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Development of a method for the determination of 9 currently used cotton pesticides by gas chromatography with electron capture detection
Available online at www.sciencedirect.com
Talanta 75 (2008) 1055–1060
Development of a method for the determination of 9 currently used cotton pesticides by gas chromatography with electron capture detection Baohong Zhang a,b,∗,1 , Xiaoping Pan a,c,1 , Louise Venne a , Suzy Dunnum a , Scott T. McMurry a , George P. Cobb a , Todd A. Anderson a a
Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX 79409-1163, United States b Department of Biology, East Carolina University, Greenville, NC 27858, United States c Department of Chemistry, Western Illinois University, Macomb, IL 61455, United States
Received 3 November 2007; received in revised form 1 January 2008; accepted 2 January 2008 Available online 20 January 2008
Abstract A reliable, sensitive, and reproducible method was developed for quantitative determination of nine new generation pesticides currently used in cotton agriculture. Injector temperature significantly affected analyte response as indicated by electron capture detector (ECD) chromatograms. A majority of the analytes had an enhanced response at injector temperatures between 240 and 260 ◦ C, especially analytes such as acephate that overall had a poor response on the ECD. The method detection limits (MDLs) were 0.13, 0.05, 0.29, 0.35, 0.08, 0.10, 0.32, 0.05, and 0.59 ng/mL for acephate, trifuralin, malathion, thiamethozam, pendimethalin, DEF6, acetamiprid, brifenthrin, and -cyhalothrin. This study provides a precision (0.17–13.1%), accuracy (recoveries = 88–107%) and good reproducible method for the analytes of interest. At relatively high concentrations, only -cyhalothrin was unstable at room temperature (20–25 ◦ C) and 4 ◦ C over 10 days. At relatively low concentrations, acephate and acetamiprid were also unstable regardless of temperature. After 10 days storage at room temperature, 30–40% degradation of -cyhalothrin was observed. It is recommended that acephate, acetamiprid, and -cyhalothrin be stored at −20 ◦ C or analyzed immediately after extraction. © 2008 Elsevier B.V. All rights reserved. Keywords: Pesticides; Gas chromatography; Electron capture detection; Cotton
1. Introduction Planted in approximately 70 tropical/subtropical and temperate countries, cotton is one of the most important fiber and economic crops; about 20 million cotton farmers grow 33.5 million hectares of cotton around the world [1,2]. Although many factors affect cotton yield, insect pests and weeds are two major constraints to cotton production. More than 1300 insect species have been reported as cotton pests worldwide [2,3]. Although transgenic Bt cotton has played an important role in pest control [1,4,5], the primary approach to pest control is still through the use of synthetic chemicals. To control pests and weeds, more than 12 kg of pesticide per ha is required annually [6].
∗
Corresponding author at: Department of Biology, East Carolina University, Greenville, NC 27858, United States. E-mail address: [email protected] (B. Zhang). 1 Both the authors contributed equally and both are co-first authors. 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.01.032
One of the most important aspects in minimizing the potential hazards of pesticides to human and environmental health is to monitor pesticide residues [7] and develop best management practices based on these monitoring data. To better monitor residues of pesticides in different media, efficient methods for extraction and determination of pesticides in soil, sediment, water, and food need to exist. As new pesticides are incorporated into agricultural practices, new methods for the determination of those new pesticides must be developed. In this paper, we report on a reliable method for the determination of nine currently used cotton pesticides at trace levels. 2. Experimental 2.1. Chemicals and reagents Pesticide standards were obtained commercially. Acephate, trifuralin, malathion, pendimethalin, DEF6, brifenthrin and
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-cyhalothrin were purchased from AccuStandard Inc. (New Haven, CT, USA) while acetamiprid and thiamethoxzam were obtained from ChemService Inc. (West Chester, PA, USA). Methanol, acetone, hexane, acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA, USA). Anhydrous Na2 SO4 was purchased from VWR (West Chester, PA, USA). C18 solid-phase extraction (SPE) cartridges (500 mg) were obtained from Fisher Scientific (Pittsburgh, PA, USA). Ultra-pure water (>18 M) was prepared by ultrafiltration with a Mili-Q purification system from Millipore (Bedford, MA, USA). All solvents were HPLC or analytical grade. 2.2. Instruments and sample analysis An HP 6890 series gas chromatograph (GC) was employed to analyze the test pesticides. The GC was equipped with an electron capture detector (ECD) and an autosampler (Agilent, Palo Alto, California, USA). The GC-ECD was controlled by Chemstation software from Hewlett–Packard (Agilent, Palo Alto, California, USA). A 30 m × 0.32 mm × 0.25 m DB-5 column from J&W Scientific (Folsom, CA, USA) was used to separate the analytes. The carrier gas was helium (99.99% purity), and was operated at a constant flow rate during the run (9.2 mL/min). The makeup gas for the detector was argon:methane (95:5) at a combined flow rate of 60.0 mL/min. The injection volume was 2 L in the splitless mode. The injector temperature was 240 ◦ C except in experiments on the optimization of analytical parameters in which different injector temperatures (100–300 ◦ C) were evaluated. The detector temperature was 270 ◦ C and was operated in the constant current mode. Although the oven temperature program varied initially during optimization experiments, the following temperature program was adopted for all subsequent studies reported herein: 110 ◦ C for 1 min, increased to 200 ◦ C at 15 ◦ C/min with a hold of 2 min, then raised to 269 ◦ C at 15 ◦ C/min, and finally held at 269 ◦ C for 6 min. 2.3. Calibration curves and method detection limit For each analyte, 10 standard concentration levels (1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL) were prepared and analyzed in order of increasing concentration. Calibration curves were constructed by plotting concentration of each pesticide versus ECD response (peak area). The method detection limit (MDL) for each pesticide was determined by the analysis of seven spiked samples at 1 ng/mL for each analyte, and was calculated using the following formula: MDL = 3.14 × S.D., where S.D. is the standard deviation of the measurements of seven spiked samples, and 3.14 is the Student’s t-value at the 99% confidence level (t = 3.14 for 6 degrees of freedom).
