Food Chemistry 145 (2014) 41–48

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Analytical Methods

Determination of eight triazine herbicide residues in cereal and vegetable by micellar electrokinetic capillary chromatography with on-line sweeping Rou Fang, Guan-hua Chen ⇑, Ling-xiao Yi, Yu-xiu Shao, Li Zhang, Qing-hong Cai, Jing Xiao College of Food and Bioengineering, Jiangsu University, Zhenjiang 212013, China

a r t i c l e

i n f o

Article history: Received 21 February 2013 Received in revised form 5 August 2013 Accepted 7 August 2013 Available online 14 August 2013 Keywords: Sweeping Micellar electrokinetic capillary chromatography Triazine herbicide residues Cereal Vegetable

a b s t r a c t A new method was developed for the determination of eight triazine herbicide residues in cereal and vegetable samples by on-line sweeping technique in micellar electrokinetic capillary chromatography (MEKC). Some factors affecting analyte enrichment and separation efficiency were examined. The optimum buffer was composed of 25 mM borate, 15 mM phosphate, 40 mM sodium dodecylsulfate (SDS) and 3% (v/v) of 1-propanol at pH 6.5. The separation voltage was 20 kV and the sample was injected at 0.5 psi for 240 s. The detection wavelength was set at 220 nm with the capillary temperature being at 25 °C. Under the optimized conditions, the enrichment factors were achieved from 479 to 610. The limits of detection (LODs, S/N = 3) ranged from 0.02 to 0.04 ng/g and the limits of quantification (LOQs) of eight triazine herbicides were all 0.1 ng/g. The average recoveries of spiked samples were 82.8–96.8%. This method has been successfully applied to the determination of the triazine herbicide residues in cereal and vegetable samples. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Triazines are widely used in agriculture around the world as selective pre- and post-emergence herbicides for the control of broadleaf and grassy weeds. With high toxicity and persistence, triazines can contaminate the environment and crops. They are also dangerous to human health causing cancers, birth defects and interruption of hormone functions (Baranowska, Barchan´ska, & Pacak, 2006). Therefore, the maximum residue limits (MRLs) of triazines in foods have been established by many countries and organizations around the world. For example, the MRL for simazine in rice set by European Union (EU) is 0.1 mg/kg and the MRL for atrazine in vegetable established by Japan is 0.02 mg/kg (WTO/ TBT-SPS notification and enquiry of China). Currently, most of the reported methods for the determination of triazines in environment and food samples mainly involve gas chromatography (GC) (Mendaš, Drevenkar, & Zupancˇicˇ-Kralj, 2001; Mendaš, Tkalcˇevic´, & Drevenkar, 2000; Stipicˇevic´, Fingler, Zupancˇicˇ, & Drevenkar, 2003), high performance liquid chromatography (HPLC) (Cheng et al., 2007; Li et al., 2010; Shah, Rasul, Ara, & Shehzad, 2011; Wang et al., 2010; Wu, Liu, Wu, Wang, & Wang, 2012), gas chromatography–mass spectrometry (GC–MS) (Nagaraju, & Huang, ⇑ Corresponding author. Address: College of Food and Bioengineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China. Tel./fax: +86 511 88780201. E-mail address: [email protected] (G.-h. Chen). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.028

2007; Rocha, Pappas, & Huang, 2008) and liquid chromatography– mass spectrometry (LC–MS) (Bichon, Dupuis, Bizec, & André, 2006; Mou, Chen, Cao, & Zhu, 2011). These methods are highly sensitive but usually require tedious clean-up procedures in order to prevent chromatographic column from contamination because the matrix components of some samples are extremely complicated. In addition, a large amount of organic solvent used in these methods makes the operation costly. Capillary electrophoresis (CE) based assays can avoid these issues, and has advantages such as short analysis time, high separation efficiency and low operation cost when compared with chromatographic techniques. However, CE with UV detection suffers from the poor sensitivity due to the small inner diameter of the capillary (25–100 lm) and the small sample injection volume (nL), which significantly limit the CE application to pesticide residue analysis. High-sensitivity detectors, such as laser-induced fluorescence detector, can be adopted as suitable approaches to solve above problems (Chen, Sun, Dai, & Dong, 2012; Guo et al., 2012). Off-line or on-line sample preconcentration techniques are also popular strategies to improve CE sensitivity. Several off-line preconcentration methods have been developed (Carabias-Martínez, RodríguezGonzalo, Domínguez-Alvárez, & Hernández-Méndez, 2002; Frías, Sánchez, & Rodríguez, 2004; Frías-García, Sánchez, & RodríguezDelado, 2004). However, the application of this method was time-consuming and complicated pretreatment was usually needed. On-line sample preconcentration by either stacking or sweeping can also be used to increase sensitivity of CE. Sweeping

