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Trace level determinations of carbamate pesticides in surface water by gas chromatography–mass spectrometry after derivatization with 9-xanthydrol
Journal of Chromatography A, 1305 (2013) 328–332
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Trace level determinations of carbamate pesticides in surface water by gas chromatography–mass spectrometry after derivatization with 9-xanthydrol Eun-Young Yang a , Ho-Sang Shin b,∗ a b
Environmental Education Graduate School, Kongju National University, Kongju, 314-701, Republic of Korea Department of Environmental Education, Kongju National University, Kongju, 314-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 March 2013 Received in revised form 13 July 2013 Accepted 15 July 2013 Available online 19 July 2013 Keywords: Carbamate pesticides Xanthydrol derivatization Water Gas chromatography–mass spectrometry
a b s t r a c t A sensitive and selective gas chromatographic mass spectrometric method, based on derivatization with 9-xanthydrol, has been established for the simultaneous determination of five carbamate pesticides (carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb) in surface water. 4-Bromo-3,5dimethylphenyl-N-methylcarbamate was chosen as the internal standard for analyzing water samples. The derivatization of carbamates was performed directly in water and the reaction conditions (9xanthydrol of 50.0 mM, HCl concentration of 0.05 M, reaction for 60 min at 60 ◦ C) were established through the optimization of various parameters. Under the established conditions, the limits of quantification were in the range of 0.007–0.028 g/L, and the intra- and inter-day relative standard deviation were each less than 15% at concentrations of 0.1, 1.0 and 10 g/L. None of the carbamate pesticides were detected in any of the sixteen surface waters analyzed. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb are pesticides of the carbamate family, with the formula C12 H11 NO2 , C12 H15 NO3 , C9 H11 NO2 , C11 H15 NO2 and C11 H15 NO2 S, respectively. They are used as insecticides, herbicides and fungicides, and are increasingly used as a replacement for organochlorine and organophosphorous pesticides, due to their lower persistence. However, some carbamates, such as carbofuran and ethiofencarb, are extremely toxic to the central nervous system [1,2] and additionally, are suspected carcinogens and mutagens [3,4]. Especially, carbofuran is a strong endocrine disruptor, and can cause temporary changes in the hormone concentrations of animals and humans even at low doses [1,5] and ethiofencarb is known to the World Health Organization as a highly hazardous pesticide [6]. Their increasing use carries a risk to aquatic and human environments. Canadian water quality guidelines for the protection of aquatic life of freshwater are known as the lowest legislative limit of various guideline values for these pesticides, and those are 0.2 g/L for carbaryl and 1.8 g/L for carbofuran [7]. In Korea, water quality criteria for carbamates in surface waters have not yet
∗ Corresponding author. Tel.: +82 41 850 8811; fax: +82 41 850 8998. E-mail address: [email protected] (H.-S. Shin). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.07.055
been established. Hence, it may be necessary to review the water quality legislation after sufficient monitoring and risk assessment have been completed. The monitoring requires a sensitive analytical method with lower detection limit than the water quality criteria established in other nations. Chromatography is convenient when combined with pretreatment techniques of extraction, concentration and derivatization, and is recommended to detect carbamate pesticides in trace amounts in water. High performance liquid chromatography (HPLC) is the most common method used to analyze carbamate pesticides in environmental water samples [8–13], and HPLC tandem mass spectrometry (MS/MS) has been used for the sensitive quantification of carbamate pesticides in environmental waters [14–16]. The well known thermal instability of carbamate pesticides, as well as their high polarity, has led to the use of HPLC in their detection, but the most commonly used detectors (for example, UV detection) have limited sensitivity, and tandem mass spectrometry has very high instrumental costs. Gas chromatographic (GC) methods [17–21] and gas chromatographic mass spectrometric (GC–MS) methods [22–30] have also been used for the analysis of carbamates. Direct GC analysis of carbamate pesticides often leads to their breakdown in the injection port to the corresponding phenols and amines, or in the column during the analysis. Therefore, derivatization of carbamate is desirable prior to GC analysis. Various derivatization reagents have been used for carbamates, including alkyl iodide [18,21], diazomethane
E.-Y. Yang, H.-S. Shin / J. Chromatogr. A 1305 (2013) 328–332
[22], acetic anhydride [19,23], pentafluoropropionic anhydride [20], trifluoroacetic anhydride [22], heptafluorobutyric anhydride [24], trimethylsulfonium hydroxide and trimethylphenylammonium hydroxide [25]. These derivatization methods have several drawbacks. Firstly, the derivatization reagents are very toxic or even potentially mutagenic [18,21,22]. Secondly, these methods have higher detection limit than Canadian water quality guidelines (0.2 g/L for carbaryl). In this study, we established a new derivatization method of carbamates, to react with xanthydrol directly in water. The formed xanthyl-carbamate derivatives were extracted with organic solvent and detected using GC–MS. The aim of this study was to optimize the parameters of derivatization of carbamate pesticides with xanthydrol, and thus, to sensitively determine these five compounds in water using GC–MS detection. 2. Experimental 2.1. Materials All organic solvents used were HPLC grade. Sodium chloride, potassium hydroxide, sodium bicarbonate, potassium carbonate, methylene chloride, ethyl acetate, methyl-tert-butyl ether, hexane, sodium sulfate, 9-xanthydrol (99.0%), carbaryl (99.5%), carbofuran (98%), metolcarb (99.5%), isoprocarb (99.9%), ethiofencarb (98.7%) and 4-bromo-3,5-dimethylphenyl-N-methylcarbamate (BDMC) (99.5%) as internal standard were obtained from Sigma–Aldrich (St. Louis, MO, USA).