determined by comparing the measured concentration to the nominal concentration. Precision was calculated by using the relative standard deviation (R.S.D.). The intra-day precision (%) was determined by repeated injections (n = 5) of the same samples on a single day. The inter-day precision (%) was determined by repeated injections on 5 different days. Accuracy was calculated by the equation: (mean measured concentration/nominal concentration) × 100. 2.5. Analyte stability in extraction solvent The stability of analytes in extraction solvent (acetonitrile) was tested at three temperatures: room temperature (∼20–25 ◦ C), 4, and −20 ◦ C. After 0, 1, 2, 4, 6, 8, and 10 days of storage at the three conditions, extracts were analyzed by GCECD. Each treatment was repeated three times, and the stability was calculated by comparing the measured analyte concentration with the nominal concentration. 2.6. Method application Natural water and sediment samples were collected from Playa lakes on the Southern High Plains of West Texas. Playa lakes were chosen because they are representative of surface water receiving pesticide runoff from cotton-growing areas. Water samples (1 L) were extracted using C18 SPE cartridges at a flow rate of 5–6 mL/min. Analytes were eluted with 6–10 mL acetonitrile, concentrated to 2 mL using nitrogen evaporation, and then filtered through 0.2 m PTFE membranes (Millipore, Bedford, MA, USA) into amber GC vials. Sediments (5 g) were air-dried, mixed with 10 g anhydrous Na2 SO4 , loaded into 22-mL cells, and extracted (hexane:acetonitrile, 1:1) using a Dionex Accelerated Solvent Extractor (Model 200, Salt Lake City, UT, USA) following a procedure previously reported [8,9]. Extracts were concentrated to 1–2 mL using nitrogen evaporation and then cleaned using 1 g Florisil® SPE cartridges. The final volume of cleaned extracts was adjusted to 2 mL with 1:1 hexane:acetonitrile, filtered through a 0.45 m PTFE membrane filter, and stored at 4 ◦ C prior to GC analysis. 2.7. Statistical analysis All collected data were processed using standard statistical software (SigmaPlot, Version 8.0, SPSS, Chicago, IL, USA). The difference in ECD response at different injector temperatures and the stability of each analyte under different storage conditions were evaluated by ANOVA. A significance level of α = 0.05 was used in all comparative statistics. 3. Results and discussion
2.4. Recovery, accuracy and precision of developed method 3.1. Optimization of analytical parameters The recovery, accuracy, and precision of the developed method were determined at three concentration levels (10, 100, and 1000 ng/mL) for each analyte. Each concentration contained five replicates (spiked samples). Recovery (%) was
The oven temperature program and carrier gas flow rate were initially optimized to provide separation of the target analytes. The analytical conditions described previously allowed for the
B. Zhang et al. / Talanta 75 (2008) 1055–1060
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Table 1 Retention times and method detection limits of analytes (mean ± S.D.) Compound
Retention time (min) (mean ± S.D.)
Acephate Trifuralin Malathion Thiamethoxzam Pendimethalin DEF6 Acetamiprid Brifenthrin -Cyhalothrin
3.62 5.58 7.73 8.20 8.53 9.91 12.13 12.34 13.17
± ± ± ± ± ± ± ± ±
0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
MDL (ng/mL)
Reporting limit (ng/mL)
Best injector temperature (◦ C)
0.13 0.05 0.29 0.35 0.08 0.10 0.32 0.05 0.59
2.0 0.5 1.0 1.0 0.5 0.5 1.0 0.5 0.5
240 260 200 140 240 240 300 240 260
separation of the nine analytes (Table 1). ECD response varied by analyte, with acetamiprid, trifuralin, DEF6, and -cyhalothrin producing the best response (Fig. 1). Injector temperature significantly affected the response of analytes on the ECD (Fig. 2). At an injector temperature of 100 ◦ C, all analytes had poor to no response. For most analytes, the detector response increased with increasing injector temperature (Fig. 2), with the highest response at injector temperatures = 240–260 ◦ C. However, the ECD response change with injector temperature was different among the different analytes. Acephate was the most sensitive analyte to injector temperature; a subtle change in injector temperature sharply changed the detector response. -Cyhalothrin, thiamethoxzam, and malathion were also sensitive to changes in injector tem-
perature. ECD response for the other analytes only varied at low injector temperatures. It appeared that an injector temperature of 240 ◦ C provided the most suitable temperature condition for detection of all nine analytes; that temperature was selected for subsequent studies including the analysis of environmental samples described below. 3.2. Calibration curve and method detection limit (MDL) Using the optimized GC-ECD conditions, a sharp peak corresponding to each analyte was obtained. For all analytes tested over a concentration range of 1–1000 ng/mL, the ECD response was linear with excellent regression coefficients (r2 > 0.99 with the exception of acetamiprid) (Table 2). The MDLs for the
Fig. 1. Representative chromatograms of (a) nine pesticide mixture; (b) Playa water sample.
