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New approach for the simultaneous determination fungicide residues in food samples by using carbon nanofiber packed microcolumn coupled with HPLC
Food Control 60 (2016) 1e6
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Food Control journal homepage: www.elsevier.com/locate/foodcont
New approach for the simultaneous determination fungicide residues in food samples by using carbon nanofiber packed microcolumn coupled with HPLC Lingling Wang, Minhui Zhang, Danfeng Zhang, Lei Zhang* College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, Liaoning, 110036, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 May 2015 Received in revised form 14 July 2015 Accepted 16 July 2015 Available online 17 July 2015
Solid-phase extraction preconcentration system using carbon nanofibers (CNFs) packed microcolumn was developed for the simultaneous determination of the fungicides carbendazim (MBC) and thiabendazole (TBZ) by high performance liquid chromatography (HPLC). CNFs exhibited a good affinity to target analytes. Important influence factors were studied in detail, such as amounts of packing material, sample solution acidity, sample flow rate and volume, eluent flow rate and volume. Under the optimal conditions, excellent linearity was obtained in a range of 2.0e500.0 ng mL1 with a correlation coefficient of r2 0.9964. The limits of quantification and detection were in the range of 1.50e1.80 ng mL1 and 0.45 e0.54 ng mL1, respectively. The present method was applied to the analysis of fruit, vegetable and juice samples, and the recoveries of analytes were in the range of 96.1e103.5% with the relative standard deviations ranging from 1.27% to 3.90% (n ¼ 3). Intra-day and inter-day repeatability values expressed as relative standard deviation were 1.08e2.74% and 2.82e3.94%, respectively. The results showed that the present method was a simply, convenient and feasible method for the determination of MBC and TBZ in real samples. © 2015 Published by Elsevier Ltd.
Keywords: Solid-phase extraction Carbon nanofibers Microcolumn Carbendazim Thiabendazole High performance liquid chromatography
1. Introduction Benzimidazole fungicides are most commonly used pesticides in agriculture for pre- and post-harvest treatment for the control of a wide range of pathogens. Carbendazim [methylbenzimidazole-2ylcarbamate (MBC)] and thiabendazole [2-(4-thiazolyl) benzimidazole (TBZ)] are main compounds of the benzimidazole family, widely used postharvest fungicides for fruit, vegetables and other agricultural products. They are either applied directly to the soil, or sprayed over crop fields. Therefore, they persist in soil, crops, and food after their application, and also are very persistent in water. Benzimidazole fungicides have attracted more and more attentions around the world, due to their significant toxicity, potential mutagenicity (Banks & Soliman, 1997) and carcinogenicity (WHO, 1989). The development of analytical methodologies for benzimidazole fungicides in food sample is an important aspect of food analytical chemistry. However, the extremely low concentrations in real samples and the complexity of their matrices make it a challenging task to detect fungicide residues.
* Corresponding author. E-mail address: [email protected] (L. Zhang). http://dx.doi.org/10.1016/j.foodcont.2015.07.024 0956-7135/© 2015 Published by Elsevier Ltd.
The effective extraction and preconcentration techniques prior to analysis of analytes are often required. The technology employed for the extraction and preconcentration of MBC and TBZ from aqueous samples includes solid phase extraction (SPE) (Al-Ebaisat, pez, Jae n-Martos et al., 2012; Guo et al., 2010; 2011; Gilbert-Lo Veneziano, Vacca, Arana, De Simone, & Rastrelli, 2004), solid pez Monzo n, Vega Moreno, phase microextraction (SPME) (Lo n, Sosa Ferrera, & Santana Rodríguez, 2007), liquidTorres Padro pez, García-Reyes, & Molinaeliquid extraction (LLE) (Gilbert-Lo Díaz, 2012) and dispersive liquideliquid microextraction (DLLME) (Wu et al., 2009). SPE are the most commonly used techniques for the preconcentration and cleanup of the selected analytes because of its simplicity, effectiveness, low cost, minimum use of solvents and excellent sample cleanup ability. Thus, the search of new material to develop solid-phase extraction preconcentration system is of continuing interest (Oliferova, Statkus, Tsysin, Shpigun, & Zolotov, 2005; Zhou, Yan, Kim, Wang, & Liu, 2006). The special and unusual properties of carbon nanostructures materials have created much interest in their use as adsorbents in a rcel, Ca rdenas, Simonet, wide variety of analytical processes (Valca Moliner-Martínez, & Lucena, 2008). Multi-walled carbon nanotubes (MWCNTs) and C18 have been proven to possess great
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potential for the enrichment of many kinds of organic pollutants (Natale, Gibis, Rodriguez-Estrada, & Weiss, 2014; Zhang, Fang, Yang, Zhang, & Wang, 2013; Zhang, Lv, et al., 2013; Zhang, Pan, et al., 2013). Recently, carbon nanofibers (CNFs) have attracted great interest because of their unique physicochemical characteristics, such as their amorphous structure, relatively large surface area, stability in acidic/basic environments, being amenable to surface functionalization, and high chemical reactivity because of the exposed edges and unsaturated bonds on their graphene layers (Klein et al., 2008; Thangaraj, Nellaiappan, Sudhakaran, & Kumar, 2014). CNFs have been used extensively as adsorbents, catalyst supports, and composite materials for battery electrodes and hydrogen storage (Bhuvaneswari, Bramnik, Ensling, Ehrenberg, & Jaegermann, 2008; Corma & Garcia, 2008; Kim, Lee, & Moon, 2011; Singhal, Sharma, & Verma, 2008). The research on the application of CNFs materials as potential SPE adsorbents is still few, it will be of great value to explore the performance of CNFs to increase the extraction efficiency, minimize organic solvents, shorten analysis time and provide automation. To our knowledge, there is no report on the application of solid-phase extraction microcolumn using CNFs for MBC and TBZ analysis by high performance liquid chromatography (HPLC). The aim of this work was to develop a new separation/preconcentration method based on CNFs packed microcolumn for the determination of fungicide residues in food samples. For this purpose, a CNFs microcolumn coupled to HPLC for analysis of two fungicides. The extraction selectivity, reproducibility and linear range were investigated. The experimental results indicated that the developed method could simultaneously determine two fungicide residues in real samples.
phase in isocratic elution mode. The detection wavelength was set at 285 nm. 2.3. SPE procedure A known quantity of the CNFs (15 mg) was packed into the column using the dry packing method with a small portion of glass wool at both ends in order to avoid adsorbent loss during SPE steps (shown in Scheme 1). Prior to use, ethanol and deionized water were passed through the column in sequence in order to clean it. A solution containing appropriate concentration of MBC and TBZ was passed through the CNFs packed microcolumn by using a peristaltic pump at a desired flow rate of 0.5 mL min1. MBC and TBZ retained by the column were eluted by 3.0 mL of methanol containing 0.2% formic acid at 0.1 mL min1. Then the eluent was evaporated to dryness under a mild nitrogen stream at room temperature. Finally, the residue was dissolved with 0.5 mL mobile phase and filtered through a 0.45 mm PTFE filter membrane before HPLC analysis. 2.4. Sample preparation Lemon, cucumber and commercially available apple juice were purchased from local supermarkets. A representative 50 g portion of previously homogenised fruits and vegetable samples were homogenised with 250 mL water, and then aliquoted into PTFE centrifuge tubes and centrifuged for 10 min at 4000 rpm. The supernatants were transferred to a glass beaker. Aliquots of the supernatants and juice samples (50 mL) were spiked with the mixed working solutions. The resulting solution was referred to as sample solution, filtered through 0.45 mm filters and then stored at 4 C.