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R. Fang et al. / Food Chemistry 145 (2014) 41–48

is an effective on-line sample preconcentration technique in MEKC. Depending on the nature of different neutral analytes, the method can provide concentration factors of 10–5000 without complicated cleanup and off-line preconcentration procedures (Quirino, & Terabe, 1998), which makes CE a feasible trace analysis technique and has a great potential for the broad application in pesticide residue analysis. To date, it has been reported in several articles that the triazine herbicides are analyzed by CE with on-line sample preconcentration technique. However, the types of the analyzed triazine herbicides were no more than four and only dozens of times enhancement in sensitivity has been obtained at most (da Silva, de Lima, & Tavares, 2003; Hinsmann, Arce, Rıos, & Valcarcel, 2000; Lin, Liu, Yang, Wang, & Yang, 2001; Turiel, Fernández, Pérez-Conde, & Cámara, 2000; Zhang et al., 2008). Higher enrichment factors can be obtained through increasing sample injection volume to the maximum. In this paper, the simultaneous determination of eight triazine herbicides including simazine, atrazine, simetryn, propazine, ametryn, terbuthylazine, prometryn, terbutryn in cereals and vegetables are studied by the on-line sweeping in MEKC yielding up to several 100-fold concentration factor. 2. Materials and methods 2.1. Chemicals and samples Simazine, atrazine, simetryn, propazine, ametryn, terbuthylazine, prometryn, terbutryn (all >97%) were supplied by Changxing First Chemical Co., (Huzhou, China). Sodium dodecylsulphate (SDS), boric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid, sodium hydroxide, 1-propanol, 2-propanol, methanol, dichloromethane, petroleum ether, sodium chloride, anhydrous sodium sulphate were analytical grade, acetonitrile was HPLC-grade, and they were all purchased from Szinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The water was deionized to the resistivity of 18.2 MX cm. The rice and wheat harvested in Zhenjiang prefecture were provided by Zhenjiang Grain and Trex Detection Institute. The fresh carrots and chives come from Zhenjiang prefecture were purchased from local supermarket (Zhenjiang, China).

temperature water bath oscillator (Zhongtan Instrument Factory, Jintan, China). 2.3. Samples and standard solutions preparation 2.3.1. Cereal sample preparation The cereal samples (husk-removed rice and wheat grain) were crushed then screened through a 100-mesh sieve. A 10.0 g of the accurately weighed sample was put into a 50-mL screw-capped glass test tube containing 30 mL methanol–water (50:50, v/v). Then the tube was shaken in a constant temperature water bath oscillator at room temperature for 30 min. The sample was centrifugated at 4000 rpm for 5 min and the supernatant was collected. The extraction steps as mentioned above were repeated for the sediment at the bottom with 10 mL methanol–water (50:50, v/v) added in the test tube. Both supernatants were incorporated and transferred to a separating funnel containing 20 mL saturated sodium chloride solution and 30 mL deionized water. Subsequently, the dichloromethane–petroleum ether (35:65, v/ v) of 20 mL was added, and the sample solution was shaken for 1 min. The supernatant dichloromethane-petroleum ether was collected. The above-mentioned steps were repeated three times for the subnatant solution. The collected solutions were incorporated and filtered into a round bottom flask through the funnel filled with 10 g anhydrous sodium sulphate. The funnel was then washed with a little dichloromethane for several times. The filtrate was evaporated to dryness in a vacuum rotary evaporator at 60 °C. The dry residue was solved in 30 mL petroleum ether and the solution was transferred to a separating funnel. The 20 mL of acetonitrile saturated with petroleum ether was added and then the solution was vigorously shaken for 1 min. After demixing, the acetonitrile phase was collected and the same operation was repeated for the petroleum ether phase. Both acetonitrile phases were combined and evaporated to dryness at 60 °C. The residue was solved and metered volume to 1.0 mL with the solution containing 20% methanol (v/v), 25 mM borate and 15 mM phosphate at pH 6.5. The sample solution was filtered through a 0.45 lm membrane for CE analysis. The sample for HPLC analysis was prepared on the basis of the same procedures as that of CE except for metering volume to 1.0 mL with methanol.