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Table 2 Parameters for the calibration curves of the analytes (Y = aX + b) Compound
a
b
r2
Concentration range (ng/mL)
Acephate Trifuralin Malathion Thiamethoxzam Pendimethalin DEF6 Acetamiprid Brifenthrin -Cyhalothrin
4.69 126.97 32.61 69.44 73.39 222.94 370.79 69.92 174.55
29.15 50.83 52.15 −54.29 72.86 44.45 −594.79 131.65 0.008
0.994 0.995 0.994 0.990 0.997 0.996 0.987 0.996 0.998
1–1000 1–1000 1–1000 2–1000 1–1000 1–1000 2–1000 1–1000 1–1000
analytes ranged from 0.05 to 0.59 ng/mL (Table 1). Although acephate had a lower MDL than other analytes, its quantitation limit (minimal detection concentration in this study) was highest (about 2 ng/mL) because of its instability (Table 1). This may be due to the stable response of the ECD to acephate compared with other analytes in this study. For a majority of the analytes, the quantitation limits were in the ppb or sub-ppb range. 3.3. Accuracy, recovery, and reproducibility of the developed method High accuracy, good precision, and good reproducibility for all nine analytes of interest were achieved at the tested concentrations (Table 3). The reproducibility of the analytical method was evaluated by determining precision at three different concentrations (10, 100, and 1000 ng/mL). The intra-day precision
(%) represented as R.S.D. was 0.37–11.23 and the inter-day precision (%) was 0.17–13.1. There was a slight difference in precision and accuracy among different analytes; pendimethalin, DEF6, and trifluralin had the highest accuracy and precision. However, we considered all accuracy and precision results for these analyses to be acceptable. 3.4. Stability of analytes in extracted solvent The stability of the nine tested pesticides in acetonitrile was investigated under three storage temperatures: room temperature (∼20–25 ◦ C), refrigeration temperature (4 ◦ C), and freezer temperature (−20 ◦ C). Concentrations of each analyte were determined by GC-ECD at different times during the 10-day study. Our results indicated that all analytes of interest were stable up to 10 days at freezer temperatures (−20 ◦ C) (data not shown). At the high concentration (1000 ng/mL), all analytes of interest except -cyhalothrin were stable at room temperature (∼20–25 ◦ C) and at 4 ◦ C for at least 10 days (Fig. 3). At the low concentration in the stability study (100 ng/mL), acephate and acetamiprid were unstable at room temperature and at 4 ◦ C. After 10 days of storage at room temperature, about 30–40% of -cyhalothrin was lost from solution. Acephate, acetamiprid, and -cyhalothrin should be stored at −20 ◦ C or analyzed immediately after extraction. 3.5. Method application To test the application of the method developed in this study on environmental samples, we tested more than 200 soil and
Fig. 2. Effect of injector temperature on ECD response to the nine analytes.
Table 3 Intra-day and inter-day recovery, precision, and accuracy of each analytea Compound
Nominal concentration (ng/mL)
Intra-day Concentration measured (ng/mL)
Inter-day Precision (R.S.D.)
Accuracy (%)
Concentration measured (ng/mL)
Precision (R.S.D.)