2. Material and methods
3. Results and discussion
2.1. Materials and reagents
3.1. Optimization of extraction conditions
MBC and TBZ standards were purchased from HEOWNS Biochemical technology Co., Ltd. (Tianjin, China). CNFs (99% purity) was purchased from Beijing Dk Nano technology Co., Ltd. (Beijing, China) and its particle size was in the 150e200 nm range. The stock solution containing the analytes was prepared by dissolving appropriate amount of them in ethanol and stored at 4 C under dark conditions. The working solutions were obtained daily by appropriately diluting the stock solution with deionized water. Analytical grade sodium chloride were purchased from Beijing Chemical Co. (Beijing, China). Deionized water used throughout experiments was purified using a Sartorius Arium 611 €ttingen, Germany). Chromatographic grade system (Sartorius, Go methanol (MeOH) was purchased from Fisher Corporation (Pittsburgh, PA, USA). HPLC-grade deionised water was obtained from a MilliQ water purification system (MilliQ Water; Molsheim, France).
To achieve the best extraction efficiency for target analytes, extraction conditions were optimised by analysing spiked samples. The experimental parameters, including the amount of adsorbents, the pH of sample solution and the type of desorption solvent, the flow rate of extraction and desorption, and the volume of extraction and desorption were investigated in detail. 3.1.1. Effect of the adsorbent amount Different amounts of the CNFs in a range of 5e30 mg were applied to extract MBC and TBZ from the sample solution (25 mL of 200 ng mL1 MBC and TBZ). The results indicated (Fig. 1A) that
2.2. Apparatus and analytical conditions A self-made glass column (20 cm 6.0 mm i.d.), a constant flow peristaltic pump with computer displaying and fraction collector (Shanghai Huxi Analysis Instrument Factory CO., LTD) were used for the SPE process. PTFE tubing with 0.5 mm i.d. was used for all connections. HPLC analyses were performed with an Agilent 1100 HPLC system (Palo Alto, CA, USA) equipped with an automatic sampler and diode array detector (DAD). Chromatographic separation of target analytes was performed on a ZOBAX Eclipse XDB-C18 (150 mm 4.6 mm, 5 mm) column (Agilent, Palo Alto, CA, USA) and the injection volume was 10 mL. A mixture of water and methanol (50:50, v/v) at a flow rate of 1.0 mL min1 was used as a mobile
Scheme 1. Schematic diagram of the microcolumn-SPE procedure.
L. Wang et al. / Food Control 60 (2016) 1e6
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15 mg CNFs were enough for the extraction, and further increasing the amount of the adsorbent gave no significant improvement for the adsorption percentage of MBC and TBZ. Therefore, 15 mg was selected as the amount of the adsorbent. 3.1.2. Effect of pH and ionic strength of sample solution One of the critical parameter in the extraction of organic analytes is the pH of sample solution because the pH value of the solution determines the present state of analytes. In order to evaluate the effect of pH, different initial pH values of sample solution ranging from 3.0 to 12.0 was studied. According to Fig. 1B, the adsorption maxima occurred in a broad range of pH value from 5 to 11.0. Generally, the pH of natural MBC and TBZ solution was close to 5.7. In this study, the sample solutions were used directly without any pH adjustment. MBC and TBZ are characterized by 2 pKa values, the two predicted pKa values of MBC and TBZ are 4.25, 11.15 and 4.75, 12.0, respectively. At pH between of 5 and 11, analytes will exist as neutral molecules, nearly all of analytes molecules carry no net electrical charge, which makes them hardly have electrostatic attraction with CNFs, so the increase of pH from 5.0 to 11.0 had no significant effect on the adsorptive affinity of MBC and TBZ in the experiment. Hence, the adsorption mechanism might be attributed to p-p interactions, H-bonds and hydrophobic occurred between the CNFs s and analytes (Wang et al., 2015; Zhang, Fang, et al., 2013; Zhang, Lv, et al., 2013; Zhang, Pan, et al., 2013). In order to investigate the influence of the ionic strength, the adsorption experiments of MBC and TBZ were carried out in the presence of 0e1.0 mol L1 NaCl, respectively. Results showed that the recoveries were no significant effect with increasing NaCl concentrations. Furthermore, the ionic strength test also confirmed that the surface electrostatic effect had no influence on the overall adsorption of MBC and TBZ on CNFs. In this experiment, the sample solution without adjusting ionic strength was adopted. 3.1.3. Effects of sample flow rate and breakthrough volume In a microcolumn preconcentration system, the enrichment factor and analytical time mainly depend on the sample flow rate and breakthrough volume. A solution containing 2.0 mg mL1 of MBC and TBZ was passed through the CNFs packed microcolumn by using a peristaltic pump. The effect of sample flow rate in the range of 0.2e1.0 mL min was also evaluated. As shown in Fig. 2, it is clear that the breakthrough of MBC and TBZ took place after 35 min (35 mL, 1.0 mL min1), 100 min (50 mL, 0.5 mL min1) and 275 min (55 mL, 0.2 mL min1), respectively. The experimental results showed the breakthrough volume was increased from 35 to 55 mL with an decrease in flow rate from 1.0 to 0.2 mL min1. This was due
Fig. 2. Breakthrough profiles and effect of the sample flow rate for MBC and TBZ with a micro-column packed with the CNFs. (CNFs: 15 mg; Ci: 2.0 mg mL1; Ci are the initial concentration of the target analytes in sample solution; Ct are the concentration of the target analytes in effluent).
to a increase in the residence time, which facilitated the contact of target analytes to the CNFs. However, it also resulted in a much longer extraction time, which is time-consuming for real-time analysis. Considering the operating time and extraction efficiency, sample flow rate of 0.5 mL min1 and sample volume of 50 mL was used. From the area of the breakthrough curve (0.5 mL min1), the loading capacities of the CNFs for MBC and TBZ were evaluated as 9.1 and 10.6 mg g1, respectively.
3.1.4. Effect of eluting solvent In order to check the recovery efficiency of MBC and TBZ, desorption of the target analytes from the adsorbents was performed. Different eluent, including methanol (MeOH), ethanol (EtOH), acetonitrile (ACN), MeOH-0.1% formic acid (FA), MeOH-0.2% FA and MeOH-0.3% FA were studied. The results (Fig. 3) indicated that MeOH-0.2% FA solution gave the highest overall desorption efficiency for the target analytes. This was because MeOH-0.2% FA could convert the target analytes into ionic forms, thus reducing their affinity for the CNFs and facilitating the elution. Therefore, MeOH-0.2% FA was selected as the desorption solvent for the subsequent studies and high desorption efficiency and stable recoveries were obtained.
Fig. 1. Optimization of extraction conditions: (A) effect of amount of the CNFs on the adsorption efficient of MBC and TBZ and (B) effect of pH on the adsorption efficient of MBC and TBZ. (MBC and TBZ concentrations: 200 ng mL1; sample solution volume: 25 mL).
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Fig. 3. Optimization of desorption conditions: effect of eluent, elution volume and flow rate on the recoveries of MBC and TBZ. (CNFs: 15 mg; MBC and TBZ concentrations: 200 ng mL1; sample solution volume: 50 mL; sample flow rate: 0.5 mL min1).
3.1.5. Effects of elution volume and elution flow rate In order to investigate the effect of the elution volume, 50 mL of sample solution containing 200 ng mL1 of MBC and TBZ was passed through the CNFs packed microcolumn at a flow rate of 0.5 mL min1. The target analytes were eluted by MeOH-0.2% formic acid solution, and the elution volume was investigated from 1.0 mL to 5.0 mL (Fig. 3). The results indicated that recoveries of target analytes were enhanced by increasing elution volume. It was found that 3.0 mL of eluent was sufficient to recover all the species quantitatively. The flow rate of eluent is also an affecting parameter for recoveries of target analytes. With eluent volume kept at 3.0 mL, the influence of elution flow rate was investigated by changing the flow rate from 0.05 to 0.2 mL min1. With the increase of flow rate, the recoveries of target analytes were slightly decreased. The results (Fig. 3) indicated that the optimal elution flow rate were in the range of 0.05e0.1 mL min1. Therefore, the elution flow rate of 0.1 mL min1 was selected for the following experiments.