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standard for the injection (50 L of 10.0 mg/L in methanol) was added to each vial in order to control for injection error. 2.4. Gas chromatography–mass spectrometry All mass spectra were obtained with an Agilent 6891/5973N instrument. The ion source was operated in the electron ionization mode (EI; 70 eV). Full-scan mass spectra (m/z 45–600) were recorded for analyte identification. An HP-5MS capillary column (60 m × 0.25 mm I.D. × 0.25 m film thickness) was used. Samples were injected in the split mode (5:1). The flow rate of helium as a carrier gas was 1.0 mL/min. The injector temperature was set at 320 ◦ C. The oven temperature programs were set as follows. The initial temperature of 200 ◦ C was held for 0 min and was then increased to 260 ◦ C at a rate of 30 ◦ C/min, held for 1 min, and was then increased to 300 ◦ C at a rate of 10 ◦ C/min (held for 2 min), and was finally increased to 320 ◦ C at a rate of 5 ◦ C/min (held for 10 min). The ions selected by SIM were m/z 381, 323 and 222 for xanthyl-carbaryl, m/z 401, 344 and 222 for xanthyl-carbofuran, m/z 345, 271 and 222 for xanthyl-metolcarb, m/z 373, 237 and 222 for xanthyl-isoprocarb and m/z 405, 343, 286 for xanthyl-ethiofencarb, and m/z 439, 363, 222 for xanthyl-BDMC (internal standard). 2.5. Calibration and quantification
Surface water samples were collected from 16 basins in the Gum River without headspace in 0.5 L bottles containing 1 drop of 2 M HCl. The sampling sites were selected to uniformly represent all streams of the River.
Calibration curves for carbamates were established by extraction and derivatization after adding carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb standard solutions to the surface water. The extraction and derivatization procedures were performed in an identical manner to those described above. The ions selected for quantification were m/z 222 for xanthyl-carbaryl, m/z 401 for xanthyl-carbofuran, m/z 345 for xanthyl-metolcarb, m/z 373 for xanthyl-isoprocarb and m/z 286 for xanthyl-ethiofencarb, and m/z 222 for xanthyl-BDMC (internal standard). The ratio of the peak area of the standard to that of the internal standard was used for quantification.