Accuracy (%)
10 100 1000
10.4 ± 0.91 100.4 ± 3.68 998.7 ± 18.1
9.08 3.68 1.81
104 100 100
10.3 ± 0.85 96.1 ± 11.3 1018.6 ± 21.6
8.51 11.27 2.16
103 96 102
Trifluralin
10 100 1000
9.9 ± 0.16 91.1 ± 0.11 1001.0 ± 4.20
1.59 1.12 0.42
99 91 100
9.9 ± 0.19 88.1 ± 0.24 1000.2 ± 6.20
1.90 0.24 0.62
99 88 100
Malathion
10 100 500
9.8 ± 0.77 99.8 ± 1.78 1003.9 ± 3.90
7.74 1.78 0.39
98 100 100
10 ± 0.57 95.9 ± 0.47 1010.9 ± 6.10
5.66 0.47 0.61
100 96 101
Thiamethoxzam
10 100 500
9.8 ± 0.19 93.9 ± 3.91 951.1 ± 3.70
1.90 3.91 0.37
98 94 95
9.9 ± 0.20 89.8 ± 2.40 922.3 ± 19.3
2.00 2.40 1.93
99 90 92
Pendimethalin
10 100 1000
10.3 ± 0.10 95.7 ± 1.08 975.8 ± 4.50
0.99 1.08 0.45
103 96 98
10.2 ± 0.13 92.9 ± 1.70 973.1 ± 7.2
1.32 0.17 0.72
102 93 97
DEF6
10 100 1000
9.5 ± 0.12 93.8 ± 1.11 977.7 ± 4.40
1.16 1.11 0.44
95 94 98
9.4 ± 0.17 90.2 ± 0.39 982 ± 7.20
1.65 0.39 0.72
94 90 98
Acetamiprid
10 100 500
9.3 ± 0.52 99.1 ± 1.12 916.9 ± 28.8
5.16 11.23 2.88
93 99 92
9.5 ± 0.14 95.4 ± 12.9 908.6 ± 130
1.36 12.89 13.01
95 95 91
Brifenthrin
10 100 1000
10.7 ± 0.13 104.3 ± 3.65 963.7 ± 4.20
1.32 3.65 0.42
107 104 96
10.7 ± 0.11 101.9 ± 1.47 984.7 ± 7.30
1.14 1.47 0.73
107 102 99
Cyhalothrin
10 100 1000
10.0 ± 0.64 97.2 ± 3.75 966.5 ± 46.1
6.37 3.75 4.61
100 97 97
9.9 ± 0.72 93.9 ± 0.41 1035.1 ± 27.2
7.22 0.41 2.72
99.0 93.9 103.5
B. Zhang et al. / Talanta 75 (2008) 1055–1060
Acephate
R.S.D. = S.D./mean × 100; accuracy (%) = (mean measured concentration/nominal concentration) × 100. a Mean ± S.D., n = 5.
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Fig. 3. Stability of nine pesticides in acetonitrile at three storage conditions. Room temperature (∼20–25 ◦ C): A, 100 ng/mL; B, 1000 ng/mL. Low temperature (4 ◦ C): C, 100 ng/mL; D, 1000 ng/mL.
water samples collected from Playa wetlands near Lubbock, TX. The method was capable of detecting cotton pesticides in several of these samples based on the retention time (Fig. 1(b)), indicating that the method is reliable for routine analysis of environmental samples. However, to confirm the result, a more reliable method, such as mass spectrum, may be employed to detect complicated samples. 4. Conclusions We developed a reliable, sensitive, and reproducible GC-ECD analysis method for quantitative determination of nine cotton pesticides currently used on cotton. Injector temperature significantly affected the analyte response on the ECD. Most analytes had an optimum response at injector temperatures = 240–260 ◦ C. High precision (0.17–13.1%), good accuracy (88–107%), and good reproducibility were obtained at all three tested concentrations for all nine analytes of interest. Good intra-day and inter-day precision also demonstrated the high reproducibility of this method. GC-ECD was highly sensitive to the nine pesticides as indicated by the low MDLs of 0.13, 0.05, 0.29, 0.35, 0.08, 0.10, 0.32, 0.05, and 0.59 (all ng/mL) for acephate, trifluralin, malathion, thiamethoxzam, pendimethalin, DEF6, acetamiprid, brifenthrin, and -cyhalothrin, respectively. All analytes of interest were stable in acetonitrile extracts at freezer temperatures (−20 ◦ C). At high concentrations, all analytes of interest except
-cyhalothrin were stable at room temperature (∼20–25 ◦ C) and 4 ◦ C for at least 10 days. The method was capable of detecting cotton pesticides in real samples. Acknowledgments The authors thank Dr. Loren Smith and Dr. Scott McMurry for partial support of this project and for help in collecting Playa samples. References [1] C. James, Global Review of Commercialized Transgenic Crops: 2001 Feature: Bt Cotton. ISAAA Briefs No. 26, ISAAA, Ithaca, NY, 2002. [2] B.H. Zhang, R. Feng, Cotton Resistance to Insect and Transgenic PestResistant Cotton, Chinese Agricultural Science and Technology Press, Beijing, 2000. [3] G.A. Matthews, in: G.A. Matthews, J.P. Tunstall (Eds.), Insect Pests of Cotton, CAB International, Wallingford, UK, 1994, p. 95. [4] B.H. Zhang, F. Liu, C.B. Yao, K.B. Wang, Curr. Sci. 79 (2000) 37. [5] B.H. Zhang, X.P. Pan, T.L. Guo, Q.L. Wang, T.A. Anderson, Mol. Biotechnol. 31 (2005) 11. [6] R.H. Coupe, E.M. Thurman, L.R. Zimmerman, Environ. Sci. Technol. 32 (1998) 3673. [7] E.M. Thurman, K.C. Bastian, T. Mollhagen, Sci. Total Environ. 248 (2000) 189. [8] B.H. Zhang, X.P. Pan, G.P. Cobb, T.A. Anderson, J. Chromatogr. B 824 (2005) 277. [9] X.P. Pan, B.H. Zhang, G.P. Cobb, Talanta 67 (2005) 816.