2.2. Apparatus All CE experiments were performed on a P/ACE MDQ Capillary Electrophoresis System (Beckman Coulter, Fullerton, CA, USA) equipped with a 32 Karat ChemStation software and a diode array detector (DAD). The dimension of the uncoated fused-silica capillary was 75 lm i.d. and the total length of 60 cm with the effective length being 50 cm (Caliola Chroma Technologies Inc., Handan, China). The HPLC experiments were carried out with a FL2000 chromatograph (Fuli Instrument Co., Ltd., Hangzhou, China) equipped with binary pumps and a UV detector. The dimension of a Weltch Ultimate™ C18 chromatographic column was 4.6 mm i.d. with the length being 250 mm, and the size of packed stationary phase particle was 5.0 lm (Welch Material Inc., Shanghai, China). The other equipments used in the experiment were as follows: a pHS-2 pH meter (Shanghai Second Analytical Instrument, China), a BS 124S electronic analytical balance (Beijing Sartorius Instrument System Inc., Beijing, China), a B5500-MT ultrasonic cleaner (Branson Ultrasonics (Shanghai) Co. Ltd., Shanghai, China), a H-180 ultra-high-speed refrigerated centrifuge (XiangYi Laboratory Instrument Development Co., Ltd., Changsha, China), a WK-1000A high-speed pulverizer (Jingcheng Machinery Co., Ltd., Qingzhou, China), a RE-2000 rotary evaporator (Yarong Biochemical Instrument Factory, Shanghai, China) and a SHA-B constant

2.3.2. Vegetable sample preparation The chives (leafy vegetable) were cut into about 0.5 cm pieces after discarding the obviously decomposed or withered leaves and rinsing lightly adhered soil with running water. The carrots (root and tuber vegetable) were chopped into thin particles about 0.5 cm after lightly rinsing adhered soil with running water and truncating tops. Then a 10.0 g of vegetable sample (carrot and chive) was weighed and ground into homogenate in a mortar. Afterwards, the homogenate was transferred to a 50 mL screwcapped glass test tube, to which 30 mL dichloromethane, 5 g anhydrous sodium sulphate and 2 g sodium chloride were added. The mixture then was vigorously shaken for 1 min. The sample solution was left static at room temperature for 30 min to separate organic phase from aqueous phase and then was centrifugated at 4000 rpm for 5 min. The supernatant was collected and the sediment was repeatedly extracted with 10.0 mL dichloromethane in accordance with the above mentioned procedures. Both dichloromethane extracts were incorporated and evaporated to dryness in a vacuum rotary evaporator at 60 °C The residue was dissolved and metered volume to 1.0 mL with the solution consisting of 20% methanol (v/ v) 25 mM borate and 15 mM phosphate at pH 6.5. After that, the sample solution was filtered through a 0.45 lm membrane for CE analysis. The sample for HPLC analysis was prepared with the same

R. Fang et al. / Food Chemistry 145 (2014) 41–48

procedures as that of CE except for metering volume to 1.0 mL with methanol.

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3. Results and discussion 3.1. Mechanism of sweeping

2.3.3. Standard solution preparation Standard solutions (0.1, 0.5, 1, 5, 10, 50 and 100 ng/g) were prepared by dissolving accurately weighed amounts of eight triazines in the blank sample (rice, wheat, carrot and chive without triazine residues) solutions for detecting triazine residues in these samples by CE. Standard solutions used in HPLC were prepared with the blank sample solutions in HPLC.

2.4. Electrophoresis procedure A new capillary was preconditioned by rinsing with 1 M HCl for 5 min, deionised water for 5 min, 1 M NaOH for 30 min, deionised water for 5 min, 0.1 M NaOH for 5 min, deionised water for 5 min and running buffer for 10 min. At the beginning of the experiment each day, the capillary was conditioned by rinsing with 0.1 M NaOH for 15 min, deionised water for 15 min and running buffer for 5 min. Between two runs, the capillary was rinsed with 0.1 M NaOH and deionised water for 2 min respectively, and running buffer for 3 min. The running buffer was composed of 25 mM borate, 15 mM phosphate, 40 mM SDS and 3% 1-propanol (v/v) at pH 6.5. The sample was hydrodynamically introduced into the capillary at 0.5 psi for 240 s. Electrophoresis was performed at 20 kV and 25 °C with DAD detection at 220 nm.

2.5. Chromatographic conditions The flow rate of the mobile phase was kept at 1 mL/min. Sample injection volume was 20 lL. The mobile phase A and B was acetonitrile and water, respectively. The gradient elution was started with 0% A for the first 0.5 min, increased linearly to 45% A within 0.5 min, and held at 45% A for 9 min, then ramped linearly to 60% A within 5 min, and equilibrated for 15 min. The absorbance was measured at 220 nm.