2.3. Extraction and derivatization procedure
3. Results and discussion
One hundred milliliters of water samples were transferred to a 250 mL separating funnel, and 20.0 mL of the extracting solvent was added to the solution after the addition of 25 L BDMC (10.0 mg/L in methanol). The sample was then shaken for 5 min using a mechanical shaker. The organic layer was induced into a glass-stopped tube and was concentrated to dryness using a rotary evaporator and a gentle stream of nitrogen below 40 ◦ C. The concentrated residue was dissolved in 100 L of 0.25 M xanthydrol (in methanol) and was then gently shaken using a vortex mixer after the additions of 25 L of 5.0 M HCl and 400 L distilled water. The derivatization reaction was conducted for 60 min at 60 ◦ C in darkness, and then the solution was then neutralized with 5.0 M KOH and the pH of the solution was controlled to 9.5 with about 100 mg of NaHCO3 /K2 CO3 (2:1, w/w). The solution was extracted with 4.0 mL of ethyl acetate and was then shaken for 15 min. The organic layer was concentrated with nitrogen gas. The concentrated residue was dissolved in 100 L of ethyl acetate and a 2.0 L sample of the solution was injected in the GC–MS system. Derivatization efficiencies were calculated at various temperatures (20, 30, 40, 50, 60 and 70 ◦ C), various 9-xanthydrol concentrations (10, 20, 30, 40, 50, 60 and 70 mM), heating times (10, 20, 30, 60, 90 and 120 min) and acid concentrations (0.01, 0.025, 0.05, 0.1, 0.2 and 0.5 M). The pH of each sample was controlled with 5.0 M HCl. The optimum derivatization conditions of carbamates with xanthydrol were determined by the amounts of the formed xanthyl-carbamate derivatives. Phenanthrene-d10 as an internal
3.1. Extraction of carbamates in samples
2.2. Water sampling
The selection of the extraction solvent and the pH on the extraction sample was of great importance in order to achieve satisfactory extraction efficiency for the target compounds. Based on the consideration of the solvent strength, methylene chloride, ethyl acetate, methyl-tert-butyl ether and hexane were selected as potential extraction solvents for use in this study. Extractions were performed with surface water samples spiked at a concentration of 20.0 g/L, at pH values of 3.0, 5.0, 7.0, 9.0, 11.0. As a result of extraction experimentations, methylene chloride at pH 7 gave the highest extraction efficiency (96%). 3.2. Optimization of the derivatization conditions in samples In order to overcome carbamate decomposition and to obtain thermally stable carbamate derivative compounds, the derivatization, including alkylation, acylation and silylation to block the CO–NH group is recommended. The NH of amide group is known to react with 9-xanthydrol in acidic water matrices [31,32]. The reaction between the amide group of carbamates and 9-xanthydrol is plausible, and is predicted to proceed according to a SN2 type reaction in water. In this study, the CO–NH group of carbamates underwent a substitution reaction with 9-xanthydrol under acidic conditions to produce corresponding xanthyl-carbamate derivatives, as shown
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reaction time were studied by the detection of the products. The reaction of carbamates with 9-xanthydrol showed good yields for 50.0 mM 9-xanthydrol, 0.05-M HCl and the optimal reaction temperature and time was found to be 60 min at 60 ◦ C. 3.3. Chromatography and mass spectrometry
Fig. 1. Xanthyl derivatization reaction from carbamate pesticides and 9-xanthydrol.
in Fig. 1, and it was possible to directly analyze the products by GC–MS. The optimum reaction conditions including 9-xanthydrol concentrations, HCl concentrations, reaction temperature, and
The optimum derivatization conditions were applied to the analysis of carbamates in water with 9-xanthydrol by GC–MS. Fig. 2 shows the GC–MS chromatogram of the extracted sample, and the derivatization of carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb. For GC separation of the derivative, the use of a nonpolar stationary phase was found to be efficient. The derivatives of carbamates showed a sharp peak, and the compound was quantified as an integration of the peak area. Extraneous peaks were not observed in the chromatograms near the retention times of the analytes. The mass spectra and fragmentation of xanthyl-carbaryl, xanthyl-carbofuran, xanthyl-metolcarb, xanthyl-isoprocarb and xanthyl-ethiofencarb by electron ionization at 70 eV were studied. A molecular ion peak (M+ ) was observed for each of the derivatives. Characteristic fragment ions were also observed at m/z 196 (M+ −COR), m/z 181 (M+ −NCH3 COR), and m/z 152
Fig. 2. (A) GC/MS (SIM) chromatogram of a standard solution of pesticides at 1.0 mg/L. (B) GC/MS (SIM) chromatogram corresponding to a spiked surface water sample (1.0 g/L). (1) Xanthyl-metolcarb; (2) xanthyl-isoprocarb; (3) xanthyl-carbofuran; (4) xanthyl-ethiofencarb; (5) xanthyl-BDMC(IS); (6) xanthyl-carbaryl.