Talanta 75 (2008) 1055–1060
Development of a method for the determination of 9 currently used cotton pesticides by gas chromatography with electron capture detection Baohong Zhang a,b,∗,1 , Xiaoping Pan a,c,1 , Louise Venne a , Suzy Dunnum a , Scott T. McMurry a , George P. Cobb a , Todd A. Anderson a a
Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX 79409-1163, United States b Department of Biology, East Carolina University, Greenville, NC 27858, United States c Department of Chemistry, Western Illinois University, Macomb, IL 61455, United States
Received 3 November 2007; received in revised form 1 January 2008; accepted 2 January 2008 Available online 20 January 2008
Abstract A reliable, sensitive, and reproducible method was developed for quantitative determination of nine new generation pesticides currently used in cotton agriculture. Injector temperature significantly affected analyte response as indicated by electron capture detector (ECD) chromatograms. A majority of the analytes had an enhanced response at injector temperatures between 240 and 260 ◦ C, especially analytes such as acephate that overall had a poor response on the ECD. The method detection limits (MDLs) were 0.13, 0.05, 0.29, 0.35, 0.08, 0.10, 0.32, 0.05, and 0.59 ng/mL for acephate, trifuralin, malathion, thiamethozam, pendimethalin, DEF6, acetamiprid, brifenthrin, and -cyhalothrin. This study provides a precision (0.17–13.1%), accuracy (recoveries = 88–107%) and good reproducible method for the analytes of interest. At relatively high concentrations, only -cyhalothrin was unstable at room temperature (20–25 ◦ C) and 4 ◦ C over 10 days. At relatively low concentrations, acephate and acetamiprid were also unstable regardless of temperature. After 10 days storage at room temperature, 30–40% degradation of -cyhalothrin was observed. It is recommended that acephate, acetamiprid, and -cyhalothrin be stored at −20 ◦ C or analyzed immediately after extraction. © 2008 Elsevier B.V. All rights reserved. Keywords: Pesticides; Gas chromatography; Electron capture detection; Cotton
1. Introduction Planted in approximately 70 tropical/subtropical and temperate countries, cotton is one of the most important fiber and economic crops; about 20 million cotton farmers grow 33.5 million hectares of cotton around the world [1,2]. Although many factors affect cotton yield, insect pests and weeds are two major constraints to cotton production. More than 1300 insect species have been reported as cotton pests worldwide [2,3]. Although transgenic Bt cotton has played an important role in pest control [1,4,5], the primary approach to pest control is still through the use of synthetic chemicals. To control pests and weeds, more than 12 kg of pesticide per ha is required annually [6].
∗
Corresponding author at: Department of Biology, East Carolina University, Greenville, NC 27858, United States. E-mail address: [email protected] (B. Zhang). 1 Both the authors contributed equally and both are co-first authors. 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.01.032
One of the most important aspects in minimizing the potential hazards of pesticides to human and environmental health is to monitor pesticide residues [7] and develop best management practices based on these monitoring data. To better monitor residues of pesticides in different media, efficient methods for extraction and determination of pesticides in soil, sediment, water, and food need to exist. As new pesticides are incorporated into agricultural practices, new methods for the determination of those new pesticides must be developed. In this paper, we report on a reliable method for the determination of nine currently used cotton pesticides at trace levels. 2. Experimental 2.1. Chemicals and reagents Pesticide standards were obtained commercially. Acephate, trifuralin, malathion, pendimethalin, DEF6, brifenthrin and
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B. Zhang et al. / Talanta 75 (2008) 1055–1060
-cyhalothrin were purchased from AccuStandard Inc. (New Haven, CT, USA) while acetamiprid and thiamethoxzam were obtained from ChemService Inc. (West Chester, PA, USA). Methanol, acetone, hexane, acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA, USA). Anhydrous Na2 SO4 was purchased from VWR (West Chester, PA, USA). C18 solid-phase extraction (SPE) cartridges (500 mg) were obtained from Fisher Scientific (Pittsburgh, PA, USA). Ultra-pure water (>18 M) was prepared by ultrafiltration with a Mili-Q purification system from Millipore (Bedford, MA, USA). All solvents were HPLC or analytical grade. 2.2. Instruments and sample analysis An HP 6890 series gas chromatograph (GC) was employed to analyze the test pesticides. The GC was equipped with an electron capture detector (ECD) and an autosampler (Agilent, Palo Alto, California, USA). The GC-ECD was controlled by Chemstation software from Hewlett–Packard (Agilent, Palo Alto, California, USA). A 30 m × 0.32 mm × 0.25 m DB-5 column from J&W Scientific (Folsom, CA, USA) was used to separate the analytes. The carrier gas was helium (99.99% purity), and was operated at a constant flow rate during the run (9.2 mL/min). The makeup gas for the detector was argon:methane (95:5) at a combined flow rate of 60.0 mL/min. The injection volume was 2 L in the splitless mode. The injector temperature was 240 ◦ C except in experiments on the optimization of analytical parameters in which different injector temperatures (100–300 ◦ C) were evaluated. The detector temperature was 270 ◦ C and was operated in the constant current mode. Although the oven temperature program varied initially during optimization experiments, the following temperature program was adopted for all subsequent studies reported herein: 110 ◦ C for 1 min, increased to 200 ◦ C at 15 ◦ C/min with a hold of 2 min, then raised to 269 ◦ C at 15 ◦ C/min, and finally held at 269 ◦ C for 6 min. 2.3. Calibration curves and method detection limit For each analyte, 10 standard concentration levels (1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL) were prepared and analyzed in order of increasing concentration. Calibration curves were constructed by plotting concentration of each pesticide versus ECD response (peak area). The method detection limit (MDL) for each pesticide was determined by the analysis of seven spiked samples at 1 ng/mL for each analyte, and was calculated using the following formula: MDL = 3.14 × S.D., where S.D. is the standard deviation of the measurements of seven spiked samples, and 3.14 is the Student’s t-value at the 99% confidence level (t = 3.14 for 6 degrees of freedom).
