Talanta 132 (2015) 416–423
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Development of gold nanoparticles modified screen-printed carbon electrode for the analysis of thiram, disulfiram and their derivative in food using ultra-high performance liquid chromatography Kanokwan Charoenkitamorn a, Orawon Chailapakul a,c,n, Weena Siangproh b,nn a Electrochemistry and Optical Spectroscopy Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok 10330, Thailand b Department of Chemistry, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, Wattana, Bangkok 10110, Thailand c National Center of Excellent of Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand
art ic l e i nf o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 10 September 2014 Accepted 11 September 2014 Available online 30 September 2014
For the first time, gold nanoparticles (AuNPs) modified screen-printed carbon electrode (SPCE) was developed as working electrode in ultra-high performance liquid chromatography (UHPLC) coupled with electrochemical detection (UHPLC-ED) for simultaneous determination of thiram, disulfiram, and N,N-diethyl-N’,N’dimethylthiuram disulfide, their derivative compound. The separation was performed in reversed-phase mode using C18 column, mobile phase consisting of 55:45 (v/v) ratio of 0.05 M phosphate buffer solution (pH 5) and acetonitrile at a flow rate of 1.5 mL min 1. For the detection part, the amperometric detection was chosen with a detection potential of 1.2 V vs. Ag/AgCl. Under the optimal conditions, the good linear relationship was obtained in the range of 0.07–15, 0.07–12, and 0.5–15 mg mL 1 (correlation coefficient more than 0.9900) for thiram, N,N-diethyl-N’,N’-dimethylthiuram disulfide, and disulfiram, respectively. The limits of detection (LODs) of thiram, N,N-diethyl-N’,N’-dimethylthiuram disulfide, and disulfiram were 0.022, 0.023, and 0.165 mg mL 1, respectively. Moreover, this method was successfully applied for the detection of these compounds in real samples (apple, grape and lettuce) with the recoveries ranging from 94.3% to 108.8%. To validate this developed method, a highly quantitative agreement was clearly observed compared to standard UHPLC–UV system. Therefore, the proposed electrode can be effectively used as an alternative electrode in UHPLC-ED for rapid, selective, highly sensitive, and simultaneous determination of thiram, disulfiram, and N,N-diethyl-N’,N’dimethylthiuram disulfide. & 2014 Elsevier B.V. All rights reserved.
Keywords: Dithiocarbamates Fungicides Screen-printed carbon electrode Gold nanoparticles Simultaneous determination Ultra-high performance liquid chromatography
1. Introduction Nowadays, the designing of electrochemical sensor has been developed in the field of electrochemistry to improve the analytical efficiency in the terms of sensitivity, selectivity, reliability, ease of fabrication and use, and low cost. Screen printing is the technology used for fabrication of biosensors and chemical sensors instead of using large-scale electrode. Their various advantages such as miniaturization, versatility, and low cost of production are really attractive [1]. In addition, many laboratories use the screen-printing for in-house production of sensors. Screen-printed carbon electrode (SPCE) is an alternative material used instead of using the traditional electrodes based on economic substrate. Recently, SPCE have been successfully used as the electrochemical sensor for various researches due to the n
Corresponding author. Tel.: þ 662 218 7615. Corresponding author. Tel.: þ 662 249 5000x18208. E-mail addresses: [email protected] (O. Chailapakul), [email protected] (W. Siangproh). nn
http://dx.doi.org/10.1016/j.talanta.2014.09.020 0039-9140/& 2014 Elsevier B.V. All rights reserved.
simplicity to produce and still give the rapid responses [2]. Moreover, the main advantage of SPCE is able to use only once and then is discarded. This advantage can be used to solve the problems of surface fouling compared with those of conventional electrodes. However, the limitation of SPCE is small surface area of working electrode leading to the lack of sensitivity [3]. Therefore, electrode modification is necessary to solve this problem. For electrode modification, metal-nanoparticles modified SPCE is focused to improve the electrochemical efficiency. Metal-nanoparticles are attractive catalyst in the electrochemical applications. Transition metals, especially precious metals, show very high catalytic abilities for catalyzing the redox process of some interested molecules, making the use of electroanalytical techniques for applications are extended [4]. Particularly, gold nanoparticles (AuNPs) have received significant attention in recent years. AuNPs are widely used in many fields due to their unique optical and physical properties, such as surface plasmon oscillations for labeling, imaging, and sensing [5]. For the electrochemical fields, AuNPs have been widely used for modification of electrode because of their benefits including catalysis, mass
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transport, and high effective surface area [1]. AuNPs have the ability to improve the detection signal, electron transfer, which make the limit of detection in electrochemical sensor is reduced. Moreover, the most importance is AuNPs showed strong specific interaction with the sulfur-containing compounds [6]. Therefore, AuNPs became an interesting nanomaterial used as modifier onto working electrode for electrochemical detection of sulfur-containing compounds. Thiram (tetramethyl thiuram) and disulfiram (tetraethyl thiuram) are oraganosulfur compounds in the group of dithiocarbamates (DTCs) fungicides. DTCs have been used in agriculture as fungicides in foliage vegetable and fruit [7]. Besides these agricultural applications, DTCs are also used in industry such as vulcanization accelerator and antioxidant in rubber. The use of DTCs results in their release into the environment causing the contamination in food and water which leads to hazardous effect in the living organisms through diverse pathways [8]. Environmental problem resulting from uncontrollable use of fungicides in agriculture is one of the most important evidence. Therefore, it is necessary to continuously develop simple, selective, and sensitive methods for analysis of fungicides [9]. Various methods have been developed for analysis of DTCs. Spectrometry [10–12] and head space gas chromatography [13] have been used as the methods for the determination of DTCs after their decomposition to carbon disulfide (CS2). From these methods, most of DTCs can degrade to CS2. Therefore, they are unable to distinguish among different DTCs. Moreover, gas chromatography [14,15], capillary electrophoresis [16–20], and high performance liquid chromatography (HPLC) were used for simultaneous determination of DTCs coupled with UV detection. Especially, HPLC is the simple technique used for easy separation of the disulfide substance. However, the drawback of the UV detection is low sensitivity. Therefore, HPLC coupled with electrochemical detection became an important choice for the determination of disulfides [21]. The main advantage of electrochemical detection compared to those of spectrometry is that the derivatization step is not needed, which makes the analysis time and cost reduced. In addition, the use of electrochemical detection after the separation by HPLC can provide more sensitive detection than UV [22]. Recently, ultra-high performance liquid chromatography coupled with electrochemical detection (UHPLC-ED) is a high potential alternative method for various analysis. UHPLC concerns to the development of short column packed with small particles for working at high pressures (4400 bar). The use of UHPLC can enhance chromatographic performances in terms of efficiency, resolution, and analysis time [23]. In the detection part, AuNPs modified electrode was used as amperometric detection. Amperometry is an attractive potential technique because of its fast response, high sensitivity, relatively low cost of equipment, no need for complicated operation, and simplicity for direct sulfur assay [24]. With such benefits from the mentioned background, the objective of this work is to develop AuNPs modified SPCE in UHPLC-ED system for simultaneous determination of DTCs with high sensitivity, specificity, and rapid analysis. Thiram (tetramethyl thiuram) and disulfiram (tetraethyl thiuram) are chosen as representative compounds of DTCs because they are importantly applied to control a variety of pest [25]. Typically, when thiram and disulfiram are mixed together, the intermediate of N,Ndiethyl-N’,N’-dimethylthiuram disulfide (DEDMTDS) appears as the derivative of thiram and disulfiram. This compound is one of DTCs that affects human health and has the same effect of thiram and disulfiram. The presence of N,N-diethyl-N’,N’-dimethylthiuram disulfide can also use to monitor thiram and disulfiram in samples. Therefore, the detection of N, N-diethyl-N’, N’-dimethylthiuram disulfide is important. This derivative compound is also determined along with the measurement of thiram and disulfiram. To the best of our knowledge, there is no publication report about the use of AuNPs modified SPCE as working electrode for ED
