was added in it. And then, 3.0 mL of PEG 6000 stock solution (20%, W/V) was added in sequence. The mixture was diluted to the 10.0 mL with distilled water. Then 2.0 g of anhydrous sodium sulfate (Na2SO4) was subsequently added. After Na2SO4 completely dissolved, the mixture solution was stirred for 15 min on the vortex and then left to centrifugate for 5 min at 4000 rpm. The surfactant-rich phase and aqueous phase were separated; the aqueous phase was sucked out by the aid of a syringe and surfactant rich phase was left in the vial and its volume was a little less than 0.5 mL, added 0.2 mL of ethyl acetate in centrifuge tube and the preconcentrated analytes were extracted into the ethyl acetate phase by applying ultrasonic waves for 20 min. This process was controlled in a hermetical condition. Two distinct layers were formed, and the upper phase was carefully transferred to a
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2.0 mL EP vial with a 0.2 mL dragon MED pipettor, and then centrifugated for 2 min at 4000 rpm for further cleanup. The clean ethyl acetate solution was then transferred to an autosample vial and analysed by GC-FPD through automatic injection. Because of using automatic injection, the study adopted the external standard method. 3. Results and discussion 3.1. Optimisation of the CPE conditions 3.1.1. Effect of surfactant concentration During CPE, it is necessary to optimise the surfactant concentration for sufficient extraction of target analytes. The effect of the surfactant concentration was determined by measuring a series of aqueous solutions containing organophosphorus pesticides, Na2SO4 (20%, W/V) and different concentrations of PEG 6000. In order to obtain the optimised extraction condition and best extraction efficiency, we used the recovery of analytes to evaluate the extraction efficiency under different conditions. We selected 5 compounds as representatives of the OPPs, and showed their behaviour under these extraction conditions. Fig. 2 shows the effect of surfactant concentration on the extraction efficiencies of the OPPs. It can be found that the extraction efficiency of five OPPs increased with concentration of the surfactants. At PEG 6000 concentration of 6% (W/V), the extraction efficiency of acephate and methamidophos was about 72%, 74%, respectively. The three others were all above 77%, and when the concentration of surfactant was beyond 6%, the efficiency remained constant. However, the increase of surfactant concentration would increase the volume and the viscidity of surfactant-rich phase, which can affect the volume of ethyl acetate in ultrasonic waves-assisted back-extraction. In this paper, in order to achieve a high preconcentration factor, 6% (W/V) of PEG 6000 was chosen as extractant. PEG 4000 and PEG 10,000 were also used as extracted reagents, through experiment, it can be found out that in the same extract conditions, the recovery of analytes was poor when using PEG 4000 and viscidity of PEG 10,000 was so high that infection of back-extraction process. On the other hand, there were many advantages of using PEG as extracted reagents. For the first; the phase balance temperature was lower while PEG was used as extracted reagents. After electrolyte addition, the two-phases were separated at room temperature. Second; the abilities of the PEGs for two-phase formation are increasing with the increased of the molecular weight (Ganong & Delmore, 1991). So it was easy for us to select extracted reagents. Third; it can achieve results similar to those obtained by traditional extraction techniques. 3.1.2. Effect of pH and temperature The physical/chemical characteristic of pH (@ 11.5°Brix) for concentrated fruit juice is 3.4–4.2. The phase behaviour was also
Fig. 2. Effect of surfactant concentration on the extraction efficiency CPE conditions: Na2SO4 20% (W/V), stir for 15 min on the vortex Back-extraction conditions: ethyl acetate 0.2 mL, ultrasonic time 20 min.