3.2. Interference studies Fruit, vegetable and juice samples contains considerable amounts of organic matter and inorganic ions. These substances may have a potentially competitive adsorption effect on MBC and TBZ in food samples. In order to assess the possible analytical applications of the proposed method, the effect of concomitant species on the determination of MBC and TBZ in real samples was examined under the optimal conditions as described above. The sample solutions containing 200 ng mL1 of MBC and TBZ and the added interfering matter were subjected to the proposed method. The tolerance ratio of each interfering matter was taken as the largest amount yielding an error in the determination of the target analytes not exceeding 5% (Table 1).
3.3. Stability of the CNFs The stability of CNFs was evaluated by checking the cycle number dependence of recoveries for 200 ng mL1 MBC and TBZ, using the same CNFs for subsequent cycles. After desorption, the regenerated CNFs was washed with deionized water to nearly neutral. The recycled adsorbents were reused, and the results (Fig. 4) indicate that the regenerated CNFs still maintained a high recoveries of the target analytes (recovery >90%) after five adsorptioneregeneration cycles. Therefore, CNFs had good reutilization and could be used as promising adsorbents for the effective extraction of benzimidazole fungicide residues in real samples. 3.4. Method evaluation 3.4.1. Analytical performances The performance of the SPE preconcentration system using CNFs packed microcolumn was evaluated under the optimized extraction conditions. A series of experiments were performed to obtain the linearity, precision, the limit of detection (LOD) and quantification (LOQ). All the experiments were performed in triplicate. As showed in Table 2, good linearities were obtained in the range of 2e500 ng mL1 with the correlation coefficients (r2)
Table 1 Effects of interfering matter on determination of 200 ng mL1 of MBC and TBZ. Interference
Interference to MBC and TBZ ratio (w/w)
Saccharose, glucose, fructose Ascorbic acid Naþ, Kþ, Fe3þ, Mg2þ, Ca2þ SO4 2 , PO4 3 , NO3 , HCO3 , CO3 2
400 800 1000 1000
Fig. 4. Reusability of the CNFs as microcolumn SPE adsorbents for extraction of MBC and TBZ (CNFs: 15 mg; MBC and TBZ concentrations: 200 ng mL1; sample solution volume: 50 mL; sample flow rate: 0.5 mL min1; eluent: 3.0 mL MeOH-0.2% FA; elution flow rate: 0.1 mL min1).
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Table 2 Analytical performance for MBC and TBZ obtained by microcolumn SPE-HPLC-DAD. Compound
Linear range (ng mL1)
Regression equation
LOD (ng mL1)
LOQ (ng mL1)
r2
EF
MBC TBZ
2.0e500.0 2.0e500.0
y ¼ 4.7316C þ 31.238 y ¼ 4.5596C þ 29.725
0.45 0.54
1.50 1.80
0.9964 0.9978
100 100
ranging from 0.9964 to 0.9978. The limit of detection (LOD, 3S/k, S: standard deviation of blank signal, n ¼ 9; k: slope of the working curve) and the limit of quantization (LOQ, 10S/k) were found ranging from 0.45 to 0.54 ng mL1 and 1.50e1.80 ng mL1, respectively. The proposed method showed good precision with the intra-day and inter-day RSDs in the range of 1.08e2.74% (n ¼ 6) and 2.82e3.94% (n ¼ 3), respectively. In order to calculate the EF for the analytes, five replicates were conducted for the sample solutions (V ¼ 50 mL) containing 200 ng mL1 of the analyte. Enrichment factor (EF) was calculated by following equations:
EF ¼ ca =cs
(1)
where ca and cs are the concentrations of the analyte in analytical solutions (V ¼ 0.5 mL) and sample solutions (V ¼ 50 mL), respectively. It was found that the EF of 100 was obtained under optimal conditions.
Fig. 5. HPLC-DAD Chromatograms obtained at 285 nm. (A) Blank apple juice sample after pretreatment by SPE; (B) The spiked apple juice sample (10.0 ng mL1) after pretreatment by SPE; (C) The standard solution of MBC and TBZ. (CNFs: 15 mg; MBC and TBZ concentrations: 200 ng mL1; sample volume: 50 mL; sample flow rate: 0.5 mL min1; eluent: 3.0 mL MeOH-0.2% FA; elution flow rate: 0.1 mL min1).
3.4.2. Analysis of samples To evaluate the applicability of the present method, fruit, vegetable and juice samples were analyzed. It can be seen (Fig. 5) that no significant interference peaks were found at the retention positions of MBC and TBZ. To evaluate the precision and accuracy of the proposed method, the spiked samples (10.0, 100.0, 200.0 ng mL1) were analyzed. The recoveries of the method were tested by five replicate experiments and the results were given in Table 3. The recoveries of MBC and TBZ were between 96.1% and 103.5%, with RSDs of 1.27 and 3.90%. It can be considered that the current method provides acceptable recoveries and precision for the determination of MBC and TBZ in real samples.
DLLME (CHCl3) (Wu et al., 2009), followed by HPLC analysis. Besides, compared to conventional absorbents or extractant, CNFs have high adsorption capacity and large special surface area, so an about 100 times high enrichment factor was obtained. Compared with SPE-HPLC-DAD, QuEChERS-LLE-LC-MS/MS methods (Gilbertpez, García-Reyes et al., 2012), the LODs of this method were Lo higher than that achieved by QuEChERS-LLE-LC-MS/MS method. Although the LODs of this method were higher than that obtained by QuEChERS-LLE-LC-MS/MS, the cost of the proposed method is lower and the operation is more convenient.
3.5. Comparing with other methods 4. Conclusions The proposed method based on microcolumn-SPE (CNFs)-HPLCDAD was compared with other methods as listed in Table 4. As can be seen from the table, the LODs of MBC and TBZ obtained by the proposed method were lower than that obtained by other sample pretreatment methods including SPE (SampliQ SCX) (Al-Ebaisat, pez Monzo n et al., 2007), 2011), SPME (CARePDMS fiber) (Lo
In this study, we proposed a new approach for simultaneous sensitive determinate of two fungicides in fruit, vegetable and juice samples by SPE using a CNFs packed microcolumn combined with HPLC-DAD. The results showed that CNFs had high enrichment capability and good stability. The packed column was reused five
Table 3 Recoveries of MBC and TBZ in food samples (mean ± SD, n ¼ 3). Samples
MBC Spike (ng mL1)
Apple juice
Lemon
Cucumber
a
0 10.0 100.0 200.0 0 10.0 100.0 200.0 0 10.0 100.0 200.0
n.d.¼ not detected.