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Table 1 Calibration curves and detection limits of carbamate pesticides in water. Compound
Linear range (g/L)
Carbaryl Carbofuran Metolcarb Isoprocarb Ethiofencarb
0.2–20 0.2–20 0.2–20 0.2–20 0.2–20
Linear curve
r
Detection limits (g/L)
y = 0.0611x − 0.0117 y = 0.1285x + 0.0252 y = 0.2765x − 0.0496 y = 0.1854x − 0.0205 y = 1.3918x − 0.2033
0.9993 0.9994 0.9993 1.000 0.9996
LOD
LOQ
0.007 0.002 0.007 0.006 0.009
0.022 0.007 0.021 0.019 0.028
Table 2 Intra- and inter-day laboratory precision and accuracy results for the analysis of carbamate pesticides in surface water (n = 5). Compounds
Spiked Conc (g/L)
Intra-day
Inter-day
Mean ± SD (g/L)
Accuracy (%)
Precision (%)
Mean ± SD (g/L)
Accuracy (%)
Precision (%)
Carbaryl
0.1 1.0 10.0
0.12 ± 0.01 0.96 ± 0.11 11.12 ± 0.39
115.0 95.7 111.2
14.7 11.8 3.5
0.12 ± 0.02 1.01 ± 0.09 11.23 ± 1.58
114.8 101.1 112.3
15.0 9.0 14.0
Carbofuran
0.1 1.0 10.0
0.11 ± 0.01 1.10 ± 0.13 9.30 ± 0.64
113.2 109.9 93.0
13.2 12.2 6.9
0.12 ± 0.02 1.17 ± 0.11 11.43 ± 1.59
114.3 117.3 114.3
14.8 8.98 13.8
Metolcarb
0.1 1.0 10.0
0.12 ± 0.02 1.12 ± 0.07 10.16 ± 1.49
115.0 112.1 101.6
13.8 6.6 14.7
0.12 ± 0.02 0.92 ± 0.12 10.22 ± 0.27
114.8 92.2 102.2
13.0 13.0 2.61
Isoprocarb
0.1 1.0 10.0
0.09 ± 0.01 0.91 ± 0.10 8.98 ± 0.79
92.2 90.7 89.9
14.9 10.6 8.8
0.09 ± 0.01 0.98 ± 0.06 7.92 ± 1.06
94.5 98.2 79.2
10.5 6.12 13.4
Ethiofencarb
0.1 1.0 10.0
0.10 ± 0.01 0.90 ± 0.13 9.61 ± 1.44
98.3 90.0 96.1
14.3 14.4 15.0
0.10 ± 0.01 1.07 ± 0.15 10.7 ± 0.71
102.3 106.5 107.0
(M+ −NCH3 CORCHO), where R is the remaining moiety besides amide group (–NHCO) of carbamates. 3.4. Validation of the assay The method was validated, establishing the limit of detection (LOD), the limit of quantification (LOQ), linearity, precision and accuracy. LOD and LOQ were defined as the analyte concentration corresponding to a signal/noise ratio of 3 and 10, respectively in surface water samples which did not contain any of the analytes. The LODs of carbamates in this study were in the concentration range of 0.002–0.009 g/L and the LOQs were in the concentration range of 0.007–0.028 g/L (Table 1). The calibration curves of carbamates were constructed by the extraction and analysis of spiked samples. Examination of the standard curve by calculation of a regression line of the peak area ratios for xanthyl-carbamate derivatives to the internal standard on concentrations, using a least-squares fit, demonstrated a linear relationship, with correlation coefficients being greater than 0.999 (Table 1). The accuracy can be assessed by determining the recovery of spiked samples: Intra-day accuracy was evaluated using five spiked samples at concentrations of 0.1, 1.0 and 10.0 g/L for carbamates. The inter-day accuracy was determined using the sample recovery on three different days. The accuracy was in range of approximately 79–113% and the precision of the assay was less than 15%, as shown in Table 2. 3.5. Method application This paper was designed to describe a method for the determination of carbamates in surface water samples, using GC–MS. When the proposed method was applied to water samples, interfering peaks were not observed in the chromatograms.
12.8 14.0 6.6
Using the proposed method, the levels of carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb were analyzed in sixteen surface water. None of the carbamate pesticides were detected in any of the samples. Although carbamate pesticides were not detected in these samples, they must be continuously monitored for surface water samples, as they can arise from a variety of sources, and pose a considerable toxicological risk. 4. Conclusions In this paper, a simple and sensitive method to detect carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb in water samples is presented, based on a new derivatization method with 9-xanthydrol. The major advantages of this method are as follows: (1) The derivatization reaction is simple and selective. (2) The proposed method has a high sensitivity of 0.007–0.028 g/L (LOQ) for surface water. (3) This method requires relatively inexpensive instrumentation. The proposed GC–MS method permits the reliable analysis of trace carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb in environmental water samples. Acknowledgment This work was supported by Korea Ministry of Environment (MOE) as “the Environmental Health Action Program”. References [1] R.T. Goad, J.T. Goad, B.H. Atieh, R.C. Gupta, Toxicology Mechanisms and Methods 14 (2004) 233. [2] R.C. Gupta, Toxicology Mechanisms and Methods 14 (2004) 103. [3] A.K. Ray, M.C. Ghosh, Aquatic toxicity of carbamates and organophosphates, in: R.C. Gupta (Ed.), Toxicology of Organophosphate and Carbamate Compounds, Academic Press, San Diego, CA, 2005.