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after the UHPLC separation to simultaneously determine thiram, DEDMTDS, and disulfiram. Moreover, this developed UHPLC-ED using AuNPs modified SPCE was applied for the first time to analyze thiram, DEDMTDS, and disulfiramthe DTCs in fruits and vegetables collected from local markets. 2. Materials and methods 2.1. Chemicals and reagents All chemicals used in this work were of analytical grade, and all solutions were prepared using Milli-Q water from Millipore (RZ18.2 M Ω cm 1). Acetonitrile (HPLC-grade), chloroform, and sulfuric acid were obtained from Merck (Darmstadt, Germany). Potassium dihydrogen phosphate (KH2PO4) was acquired from BDH laboratory supplies (VWR International Ltd., England). Disodium hydrogen phosphate (Na2HPO4) and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany). Standard thiram (tetramethyl thiuram) was obtained from Chem Service (West chester, PA, USA). Standard disulfiram (tetraethyl thiuram) and gold (III) chloride solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). All of stock standard solutions (1000 mg mL 1) were prepared by dissolving 10 mg of each analyte in acetonitrile. The solutions were then placed in an amber bottle and stored at 4 1C. To prepare working standard solution, the standard solution was mixed and diluted in suitable proportions of acetonitrile:Milli-Q water (50:50; v/v), and kept for 5 min before measurement. All solutions and solvents were filtered by 0.22 mm nylon membranes prior to use in UHPLC separation. 2.2. Fabrication and modification of screen-printed carbon electrode The working electrode was fabricated using screen printing and modified by electrodeposition techniques. The Ag/AgCl electrode and Pt wire was used as reference and auxiliary electrodes, respectively. To fabricate the in-house screen-printed carbon electrode (Fig. S1), the Ag/AgCl ink was first printed on the PVC substrate for using as a conductive pad. Then, the carbon ink was printed to create a working electrode. The each step of printing electrode was allowed to dry in an oven at 55 1C for 1 h. The screen-printed carbon electrode was ready to modify in the next step. For the electrodes modification, the 10 mM of gold (III) solution was used to modify onto electrode surface by electrodeposition technique. The three-electrode system was placed into cell containing 2 mL of solution of 10 mM of gold (III) solution in 0.5 M H2SO4 followed by deposition of gold onto the SPCE at a deposition potential of –0.6 V (vs. Ag/AgCl) for 50 s while the solution was stirred (by means of a mechanical stirrer bar). The gold nanoparticles (AuNPs) were obtained onto the electrode surface. After the modification, the AuNPs modified SPCE was carefully rinsed with distilled water and dried under N2 gas prior to use. 2.3. Cyclic voltammetry The electrochemical reaction and optimal parameters for electrodeposition step were studied by cyclic voltammetry (CV) using a CH instrument potentiostat 1232 A (CH Instrument, Inc., USA). The working electrodes used in this work were bare SPCE and AuNPs modified SPCE. The reference and auxiliary electrode were silver/silver chloride electrode and Pt wire, respectively. All experiments were done at room temperature. 2.4. UHPLC experiment and apparatus The UHPLC system was performed in reversed-phase mode using an LC-20ADXR solvent deliver unit (Shimadzu Corporation,
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Japan), an autosampler (SIL-20 A) with 0.1–100 mL loop and a KinetexTM core-shell C18 column (50 mm x 4.6 mm i.d.; particle size, 2.6 mm, Phenomenex). The UHPLC-electrochemical measurement was carried out in 45:55 (v/v) ratio acetonitrile: 0.05 M phosphate buffer solution (pH 5), which was adjusted to pH 5 with 0.1 M sodium hydroxide solution, as mobile phase with an applied potential of 1.2 V vs. Ag/AgCl, an injection volume of 50 mL, and a flow rate of 1.5 mL min 1. All chromatographic separations were performed at room temperature. 2.5. Electrochemical measurement The thin-layer flow cell consisted of a working AuNPs modified SPCE, a reference Ag/AgCl electrode (Bioanalytical system Inc., USA), and a stainless steel tube counter electrode. The geometric area of the AuNPs modified SPCE in the flow cell was estimated to be 0.40 cm2 with 1 mm thick silicon rubber gasket as a spacer. An electrochemical measurement for amperometric control and signal processing was performed using CH instrument potentiostat 1232 A. 2.6. Sample preparation The three real samples including apple, grape and lettuce were obtained from local markets in Thailand. For analysis, 50 g of cut sample was weighted accurately and transferred to a blender. Then 50 mL of 500 mg mL 1 of standard mixture solution and chloroform were added. The mixture was homogenized with blender and transferred into centrifuge tubes. After centrifugation at 4000 rpm for 10 min, all of the organic phases were filtered through a Buchner funnel with Whatman filter paper No. 4 and transferred to 250 mL vessel of the rotary vacuum evaporator for evaporation to dryness at room temperature. The residues were dissolved in 4.5 mL of acetonitrile using ultrasonic stirring, filtered through the 0.2 mm pore size of nylon membrane, and collected in 5 mL of volumetric flask. After that, acetonitrile filtered through the 0.2 mm pore size of nylon membrane was added to adjust volume to 5 mL of solution. Finally, 50 mL of the solution was injected into the UHPLC system. The determination of DTCs in samples was analyzed within the linear dynamic range that obtained by adding different volume of 500 mg mL 1 of standard mixture solution into the blank sample and prepared following the above treatment. 2.7. Method validation Standard solutions and samples were analyzed, and the currents were integrated. A calibration curve of standard solutions was performed under optimal conditions and treated with linear least square regression analysis using the Excel software package. The limit of detection (LOD) and limit of quantification (LOQ) were determined from the 3 Sbl/S and 10 Sbl/S, respectively, where Sbl is standard deviation of blank (n ¼10), and S is sensitivity of method, obtained from the slope of the linearity. For the precision of intraday and inter-day, three concentrations of DCTs (1, 6, and 12 mg mL 1) were observed for three times within a day and three different days. To validate the developed method, UHPLC-ED was compared to the standard method of UHPLC coupled with ultraviolet detection (UHPLC–UV) using the paired t-test.
3. Results and discussion 3.1. Surface morphology of AuNPs modified SPCE Gold (III) solution was used to generate the AuNPs on the electrode surface by electrodeposition. The surface morphology of bare SPCE and AuNPs modified SPCE was investigated using
Fig. 1. SEM images of (A) bare SPCE and (B) AuNPs modified SPCE prepared by electrodeposition of 10 mM gold (III) solution at applied potential of -0.6 V for 50 s.
scanning electron microscope (SEM) as shown in Fig. 1A and B, respectively. After the modification, the well dispersed gold particles in nanoscale was observed (Fig. 1B). The distribution of AuNPs on the electrode surface led to increase the surface area and improve the electrochemical sensitivity of the modified electrode. 3.2. Optimization of the electrode modification For the electrode modification using electrodeposition technique, the deposition potential and deposition time were optimized because these factors can effect to the analytical results. First, thiram was selected as the representative compounds because it is the best electrochemically active species at bare carbon electrode. Therefore, it is easy to observe the signal obtained from modified and unmodified electrode. To obtain the optimal condition of AuNPs modified SPCE, the dependence of deposition potential and
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Fig. 2. Effect of the (A) deposition potential and (B) deposition time of thiram at AuNPs modified SPCE. Data are shown as the mean7 SD derived from three replicates.
deposition time were studied using cyclic voltammetry, and the relationship between the studied parameter and current response of thiram was plotted. The deposition potential was studied in the range of –1.0 to 0.2 V. The current signal of thiram was initially increased as the function of deposition potential from –1.0 to – 0.6 V and then decreased (Fig. 2A). For the deposition time, the current signal of thiram increased from 10 s to 50 s and then decreased (Fig. 2B). Therefore, the deposition potential of –0.6 V and the deposition time of 50 s were chosen as optimal parameters for modification of AuNPs onto the electrode surface.