TBZ Found (ng mL1) a
n.d. 10.12 ± 0.18 97.2 ± 1.95 195.6 ± 4.05 n.d. 10.35 ± 0.36 96.1 ± 3.75 194.9 ± 3.01 n.d. 10.23 ± 0.30 99.1 ± 2.86 197.3 ± 3.00
Recovery (%)
RSD (%)
Spike (ng mL1)
Found (ng mL1)
Recovery (%)
RSD (%)
e 101.2 97.2 97.8 e 103.5 96.1 97.4 e 102.3 99.1 98.6
e 1.82 2.01 2.07 e 3.52 3.90 1.54 e 2.99 2.88 1.52
0 10.0 100.0 200.0 0 10.0 100.0 200.0 0 10.0 100.0 200.0
n.d. 9.86 ± 0.34 101.3 ± 2.31 194.8 ± 3.41 n.d. 9.93 ± 0.23 97.2 ± 2.36 193.8 ± 2.45 n.d. 9.94 ± 0.26 100.9 ± 2.11 196.3 ± 3.05
e 98.6 101.3 97.4 e 99.3 97.2 96.9 e 99.4 100.9 98.2
e 3.42 2.28 1.75 e 2.32 2.42 1.27 e 2.66 2.09 1.55
6
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Table 4 Comparison of the proposed method with other method for the determination of MBC and TBZ. Analytical method
SPE-HPLC-VWD SPME-HPLC-FLD DLLME-HPLC-FLD QuEChERS-LLE-LC-MS/MS SPE-HPLC-DAD (this work)
Adsorbent/extractant
SampliQ SCX CARePDMS fiber CHCl3 acetonitrile CNFs Microcolumn
LOD (ng mL1) MBC
TBZ
3.55 1.30 0.5 0.003 0.45
e 0.04 1.0 0.003 0.54
times without loss in column performance. The proposed method demonstrated good linearity, low LOD, excellent repeatability and satisfactory recovery, and was proved to be sensitive, accurate and reliable in determining the fungicides in real samples. In addition, this method is characterized with simplicity of operation, minimum use of solvents and low cost. Therefore, it is optimistic about the prospect of the developed method in determining the fungicide residues in food sample. Acknowledgements This project was supported by the National Nature Science Foundation of China (NSFC51178212), the Science and Technology Foundation of Ocean and Fisheries of Liaoning Province (201408, 201406), the General project of scientific research of the Education Department of Liaoning Province (L2015206) and the Foundation of 211 project for Innovati-ve Talent Training, Liaoning University. The authors also thank their colleagues and other students who participated in this study. References Al-Ebaisat, H. (2011). Determination of some benzimidazole fungicides in tomato puree by high performance liquid chromatography with SampliQ polymer SCX solid phase extraction. Arabian Journal of Chemistry, 4(1), 115e117. Banks, D., & Soliman, M. R. (1997). Protective effects of antioxidants against benomyl-induced lipid peroxidation and glutathione depletion in rats. Toxicology, 116(1e3), 177e181. Bhuvaneswari, M. S., Bramnik, N. N., Ensling, D., Ehrenberg, H., & Jaegermann, W. (2008). Synthesis and characterization of carbon nano fiber/LiFePO4 composites for Li-ion batteries. Journal of Power Sources, 180(1), 553e560. Corma, A., & Garcia, H. (2008). Supported gold nanoparticles as catalysts for organic reactions. Chemical Society Reviews, 37(9), 2096e2126. pez, B., García-Reyes, J. F., & Molina-Díaz, A. (2012). Determination of Gilbert-Lo fungicide residues in baby food by liquid chromatographyeion trap tandem mass spectrometry. Food Chemistry, 135(2), 780e786. pez, B., Jae n-Martos, L., García-Reyes, J. F., Villar-Pulido, M., Polgar, L., Gilbert-Lo Ramos-Martos, N., et al. (2012). Study on the occurrence of pesticide residues in fruit-based soft drinks from the EU market and morocco using liquid
chromatographyemass spectrometry. Food Control, 26(2), 341e346. Guo, B., Huang, Z., Wang, M., Wang, X., Zhang, Y., Chen, B., et al. (2010). Simultaneous direct analysis of benzimidazole fungicides and relevant metabolites in agricultural products based on multifunction dispersive solid-phase extraction and liquid chromatographyemass spectrometry. Journal of Chromatography A, 1217(29), 4796e4807. Kim, H., Lee, D., & Moon, J. (2011). Co-electrospun Pd-coated porous carbon nanofibers for hydrogen storage applications. International Journal of Hydrogen Energy, 36(5), 3566e3573. Klein, K. L., Melechko, A. V., McKnight, T. E., Retterer, S. T., Rack, P. D., Fowlkes, J. D., et al. (2008). Surface characterization and functionalization of carbon nanofibers. Journal of Applied Physics, 103(6), 061301. pez Monzo n, A., Vega Moreno, D., Torres Padro n, M. E., Sosa Ferrera, Z., & Santana Lo Rodríguez, J. J. (2007). Solid-phase microextraction of benzimidazole fungicides in environmental liquid samples and HPLCefluorescence determination. Analytical and Bioanalytical Chemistry, 387(6), 1957e1963. Natale, D., Gibis, M., Rodriguez-Estrada, M. T., & Weiss, J. (2014). Inhibitory effect of liposomal solutions of grape seed extract on the formation of heterocyclic aromatic amines. Journal of Agricultural and Food Chemistry, 62(1), 279e287. Oliferova, L., Statkus, M., Tsysin, G., Shpigun, O., & Zolotov, Y. (2005). On-line solidphase extraction and HPLC determination of polycyclic aromatic hydrocarbons in water using fluorocarbon polymer sorbents. Analytica Chimica Acta, 538(1e2), 35e40. Singhal, R. M., Sharma, A., & Verma, N. (2008). Micronano hierarchal web of activated carbon fibers for catalytic gas adsorption and reaction. Industrial & Engineering Chemistry Research, 47(10), 3700e3707. Thangaraj, R., Nellaiappan, S., Sudhakaran, R., & Kumar, A. S. (2014). A flow injection analysis coupled dual electrochemical detector for selective and simultaneous detection of guanine and adenine. Electrochimica Acta, 123(0), 485e493. Valc arcel, M., C ardenas, S., Simonet, B. M., Moliner-Martínez, Y., & Lucena, R. (2008). Carbon nanostructures as sorbent materials in analytical processes. TrAC Trends in Analytical Chemistry, 27(1), 34e43. Veneziano, A., Vacca, G., Arana, S., De Simone, F., & Rastrelli, L. (2004). Determination of carbendazim, thiabendazole and thiophanate-methyl in banana (Musa acuminata) samples imported to Italy. Food Chemistry, 87(3), 383e386. Wang, L., Zhang, Z., Xu, X., Zhang, D., Wang, F., & Zhang, L. (2015). Simultaneous determination of four trace level endocrine disrupting compounds in environmental samples by solid-phase microextraction coupled with HPLC. Talanta, 142(0), 97e103. WHO, C. (1989). Environmental health criteria. Geneva, Switzerland. Wu, Q., Li, Y., Wang, C., Liu, Z., Zang, X., Zhou, X., et al. (2009). Dispersive liquideliquid microextraction combined with high performance liquid chromatographyefluorescence detection for the determination of carbendazim and thiabendazole in environmental samples. Analytica Chimica Acta, 638(2), 139e145. Zhang, L., Fang, P., Yang, L., Zhang, J., & Wang, X. (2013). Rapid method for the separation and recovery of endocrine-disrupting compound bisphenol AP from wastewater. Langmuir, 29(12), 3968e3975. Zhang, L., Lv, J., Xu, T., Yang, L., Jiang, X., & Li, Q. (2013). High efficiency removal and recovery of an endocrine disrupting compoundebisphenol AF from wastewaters. Separation and Purification Technology, 116(0), 145e153. Zhang, L., Pan, F., Liu, X., Yang, L., Jiang, X., Yang, J., et al. (2013). Multi-walled carbon nanotubes as sorbent for recovery of endocrine disrupting compoundbisphenol F from wastewater. Chemical Engineering Journal, 218(0), 238e246. Zhou, Y.-Y., Yan, X.-P., Kim, K.-N., Wang, S.-W., & Liu, M.-G. (2006). Exploration of coordination polymer as sorbent for flow injection solid-phase extraction online coupled with high-performance liquid chromatography for determination of polycyclic aromatic hydrocarbons in environmental materials. Journal of Chromatography A, 1116(1e2), 172e178.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
New approach for the simultaneous determination fungicide residues in food samples by using carbon nanofiber packed microcolumn coupled with HPLC Lingling Wang, Minhui Zhang, Danfeng Zhang, Lei Zhang* College of Chemistry, Liaoning University, 66 Chongshan Middle Road, Shenyang, Liaoning, 110036, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 May 2015 Received in revised form 14 July 2015 Accepted 16 July 2015 Available online 17 July 2015
Solid-phase extraction preconcentration system using carbon nanofibers (CNFs) packed microcolumn was developed for the simultaneous determination of the fungicides carbendazim (MBC) and thiabendazole (TBZ) by high performance liquid chromatography (HPLC). CNFs exhibited a good affinity to target analytes. Important influence factors were studied in detail, such as amounts of packing material, sample solution acidity, sample flow rate and volume, eluent flow rate and volume. Under the optimal conditions, excellent linearity was obtained in a range of 2.0e500.0 ng mL1 with a correlation coefficient of r2 0.9964. The limits of quantification and detection were in the range of 1.50e1.80 ng mL1 and 0.45 e0.54 ng mL1, respectively. The present method was applied to the analysis of fruit, vegetable and juice samples, and the recoveries of analytes were in the range of 96.1e103.5% with the relative standard deviations ranging from 1.27% to 3.90% (n ¼ 3). Intra-day and inter-day repeatability values expressed as relative standard deviation were 1.08e2.74% and 2.82e3.94%, respectively. The results showed that the present method was a simply, convenient and feasible method for the determination of MBC and TBZ in real samples. © 2015 Published by Elsevier Ltd.