determined by comparing the measured concentration to the nominal concentration. Precision was calculated by using the relative standard deviation (R.S.D.). The intra-day precision (%) was determined by repeated injections (n = 5) of the same samples on a single day. The inter-day precision (%) was determined by repeated injections on 5 different days. Accuracy was calculated by the equation: (mean measured concentration/nominal concentration) × 100. 2.5. Analyte stability in extraction solvent The stability of analytes in extraction solvent (acetonitrile) was tested at three temperatures: room temperature (∼20–25 ◦ C), 4, and −20 ◦ C. After 0, 1, 2, 4, 6, 8, and 10 days of storage at the three conditions, extracts were analyzed by GCECD. Each treatment was repeated three times, and the stability was calculated by comparing the measured analyte concentration with the nominal concentration. 2.6. Method application Natural water and sediment samples were collected from Playa lakes on the Southern High Plains of West Texas. Playa lakes were chosen because they are representative of surface water receiving pesticide runoff from cotton-growing areas. Water samples (1 L) were extracted using C18 SPE cartridges at a flow rate of 5–6 mL/min. Analytes were eluted with 6–10 mL acetonitrile, concentrated to 2 mL using nitrogen evaporation, and then filtered through 0.2 m PTFE membranes (Millipore, Bedford, MA, USA) into amber GC vials. Sediments (5 g) were air-dried, mixed with 10 g anhydrous Na2 SO4 , loaded into 22-mL cells, and extracted (hexane:acetonitrile, 1:1) using a Dionex Accelerated Solvent Extractor (Model 200, Salt Lake City, UT, USA) following a procedure previously reported [8,9]. Extracts were concentrated to 1–2 mL using nitrogen evaporation and then cleaned using 1 g Florisil® SPE cartridges. The final volume of cleaned extracts was adjusted to 2 mL with 1:1 hexane:acetonitrile, filtered through a 0.45 m PTFE membrane filter, and stored at 4 ◦ C prior to GC analysis. 2.7. Statistical analysis All collected data were processed using standard statistical software (SigmaPlot, Version 8.0, SPSS, Chicago, IL, USA). The difference in ECD response at different injector temperatures and the stability of each analyte under different storage conditions were evaluated by ANOVA. A significance level of α = 0.05 was used in all comparative statistics. 3. Results and discussion
2.4. Recovery, accuracy and precision of developed method 3.1. Optimization of analytical parameters The recovery, accuracy, and precision of the developed method were determined at three concentration levels (10, 100, and 1000 ng/mL) for each analyte. Each concentration contained five replicates (spiked samples). Recovery (%) was
The oven temperature program and carrier gas flow rate were initially optimized to provide separation of the target analytes. The analytical conditions described previously allowed for the
B. Zhang et al. / Talanta 75 (2008) 1055–1060
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Table 1 Retention times and method detection limits of analytes (mean ± S.D.) Compound
Retention time (min) (mean ± S.D.)
Acephate Trifuralin Malathion Thiamethoxzam Pendimethalin DEF6 Acetamiprid Brifenthrin -Cyhalothrin
3.62 5.58 7.73 8.20 8.53 9.91 12.13 12.34 13.17
± ± ± ± ± ± ± ± ±
0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
MDL (ng/mL)
Reporting limit (ng/mL)
Best injector temperature (◦ C)
0.13 0.05 0.29 0.35 0.08 0.10 0.32 0.05 0.59
2.0 0.5 1.0 1.0 0.5 0.5 1.0 0.5 0.5
240 260 200 140 240 240 300 240 260
separation of the nine analytes (Table 1). ECD response varied by analyte, with acetamiprid, trifuralin, DEF6, and -cyhalothrin producing the best response (Fig. 1). Injector temperature significantly affected the response of analytes on the ECD (Fig. 2). At an injector temperature of 100 ◦ C, all analytes had poor to no response. For most analytes, the detector response increased with increasing injector temperature (Fig. 2), with the highest response at injector temperatures = 240–260 ◦ C. However, the ECD response change with injector temperature was different among the different analytes. Acephate was the most sensitive analyte to injector temperature; a subtle change in injector temperature sharply changed the detector response. -Cyhalothrin, thiamethoxzam, and malathion were also sensitive to changes in injector tem-
perature. ECD response for the other analytes only varied at low injector temperatures. It appeared that an injector temperature of 240 ◦ C provided the most suitable temperature condition for detection of all nine analytes; that temperature was selected for subsequent studies including the analysis of environmental samples described below. 3.2. Calibration curve and method detection limit (MDL) Using the optimized GC-ECD conditions, a sharp peak corresponding to each analyte was obtained. For all analytes tested over a concentration range of 1–1000 ng/mL, the ECD response was linear with excellent regression coefficients (r2 > 0.99 with the exception of acetamiprid) (Table 2). The MDLs for the
Fig. 1. Representative chromatograms of (a) nine pesticide mixture; (b) Playa water sample.