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Fig. 3. (A) Cyclic voltammogram of thiram at the AuNPs modified SPCE (red line) compared to bare SPCE (blue line) and (B) comparison of oxidation current density between AuNPs modified SPCE (red rod) and bare SPCE (blue rod) vs. Ag/AgCl at the concentration of 10 mg mL 1 in 55:45 of 0.05 M phosphate solution pH 5: acetonitrile, scan rate 100 mV s 1.
3.3. Electrochemical response of thiram at AuNPs modified SPCE As mentioned above, thiram is the best electrochemically active species. Thus, it was used as the representative of DTCs to study the electrochemical response. The cyclic voltammogram (CV) of 10 mg mL 1 of thiram obtained from bare SPCE and AuNPs modified SPCE at the potential range of 0.0 to 1.4 V was shown in Fig. 3. From CV results, the anodic current of thiram was investigated, and the current at AuNPs modified SPCE was significantly higher than the current obtained from bare SPCE due to the distribution of AuNPs on the electrode surface. We believe that the AuNPs on the electrode surface can enhance electron transfer and also improve the electrochemical determination of DTCs. For the comparison of oxidation current density between AuNPs modified SPCE and bare SPCE (Fig. 3B), the higher current density about nine times was observed at AuNPs modified SPCE. This result indicated that the AuNPs modified SPCE can be assuring electrode for the detection of thiram, disulfiram, and DEDMTDS. Therefore, this modified electrode can be an alternative electrode
Fig. 4. Representative UHPLC-ED chromatogram of 12 mg mL 1 of thiram (a), DEDMTDS (b), and disulfiram (c) in a mobile phase (0.05 M phosphate solution (pH 5): acetonitrile), the detection potential of 1.2 V vs. Ag/AgCl using AuNPs modified SPCE, the injection volume of 50 mL, and the flow rate of 1.5 mL min 1. Chromatograms are representative of at least three independent repetitions.
for using as working electrode with the advantages of low-cost material, simple fabrication and high sensitivity in case of detection DTCs. 3.4. UHPLC separation In this work, C18 column was used to separate thiram, DEDMTDS, and disulfiram by isocratic elution. The effect of percentage of acetonitrile used in the mobile phase on the
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retention characteristics was first evaluated. When the percentage of acetonitrile r40 was used, the separation time was higher than 20 min and peak broadening of disulfiram was observed. On the other hand, at higher content of acetonitrile in mobile phase, the current signal of thiram, DEDMTDS, and disulfiram were decreased due to the low of diffusion coefficient with changing ionic strength of the medium. Therefore, the acetonitrile of 45% was selected as
compromise to achieve the completely separated them within 6 min with good sensitivity. In addition, 0.05 M phosphate buffer solution (pH 5) was chosen as the electrolyte solution because the high concentration of salt in buffer solution was not suitable for the UHPLC system. The chromatogram for the separation of standard mixture solution was shown in Fig. 4. The retention time of thiram, DEDMTDS, and disulfiram was 1.5, 2.6, and 5.4 min, respectively, and the retention time stability was found in the range of 0.1% to 1.5%. The peak widths were 11.6 s for thiram, 14.2 s for DEDMTDS, and 18.8 s for disulfiram. Therefore, this proposed system offers the rapid time for separating DTCs. 3.5. Hydrodynamic voltammetry Hydrodynamic voltammetry was used to optimize the detection potential. The potential was studied in the range of 0.8 to 1.4 V. The peak current after each injection at different potential was recorded corresponding to the background current. These data were plotted as a function of applied potential to obtain the hydrodynamic curves as shown in Fig. 5. The detection potentials significantly affected the oxidation current of the thiram, DEDMTDS, and disulfiram as well as the background current (Fig. 5A). Therefore, the net current after background subtraction was studied, and signal-to-background (S/B) ratios were plotted corresponding to the detection potential. It was found that the current increased when the potential increased up to 1.2 V for all compounds (Fig. 5B). Therefore, the detection potential of 1.2 V was selected as optimal detection potential for amperometric detection of thiram, DEDMTDS, and disulfiram after their separation with UHPLC. 3.6. Method validation
Fig. 5. Hydrodynamic voltammetric results at the AuNPs modified SPCE for a 10 mg mL 1 each mixture (50 mL total) of thiram and disulfiram. (A) thiram (blue line), DEDMTDS (green line), disulfiram (red line), and background (purple line); (B) hydrodynamic voltammogram of signal-to-background ratios. The other conditions are the same as in Fig. 4. Data are shown as the mean 7 1 SD derived from three independent repetitions.
3.6.1. Analytical performance The analytical performance of this proposed system was studied. The calibration curve between the concentration and current response of analytes was plotted as shown in Fig. 6. Under the optimal conditions, the linearity was observed in the range of 0.07 to 15 mg mL 1 for thiram, 0.07 to 12 mg mL 1 for DEDMTDS, and 0.5 to 15 mg mL 1 for disulfiram. The limit of detection (LOD) and limit of quantification (LOQ) were calculated from 3 Sbl/S and 10 Sbl/S, where Sbl is the standard deviation of blank measurements (n¼10), and S is the sensitivity of the method or the slope of linearity. Good linearity value with r2 4 0.99 was obtained. LODs and LOQs were summarized in Table 1. In addition, when the performance of AuNPs
Fig. 6. The amperometric voltammogram for different concentrations of thiram (a), DEDMTDS (b), and disulfiram (c) (0.07 to 15 mg mL 1) using AuNPs modified SPCE and the corresponding calibration plots (inset). Measurements were performed under the optimal conditions.
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Table 1 Linearity, limit of detection (LOD) and limit of quantitation (LOQ) of the UHPLC-ED method (n¼3). Analyte
Linear range (mg mL 1 )
Slope (peak height) (mA/mg mL 1 )
Intercept (mA)
r2
LOD (mg mL 1 )
LOQ (mg mL 1 )
Thiram DEDMTDS Disulfiram
0.07–15 0.07–12 0.5–15
0.2585 0.3247 0.1283
0.1636 0.129 0.0406
0.9905 0.9945 0.9948
0.022 0.023 0.165
0.074 0.076 0.551
Table 2 Recent report using different types of electrodes for electrochemical determination of DTCs. Modified electrode
Detection method
Linearity range (mg mL 1)
LOD (mg mL 1)
Analyte
Real sample
Reference
Cylindrical carbon fiber microelectrode Graphite-poly (tetrafluoroethylene) Hanging mercury drop electrode
Square-wave voltammetry
0.24–144
0.10
Thiram
Grape
[7]
2–100 0.01–0.6
0.14–1.0 0.01
Thiram and disulfiram Ziram
Apple rice
[26] [27]
Hanging mercury drop electrode
Amperometry Differential pulse voltammetry Square-wave voltammetry
0.01–0.19
0.0072
Ziram
[28]
AuNPs modified SPCE
Amperometry
0.07–15
0.022– 0.165
Thiram, DEDMTDS and disulfiram
Potato, cabbage and tomato Grape, apple and lettuce
This work
Table 3 The intra- and inter-precisions and recoveries of the UHPLC-ED method (n¼3). Samples
Apple
Spiked level (mg mL 1)
1
3
6
9
12
Grape
1
3
6
9
12
Lettuce
1
3
6
9
12
Analyte
Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram
Intra-day
Inter-day
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
97.1 101.5 104.1 102.7 102.7 108.5 109.0 109.2 97.4 109.3 109.2 103.6 101.5 106.5 102.6 100.1 101.5 108.8 103.9 105.2 107.9 103.5 98.9 98.4 107.4 100.4 97.5 106.1 102.1 100.5 98.8 102.6 102.4 105.1 102.4 104.1 98.9 98.7 106.0 108.0 100.5 103.1 102.6 100.5 100.1
1.1 0.2 3.3 0.1 0.3 0.3 0.9 1.3 0.6 0.6 0.3 0.6 0.2 0.4 0.2 0.2 2.3 0.7 0.6 0.2 0.3 1.0 1.5 0.2 0.5 1.0 0.4 0.4 0.7 0.3 0.8 1.4 1.7 0.9 1.0 1.9 0.2 0.5 0.5 1.8 3.7 0.8 1.3 2.9 0.6
99.0 100.6 100.6 100.7 102.0 103.3 104.0 104.8 98.1 107.0 107.7 104.2 101.9 102.5 101.4 102.3 99.7 104.2 100.7 102.2 104.7 103.3 102.6 100.6 107.2 102.6 98.0 103.3 101.9 100.9 101.9 101.6 100.8 99.7 103.4 99.0 96.7 101.7 101.3 105.8 101.8 102.6 101.6 101.1 102.5
3.7 1.0 4.9 5.7 1.4 4.4 4.4 4.0 2.4 1.8 1.7 4.9 0.7 3.4 2.5 3.3 1.9 7.7 2.8 2.5 2.7 2.9 4.9 3.4 0.1 4.7 2.6 2.4 0.8 5.0 3.4 1.4 4.3 4.8 3.6 4.9 4.4 4.7 4.1 2.5 3.8 4.2 2.5 0.8 2.3
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Table 4 Determination of DTCs level in different samples (n ¼3) by traditional UHPLC-UV method and the developed UHPLC-ED method reported here. Samples
Apple
Spiked level (mg mL 1)
1
6
12
Grape
1
6
12
Lettuce
1
6
12
Paired two-tail test
t values (at 1 mg mL 1)
t values (at 6 mg mL 1)
t values (at 12 mg mL 1)
Analyte
Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram
t critical
modified SPCE was compared to the other previous electrodes for the detection of DTCs as shown in Table 2. It was found that the LODs obtained from the proposed electrode were lower than using cylindrical carbon fiber [7] and graphite-poly (tetrafluoroethylene) composite electrode [26]. However, few researches used the mercury electrode to provide lower LOD than this method but the mercury is toxic [27,28]. Therefore, the proposed electrode offers less toxicity, uncomplicated, and low cost. Additionally, the proposed electrode shows good electrocatalytic properties for DTCs detection to obtain a high electrochemical sensitivity.