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investigated as a function of pH. The effect of pH on the extraction efficiency of nine OPPs was studied over the pH range 3.5–8.0. The experimental results indicated that exclusive of parathion-methyl and diazinon, the extraction efficiencies of all other pesticides were reduced. The Effect of pH on the extraction efficiency was shown in Fig. 3. The main reason of reduction was these pesticides can be hydrolysed in alkalescent solution. Jia et al. (2008) used pH 6.0 phosphate buffer to adjust urine specimen, we adopt this method, but the result indicated that the recovery of all pesticides had not obviously changed when compared with non-addition of phosphate buffer solution. So pH was not adjusted when using CPE. The temperature was also investigated. As a universal observation, an elevated temperature leads to dehydration of the micelle and increase of the phase-volume ratio, therefore, the pre-concentration efficiency can be enhanced. On the other hand, it was proved by experiments that the stability of most organophosphorus pesticides reduced with the rise of temperature. The surfactant of PEG 6000 can be dehydration at room temperature. Therefore, room temperature was adequate for the CPE procedure. 3.1.3. Effect of salt type and concentration Addition of a salt can increase the incompatibility between the water structures in hydration shells of ions and surfactant macromolecules which can reduce the concentration of ‘‘free water’’ in surfactant-rich phase and consequently reduce the volume of the phase. Fig. 4 shows that the salt (Na2SO4) impacts on the extraction efficiencies of pesticides. The extraction recovery varied from 15% to 93% when concentration of salt increased from 5% to 25%. Addition of salt can accelerate phase separation by enhancing the micellar concentration in the surfactant rich phase, and high concentration of salt can induce a fall of temperature of cloud point in CPE procedure. The volume of surfactant rich phase can be decreased by adding of salt, which can allow increasing pre-concentration factor, but the phase would become too sticky to be dealt with. Taking the above into account, 20% (W/V) Na2SO4 was chosen for cloud point extraction. CPE-NaCl system was also investigated, between above two CPE systems, the extraction efficiency of CPENa2SO4 system is the highest; on the other hand, when Na2SO4 was added in surfactant solution, the time achieved phase separation was the shortest. 3.1.4. Ultrasonic-assisted back-extraction In this paper, ethyl acetate was selected as back-extraction reagent. This process was controlled in a hermetical condition, which can reduce the volatilisation of ethyl acetate. Then ultrasonic was applied to back-extraction the analytes from the surfactant-rich phase into ethyl acetate. Through experiment, it can be found that ethyl acetate can provide good extraction efficiency and reproducibility. When the volume of ethyl acetate was 0.2 mL, the pesticides were quantitatively extracted into ethyl acetate within 20 min. Further application of ultrasonic up to 30 min gave no significant enhancement in the analytical response. And the two phases (ethyl
Fig. 3. Effect of pH on the extraction efficiency CPE conditions: PEG 6000 6% (W/V), Na2SO4 20% (W/V), stir for 15 min on the vortex. Back-extraction conditions: ethyl acetate 0.2 mL, ultrasonic time 20 min.
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Fig. 4. Effect of salt on the extraction efficiency CPE conditions: PEG 6000 6% (W/V), stir for 15 min on the vortex. Back-extraction conditions: ethyl acetate 0.2 mL, ultrasonic time 20 min.
acetate and surfactant-rich phase) were separated completely. Therefore, a 20 min application of ultrasonic was selected for further study. There are two important factors to be taken into account in optimising the volume of ethyl acetate: the preconcentration factor and the required volume of the automated injection. Pre-concentration factor calculated as the ratio of the volume of ethyl acetate (Vu) for back-extraction of surfactant-rich phase to the total volume of the solution (VL) used for CPE. Regarding VL/Vu, the general tendency expected would be a decrease of volume of rich phase. It can be seen from Fig. 5, the extraction recovery was relatively high when 0.2 mL of ethyl acetate was used as back-extraction, beyond 0.2 mL, the recovery of five OPPs become constant. On the other hand, the preconcentration factor reduced with increased the volume of the ethyl acetate. Taking in above two aspects, a volume of 0.2 mL was finally selected as the optimum and the preconcentration factor calculated theoretically was 50 for 10 mL of a sample. 3.2. Centrifugation cleanup During the experiment, it was found that the upper ethyl acetate phase was not clear after ultrasonic-assisted back-extraction. In order to avoid blocking of the capillary column and achieving automated injection successfully, centrifugation was adopted for further cleanup. After back-extraction, the upper ethyl acetate phase was centrifuged for 5 min at 4000 rpm, and then immediately for analysis.