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Table 2 Parameters for the calibration curves of the analytes (Y = aX + b) Compound
a
b
r2
Concentration range (ng/mL)
Acephate Trifuralin Malathion Thiamethoxzam Pendimethalin DEF6 Acetamiprid Brifenthrin -Cyhalothrin
4.69 126.97 32.61 69.44 73.39 222.94 370.79 69.92 174.55
29.15 50.83 52.15 −54.29 72.86 44.45 −594.79 131.65 0.008
0.994 0.995 0.994 0.990 0.997 0.996 0.987 0.996 0.998
1–1000 1–1000 1–1000 2–1000 1–1000 1–1000 2–1000 1–1000 1–1000
analytes ranged from 0.05 to 0.59 ng/mL (Table 1). Although acephate had a lower MDL than other analytes, its quantitation limit (minimal detection concentration in this study) was highest (about 2 ng/mL) because of its instability (Table 1). This may be due to the stable response of the ECD to acephate compared with other analytes in this study. For a majority of the analytes, the quantitation limits were in the ppb or sub-ppb range. 3.3. Accuracy, recovery, and reproducibility of the developed method High accuracy, good precision, and good reproducibility for all nine analytes of interest were achieved at the tested concentrations (Table 3). The reproducibility of the analytical method was evaluated by determining precision at three different concentrations (10, 100, and 1000 ng/mL). The intra-day precision
(%) represented as R.S.D. was 0.37–11.23 and the inter-day precision (%) was 0.17–13.1. There was a slight difference in precision and accuracy among different analytes; pendimethalin, DEF6, and trifluralin had the highest accuracy and precision. However, we considered all accuracy and precision results for these analyses to be acceptable. 3.4. Stability of analytes in extracted solvent The stability of the nine tested pesticides in acetonitrile was investigated under three storage temperatures: room temperature (∼20–25 ◦ C), refrigeration temperature (4 ◦ C), and freezer temperature (−20 ◦ C). Concentrations of each analyte were determined by GC-ECD at different times during the 10-day study. Our results indicated that all analytes of interest were stable up to 10 days at freezer temperatures (−20 ◦ C) (data not shown). At the high concentration (1000 ng/mL), all analytes of interest except -cyhalothrin were stable at room temperature (∼20–25 ◦ C) and at 4 ◦ C for at least 10 days (Fig. 3). At the low concentration in the stability study (100 ng/mL), acephate and acetamiprid were unstable at room temperature and at 4 ◦ C. After 10 days of storage at room temperature, about 30–40% of -cyhalothrin was lost from solution. Acephate, acetamiprid, and -cyhalothrin should be stored at −20 ◦ C or analyzed immediately after extraction. 3.5. Method application To test the application of the method developed in this study on environmental samples, we tested more than 200 soil and
Fig. 2. Effect of injector temperature on ECD response to the nine analytes.
Table 3 Intra-day and inter-day recovery, precision, and accuracy of each analytea Compound
Nominal concentration (ng/mL)
Intra-day Concentration measured (ng/mL)
Inter-day Precision (R.S.D.)
Accuracy (%)
Concentration measured (ng/mL)
Precision (R.S.D.)