3.6.2. Application to real samples To assess the applicability of the proposed method, target compounds in samples from local supermarkets were investigated. The proposed method was applied for the detection of three different samples, including apple, grape, and lettuce. The standard addition method was chosen to investigate the reliability of this proposed system. The precision of the analytical process was evaluated by the repeatability of the process. The spiked concentrations in the dynamic linearity between 1 to 15 mg mL 1 were studied to calculate the RSD percentage. The summary of intraand inter-day precision, and recovery that obtained from the proposed method was shown in Table 3. The RSDs and recoveries of intra-day were found in the range of 0.1–3.7% and 94.3–108.8%, respectively, while the inter-day RSDs and recoveries were found in the range of 0.1–5.7% and 95.8–107.7%, respectively. Therefore,
Amount found (mg mL 1) (x7 SD)
% recovery
UHPLC-ED
UHPLC-UV
UHPLC-ED
UHPLC-UV
0.99 70.04 1.017 0.01 0.98 70.05 6.247 0.27 6.15 70.10 5.89 70.14 12.23 70.50 12.30 70.49 12.42 70.25 1.02 7 0.03 1.017 0.02 1.047 0.08 6.20 70.18 6.15 70.30 6.04 7 0.21 12.177 0.34 12.23 70.60 12.40 70.41 1.02 7 0.03 1.02 7 0.01 1.017 0.04 5.80 70.26 6.10 70.28 6.08 70.25 12.20 70.41 12.137 0.47 12.30 70.20 0.104 0.898 0.792 2.293 3.029 1.747 2.808 2.989 0.243 4.303
1.02 7 0.03 1.01 70.02 1.01 70.05 5.79 7 0.26 6.12 70.19 5.72 70.13 11.90 7 0.36 11.78 7 0.62 12.26 7 0.23 1.02 7 0.02 1.007 0.01 0.99 7 0.01 5.92 7 0.19 6.017 0.22 6.047 0.12 12.09 7 0.41 12.00 70.60 12.30 7 0.20 0.98 7 0.03 1.02 7 0.04 0.97 70.09 5.75 7 0.25 5.99 7 0.22 5.99 7 0.13 11.98 7 0.27 11.94 7 0.62 12.117 0.35
99.0 100.6 97.5 104.0 102.5 98.1 101.9 102.5 103.5 102.3 100.8 104.2 103.3 102.6 100.6 101.4 101.9 103.3 101.9 102.2 100.8 96.7 101.7 101.3 101.6 101.1 102.5
102.2 101.1 100.6 96.5 101.9 95.4 99.2 98.1 102.1 102.2 99.7 98.7 98.7 100.1 100.7 100.8 100.0 102.5 98.1 101.6 97.1 95.8 99.8 99.8 99.9 99.5 100.9
this method is an alternative and suitable for rapid separation and simultaneous determination of DTCs. To validate the proposed method, UHPLC-UV was used as a standard method to compare the acceptable and reliable. The results obtained from UHPLC-ED and UHPLC-UV were compared by a paired t-test at the 95% confidence for three samples that spiked with three standard concentrations (1, 6, and 12 mg mL 1 to represent the low, medium, and high level, respectively). The critical t-value was significantly higher than calculated t-values between two assays. From the results shown in Table 4, the calculated t-values of three concentrations were found in the range of –0.243 to 3.029 and lower than critical t-values (4.303). It can be concluded that there is no significant difference between UHPLC–ED and conventional UHPLC– UV method. Therefore, the results obtained from UHPLC coupled with amperometric detection using AuNPs modified SPCE is acceptable and reliable for applying to simultaneous determination of DTCs in food.
4. Conclusions AuNPs modified SPCE was firstly developed for the determination of thiram, disulfiram, and DEDMTDS after their separation with UHPLC system. Under the optimal conditions, the separation was complete within 6 min, and the high current response signal was obtained at AuNPs modified SPCE. The main advantages for the use of AuNPs modified SPCE are in term of high sensitivity, low-cost and simple fabrication-based material. Reproducible signal from %RSD for intra- and inter-day were below 5%. These
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results indicated that the high reproducibility was obtained using this system for long time. Moreover, the proposed UHPLC-ED system is an acceptable and reliable method compared to standard UHPLC-UV system using paired t-test. Ultimately, the UHPLC coupled with AuNPs modified SPCE amperometry is successfully applied for real samples. This proposed electrode could be a novel or an alternative electrode in UHPLC-ED system for the simultaneous determination of DTCs in fruits and vegetables with low cost material, simple fabrication, and high sensitivity. Acknowledgments KC gratefully acknowledges the partially financial supports from Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. program (Grant Number PHD/0049/2553). OC and WS greatly thank the Thailand Research Fund through Research Team Promotion Grant (RTA5780005), the Thai Government Stimulus Package 2 (TKK2555), under the Project for Establishment of Comprehensive Center for Innovative Food, Health Products and Agriculture, Chulalongkorn University. We also thank the financial support from the 90th Anniversary of Chulalongkorn University Fund (Ratchadphiseksomphot Endowment Fund). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.09. 020. References [1] M. Albareda-Sirvent, A. Merkoci, S. Alegret, Sensor Actuat. B-Chem. 69 (2000) 153–163. [2] J. Li, H. Xie, L. Chen, Sensor Actuat. B-Chem. 153 (2011) 239–245. [3] K.C. Honeychurch, J.P. Hart, TrAC, Trends Anal. Chem. 22 (2003) 456–469. [4] D. Hernandez-Santos, M.B. Gonzalez-Garcia, A.C. Garcia, Electroanalysis 14 (2002) 1225–1235.