Sweeping was an on-line sample preconcentration technique for neutral molecules in MEKC. The eight triazines were proved to be netural in the running buffer. The focusing mechanism of sweeping was illustrated in Fig. 1. A large-volume sample prepared in a matrix void of the micelles used was hydrodynamically injected into the capillary filled with micellar running buffer (Fig. 1A). The conductivity of the sample matrix was similar to that of the running buffer. The neutral analytes together with matrix in the sample plug migrated toward the detector by electroosmotic flow (EOF) under the action of voltage (Fig. 1B). Because the electrophoretic mobility of charged micelles at the front boundary of sample plug was opposite to and lower than that of EOF, the micelles, while moving toward the detector, migrated slower than the neutral analytes. Therefore, the analytes were incorporated and picked up by the micelles. Meanwhile the sample matrix crossed the boundary between sample and running buffer and gradually migrated into the running buffer by EOF. When the sample matrix completely entered the running buffer, the sweeping was accomplished (Sun, Chen, Wang, Dong, & Dai, 2010) and the analytes in a large sample zone were totally accumulated by micelles (Fig. 1C). Subsequantly, analytes were separated under applied voltage (Fig. 1D).

3.2. Optimization of the sweeping conditions 3.2.1. Effect of buffer parameters Under the condition of large sample size, the length of separation path was accordingly shortened, and therefore the sample size had an impact on the resolution of analytes. However, when the injection time was more than 30 s at 0.5 psi, the experiment showed that the buffer parameters, except organic solvent, could not significantly affect resolution. Therefore, these buffer parameters were optimized under the condition of the sample size of 0.5 psi  30 s.

Fig. 1. Sweeping steps of neutral analytes.

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R. Fang et al. / Food Chemistry 145 (2014) 41–48

3.2.1.1. Effect of buffer pH. The pH value of buffer is one of the most important parameters affecting CE separation since it determines the direction and magnitude of EOF and thus has an effect on the separation efficiency of analytes. The influence of buffer pH on the separation of eight triazines was studied under the conditions of 0.5 psi  30 s sample size and 20 kV separation voltage. Running buffer consisted of 15 mM borate, 15 mM phosphate and 40 mM SDS and the concentration of eight triazine mixture solution was 0.1 mg/L. The buffer pH varied from 6.0 to 9.5 at 0.5 pH interval. Each pH value was examined three times by replicate injections of the eight triazine mixture solution as mentioned above. Fig. 2A showed the electropherograms of eight triazines at four pH values. The resolution was very poor at pH 7.5 and only simazine and terbutryn were well resolved with the rest of triazines being observably overlapped. When the pH was higher than 7.5, atrazine and simetryn still overlapped. The best resolution was obtained at pH 6.5 and the relative standard deviation (RSD) was below 1.5%. The resolution was not changed but the analysis time was prolonged at the pH value lower than 6.5. Consequently, pH 6.5 was chosen for the further optimization of buffer parameters. 3.2.1.2. Effect of SDS concentration. SDS concentration could change the amount of analyte incorporated into micelles by affecting the retention factor k of analyte. Thereby SDS concentration could influence enrichment effect. The experiment result proved that SDS concentration affected peak area but did not have significant impact on resolution. The effect of SDS concentration was investigated under the conditions of 20 kV separation voltage and the buffer consisting of 15 mM borate, 15 mM phosphate and SDS in the range of 20 50 mM at pH 6.5. The eight triazine mixture solution of 0.1 mg/L was injected in triplicate at each SDS concentration level. In all case the RSD values of peak areas were acceptable and below 2.0%. When SDS concentration changed from 20 to 40 mM, the peak areas of analytes were gradually increased. When SDS concentration was higher than 40 mM, the peak areas were almost constant but the analysis time was increased significantly. Therefore, the best SDS concentration was 40 mM. 3.2.1.3. Effect of electrolyte concentration. As electrolyte concentration increased within a certain range, the zeta potential at the capillary wall was gradually reduced, and thereby the EOF velocity was decreased. Meanwhile the longitudinal diffusion of analytes

was also suppressed. Therefore, electrolyte concentration affected migration time and increased peak height through suppressing peak broadening. The influence of borate concentration on the separation of eight triazines was examined under the conditions of 20 kV separation voltage and the buffer consisting of 15 mM phosphate, 40 mM SDS as well as borate in the range of 10–35 mM at pH 6.5. The eight triazine mixture solution of 0.1 mg/L was injected in triplicate at each borate concentration level. The result demonstrated that migration time and peak height were increased with the increase of borate concentration varing from 10 to 25 mM. However, when borate concentration was more than 25 mM, the peak height had no change but migration time was prolonged. Thus, borate concentration was selected as 25 mM for subsequent studies and the RSD of peak height was below 1.5% at this concentration. The effect of phosphate concentration in the range of 5 30 mM was also examined by three replicate injections. Ametryn and terbuthylazine overlapped when phosphate concentration was lower than 15 mM. The baseline separation of eight triazines was achieved but the peak height was not significantly changed when phosphate concentration was within the range of 15–30 mM. However, with the increase of phosphate concentration, migration time was prolonged and the effect of Joule heat became more obvious which would cause the noisier baseline. Therefore, 15 mM phosphate concentration was selected as optimal value and the RSD of migration time was also below 1.3% at this concentration.