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[4] Q.X. Li, B.D. Hammock, J.N. Seiber, Journal of Agricultural and Food Chemistry 39 (1991) 1537. [5] T.K. Lau, W. Chu, N. Graham, Water Science and Technology 55 (2007) 275. [6] Ethiofencarb Powered by Atlassian Confluence and Zen Foundation, http://toxipedia.org/display/toxipedia/Ethiofencarb, 2012. [7] Canadian Federal Environment Minister, Canadian Environmental Quality Guidelines, http://ceqg-rcqe.ccme.ca/ [8] Q. Wu, Q. Chang, C. Wu, Journal of Chromatography A 1217 (2010) 1773. [9] L.j. He, C.J. Wang, Y.J. Sun, International Journal of Environmental Analytical Chemistry 89 (2009) 439. [10] Q.H. Wu, X. Zhou, Y.M. Li, Analytical and Bioanalytical Chemistry 393 (2009) 1755. [11] K. Saeid, H. Mohammadreza, Analytica Chimica Acta 699 (2011) 113. [12] S.M. Goulart, R.D. Alves, A.A. Neves, Analytica Chimica Acta 671 (2010) 41. [13] P. Morrica, P. Fidente, S. Seccia, Biomedical Chromatography 19 (2005) 107. [14] M.G. Cahill, G. Caprioli, M. Stack, S. Vittori, K.J. James, Analytical and Bioanalytical Chemistry 400 (2011) 587. [15] C.Y. Hao, B. Nguyen, X.M. Zhiao, Journal of AOAC International 93 (2010) 400. [16] F. Boujelbane, F. Oueslati, N.B. Hamida, Desalination 250 (2010) 473. [17] K. Nagasawa, H. Uchiyama, A. Ogamo, Journal of Chromatography 144 (1977) 77.
[18] E. Crespo-Corral, M.J. Santos-Delgado, L.M. Polo-Diez, Journal of Chromatography A 1209 (2008) 22. [19] E. Ballesteros, M. Gallego, M. Valcarcel, Journal of Chromatography 633 (1993) 169. [20] Y.S. Oh-Shin, M. Ko, H.S. Shin, Journal of Chromatography A 769 (1997) 285. [21] E. Crespo-Corral, M.J. Santos-Delgado, L.M. Polo-Díez, J. Sanz-Perucha, Journal of Chromatography A 1132 (2006) 241. [22] T. Okumura, K. Imamura, Y. Nishikawa, Analyst 120 (1995) 2675. [23] M. Saraji, N. Esteki, Analytical and Bioanalytical Chemistry 391 (2008) 1091. [24] J.W. King, Z. Zhang, Analytical and Bioanalytical Chemistry 374 (2002) 88. [25] J. Zhang, H.K. Lee, Journal of Chromatography A 1117 (2006) 31. [26] J.Y. Lee, H.K. Lee, Analytical Chemistry 83 (2011) 6856. [27] M.J. Santos-Delgado, B.S. Rubio, G.T. Fernandez-Tostado, L.M. Polo-Diez, Journal of Separation Science 28 (2005) 2130. [28] R. Carabias-Martínez, C. García-Hermida, E. Rodríguez-Gonzalo, L. RuanoMiguel, Journal of Chromatography A 921 (2001) 287. [29] A.M. Filho, F.N. dos Santos, P.A. de P. Pereira, Microchemical Journal 96 (2010) 139. [30] H. Chen, R. Chen, S. Li, Journal of Chromatography A 1217 (2010) 1244. [31] P. Herbert, L. Santos, M. Bastos, M. Barros, A. Alves, Journal of Food Science 67 (2002) 1616. [32] H.S. Shin, E.Y. Yang, Chemistry Central Journal 6 (2012) 157.