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Development of gold nanoparticles modified screen-printed carbon electrode for the analysis of thiram, disulfiram and their derivative in food using ultra-high performance liquid chromatography Kanokwan Charoenkitamorn a, Orawon Chailapakul a,c,n, Weena Siangproh b,nn a Electrochemistry and Optical Spectroscopy Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok 10330, Thailand b Department of Chemistry, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, Wattana, Bangkok 10110, Thailand c National Center of Excellent of Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand
art ic l e i nf o
a b s t r a c t
Article history: Received 16 July 2014 Received in revised form 10 September 2014 Accepted 11 September 2014 Available online 30 September 2014
For the first time, gold nanoparticles (AuNPs) modified screen-printed carbon electrode (SPCE) was developed as working electrode in ultra-high performance liquid chromatography (UHPLC) coupled with electrochemical detection (UHPLC-ED) for simultaneous determination of thiram, disulfiram, and N,N-diethyl-N’,N’dimethylthiuram disulfide, their derivative compound. The separation was performed in reversed-phase mode using C18 column, mobile phase consisting of 55:45 (v/v) ratio of 0.05 M phosphate buffer solution (pH 5) and acetonitrile at a flow rate of 1.5 mL min 1. For the detection part, the amperometric detection was chosen with a detection potential of 1.2 V vs. Ag/AgCl. Under the optimal conditions, the good linear relationship was obtained in the range of 0.07–15, 0.07–12, and 0.5–15 mg mL 1 (correlation coefficient more than 0.9900) for thiram, N,N-diethyl-N’,N’-dimethylthiuram disulfide, and disulfiram, respectively. The limits of detection (LODs) of thiram, N,N-diethyl-N’,N’-dimethylthiuram disulfide, and disulfiram were 0.022, 0.023, and 0.165 mg mL 1, respectively. Moreover, this method was successfully applied for the detection of these compounds in real samples (apple, grape and lettuce) with the recoveries ranging from 94.3% to 108.8%. To validate this developed method, a highly quantitative agreement was clearly observed compared to standard UHPLC–UV system. Therefore, the proposed electrode can be effectively used as an alternative electrode in UHPLC-ED for rapid, selective, highly sensitive, and simultaneous determination of thiram, disulfiram, and N,N-diethyl-N’,N’dimethylthiuram disulfide. & 2014 Elsevier B.V. All rights reserved.
Keywords: Dithiocarbamates Fungicides Screen-printed carbon electrode Gold nanoparticles Simultaneous determination Ultra-high performance liquid chromatography
1. Introduction Nowadays, the designing of electrochemical sensor has been developed in the field of electrochemistry to improve the analytical efficiency in the terms of sensitivity, selectivity, reliability, ease of fabrication and use, and low cost. Screen printing is the technology used for fabrication of biosensors and chemical sensors instead of using large-scale electrode. Their various advantages such as miniaturization, versatility, and low cost of production are really attractive [1]. In addition, many laboratories use the screen-printing for in-house production of sensors. Screen-printed carbon electrode (SPCE) is an alternative material used instead of using the traditional electrodes based on economic substrate. Recently, SPCE have been successfully used as the electrochemical sensor for various researches due to the n
Corresponding author. Tel.: þ 662 218 7615. Corresponding author. Tel.: þ 662 249 5000x18208. E-mail addresses: [email protected] (O. Chailapakul), [email protected] (W. Siangproh). nn
http://dx.doi.org/10.1016/j.talanta.2014.09.020 0039-9140/& 2014 Elsevier B.V. All rights reserved.
simplicity to produce and still give the rapid responses [2]. Moreover, the main advantage of SPCE is able to use only once and then is discarded. This advantage can be used to solve the problems of surface fouling compared with those of conventional electrodes. However, the limitation of SPCE is small surface area of working electrode leading to the lack of sensitivity [3]. Therefore, electrode modification is necessary to solve this problem. For electrode modification, metal-nanoparticles modified SPCE is focused to improve the electrochemical efficiency. Metal-nanoparticles are attractive catalyst in the electrochemical applications. Transition metals, especially precious metals, show very high catalytic abilities for catalyzing the redox process of some interested molecules, making the use of electroanalytical techniques for applications are extended [4]. Particularly, gold nanoparticles (AuNPs) have received significant attention in recent years. AuNPs are widely used in many fields due to their unique optical and physical properties, such as surface plasmon oscillations for labeling, imaging, and sensing [5]. For the electrochemical fields, AuNPs have been widely used for modification of electrode because of their benefits including catalysis, mass
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transport, and high effective surface area [1]. AuNPs have the ability to improve the detection signal, electron transfer, which make the limit of detection in electrochemical sensor is reduced. Moreover, the most importance is AuNPs showed strong specific interaction with the sulfur-containing compounds [6]. Therefore, AuNPs became an interesting nanomaterial used as modifier onto working electrode for electrochemical detection of sulfur-containing compounds. Thiram (tetramethyl thiuram) and disulfiram (tetraethyl thiuram) are oraganosulfur compounds in the group of dithiocarbamates (DTCs) fungicides. DTCs have been used in agriculture as fungicides in foliage vegetable and fruit [7]. Besides these agricultural applications, DTCs are also used in industry such as vulcanization accelerator and antioxidant in rubber. The use of DTCs results in their release into the environment causing the contamination in food and water which leads to hazardous effect in the living organisms through diverse pathways [8]. Environmental problem resulting from uncontrollable use of fungicides in agriculture is one of the most important evidence. Therefore, it is necessary to continuously develop simple, selective, and sensitive methods for analysis of fungicides [9]. Various methods have been developed for analysis of DTCs. Spectrometry [10–12] and head space gas chromatography [13] have been used as the methods for the determination of DTCs after their decomposition to carbon disulfide (CS2). From these methods, most of DTCs can degrade to CS2. Therefore, they are unable to distinguish among different DTCs. Moreover, gas chromatography [14,15], capillary electrophoresis [16–20], and high performance liquid chromatography (HPLC) were used for simultaneous determination of DTCs coupled with UV detection. Especially, HPLC is the simple technique used for easy separation of the disulfide substance. However, the drawback of the UV detection is low sensitivity. Therefore, HPLC coupled with electrochemical detection became an important choice for the determination of disulfides [21]. The main advantage of electrochemical detection compared to those of spectrometry is that the derivatization step is not needed, which makes the analysis time and cost reduced. In addition, the use of electrochemical detection after the separation by HPLC can provide more sensitive detection than UV [22]. Recently, ultra-high performance liquid chromatography coupled with electrochemical detection (UHPLC-ED) is a high potential alternative method for various analysis. UHPLC concerns to the development of short column packed with small particles for working at high pressures (4400 bar). The use of UHPLC can enhance chromatographic performances in terms of efficiency, resolution, and analysis time [23]. In the detection part, AuNPs modified electrode was used as amperometric detection. Amperometry is an attractive potential technique because of its fast response, high sensitivity, relatively low cost of equipment, no need for complicated operation, and simplicity for direct sulfur assay [24]. With such benefits from the mentioned background, the objective of this work is to develop AuNPs modified SPCE in UHPLC-ED system for simultaneous determination of DTCs with high sensitivity, specificity, and rapid analysis. Thiram (tetramethyl thiuram) and disulfiram (tetraethyl thiuram) are chosen as representative compounds of DTCs because they are importantly applied to control a variety of pest [25]. Typically, when thiram and disulfiram are mixed together, the intermediate of N,Ndiethyl-N’,N’-dimethylthiuram disulfide (DEDMTDS) appears as the derivative of thiram and disulfiram. This compound is one of DTCs that affects human health and has the same effect of thiram and disulfiram. The presence of N,N-diethyl-N’,N’-dimethylthiuram disulfide can also use to monitor thiram and disulfiram in samples. Therefore, the detection of N, N-diethyl-N’, N’-dimethylthiuram disulfide is important. This derivative compound is also determined along with the measurement of thiram and disulfiram. To the best of our knowledge, there is no publication report about the use of AuNPs modified SPCE as working electrode for ED