3.2.1.4. Effect of organic modifier. The organic modifier added to running buffer improved resolution by changing separation selectivity since they could make the alteration of interaction between micelles and analytes. In this experiment, when injection time of sample was longer than 30 s at 0.5 psi, the baseline separation of eight triazines could not be attained under the optimized buffer condition. The further improvement of resolution was necessary for achieving the baseline separation of eight triazines under the condition of the larger sample size. For this purpose, organic solvents were added to the optimal buffer. Methanol, acetonitrile, 1-propanol and 2-propanol of 3% (v/v) were all tried. The eight triazine mixture solution of 1 mg/L was injected at 0.5 psi for 120 s. The result demonstrated that resolution was very poor using methanol and acetonitrile as modifier. 2-Propanol could improve resolution, but the baseline separation of some analytes could not be still

Fig. 2. Effects of buffer pH (A), 1-propanol volume percentage (B) and injection time (C) on the separation of eight triazines (1) simazine, (2) atrazine, (3) simetryn, (4) propazine, (5) ametryn, (6) terbuthylazine, (7) prometryn, and (8) terbutryn.

R. Fang et al. / Food Chemistry 145 (2014) 41–48

achieved. However the baseline separation for all of analytes was obtained by using 1-propanol as modifier. Hence 1-propanol was chosen as organic modifier. The effect of 1-propanol volume percentage was further investigated by changing its volume percentage within 1–8%. The eight triazine mixture solution of 0.1 mg/L was injected in triplicate at each 1-propanol volume percentage. In all cases the RSDs of resolutions were within 1.8%. The results are shown in Fig. 2B. When the 1-propanol volume percentage was 3%, the baseline separation of eight triazines was gained. The resolutions of two pairs of analyte – simetryn and propazine, ametryn and terbuthylazine – were very poor when the 1-propanol concentration was lower than 3%. But when its concentration was higher than 3%, peak width became broadened and resolution decreased because the excessive addition of organic solvent diminished the amount of analytes dissolved in micelles, which caused the poor peak compression effect by sweeping. The excessive addition of organic solvent also increased residence time of neutral analytes in running buffer during separation, but the neutral analytes could not be separated only staying in aqueous phase without micelles. Thus, the volume percentage of 3% was selected. 3.2.2. Effect of applied voltage The effect of applied voltage was examined in the range 10– 30 kV by three replicate injections at each voltage using the optimized buffer (25 mM borate, 15 mM phosphate, 40 mM SDS, 3% of 1-propanol, at pH 6.5). As applied voltage increased, migration time was shortened and peak height was increased, but the resolution was decreased. When applied voltage was more than 20 kV, the peaks of atrazine and simetryn were partially overlapped and the electric current was more than 90 lA at which the effect of Joule heat became more obvious. Therefore, the applied voltage of 20 kV was selected for the experiment with satisfying migration time RSD below 1.1%. 3.2.3. Effect of sample injection time and sample matrix The length of sample plug in a capillary is in direct proportion to the product of the injection pressure and sample injection time. Thus, injection time was optimized by injecting the eight triazines mixture solution of 0.1 mg/L at 0.5 psi for 30, 60, 100, 200, 240, 280 and 300 s (three replicate injections for each injection time). In all cases, peak area RSD values were within 2.4%. Fig. 2C showed the effect of injection time on peak area and peak height. As illustrated, with the increase of injection time, peak heights and peak areas were dramatically increased. However, when the injection time was more than 240 s, peak width was also increased and two peaks (atrazine/simetryn and ametryn/terbuthylazine) were partially overlapped. Undoubtedly, the overloaded sample made the sweeping time be so long that analytes incorporated into the micelles had begun to be separated before sweeping was finished. Therefore, the injection time of 240 s at 0.5 psi was chosen as the optimum condition. When the analyte in the large sample plug was picked up by micelles, its apparent velocity was slower than that of sample matrix driven by EOF. Therefore, when the swept analyte entered the separation path, the electrolyte concentration around the analyte was mainly affected by the electrolyte concentration of sample matrix for a considerablely long distance. The influence of sample matrix on the sweeping efficiency and the detection sensitivity of analytes were identical with that of running buffer. This influence was examined with triazine mixture dissolved in the aqueous solution containing either borate in the range of 20–60 mM and phosphate in the range of 10–40 mM at pH 6.5. Each of triazine mixture with different electrolyte concentration was injected in triplicate. The selected injection time was 240 s at 0.5 psi and the applied voltage was 20 kV. The result indicated that when the concentration of