Sakkas, Albanis, & Lampropoulou, 2005; Mo, Zheng, Chen, Zhao, & Yang, 2009; Sikalos & Paleologos, 2005; Zygoura, Paleologos, Riganakos, & Kontominas, 2005). In our work, we apply this technique to GC-FPD with automatic injection. According to the optimal extraction procedure, a blank concentrated fruit juice and a concentrated fruit juice spiked with 50 lg kg1 of each organophosphorous pesticides were subjected to the CPE-back extraction produce. The chromatograms were shown in Fig. 6. The blank chromatogram (Fig. 6a) presented a smooth baseline. As can be seen from the Fig. 6b, nine pesticides were separated under 14 min, and their peaks appeared free from any interference. Fig. 6c indicated in the case of the extract obtained from concentrated fruit juice spiked with 50 lg kg1 of each organophosphorus pesticide. There were nine peaks corresponding with the peaks of pesticides in Fig. 6b.
3.3.2. Linear range, detection limit and repeatability of this method Based on the method development described above, the linear range of the method was tested by varying the different concentration of the mixed nine standard solutions. The peak areas for the OPPs were proportional to their concentrations (lg kg1) in good linear relationships. The relative standard deviations (RSD) ranged between 1.03% and 8.41%. The limit of detection (LOD) of each OPPs was calculated based on a signal-to-noise ratio 3:1. The limit of quantification (LOQ) was defined as three times the LOD, the results were shown in Table 1.
3.3.3. Comparison of this method with others In order to estimate the GC-FPD method of determination of nine OPPs involving CPE-ultrasonic back extraction, the determination results in concentrated fruit juice sample were compared with this obtained by the method of S/V 0334-1995 (SN 0334-1995), which was the official method about import and export for concentrated fruit juice of determination of OPPs in China. The comparison results were shown in Table 2. Through comparison, it verified the applicability of the proposed method. To further confirm the reliability of the method, we prove the method in three different laboratories. They all verify this proposed
3.3. Analytical characteristic 3.3.1. GC-FPD analysis Cloud point extraction coupled with the assisted back extraction has been combined with GC analysis successfully applied in many studies concerning the extraction of analytes (Giokas,
Fig. 5. Effect of the volume of ethyl acetate on the extraction efficiency CPE conditions: PEG 6000 6% (W/V), Na2SO4 20% (W/V), stir for 15 min on the vortex. Back-extraction conditions: Ultrasonic time 20 min.
Fig. 6. GC-FPD chromatograms: (a) blank concentrated fruit juice, (b) standards, (c) concentrated fruit juice spiked with working mixed standard solutions (50 lg kg1). Peak No: (1) Dichlorvos; (2) Methamidophos; (3) Acephate; (4) Diazinon; (5) Dimethoate; (6) Chlorpyrifos; (7) Parathion-methyl; (8) Malathion; (9) Parathion-ethyl.
W.-j. Zhao et al. / Food Chemistry 127 (2011) 683–688 Table 1 Analytical parametres of the cloud point extraction (CPE) – back extraction method.
a b c
Organophosphorus pesticides (OPPs)
R2
RSDa (%)
Linear range (lg kg1)
(LOD)b (lg kg1)
(LOQ)c (lg kg1)
Dichlorvos Methamidophos Acephate Diazinon Dimethoate Chlorpyrifos Parathion-methyl Malathion Parathion-ethyl
0.9995 0.9994 0.9985 0.9995 0.9984 0.9999 0.9996 0.9983 0.9997
4.56 2.98 8.41 1.51 6.57 2.27 1.03 1.88 4.39
5.0–200 5.5–200 8.0–200 4.0–200 5.5–200 4.0–200 4.0–200 4.0–200 5.0–200
1.5 2.0 3.0 0.5 2.0 1.0 1.0 1.0 1.5
4.5 5.0 7.0 1.5 5.5 3.0 3.0 3.0 4.5
Relative standard deviation, n = 5. Represents of limit of detection. Represents of limit of quantification.