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after the UHPLC separation to simultaneously determine thiram, DEDMTDS, and disulfiram. Moreover, this developed UHPLC-ED using AuNPs modified SPCE was applied for the first time to analyze thiram, DEDMTDS, and disulfiramthe DTCs in fruits and vegetables collected from local markets. 2. Materials and methods 2.1. Chemicals and reagents All chemicals used in this work were of analytical grade, and all solutions were prepared using Milli-Q water from Millipore (RZ18.2 M Ω cm 1). Acetonitrile (HPLC-grade), chloroform, and sulfuric acid were obtained from Merck (Darmstadt, Germany). Potassium dihydrogen phosphate (KH2PO4) was acquired from BDH laboratory supplies (VWR International Ltd., England). Disodium hydrogen phosphate (Na2HPO4) and sodium hydroxide (NaOH) were purchased from Merck (Darmstadt, Germany). Standard thiram (tetramethyl thiuram) was obtained from Chem Service (West chester, PA, USA). Standard disulfiram (tetraethyl thiuram) and gold (III) chloride solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). All of stock standard solutions (1000 mg mL 1) were prepared by dissolving 10 mg of each analyte in acetonitrile. The solutions were then placed in an amber bottle and stored at 4 1C. To prepare working standard solution, the standard solution was mixed and diluted in suitable proportions of acetonitrile:Milli-Q water (50:50; v/v), and kept for 5 min before measurement. All solutions and solvents were filtered by 0.22 mm nylon membranes prior to use in UHPLC separation. 2.2. Fabrication and modification of screen-printed carbon electrode The working electrode was fabricated using screen printing and modified by electrodeposition techniques. The Ag/AgCl electrode and Pt wire was used as reference and auxiliary electrodes, respectively. To fabricate the in-house screen-printed carbon electrode (Fig. S1), the Ag/AgCl ink was first printed on the PVC substrate for using as a conductive pad. Then, the carbon ink was printed to create a working electrode. The each step of printing electrode was allowed to dry in an oven at 55 1C for 1 h. The screen-printed carbon electrode was ready to modify in the next step. For the electrodes modification, the 10 mM of gold (III) solution was used to modify onto electrode surface by electrodeposition technique. The three-electrode system was placed into cell containing 2 mL of solution of 10 mM of gold (III) solution in 0.5 M H2SO4 followed by deposition of gold onto the SPCE at a deposition potential of –0.6 V (vs. Ag/AgCl) for 50 s while the solution was stirred (by means of a mechanical stirrer bar). The gold nanoparticles (AuNPs) were obtained onto the electrode surface. After the modification, the AuNPs modified SPCE was carefully rinsed with distilled water and dried under N2 gas prior to use. 2.3. Cyclic voltammetry The electrochemical reaction and optimal parameters for electrodeposition step were studied by cyclic voltammetry (CV) using a CH instrument potentiostat 1232 A (CH Instrument, Inc., USA). The working electrodes used in this work were bare SPCE and AuNPs modified SPCE. The reference and auxiliary electrode were silver/silver chloride electrode and Pt wire, respectively. All experiments were done at room temperature. 2.4. UHPLC experiment and apparatus The UHPLC system was performed in reversed-phase mode using an LC-20ADXR solvent deliver unit (Shimadzu Corporation,
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Japan), an autosampler (SIL-20 A) with 0.1–100 mL loop and a KinetexTM core-shell C18 column (50 mm x 4.6 mm i.d.; particle size, 2.6 mm, Phenomenex). The UHPLC-electrochemical measurement was carried out in 45:55 (v/v) ratio acetonitrile: 0.05 M phosphate buffer solution (pH 5), which was adjusted to pH 5 with 0.1 M sodium hydroxide solution, as mobile phase with an applied potential of 1.2 V vs. Ag/AgCl, an injection volume of 50 mL, and a flow rate of 1.5 mL min 1. All chromatographic separations were performed at room temperature. 2.5. Electrochemical measurement The thin-layer flow cell consisted of a working AuNPs modified SPCE, a reference Ag/AgCl electrode (Bioanalytical system Inc., USA), and a stainless steel tube counter electrode. The geometric area of the AuNPs modified SPCE in the flow cell was estimated to be 0.40 cm2 with 1 mm thick silicon rubber gasket as a spacer. An electrochemical measurement for amperometric control and signal processing was performed using CH instrument potentiostat 1232 A. 2.6. Sample preparation The three real samples including apple, grape and lettuce were obtained from local markets in Thailand. For analysis, 50 g of cut sample was weighted accurately and transferred to a blender. Then 50 mL of 500 mg mL 1 of standard mixture solution and chloroform were added. The mixture was homogenized with blender and transferred into centrifuge tubes. After centrifugation at 4000 rpm for 10 min, all of the organic phases were filtered through a Buchner funnel with Whatman filter paper No. 4 and transferred to 250 mL vessel of the rotary vacuum evaporator for evaporation to dryness at room temperature. The residues were dissolved in 4.5 mL of acetonitrile using ultrasonic stirring, filtered through the 0.2 mm pore size of nylon membrane, and collected in 5 mL of volumetric flask. After that, acetonitrile filtered through the 0.2 mm pore size of nylon membrane was added to adjust volume to 5 mL of solution. Finally, 50 mL of the solution was injected into the UHPLC system. The determination of DTCs in samples was analyzed within the linear dynamic range that obtained by adding different volume of 500 mg mL 1 of standard mixture solution into the blank sample and prepared following the above treatment. 2.7. Method validation Standard solutions and samples were analyzed, and the currents were integrated. A calibration curve of standard solutions was performed under optimal conditions and treated with linear least square regression analysis using the Excel software package. The limit of detection (LOD) and limit of quantification (LOQ) were determined from the 3 Sbl/S and 10 Sbl/S, respectively, where Sbl is standard deviation of blank (n ¼10), and S is sensitivity of method, obtained from the slope of the linearity. For the precision of intraday and inter-day, three concentrations of DCTs (1, 6, and 12 mg mL 1) were observed for three times within a day and three different days. To validate the developed method, UHPLC-ED was compared to the standard method of UHPLC coupled with ultraviolet detection (UHPLC–UV) using the paired t-test.
3. Results and discussion 3.1. Surface morphology of AuNPs modified SPCE Gold (III) solution was used to generate the AuNPs on the electrode surface by electrodeposition. The surface morphology of bare SPCE and AuNPs modified SPCE was investigated using
Fig. 1. SEM images of (A) bare SPCE and (B) AuNPs modified SPCE prepared by electrodeposition of 10 mM gold (III) solution at applied potential of -0.6 V for 50 s.
scanning electron microscope (SEM) as shown in Fig. 1A and B, respectively. After the modification, the well dispersed gold particles in nanoscale was observed (Fig. 1B). The distribution of AuNPs on the electrode surface led to increase the surface area and improve the electrochemical sensitivity of the modified electrode. 3.2. Optimization of the electrode modification For the electrode modification using electrodeposition technique, the deposition potential and deposition time were optimized because these factors can effect to the analytical results. First, thiram was selected as the representative compounds because it is the best electrochemically active species at bare carbon electrode. Therefore, it is easy to observe the signal obtained from modified and unmodified electrode. To obtain the optimal condition of AuNPs modified SPCE, the dependence of deposition potential and
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Fig. 2. Effect of the (A) deposition potential and (B) deposition time of thiram at AuNPs modified SPCE. Data are shown as the mean7 SD derived from three replicates.
deposition time were studied using cyclic voltammetry, and the relationship between the studied parameter and current response of thiram was plotted. The deposition potential was studied in the range of –1.0 to 0.2 V. The current signal of thiram was initially increased as the function of deposition potential from –1.0 to – 0.6 V and then decreased (Fig. 2A). For the deposition time, the current signal of thiram increased from 10 s to 50 s and then decreased (Fig. 2B). Therefore, the deposition potential of –0.6 V and the deposition time of 50 s were chosen as optimal parameters for modification of AuNPs onto the electrode surface.