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borate and phosphate in sample zone was identical with those of running buffer, the best separation efficiency was attained. The above mentioned inference was confirmed. The 25 mM borate and 15 mM phosphate were chosen as the optimum electrolye concentration in sample with acceptable peak height reproducibility (RSD 6 1.7%). 3.3. Enrichment factor As discussed above, the optimal conditions for sweeping in MEKC were chosen as follows: the running buffer consisted of 25 mM borate, 15 mM phosphate, 40 mM SDS and 3% of 1-propanol at pH 6.5; the separation voltage was 20 kV; the sample dissolved in buffer containing 25 mM borate and 15 mM phosphate (pH 6.5) was injected at 0.5 psi for 240 s. The eight triazine mixture solution of 0.01 mg/L was separated under the optimized conditions, and their peak areas Ais were measured. The eight triazine mixture solution of 30 mg/L was also separated under the same conditions except for the sample size being 0.5 psi  3 s and their peak areas ais were determined. Each solution was injected in triplicate. The enrichment factors calculated according to the formula of fi = (Ai/ai)(30/0.01) were 496, 479, 566, 610, 503, 595, 555, 603 and the corresponding RSD values were 1.2%, 1.0%, 1.6%, 0.8%, 1.1%, 0.9%, 1.5%, 1.7% for simazine, atrazine, simetryn, propazine, ametryn, terbuthylazine, prometryn and terbutryn respectively. 3.4. Method evaluation Under the optimized conditions, the four sets of standard solution for eight triazines were determined for the calibration curves corresponding to rice, wheat, chive, and carrot matrix (three replicate injections for each concentration) and the four sets of blank samples (11 per set) were tested for the limits of detection (LODs) corresponding to these four sample matrix. The peak areas of the eight triazines were used for quantification signals as ordinate (Y) and their concentration (ng/g) as abscissa (X). The linearity of calibration curves for eight triazines was checked in a wide range of concentrations (0.1–100 ng/g) by F-test. Each standard solution was injected in triplicate. The results showed that all of the calculated F-values for eight triazines were higher than theoretical F-value 6.61 (P = 0.05) in these four sample matrix. Thus, the linearity of the method was significant. The calibration curves, linear ranges (LRs), determination coefficients (r2), LODs (S/N = 3) were summarized in Table 1. The repeatability of the method was evaluated in terms of intraday and interday precision. Intraday precision was examined by five replicate injections of the sample spiked at 20 ng/g for each triazine on the same day. Interday precision was assessed by injecting the same sample for three consecutive days (five replicate injections per each day). The RSDs of the peak areas in terms of intraday and interday precision were also shown in Table 1. As shown in the table, an acceptable precision was obtained with intraday RSD values below 1.7%, while interday RSD values within 3.3%. These results indicated that this method was repeatable and sensitive. 3.5. Analysis of sample and recoveries of spiked sample Under the optimum condition, the cereal and vegetable samples were determined and the eight triazine residues were not detected in all samples. The negative results originated from two possible reasons. One was that 80–90% of the sprayed herbicides entered the soil (Zheng, Fang, Zhou, & Liu, 2007). Triazines, such as atrazine, have relatively low absorption in soil and thus leach into groundwater for the most part (Ma, Fu, Cai, & Jiang, 2003). These facts meant that only the very small part of sprayed triazines could

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R. Fang et al. / Food Chemistry 145 (2014) 41–48

Table 1 Calibration curves, LODs and repeatabilities in the cereal and vegetable samples by sweeping-MEKC Pesticide

Sample matrix

Calibration curve

r2

LR (lg/kg)

LOD (lg/kg)

Intraday RSD (%)

Interday RSD (%)

Simazine

Rice Wheat Chive Carrot Rice Wheat Chive Carrot Rice Wheat Chive Carrot Rice Wheat Chive Carrot Rice Wheat Chive Carrot Rice Wheat Chive Carrot Rice Wheat Chive Carrot Rice Wheat Chive Carrot