Keywords: Solid-phase extraction Carbon nanofibers Microcolumn Carbendazim Thiabendazole High performance liquid chromatography
1. Introduction Benzimidazole fungicides are most commonly used pesticides in agriculture for pre- and post-harvest treatment for the control of a wide range of pathogens. Carbendazim [methylbenzimidazole-2ylcarbamate (MBC)] and thiabendazole [2-(4-thiazolyl) benzimidazole (TBZ)] are main compounds of the benzimidazole family, widely used postharvest fungicides for fruit, vegetables and other agricultural products. They are either applied directly to the soil, or sprayed over crop fields. Therefore, they persist in soil, crops, and food after their application, and also are very persistent in water. Benzimidazole fungicides have attracted more and more attentions around the world, due to their significant toxicity, potential mutagenicity (Banks & Soliman, 1997) and carcinogenicity (WHO, 1989). The development of analytical methodologies for benzimidazole fungicides in food sample is an important aspect of food analytical chemistry. However, the extremely low concentrations in real samples and the complexity of their matrices make it a challenging task to detect fungicide residues.
* Corresponding author. E-mail address: [email protected] (L. Zhang). http://dx.doi.org/10.1016/j.foodcont.2015.07.024 0956-7135/© 2015 Published by Elsevier Ltd.
The effective extraction and preconcentration techniques prior to analysis of analytes are often required. The technology employed for the extraction and preconcentration of MBC and TBZ from aqueous samples includes solid phase extraction (SPE) (Al-Ebaisat, pez, Jae n-Martos et al., 2012; Guo et al., 2010; 2011; Gilbert-Lo Veneziano, Vacca, Arana, De Simone, & Rastrelli, 2004), solid pez Monzo n, Vega Moreno, phase microextraction (SPME) (Lo n, Sosa Ferrera, & Santana Rodríguez, 2007), liquidTorres Padro pez, García-Reyes, & Molinaeliquid extraction (LLE) (Gilbert-Lo Díaz, 2012) and dispersive liquideliquid microextraction (DLLME) (Wu et al., 2009). SPE are the most commonly used techniques for the preconcentration and cleanup of the selected analytes because of its simplicity, effectiveness, low cost, minimum use of solvents and excellent sample cleanup ability. Thus, the search of new material to develop solid-phase extraction preconcentration system is of continuing interest (Oliferova, Statkus, Tsysin, Shpigun, & Zolotov, 2005; Zhou, Yan, Kim, Wang, & Liu, 2006). The special and unusual properties of carbon nanostructures materials have created much interest in their use as adsorbents in a rcel, Ca rdenas, Simonet, wide variety of analytical processes (Valca Moliner-Martínez, & Lucena, 2008). Multi-walled carbon nanotubes (MWCNTs) and C18 have been proven to possess great
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potential for the enrichment of many kinds of organic pollutants (Natale, Gibis, Rodriguez-Estrada, & Weiss, 2014; Zhang, Fang, Yang, Zhang, & Wang, 2013; Zhang, Lv, et al., 2013; Zhang, Pan, et al., 2013). Recently, carbon nanofibers (CNFs) have attracted great interest because of their unique physicochemical characteristics, such as their amorphous structure, relatively large surface area, stability in acidic/basic environments, being amenable to surface functionalization, and high chemical reactivity because of the exposed edges and unsaturated bonds on their graphene layers (Klein et al., 2008; Thangaraj, Nellaiappan, Sudhakaran, & Kumar, 2014). CNFs have been used extensively as adsorbents, catalyst supports, and composite materials for battery electrodes and hydrogen storage (Bhuvaneswari, Bramnik, Ensling, Ehrenberg, & Jaegermann, 2008; Corma & Garcia, 2008; Kim, Lee, & Moon, 2011; Singhal, Sharma, & Verma, 2008). The research on the application of CNFs materials as potential SPE adsorbents is still few, it will be of great value to explore the performance of CNFs to increase the extraction efficiency, minimize organic solvents, shorten analysis time and provide automation. To our knowledge, there is no report on the application of solid-phase extraction microcolumn using CNFs for MBC and TBZ analysis by high performance liquid chromatography (HPLC). The aim of this work was to develop a new separation/preconcentration method based on CNFs packed microcolumn for the determination of fungicide residues in food samples. For this purpose, a CNFs microcolumn coupled to HPLC for analysis of two fungicides. The extraction selectivity, reproducibility and linear range were investigated. The experimental results indicated that the developed method could simultaneously determine two fungicide residues in real samples.
phase in isocratic elution mode. The detection wavelength was set at 285 nm. 2.3. SPE procedure A known quantity of the CNFs (15 mg) was packed into the column using the dry packing method with a small portion of glass wool at both ends in order to avoid adsorbent loss during SPE steps (shown in Scheme 1). Prior to use, ethanol and deionized water were passed through the column in sequence in order to clean it. A solution containing appropriate concentration of MBC and TBZ was passed through the CNFs packed microcolumn by using a peristaltic pump at a desired flow rate of 0.5 mL min1. MBC and TBZ retained by the column were eluted by 3.0 mL of methanol containing 0.2% formic acid at 0.1 mL min1. Then the eluent was evaporated to dryness under a mild nitrogen stream at room temperature. Finally, the residue was dissolved with 0.5 mL mobile phase and filtered through a 0.45 mm PTFE filter membrane before HPLC analysis. 2.4. Sample preparation Lemon, cucumber and commercially available apple juice were purchased from local supermarkets. A representative 50 g portion of previously homogenised fruits and vegetable samples were homogenised with 250 mL water, and then aliquoted into PTFE centrifuge tubes and centrifuged for 10 min at 4000 rpm. The supernatants were transferred to a glass beaker. Aliquots of the supernatants and juice samples (50 mL) were spiked with the mixed working solutions. The resulting solution was referred to as sample solution, filtered through 0.45 mm filters and then stored at 4 C.