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Fig. 3. (A) Cyclic voltammogram of thiram at the AuNPs modified SPCE (red line) compared to bare SPCE (blue line) and (B) comparison of oxidation current density between AuNPs modified SPCE (red rod) and bare SPCE (blue rod) vs. Ag/AgCl at the concentration of 10 mg mL 1 in 55:45 of 0.05 M phosphate solution pH 5: acetonitrile, scan rate 100 mV s 1.
3.3. Electrochemical response of thiram at AuNPs modified SPCE As mentioned above, thiram is the best electrochemically active species. Thus, it was used as the representative of DTCs to study the electrochemical response. The cyclic voltammogram (CV) of 10 mg mL 1 of thiram obtained from bare SPCE and AuNPs modified SPCE at the potential range of 0.0 to 1.4 V was shown in Fig. 3. From CV results, the anodic current of thiram was investigated, and the current at AuNPs modified SPCE was significantly higher than the current obtained from bare SPCE due to the distribution of AuNPs on the electrode surface. We believe that the AuNPs on the electrode surface can enhance electron transfer and also improve the electrochemical determination of DTCs. For the comparison of oxidation current density between AuNPs modified SPCE and bare SPCE (Fig. 3B), the higher current density about nine times was observed at AuNPs modified SPCE. This result indicated that the AuNPs modified SPCE can be assuring electrode for the detection of thiram, disulfiram, and DEDMTDS. Therefore, this modified electrode can be an alternative electrode
Fig. 4. Representative UHPLC-ED chromatogram of 12 mg mL 1 of thiram (a), DEDMTDS (b), and disulfiram (c) in a mobile phase (0.05 M phosphate solution (pH 5): acetonitrile), the detection potential of 1.2 V vs. Ag/AgCl using AuNPs modified SPCE, the injection volume of 50 mL, and the flow rate of 1.5 mL min 1. Chromatograms are representative of at least three independent repetitions.
for using as working electrode with the advantages of low-cost material, simple fabrication and high sensitivity in case of detection DTCs. 3.4. UHPLC separation In this work, C18 column was used to separate thiram, DEDMTDS, and disulfiram by isocratic elution. The effect of percentage of acetonitrile used in the mobile phase on the
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retention characteristics was first evaluated. When the percentage of acetonitrile r40 was used, the separation time was higher than 20 min and peak broadening of disulfiram was observed. On the other hand, at higher content of acetonitrile in mobile phase, the current signal of thiram, DEDMTDS, and disulfiram were decreased due to the low of diffusion coefficient with changing ionic strength of the medium. Therefore, the acetonitrile of 45% was selected as
compromise to achieve the completely separated them within 6 min with good sensitivity. In addition, 0.05 M phosphate buffer solution (pH 5) was chosen as the electrolyte solution because the high concentration of salt in buffer solution was not suitable for the UHPLC system. The chromatogram for the separation of standard mixture solution was shown in Fig. 4. The retention time of thiram, DEDMTDS, and disulfiram was 1.5, 2.6, and 5.4 min, respectively, and the retention time stability was found in the range of 0.1% to 1.5%. The peak widths were 11.6 s for thiram, 14.2 s for DEDMTDS, and 18.8 s for disulfiram. Therefore, this proposed system offers the rapid time for separating DTCs. 3.5. Hydrodynamic voltammetry Hydrodynamic voltammetry was used to optimize the detection potential. The potential was studied in the range of 0.8 to 1.4 V. The peak current after each injection at different potential was recorded corresponding to the background current. These data were plotted as a function of applied potential to obtain the hydrodynamic curves as shown in Fig. 5. The detection potentials significantly affected the oxidation current of the thiram, DEDMTDS, and disulfiram as well as the background current (Fig. 5A). Therefore, the net current after background subtraction was studied, and signal-to-background (S/B) ratios were plotted corresponding to the detection potential. It was found that the current increased when the potential increased up to 1.2 V for all compounds (Fig. 5B). Therefore, the detection potential of 1.2 V was selected as optimal detection potential for amperometric detection of thiram, DEDMTDS, and disulfiram after their separation with UHPLC. 3.6. Method validation
Fig. 5. Hydrodynamic voltammetric results at the AuNPs modified SPCE for a 10 mg mL 1 each mixture (50 mL total) of thiram and disulfiram. (A) thiram (blue line), DEDMTDS (green line), disulfiram (red line), and background (purple line); (B) hydrodynamic voltammogram of signal-to-background ratios. The other conditions are the same as in Fig. 4. Data are shown as the mean 7 1 SD derived from three independent repetitions.
3.6.1. Analytical performance The analytical performance of this proposed system was studied. The calibration curve between the concentration and current response of analytes was plotted as shown in Fig. 6. Under the optimal conditions, the linearity was observed in the range of 0.07 to 15 mg mL 1 for thiram, 0.07 to 12 mg mL 1 for DEDMTDS, and 0.5 to 15 mg mL 1 for disulfiram. The limit of detection (LOD) and limit of quantification (LOQ) were calculated from 3 Sbl/S and 10 Sbl/S, where Sbl is the standard deviation of blank measurements (n¼10), and S is the sensitivity of the method or the slope of linearity. Good linearity value with r2 4 0.99 was obtained. LODs and LOQs were summarized in Table 1. In addition, when the performance of AuNPs
Fig. 6. The amperometric voltammogram for different concentrations of thiram (a), DEDMTDS (b), and disulfiram (c) (0.07 to 15 mg mL 1) using AuNPs modified SPCE and the corresponding calibration plots (inset). Measurements were performed under the optimal conditions.
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421
Table 1 Linearity, limit of detection (LOD) and limit of quantitation (LOQ) of the UHPLC-ED method (n¼3). Analyte
Linear range (mg mL 1 )
Slope (peak height) (mA/mg mL 1 )
Intercept (mA)
r2
LOD (mg mL 1 )
LOQ (mg mL 1 )
Thiram DEDMTDS Disulfiram
0.07–15 0.07–12 0.5–15
0.2585 0.3247 0.1283
0.1636 0.129 0.0406
0.9905 0.9945 0.9948
0.022 0.023 0.165
0.074 0.076 0.551
Table 2 Recent report using different types of electrodes for electrochemical determination of DTCs. Modified electrode
Detection method
Linearity range (mg mL 1)
LOD (mg mL 1)
Analyte
Real sample
Reference
Cylindrical carbon fiber microelectrode Graphite-poly (tetrafluoroethylene) Hanging mercury drop electrode
Square-wave voltammetry
0.24–144
0.10
Thiram
Grape
[7]
2–100 0.01–0.6
0.14–1.0 0.01
Thiram and disulfiram Ziram
Apple rice
[26] [27]
Hanging mercury drop electrode
Amperometry Differential pulse voltammetry Square-wave voltammetry
0.01–0.19
0.0072
Ziram
[28]
AuNPs modified SPCE
Amperometry
0.07–15
0.022– 0.165
Thiram, DEDMTDS and disulfiram
Potato, cabbage and tomato Grape, apple and lettuce
This work
Table 3 The intra- and inter-precisions and recoveries of the UHPLC-ED method (n¼3). Samples
Apple
Spiked level (mg mL 1)
1
3
6
9
12
Grape
1
3
6
9
12
Lettuce
1
3
6
9
12
Analyte
Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram
Intra-day
Inter-day
Recovery (%)
RSD (%)
Recovery (%)
RSD (%)
97.1 101.5 104.1 102.7 102.7 108.5 109.0 109.2 97.4 109.3 109.2 103.6 101.5 106.5 102.6 100.1 101.5 108.8 103.9 105.2 107.9 103.5 98.9 98.4 107.4 100.4 97.5 106.1 102.1 100.5 98.8 102.6 102.4 105.1 102.4 104.1 98.9 98.7 106.0 108.0 100.5 103.1 102.6 100.5 100.1
1.1 0.2 3.3 0.1 0.3 0.3 0.9 1.3 0.6 0.6 0.3 0.6 0.2 0.4 0.2 0.2 2.3 0.7 0.6 0.2 0.3 1.0 1.5 0.2 0.5 1.0 0.4 0.4 0.7 0.3 0.8 1.4 1.7 0.9 1.0 1.9 0.2 0.5 0.5 1.8 3.7 0.8 1.3 2.9 0.6
99.0 100.6 100.6 100.7 102.0 103.3 104.0 104.8 98.1 107.0 107.7 104.2 101.9 102.5 101.4 102.3 99.7 104.2 100.7 102.2 104.7 103.3 102.6 100.6 107.2 102.6 98.0 103.3 101.9 100.9 101.9 101.6 100.8 99.7 103.4 99.0 96.7 101.7 101.3 105.8 101.8 102.6 101.6 101.1 102.5
3.7 1.0 4.9 5.7 1.4 4.4 4.4 4.0 2.4 1.8 1.7 4.9 0.7 3.4 2.5 3.3 1.9 7.7 2.8 2.5 2.7 2.9 4.9 3.4 0.1 4.7 2.6 2.4 0.8 5.0 3.4 1.4 4.3 4.8 3.6 4.9 4.4 4.7 4.1 2.5 3.8 4.2 2.5 0.8 2.3
422
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Table 4 Determination of DTCs level in different samples (n ¼3) by traditional UHPLC-UV method and the developed UHPLC-ED method reported here. Samples
Apple
Spiked level (mg mL 1)
1
6
12
Grape
1
6
12
Lettuce
1
6
12
Paired two-tail test
t values (at 1 mg mL 1)
t values (at 6 mg mL 1)
t values (at 12 mg mL 1)
Analyte
Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram Thiram DEDMTDS Disulfiram
t critical
modified SPCE was compared to the other previous electrodes for the detection of DTCs as shown in Table 2. It was found that the LODs obtained from the proposed electrode were lower than using cylindrical carbon fiber [7] and graphite-poly (tetrafluoroethylene) composite electrode [26]. However, few researches used the mercury electrode to provide lower LOD than this method but the mercury is toxic [27,28]. Therefore, the proposed electrode offers less toxicity, uncomplicated, and low cost. Additionally, the proposed electrode shows good electrocatalytic properties for DTCs detection to obtain a high electrochemical sensitivity.