Y = 4776.8X + 1383.8 Y = 4541.9X + 1649.3 Y = 5344.1X + 1908.4 Y = 5621.2X + 1638.7 Y = 5876.6X + 785.51 Y = 5561.8X + 296.21 Y = 5459.1X + 741.48 Y = 5886.2X + 1589.4 Y = 5229.5X + 189.29 Y = 5098.1X 377.93 Y = 5278.1X + 432.79 Y = 5995.9X + 170.84 Y = 7072.2X + 475.38 Y = 5563.7X + 125.46 Y = 5685.1X + 779.64 Y = 6290.1X + 628.77 Y = 7027.2X + 475.38 Y = 5626.4X + 455.40 Y = 5835.1X + 693.32 Y = 6343.1X + 615.84 Y = 8120.9X + 656.84 Y = 5292.3X + 449.38 Y = 5959.9X + 2.0432 Y = 6666.6X + 161.08 Y = 6614.7X + 659.52 Y = 5916.5X + 985.97 Y = 6359.9X + 675.98 Y = 7065.0X + 45.421 Y = 6837.5X + 641.08 Y = 6121.5X + 496.06 Y = 6879.1X + 430.10 Y = 6368.1X + 843.21

0.9997 0.9996 0.9998 0.9994 0.9997 1.0000 0.9996 0.9998 0.9995 0.9999 1.0000 1.0000 0.9999 1.0000 0.9999 0.9999 0.9991 1.0000 0.9994 1.0000 0.9999 0.9999 0.9998 0.9998 0.9996 0.9999 0.9999 0.9999 0.9998 0.9999 0.9999 0.9995

0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1-100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100 0.1–100

0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.03 0.03 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02

1.2 1.5 1.0 1.2 1.3 1.2 1.3 1.3 1.7 1.4 1.5 1.1 1.0 1.2 0.9 1.5 1.4 1.4 1.3 0.8 0.8 1.0 1.0 1.3 1.2 1.3 1.4 1.2 1.6 1.7 1.3 1.2

3.0 2.8 2.4 2.5 2.4 2.2 2.7 2.7 2.2 3.3 3.1 2.0 1.7 2.8 2.1 2.3 2.1 2.2 2.5 1.7 2.4 2.7 2.9 2.1 2.0 2.7 3.0 2.2 2.7 3.1 2.6 2.4

Atrazine

Simetryn

Propazine

Ametryn

Terbuthylazine

Prometryn

Terbutryn

directly be absorbed by crops. The other reason was that triazines could be degraded in some cereals and vegetables. GlutathioneS-transferase in some plants catalyzed the glutathione conjugated with triazines (Si, & Meng, 2007). This meant the metabolism of triazines was rapid in some plant, which was proved in corn (Fang, Li, Ding, & Wang, 2012).

The recoveries of eight triazines were measured in the cereal and vegetable samples spiked at 5, 10 and 50 ng/g respectively. Each sample spiked at different levels was analyzed five times using the proposed method. The results were compared with those of HPLC methods and summarized in Table 2. The average recoveries of the sweeping-MEKC method for triazines in the

Table 2 Average recoveries of eight triazine herbicides in cereal and vegetable samples. Pesticide

Simazine

Spiked (lg/kg)

50 10 5 Atrazine 50 10 5 Simetryn 50 10 5 Propazine 50 10 5 Ametryn 50 10 5 Terbuthylazine 50 10 5 Prometryn 50 10 5 Terbutryn 50 10 5

Rice

Wheat

CE

HPLC

Chive

CE

HPLC

Carrot

CE

HPLC

CE

HPLC

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

90.3 86.3 82.1 91.9 87.1 83.7 89.8 87.5 84.8 93.5 87.9 84.7 91.1 89.6 85.4 92.5 90.1 85.8 93.8 87.1 85.6 92.2 88.2 84.9

3.1 3.7 4.3 3.6 4.5 5.0 3.0 3.2 5.6 2.4 3.2 4.4 3.3 3.5 5.1 3.0 4.3 5.1 3.1 4.1 4.4 2.5 4.3 4.9

89.3 87.3 80.2 90.5 85.0 82.9 91.3 89.1 81.9 90.0 86.9 85.1 89.6 91.8 85.1 95.1 90.9 83.3 92.3 85.9 88.2 88.2 89.1 81.1

3.4 3.7 3.8 3.8 4.5 4.8 2.6 4.7 5.1 2.1 3.6 3.9 2.8 3.3 5.8 2.9 4.5 4.9 2.4 4.2 5.0 2.3 3.6 5.1

93.2 90.7 84.1 90.7 85.6 82.8 88.3 85.6 86.0 90.2 86. 83.0 89.1 87.0 84.9 94.6 89.9 86.1 92.7 89.4 83.1 92.8 89.4 83.2