Table 2 Comparing the performance of this method with that of the official authority method about import and export of China. Organophosphorus pesticides (OPPs)
Added (lg kg1)
Found, lg kg1/ RSDa, % CPE-GC method
Found, lg kg1/RSDa, % SN 0334-1995 method
Dichlorvos
10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0 10.0 50.0
7.65/3.56 36.4/4.44 7.45/3.53 36.8/3.01 7.31/5.49 36.2/3.97 9.15/0.53 46.2/1.47 7.15/3.97 36.2/3.71 8.54/2.03 41.5/2.55 9.00/2.62 45.4/2.52 9.36/2.87 47.1/1.09 8.97/1.88 45.1/1.13
8.31/4.44 41.4/3.87 7.65/2.59 37.4/2.16 7.24/3.10 35.4/2.01 8.87/0.56 45.8/1.07 8.47/2.31 42.5/1.97 9.71/4.79 47.5/5.19 9.06/6.55 44.2/4.39 9.11/2.33 44.7/1.06 9.28/1.68 46.6/1.47
Methamidophos Acephate Diazinon Dimethoate Chlorpyrifos Parathion-methyl Malathion Parathion-ethyl a
Relative standard deviation, n = 5.
Table 3 Recovery of the cloud point extraction (CPE) – back extraction method.
a
Organophosphorus pesticides (OPPs)
Added (lg kg1)
Found, lg kg1/ RSDa, (%)
Average recovery (%)
Dichlorvos Methamidophos Acephate Diazinon Dimethoate Chlorpyrifos Parathion-methyl Malathion Parathion-ethyl
50 50 50 50 50 50 50 50 50
35.8/3.98 38.7/2.07 36.0/5.14 46.8/3.55 38.2/1.86 40.7/2.89 44.6/2.04 47.3/0.98 46.5/1.90
71.6 77.4 72.0 93.6 76.4 81.4 89.2 94.6 93.0
Relative standard deviation, n = 5.
method for determination of nine organophosphorus pesticides in concentrated fruit juice. 3.3.4. Interference test For the above mentioned experiment, concerning interference of foreign chlororganic pesticides with OPPs was necessary. 20.00 mg L1 mixed standard solution containing the a-HCH, bHCH, c-HCH, d-HCH, heptachlor, aldrin, dieldrin, O,P0 -DDT, P,P0 DDE, P,P0 -DDD and P,P0 -DDT, were prepared in petroleum ether. Working mixed standard solutions 2.0 mg L1 were obtained by diluting mixed standard solution with petroleum ether. Before extraction experiment, 50 lL of working mixed standard solutions
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contained chlororganic pesticides was added in 2.0 g of concentrated fruit juice, then the pre-processing and analytical process were done according to the conditions mentioned above. The recovery results of OPPs were shown in Table 3. It can be seen that the recovery of OPPs had not changed when compared with nonaddition of chlororganic pesticides.
4. Conclusions In order to overcome the problems arising from the use of organic solvents in an extraction procedure, CPE coupled with ultrasonic-assisted back-extraction for determination of nine OPPs in concentrated fruit juice by GC-FPD with automated injection was successfully developed. The upper ethyl acetate phase obtained from back-extraction was centrifugated for further cleanup to achieve automatic injection. The performance of the experiment indicates that for determination of nine OPPs in concentrated fruit juice, it can be used as a rapid, easy, sensitive, convenient and practical method.
Acknowledgements The authors acknowledge the financial support from the Shannxi Haisheng Fresh Fruit Juice Co. Ltd of Qianxian Plant in China. The authors also acknowledge the support of Qianxian Plant for providing the sample of concentrated fruit (apple, pear) juice. The author thanks three other laboratories for further confirmation of this method.
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