3.6.2. Application to real samples To assess the applicability of the proposed method, target compounds in samples from local supermarkets were investigated. The proposed method was applied for the detection of three different samples, including apple, grape, and lettuce. The standard addition method was chosen to investigate the reliability of this proposed system. The precision of the analytical process was evaluated by the repeatability of the process. The spiked concentrations in the dynamic linearity between 1 to 15 mg mL 1 were studied to calculate the RSD percentage. The summary of intraand inter-day precision, and recovery that obtained from the proposed method was shown in Table 3. The RSDs and recoveries of intra-day were found in the range of 0.1–3.7% and 94.3–108.8%, respectively, while the inter-day RSDs and recoveries were found in the range of 0.1–5.7% and 95.8–107.7%, respectively. Therefore,
Amount found (mg mL 1) (x7 SD)
% recovery
UHPLC-ED
UHPLC-UV
UHPLC-ED
UHPLC-UV
0.99 70.04 1.017 0.01 0.98 70.05 6.247 0.27 6.15 70.10 5.89 70.14 12.23 70.50 12.30 70.49 12.42 70.25 1.02 7 0.03 1.017 0.02 1.047 0.08 6.20 70.18 6.15 70.30 6.04 7 0.21 12.177 0.34 12.23 70.60 12.40 70.41 1.02 7 0.03 1.02 7 0.01 1.017 0.04 5.80 70.26 6.10 70.28 6.08 70.25 12.20 70.41 12.137 0.47 12.30 70.20 0.104 0.898 0.792 2.293 3.029 1.747 2.808 2.989 0.243 4.303
1.02 7 0.03 1.01 70.02 1.01 70.05 5.79 7 0.26 6.12 70.19 5.72 70.13 11.90 7 0.36 11.78 7 0.62 12.26 7 0.23 1.02 7 0.02 1.007 0.01 0.99 7 0.01 5.92 7 0.19 6.017 0.22 6.047 0.12 12.09 7 0.41 12.00 70.60 12.30 7 0.20 0.98 7 0.03 1.02 7 0.04 0.97 70.09 5.75 7 0.25 5.99 7 0.22 5.99 7 0.13 11.98 7 0.27 11.94 7 0.62 12.117 0.35
99.0 100.6 97.5 104.0 102.5 98.1 101.9 102.5 103.5 102.3 100.8 104.2 103.3 102.6 100.6 101.4 101.9 103.3 101.9 102.2 100.8 96.7 101.7 101.3 101.6 101.1 102.5
102.2 101.1 100.6 96.5 101.9 95.4 99.2 98.1 102.1 102.2 99.7 98.7 98.7 100.1 100.7 100.8 100.0 102.5 98.1 101.6 97.1 95.8 99.8 99.8 99.9 99.5 100.9
this method is an alternative and suitable for rapid separation and simultaneous determination of DTCs. To validate the proposed method, UHPLC-UV was used as a standard method to compare the acceptable and reliable. The results obtained from UHPLC-ED and UHPLC-UV were compared by a paired t-test at the 95% confidence for three samples that spiked with three standard concentrations (1, 6, and 12 mg mL 1 to represent the low, medium, and high level, respectively). The critical t-value was significantly higher than calculated t-values between two assays. From the results shown in Table 4, the calculated t-values of three concentrations were found in the range of –0.243 to 3.029 and lower than critical t-values (4.303). It can be concluded that there is no significant difference between UHPLC–ED and conventional UHPLC– UV method. Therefore, the results obtained from UHPLC coupled with amperometric detection using AuNPs modified SPCE is acceptable and reliable for applying to simultaneous determination of DTCs in food.
4. Conclusions AuNPs modified SPCE was firstly developed for the determination of thiram, disulfiram, and DEDMTDS after their separation with UHPLC system. Under the optimal conditions, the separation was complete within 6 min, and the high current response signal was obtained at AuNPs modified SPCE. The main advantages for the use of AuNPs modified SPCE are in term of high sensitivity, low-cost and simple fabrication-based material. Reproducible signal from %RSD for intra- and inter-day were below 5%. These
K. Charoenkitamorn et al. / Talanta 132 (2015) 416–423
results indicated that the high reproducibility was obtained using this system for long time. Moreover, the proposed UHPLC-ED system is an acceptable and reliable method compared to standard UHPLC-UV system using paired t-test. Ultimately, the UHPLC coupled with AuNPs modified SPCE amperometry is successfully applied for real samples. This proposed electrode could be a novel or an alternative electrode in UHPLC-ED system for the simultaneous determination of DTCs in fruits and vegetables with low cost material, simple fabrication, and high sensitivity. Acknowledgments KC gratefully acknowledges the partially financial supports from Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. program (Grant Number PHD/0049/2553). OC and WS greatly thank the Thailand Research Fund through Research Team Promotion Grant (RTA5780005), the Thai Government Stimulus Package 2 (TKK2555), under the Project for Establishment of Comprehensive Center for Innovative Food, Health Products and Agriculture, Chulalongkorn University. We also thank the financial support from the 90th Anniversary of Chulalongkorn University Fund (Ratchadphiseksomphot Endowment Fund). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.09. 020. References [1] M. Albareda-Sirvent, A. Merkoci, S. Alegret, Sensor Actuat. B-Chem. 69 (2000) 153–163. [2] J. Li, H. Xie, L. Chen, Sensor Actuat. B-Chem. 153 (2011) 239–245. [3] K.C. Honeychurch, J.P. Hart, TrAC, Trends Anal. Chem. 22 (2003) 456–469. [4] D. Hernandez-Santos, M.B. Gonzalez-Garcia, A.C. Garcia, Electroanalysis 14 (2002) 1225–1235.
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