3.8 3.8 4.8 3.4 3.4 5.4 3.5 3.5 4.7 3.1 4.1 4.8 3.0 3.5 5.1 2.9 3.4 4.5 3.9 3.5 4.8 3.9 4.4 5.3

91.6 93.0 82.8 88.9 84.9 81.1 88.5 90.0 85.1 89.1 83.6 82.2 88.9 87.6 85.9 90.7 90.1 86.4 90.4 86.5 84.0 86.6 87.1 83.0

3.1 3.6 4.3 2.9 4.0 5.6 3.1 4.1 3.2 2.9 4.0 4.4 3.2 3.3 4.7 4.0 2.8 4.1 3.3 3.1 5.0 4.0 3.8 4.8

94.4 89.1 86.0 94.1 88.4 85.9 92.2 87.5 88.1 95.4 89.6 86.0 93.5 89.9 86.7 94.6 89.2 86.8 96.8 88.2 91.0 94.5 89.5 87.3

2.7 3.2 3.6 2.8 3.6 3.6 2.8 2.6 2.5 2.1 3.2 3.8 2.8 3.2 3.8 1.5 2.7 3.9 2.3 3.2 3.8 2.0 3.7 4.3

93.9 90.1 83.7 93.2 86.5 82.3 93.1 85.2 85.9 91.1 92.7 83.5 94.9 85.3 85.0 93.0 90.2 83.9 93.3 88.8 87.2 92.8 84.5 85.1

2.4 2.6 3.1 2.0 3.3 3.7 1.9 3.5 2.4 2.5 4.6 3.3 2.5 2.5 3.4 2.4 2.5 3.8 2.2 3.3 4.1 1.8 3.3 3.1

93.5 89.4 91.1 93.2 87.0 87.2 95.4 88.7 87.2 92.0 93.0 85.8 96.8 90.1 87.3 95.4 88.6 87.0 94.6 88.0 89.6 95.1 90.2 87.3

1.7 2.2 2.1 2.4 3.3 3.8 2.0 3.3 3.8 2.8 2.5 3.9 2.5 3.3 3.7 2.0 2.8 2.6 2.7 3.3 3.4 2.9 3.6 4.3

93.2 90.7 84.1 95.7 85.6 82.8 88.3 85.6 89.0 92.5 90.5 85.4 94.4 86.7 81.4 95.8 87.3 83.6 96.0 86.4 87.8 91.5 85.9 82.5

2.4 2.7 2.8 2.8 3.5 4.1 2.6 3.7 4.1 2.1 2.4 4.1 3.0 3.0 3.4 2.5 3.8 3.2 3.0 2.5 4.0 3.4 4.2 4.0

R. Fang et al. / Food Chemistry 145 (2014) 41–48

47

References

Fig. 3. Electropherograms of rice (A) and spiked rice (B) (1) simazine, (2) atrazine, (3) simetryn, (4) propazine, (5) ametryn, (6) terbuthylazine, (7) prometryn, and (8) terbutryn.

cereal and vegetable samples were 82.1–96.8% and the RSDs were 1.46–4.98%. The results of HPLC method were similar to those of sweeping-MEKC method. Fig. 3 shows the typical electropherograms of rice sample with and without being spiked with 10 ng/g of each of the eight triazines. It would be specially mentioned that the LOQ of the method, 0.1 ng/g, was not detected by HPLC. The fact proved that this sweeping-MEKC method was much more sensitive than the HPLC. 4. Conclusions In this work, on-line sweeping preconcentration in MEKC is proposed for the analysis of a group of eight triazine residues in cereal and vegetable samples. The results reveal that the method has high enrichment factors on the basis of high separation efficiency. The enrichment factors were achieved in the range of 479–610. The LODs ranged from 0.02 to 0.04 ng/g. The LOQs of eight triazine herbicides in cereal and vegetable samples was 1/ 25 of MRLs established by European Union legislation, 1/50 of MRLs established by USA legislation and 1/20 of MRLs established by Japan legislation. Compared with the traditional chromatographic technique, this sweeping-MEKC method can be employed directly to determine the sample without relatively complex offline cleanup and preconcentration procedure, which can reduce the operation costs. The LOQ of 0.1 ng/g is not detected by HPLCUV. All of these prove that the developed method is better than HPLC-UV and can be used for the determination of the triazine herbicides in cereal and vegetable samples. Acknowledgements This work is supported by Specialized Research Fund for the Doctoral Program of Higher Education, Ministry of Education, China, (Grant No. 20093227110010) and The Science Fund of Jiangsu University (Grant No. 08JDG001). The authors are grateful to A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 08.028.

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