2. Material and methods
3. Results and discussion
2.1. Materials and reagents
3.1. Optimization of extraction conditions
MBC and TBZ standards were purchased from HEOWNS Biochemical technology Co., Ltd. (Tianjin, China). CNFs (99% purity) was purchased from Beijing Dk Nano technology Co., Ltd. (Beijing, China) and its particle size was in the 150e200 nm range. The stock solution containing the analytes was prepared by dissolving appropriate amount of them in ethanol and stored at 4 C under dark conditions. The working solutions were obtained daily by appropriately diluting the stock solution with deionized water. Analytical grade sodium chloride were purchased from Beijing Chemical Co. (Beijing, China). Deionized water used throughout experiments was purified using a Sartorius Arium 611 €ttingen, Germany). Chromatographic grade system (Sartorius, Go methanol (MeOH) was purchased from Fisher Corporation (Pittsburgh, PA, USA). HPLC-grade deionised water was obtained from a MilliQ water purification system (MilliQ Water; Molsheim, France).
To achieve the best extraction efficiency for target analytes, extraction conditions were optimised by analysing spiked samples. The experimental parameters, including the amount of adsorbents, the pH of sample solution and the type of desorption solvent, the flow rate of extraction and desorption, and the volume of extraction and desorption were investigated in detail. 3.1.1. Effect of the adsorbent amount Different amounts of the CNFs in a range of 5e30 mg were applied to extract MBC and TBZ from the sample solution (25 mL of 200 ng mL1 MBC and TBZ). The results indicated (Fig. 1A) that
2.2. Apparatus and analytical conditions A self-made glass column (20 cm 6.0 mm i.d.), a constant flow peristaltic pump with computer displaying and fraction collector (Shanghai Huxi Analysis Instrument Factory CO., LTD) were used for the SPE process. PTFE tubing with 0.5 mm i.d. was used for all connections. HPLC analyses were performed with an Agilent 1100 HPLC system (Palo Alto, CA, USA) equipped with an automatic sampler and diode array detector (DAD). Chromatographic separation of target analytes was performed on a ZOBAX Eclipse XDB-C18 (150 mm 4.6 mm, 5 mm) column (Agilent, Palo Alto, CA, USA) and the injection volume was 10 mL. A mixture of water and methanol (50:50, v/v) at a flow rate of 1.0 mL min1 was used as a mobile
Scheme 1. Schematic diagram of the microcolumn-SPE procedure.
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15 mg CNFs were enough for the extraction, and further increasing the amount of the adsorbent gave no significant improvement for the adsorption percentage of MBC and TBZ. Therefore, 15 mg was selected as the amount of the adsorbent. 3.1.2. Effect of pH and ionic strength of sample solution One of the critical parameter in the extraction of organic analytes is the pH of sample solution because the pH value of the solution determines the present state of analytes. In order to evaluate the effect of pH, different initial pH values of sample solution ranging from 3.0 to 12.0 was studied. According to Fig. 1B, the adsorption maxima occurred in a broad range of pH value from 5 to 11.0. Generally, the pH of natural MBC and TBZ solution was close to 5.7. In this study, the sample solutions were used directly without any pH adjustment. MBC and TBZ are characterized by 2 pKa values, the two predicted pKa values of MBC and TBZ are 4.25, 11.15 and 4.75, 12.0, respectively. At pH between of 5 and 11, analytes will exist as neutral molecules, nearly all of analytes molecules carry no net electrical charge, which makes them hardly have electrostatic attraction with CNFs, so the increase of pH from 5.0 to 11.0 had no significant effect on the adsorptive affinity of MBC and TBZ in the experiment. Hence, the adsorption mechanism might be attributed to p-p interactions, H-bonds and hydrophobic occurred between the CNFs s and analytes (Wang et al., 2015; Zhang, Fang, et al., 2013; Zhang, Lv, et al., 2013; Zhang, Pan, et al., 2013). In order to investigate the influence of the ionic strength, the adsorption experiments of MBC and TBZ were carried out in the presence of 0e1.0 mol L1 NaCl, respectively. Results showed that the recoveries were no significant effect with increasing NaCl concentrations. Furthermore, the ionic strength test also confirmed that the surface electrostatic effect had no influence on the overall adsorption of MBC and TBZ on CNFs. In this experiment, the sample solution without adjusting ionic strength was adopted. 3.1.3. Effects of sample flow rate and breakthrough volume In a microcolumn preconcentration system, the enrichment factor and analytical time mainly depend on the sample flow rate and breakthrough volume. A solution containing 2.0 mg mL1 of MBC and TBZ was passed through the CNFs packed microcolumn by using a peristaltic pump. The effect of sample flow rate in the range of 0.2e1.0 mL min was also evaluated. As shown in Fig. 2, it is clear that the breakthrough of MBC and TBZ took place after 35 min (35 mL, 1.0 mL min1), 100 min (50 mL, 0.5 mL min1) and 275 min (55 mL, 0.2 mL min1), respectively. The experimental results showed the breakthrough volume was increased from 35 to 55 mL with an decrease in flow rate from 1.0 to 0.2 mL min1. This was due
Fig. 2. Breakthrough profiles and effect of the sample flow rate for MBC and TBZ with a micro-column packed with the CNFs. (CNFs: 15 mg; Ci: 2.0 mg mL1; Ci are the initial concentration of the target analytes in sample solution; Ct are the concentration of the target analytes in effluent).
to a increase in the residence time, which facilitated the contact of target analytes to the CNFs. However, it also resulted in a much longer extraction time, which is time-consuming for real-time analysis. Considering the operating time and extraction efficiency, sample flow rate of 0.5 mL min1 and sample volume of 50 mL was used. From the area of the breakthrough curve (0.5 mL min1), the loading capacities of the CNFs for MBC and TBZ were evaluated as 9.1 and 10.6 mg g1, respectively.
3.1.4. Effect of eluting solvent In order to check the recovery efficiency of MBC and TBZ, desorption of the target analytes from the adsorbents was performed. Different eluent, including methanol (MeOH), ethanol (EtOH), acetonitrile (ACN), MeOH-0.1% formic acid (FA), MeOH-0.2% FA and MeOH-0.3% FA were studied. The results (Fig. 3) indicated that MeOH-0.2% FA solution gave the highest overall desorption efficiency for the target analytes. This was because MeOH-0.2% FA could convert the target analytes into ionic forms, thus reducing their affinity for the CNFs and facilitating the elution. Therefore, MeOH-0.2% FA was selected as the desorption solvent for the subsequent studies and high desorption efficiency and stable recoveries were obtained.
Fig. 1. Optimization of extraction conditions: (A) effect of amount of the CNFs on the adsorption efficient of MBC and TBZ and (B) effect of pH on the adsorption efficient of MBC and TBZ. (MBC and TBZ concentrations: 200 ng mL1; sample solution volume: 25 mL).
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Fig. 3. Optimization of desorption conditions: effect of eluent, elution volume and flow rate on the recoveries of MBC and TBZ. (CNFs: 15 mg; MBC and TBZ concentrations: 200 ng mL1; sample solution volume: 50 mL; sample flow rate: 0.5 mL min1).
3.1.5. Effects of elution volume and elution flow rate In order to investigate the effect of the elution volume, 50 mL of sample solution containing 200 ng mL1 of MBC and TBZ was passed through the CNFs packed microcolumn at a flow rate of 0.5 mL min1. The target analytes were eluted by MeOH-0.2% formic acid solution, and the elution volume was investigated from 1.0 mL to 5.0 mL (Fig. 3). The results indicated that recoveries of target analytes were enhanced by increasing elution volume. It was found that 3.0 mL of eluent was sufficient to recover all the species quantitatively. The flow rate of eluent is also an affecting parameter for recoveries of target analytes. With eluent volume kept at 3.0 mL, the influence of elution flow rate was investigated by changing the flow rate from 0.05 to 0.2 mL min1. With the increase of flow rate, the recoveries of target analytes were slightly decreased. The results (Fig. 3) indicated that the optimal elution flow rate were in the range of 0.05e0.1 mL min1. Therefore, the elution flow rate of 0.1 mL min1 was selected for the following experiments.