Accuracy (%)
10 100 1000
10.4 ± 0.91 100.4 ± 3.68 998.7 ± 18.1
9.08 3.68 1.81
104 100 100
10.3 ± 0.85 96.1 ± 11.3 1018.6 ± 21.6
8.51 11.27 2.16
103 96 102
Trifluralin
10 100 1000
9.9 ± 0.16 91.1 ± 0.11 1001.0 ± 4.20
1.59 1.12 0.42
99 91 100
9.9 ± 0.19 88.1 ± 0.24 1000.2 ± 6.20
1.90 0.24 0.62
99 88 100
Malathion
10 100 500
9.8 ± 0.77 99.8 ± 1.78 1003.9 ± 3.90
7.74 1.78 0.39
98 100 100
10 ± 0.57 95.9 ± 0.47 1010.9 ± 6.10
5.66 0.47 0.61
100 96 101
Thiamethoxzam
10 100 500
9.8 ± 0.19 93.9 ± 3.91 951.1 ± 3.70
1.90 3.91 0.37
98 94 95
9.9 ± 0.20 89.8 ± 2.40 922.3 ± 19.3
2.00 2.40 1.93
99 90 92
Pendimethalin
10 100 1000
10.3 ± 0.10 95.7 ± 1.08 975.8 ± 4.50
0.99 1.08 0.45
103 96 98
10.2 ± 0.13 92.9 ± 1.70 973.1 ± 7.2
1.32 0.17 0.72
102 93 97
DEF6
10 100 1000
9.5 ± 0.12 93.8 ± 1.11 977.7 ± 4.40
1.16 1.11 0.44
95 94 98
9.4 ± 0.17 90.2 ± 0.39 982 ± 7.20
1.65 0.39 0.72
94 90 98
Acetamiprid
10 100 500
9.3 ± 0.52 99.1 ± 1.12 916.9 ± 28.8
5.16 11.23 2.88
93 99 92
9.5 ± 0.14 95.4 ± 12.9 908.6 ± 130
1.36 12.89 13.01
95 95 91
Brifenthrin
10 100 1000
10.7 ± 0.13 104.3 ± 3.65 963.7 ± 4.20
1.32 3.65 0.42
107 104 96
10.7 ± 0.11 101.9 ± 1.47 984.7 ± 7.30
1.14 1.47 0.73
107 102 99
Cyhalothrin
10 100 1000
10.0 ± 0.64 97.2 ± 3.75 966.5 ± 46.1
6.37 3.75 4.61
100 97 97
9.9 ± 0.72 93.9 ± 0.41 1035.1 ± 27.2
7.22 0.41 2.72
99.0 93.9 103.5
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Acephate
R.S.D. = S.D./mean × 100; accuracy (%) = (mean measured concentration/nominal concentration) × 100. a Mean ± S.D., n = 5.
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Fig. 3. Stability of nine pesticides in acetonitrile at three storage conditions. Room temperature (∼20–25 ◦ C): A, 100 ng/mL; B, 1000 ng/mL. Low temperature (4 ◦ C): C, 100 ng/mL; D, 1000 ng/mL.
water samples collected from Playa wetlands near Lubbock, TX. The method was capable of detecting cotton pesticides in several of these samples based on the retention time (Fig. 1(b)), indicating that the method is reliable for routine analysis of environmental samples. However, to confirm the result, a more reliable method, such as mass spectrum, may be employed to detect complicated samples. 4. Conclusions We developed a reliable, sensitive, and reproducible GC-ECD analysis method for quantitative determination of nine cotton pesticides currently used on cotton. Injector temperature significantly affected the analyte response on the ECD. Most analytes had an optimum response at injector temperatures = 240–260 ◦ C. High precision (0.17–13.1%), good accuracy (88–107%), and good reproducibility were obtained at all three tested concentrations for all nine analytes of interest. Good intra-day and inter-day precision also demonstrated the high reproducibility of this method. GC-ECD was highly sensitive to the nine pesticides as indicated by the low MDLs of 0.13, 0.05, 0.29, 0.35, 0.08, 0.10, 0.32, 0.05, and 0.59 (all ng/mL) for acephate, trifluralin, malathion, thiamethoxzam, pendimethalin, DEF6, acetamiprid, brifenthrin, and -cyhalothrin, respectively. All analytes of interest were stable in acetonitrile extracts at freezer temperatures (−20 ◦ C). At high concentrations, all analytes of interest except
-cyhalothrin were stable at room temperature (∼20–25 ◦ C) and 4 ◦ C for at least 10 days. The method was capable of detecting cotton pesticides in real samples. Acknowledgments The authors thank Dr. Loren Smith and Dr. Scott McMurry for partial support of this project and for help in collecting Playa samples. References [1] C. James, Global Review of Commercialized Transgenic Crops: 2001 Feature: Bt Cotton. ISAAA Briefs No. 26, ISAAA, Ithaca, NY, 2002. [2] B.H. Zhang, R. Feng, Cotton Resistance to Insect and Transgenic PestResistant Cotton, Chinese Agricultural Science and Technology Press, Beijing, 2000. [3] G.A. Matthews, in: G.A. Matthews, J.P. Tunstall (Eds.), Insect Pests of Cotton, CAB International, Wallingford, UK, 1994, p. 95. [4] B.H. Zhang, F. Liu, C.B. Yao, K.B. Wang, Curr. Sci. 79 (2000) 37. [5] B.H. Zhang, X.P. Pan, T.L. Guo, Q.L. Wang, T.A. Anderson, Mol. Biotechnol. 31 (2005) 11. [6] R.H. Coupe, E.M. Thurman, L.R. Zimmerman, Environ. Sci. Technol. 32 (1998) 3673. [7] E.M. Thurman, K.C. Bastian, T. Mollhagen, Sci. Total Environ. 248 (2000) 189. [8] B.H. Zhang, X.P. Pan, G.P. Cobb, T.A. Anderson, J. Chromatogr. B 824 (2005) 277. [9] X.P. Pan, B.H. Zhang, G.P. Cobb, Talanta 67 (2005) 816.