3.2. Interference studies Fruit, vegetable and juice samples contains considerable amounts of organic matter and inorganic ions. These substances may have a potentially competitive adsorption effect on MBC and TBZ in food samples. In order to assess the possible analytical applications of the proposed method, the effect of concomitant species on the determination of MBC and TBZ in real samples was examined under the optimal conditions as described above. The sample solutions containing 200 ng mL1 of MBC and TBZ and the added interfering matter were subjected to the proposed method. The tolerance ratio of each interfering matter was taken as the largest amount yielding an error in the determination of the target analytes not exceeding 5% (Table 1).
3.3. Stability of the CNFs The stability of CNFs was evaluated by checking the cycle number dependence of recoveries for 200 ng mL1 MBC and TBZ, using the same CNFs for subsequent cycles. After desorption, the regenerated CNFs was washed with deionized water to nearly neutral. The recycled adsorbents were reused, and the results (Fig. 4) indicate that the regenerated CNFs still maintained a high recoveries of the target analytes (recovery >90%) after five adsorptioneregeneration cycles. Therefore, CNFs had good reutilization and could be used as promising adsorbents for the effective extraction of benzimidazole fungicide residues in real samples. 3.4. Method evaluation 3.4.1. Analytical performances The performance of the SPE preconcentration system using CNFs packed microcolumn was evaluated under the optimized extraction conditions. A series of experiments were performed to obtain the linearity, precision, the limit of detection (LOD) and quantification (LOQ). All the experiments were performed in triplicate. As showed in Table 2, good linearities were obtained in the range of 2e500 ng mL1 with the correlation coefficients (r2)
Table 1 Effects of interfering matter on determination of 200 ng mL1 of MBC and TBZ. Interference
Interference to MBC and TBZ ratio (w/w)
Saccharose, glucose, fructose Ascorbic acid Naþ, Kþ, Fe3þ, Mg2þ, Ca2þ SO4 2 , PO4 3 , NO3 , HCO3 , CO3 2
400 800 1000 1000
Fig. 4. Reusability of the CNFs as microcolumn SPE adsorbents for extraction of MBC and TBZ (CNFs: 15 mg; MBC and TBZ concentrations: 200 ng mL1; sample solution volume: 50 mL; sample flow rate: 0.5 mL min1; eluent: 3.0 mL MeOH-0.2% FA; elution flow rate: 0.1 mL min1).
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Table 2 Analytical performance for MBC and TBZ obtained by microcolumn SPE-HPLC-DAD. Compound
Linear range (ng mL1)
Regression equation
LOD (ng mL1)
LOQ (ng mL1)
r2
EF
MBC TBZ
2.0e500.0 2.0e500.0
y ¼ 4.7316C þ 31.238 y ¼ 4.5596C þ 29.725
0.45 0.54
1.50 1.80
0.9964 0.9978
100 100
ranging from 0.9964 to 0.9978. The limit of detection (LOD, 3S/k, S: standard deviation of blank signal, n ¼ 9; k: slope of the working curve) and the limit of quantization (LOQ, 10S/k) were found ranging from 0.45 to 0.54 ng mL1 and 1.50e1.80 ng mL1, respectively. The proposed method showed good precision with the intra-day and inter-day RSDs in the range of 1.08e2.74% (n ¼ 6) and 2.82e3.94% (n ¼ 3), respectively. In order to calculate the EF for the analytes, five replicates were conducted for the sample solutions (V ¼ 50 mL) containing 200 ng mL1 of the analyte. Enrichment factor (EF) was calculated by following equations:
EF ¼ ca =cs
(1)
where ca and cs are the concentrations of the analyte in analytical solutions (V ¼ 0.5 mL) and sample solutions (V ¼ 50 mL), respectively. It was found that the EF of 100 was obtained under optimal conditions.
Fig. 5. HPLC-DAD Chromatograms obtained at 285 nm. (A) Blank apple juice sample after pretreatment by SPE; (B) The spiked apple juice sample (10.0 ng mL1) after pretreatment by SPE; (C) The standard solution of MBC and TBZ. (CNFs: 15 mg; MBC and TBZ concentrations: 200 ng mL1; sample volume: 50 mL; sample flow rate: 0.5 mL min1; eluent: 3.0 mL MeOH-0.2% FA; elution flow rate: 0.1 mL min1).
3.4.2. Analysis of samples To evaluate the applicability of the present method, fruit, vegetable and juice samples were analyzed. It can be seen (Fig. 5) that no significant interference peaks were found at the retention positions of MBC and TBZ. To evaluate the precision and accuracy of the proposed method, the spiked samples (10.0, 100.0, 200.0 ng mL1) were analyzed. The recoveries of the method were tested by five replicate experiments and the results were given in Table 3. The recoveries of MBC and TBZ were between 96.1% and 103.5%, with RSDs of 1.27 and 3.90%. It can be considered that the current method provides acceptable recoveries and precision for the determination of MBC and TBZ in real samples.
DLLME (CHCl3) (Wu et al., 2009), followed by HPLC analysis. Besides, compared to conventional absorbents or extractant, CNFs have high adsorption capacity and large special surface area, so an about 100 times high enrichment factor was obtained. Compared with SPE-HPLC-DAD, QuEChERS-LLE-LC-MS/MS methods (Gilbertpez, García-Reyes et al., 2012), the LODs of this method were Lo higher than that achieved by QuEChERS-LLE-LC-MS/MS method. Although the LODs of this method were higher than that obtained by QuEChERS-LLE-LC-MS/MS, the cost of the proposed method is lower and the operation is more convenient.
3.5. Comparing with other methods 4. Conclusions The proposed method based on microcolumn-SPE (CNFs)-HPLCDAD was compared with other methods as listed in Table 4. As can be seen from the table, the LODs of MBC and TBZ obtained by the proposed method were lower than that obtained by other sample pretreatment methods including SPE (SampliQ SCX) (Al-Ebaisat, pez Monzo n et al., 2007), 2011), SPME (CARePDMS fiber) (Lo
In this study, we proposed a new approach for simultaneous sensitive determinate of two fungicides in fruit, vegetable and juice samples by SPE using a CNFs packed microcolumn combined with HPLC-DAD. The results showed that CNFs had high enrichment capability and good stability. The packed column was reused five
Table 3 Recoveries of MBC and TBZ in food samples (mean ± SD, n ¼ 3). Samples
MBC Spike (ng mL1)
Apple juice
Lemon
Cucumber
a
0 10.0 100.0 200.0 0 10.0 100.0 200.0 0 10.0 100.0 200.0
n.d.¼ not detected.
TBZ Found (ng mL1) a
n.d. 10.12 ± 0.18 97.2 ± 1.95 195.6 ± 4.05 n.d. 10.35 ± 0.36 96.1 ± 3.75 194.9 ± 3.01 n.d. 10.23 ± 0.30 99.1 ± 2.86 197.3 ± 3.00
Recovery (%)
RSD (%)
Spike (ng mL1)
Found (ng mL1)
Recovery (%)
RSD (%)
e 101.2 97.2 97.8 e 103.5 96.1 97.4 e 102.3 99.1 98.6
e 1.82 2.01 2.07 e 3.52 3.90 1.54 e 2.99 2.88 1.52
0 10.0 100.0 200.0 0 10.0 100.0 200.0 0 10.0 100.0 200.0
n.d. 9.86 ± 0.34 101.3 ± 2.31 194.8 ± 3.41 n.d. 9.93 ± 0.23 97.2 ± 2.36 193.8 ± 2.45 n.d. 9.94 ± 0.26 100.9 ± 2.11 196.3 ± 3.05
e 98.6 101.3 97.4 e 99.3 97.2 96.9 e 99.4 100.9 98.2
e 3.42 2.28 1.75 e 2.32 2.42 1.27 e 2.66 2.09 1.55
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Table 4 Comparison of the proposed method with other method for the determination of MBC and TBZ. Analytical method
SPE-HPLC-VWD SPME-HPLC-FLD DLLME-HPLC-FLD QuEChERS-LLE-LC-MS/MS SPE-HPLC-DAD (this work)
Adsorbent/extractant
SampliQ SCX CARePDMS fiber CHCl3 acetonitrile CNFs Microcolumn
LOD (ng mL1) MBC
TBZ
3.55 1.30 0.5 0.003 0.45
e 0.04 1.0 0.003 0.54
times without loss in column performance. The proposed method demonstrated good linearity, low LOD, excellent repeatability and satisfactory recovery, and was proved to be sensitive, accurate and reliable in determining the fungicides in real samples. In addition, this method is characterized with simplicity of operation, minimum use of solvents and low cost. Therefore, it is optimistic about the prospect of the developed method in determining the fungicide residues in food sample. Acknowledgements This project was supported by the National Nature Science Foundation of China (NSFC51178212), the Science and Technology Foundation of Ocean and Fisheries of Liaoning Province (201408, 201406), the General project of scientific research of the Education Department of Liaoning Province (L2015206) and the Foundation of 211 project for Innovati-ve Talent Training, Liaoning University. The authors also thank their colleagues and other students who participated in this study. References Al-Ebaisat, H. (2011). Determination of some benzimidazole fungicides in tomato puree by high performance liquid chromatography with SampliQ polymer SCX solid phase extraction. Arabian Journal of Chemistry, 4(1), 115e117. Banks, D., & Soliman, M. R. (1997). Protective effects of antioxidants against benomyl-induced lipid peroxidation and glutathione depletion in rats. Toxicology, 116(1e3), 177e181. Bhuvaneswari, M. S., Bramnik, N. N., Ensling, D., Ehrenberg, H., & Jaegermann, W. (2008). Synthesis and characterization of carbon nano fiber/LiFePO4 composites for Li-ion batteries. Journal of Power Sources, 180(1), 553e560. Corma, A., & Garcia, H. (2008). Supported gold nanoparticles as catalysts for organic reactions. Chemical Society Reviews, 37(9), 2096e2126. pez, B., García-Reyes, J. F., & Molina-Díaz, A. (2012). Determination of Gilbert-Lo fungicide residues in baby food by liquid chromatographyeion trap tandem mass spectrometry. Food Chemistry, 135(2), 780e786. pez, B., Jae n-Martos, L., García-Reyes, J. F., Villar-Pulido, M., Polgar, L., Gilbert-Lo Ramos-Martos, N., et al. (2012). Study on the occurrence of pesticide residues in fruit-based soft drinks from the EU market and morocco using liquid
chromatographyemass spectrometry. Food Control, 26(2), 341e346. Guo, B., Huang, Z., Wang, M., Wang, X., Zhang, Y., Chen, B., et al. (2010). Simultaneous direct analysis of benzimidazole fungicides and relevant metabolites in agricultural products based on multifunction dispersive solid-phase extraction and liquid chromatographyemass spectrometry. Journal of Chromatography A, 1217(29), 4796e4807. Kim, H., Lee, D., & Moon, J. (2011). Co-electrospun Pd-coated porous carbon nanofibers for hydrogen storage applications. International Journal of Hydrogen Energy, 36(5), 3566e3573. Klein, K. L., Melechko, A. V., McKnight, T. E., Retterer, S. T., Rack, P. D., Fowlkes, J. D., et al. (2008). Surface characterization and functionalization of carbon nanofibers. Journal of Applied Physics, 103(6), 061301. pez Monzo n, A., Vega Moreno, D., Torres Padro n, M. E., Sosa Ferrera, Z., & Santana Lo Rodríguez, J. J. (2007). Solid-phase microextraction of benzimidazole fungicides in environmental liquid samples and HPLCefluorescence determination. Analytical and Bioanalytical Chemistry, 387(6), 1957e1963. Natale, D., Gibis, M., Rodriguez-Estrada, M. T., & Weiss, J. (2014). Inhibitory effect of liposomal solutions of grape seed extract on the formation of heterocyclic aromatic amines. Journal of Agricultural and Food Chemistry, 62(1), 279e287. Oliferova, L., Statkus, M., Tsysin, G., Shpigun, O., & Zolotov, Y. (2005). On-line solidphase extraction and HPLC determination of polycyclic aromatic hydrocarbons in water using fluorocarbon polymer sorbents. Analytica Chimica Acta, 538(1e2), 35e40. Singhal, R. M., Sharma, A., & Verma, N. (2008). Micronano hierarchal web of activated carbon fibers for catalytic gas adsorption and reaction. Industrial & Engineering Chemistry Research, 47(10), 3700e3707. Thangaraj, R., Nellaiappan, S., Sudhakaran, R., & Kumar, A. S. (2014). A flow injection analysis coupled dual electrochemical detector for selective and simultaneous detection of guanine and adenine. Electrochimica Acta, 123(0), 485e493. Valc arcel, M., C ardenas, S., Simonet, B. M., Moliner-Martínez, Y., & Lucena, R. (2008). Carbon nanostructures as sorbent materials in analytical processes. TrAC Trends in Analytical Chemistry, 27(1), 34e43. Veneziano, A., Vacca, G., Arana, S., De Simone, F., & Rastrelli, L. (2004). Determination of carbendazim, thiabendazole and thiophanate-methyl in banana (Musa acuminata) samples imported to Italy. Food Chemistry, 87(3), 383e386. Wang, L., Zhang, Z., Xu, X., Zhang, D., Wang, F., & Zhang, L. (2015). Simultaneous determination of four trace level endocrine disrupting compounds in environmental samples by solid-phase microextraction coupled with HPLC. Talanta, 142(0), 97e103. WHO, C. (1989). Environmental health criteria. Geneva, Switzerland. Wu, Q., Li, Y., Wang, C., Liu, Z., Zang, X., Zhou, X., et al. (2009). Dispersive liquideliquid microextraction combined with high performance liquid chromatographyefluorescence detection for the determination of carbendazim and thiabendazole in environmental samples. Analytica Chimica Acta, 638(2), 139e145. Zhang, L., Fang, P., Yang, L., Zhang, J., & Wang, X. (2013). Rapid method for the separation and recovery of endocrine-disrupting compound bisphenol AP from wastewater. Langmuir, 29(12), 3968e3975. Zhang, L., Lv, J., Xu, T., Yang, L., Jiang, X., & Li, Q. (2013). High efficiency removal and recovery of an endocrine disrupting compoundebisphenol AF from wastewaters. Separation and Purification Technology, 116(0), 145e153. Zhang, L., Pan, F., Liu, X., Yang, L., Jiang, X., Yang, J., et al. (2013). Multi-walled carbon nanotubes as sorbent for recovery of endocrine disrupting compoundbisphenol F from wastewater. Chemical Engineering Journal, 218(0), 238e246. Zhou, Y.-Y., Yan, X.-P., Kim, K.-N., Wang, S.-W., & Liu, M.-G. (2006). Exploration of coordination polymer as sorbent for flow injection solid-phase extraction online coupled with high-performance liquid chromatography for determination of polycyclic aromatic hydrocarbons in environmental materials. Journal of Chromatography A, 1116(1e2), 172e178.