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Short communication
Trace level determinations of carbamate pesticides in surface water by gas chromatography–mass spectrometry after derivatization with 9-xanthydrol Eun-Young Yang a , Ho-Sang Shin b,∗ a b
Environmental Education Graduate School, Kongju National University, Kongju, 314-701, Republic of Korea Department of Environmental Education, Kongju National University, Kongju, 314-701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 March 2013 Received in revised form 13 July 2013 Accepted 15 July 2013 Available online 19 July 2013 Keywords: Carbamate pesticides Xanthydrol derivatization Water Gas chromatography–mass spectrometry
a b s t r a c t A sensitive and selective gas chromatographic mass spectrometric method, based on derivatization with 9-xanthydrol, has been established for the simultaneous determination of five carbamate pesticides (carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb) in surface water. 4-Bromo-3,5dimethylphenyl-N-methylcarbamate was chosen as the internal standard for analyzing water samples. The derivatization of carbamates was performed directly in water and the reaction conditions (9xanthydrol of 50.0 mM, HCl concentration of 0.05 M, reaction for 60 min at 60 ◦ C) were established through the optimization of various parameters. Under the established conditions, the limits of quantification were in the range of 0.007–0.028 g/L, and the intra- and inter-day relative standard deviation were each less than 15% at concentrations of 0.1, 1.0 and 10 g/L. None of the carbamate pesticides were detected in any of the sixteen surface waters analyzed. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb are pesticides of the carbamate family, with the formula C12 H11 NO2 , C12 H15 NO3 , C9 H11 NO2 , C11 H15 NO2 and C11 H15 NO2 S, respectively. They are used as insecticides, herbicides and fungicides, and are increasingly used as a replacement for organochlorine and organophosphorous pesticides, due to their lower persistence. However, some carbamates, such as carbofuran and ethiofencarb, are extremely toxic to the central nervous system [1,2] and additionally, are suspected carcinogens and mutagens [3,4]. Especially, carbofuran is a strong endocrine disruptor, and can cause temporary changes in the hormone concentrations of animals and humans even at low doses [1,5] and ethiofencarb is known to the World Health Organization as a highly hazardous pesticide [6]. Their increasing use carries a risk to aquatic and human environments. Canadian water quality guidelines for the protection of aquatic life of freshwater are known as the lowest legislative limit of various guideline values for these pesticides, and those are 0.2 g/L for carbaryl and 1.8 g/L for carbofuran [7]. In Korea, water quality criteria for carbamates in surface waters have not yet
∗ Corresponding author. Tel.: +82 41 850 8811; fax: +82 41 850 8998. E-mail address: [email protected] (H.-S. Shin). 0021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2013.07.055
been established. Hence, it may be necessary to review the water quality legislation after sufficient monitoring and risk assessment have been completed. The monitoring requires a sensitive analytical method with lower detection limit than the water quality criteria established in other nations. Chromatography is convenient when combined with pretreatment techniques of extraction, concentration and derivatization, and is recommended to detect carbamate pesticides in trace amounts in water. High performance liquid chromatography (HPLC) is the most common method used to analyze carbamate pesticides in environmental water samples [8–13], and HPLC tandem mass spectrometry (MS/MS) has been used for the sensitive quantification of carbamate pesticides in environmental waters [14–16]. The well known thermal instability of carbamate pesticides, as well as their high polarity, has led to the use of HPLC in their detection, but the most commonly used detectors (for example, UV detection) have limited sensitivity, and tandem mass spectrometry has very high instrumental costs. Gas chromatographic (GC) methods [17–21] and gas chromatographic mass spectrometric (GC–MS) methods [22–30] have also been used for the analysis of carbamates. Direct GC analysis of carbamate pesticides often leads to their breakdown in the injection port to the corresponding phenols and amines, or in the column during the analysis. Therefore, derivatization of carbamate is desirable prior to GC analysis. Various derivatization reagents have been used for carbamates, including alkyl iodide [18,21], diazomethane
E.-Y. Yang, H.-S. Shin / J. Chromatogr. A 1305 (2013) 328–332
[22], acetic anhydride [19,23], pentafluoropropionic anhydride [20], trifluoroacetic anhydride [22], heptafluorobutyric anhydride [24], trimethylsulfonium hydroxide and trimethylphenylammonium hydroxide [25]. These derivatization methods have several drawbacks. Firstly, the derivatization reagents are very toxic or even potentially mutagenic [18,21,22]. Secondly, these methods have higher detection limit than Canadian water quality guidelines (0.2 g/L for carbaryl). In this study, we established a new derivatization method of carbamates, to react with xanthydrol directly in water. The formed xanthyl-carbamate derivatives were extracted with organic solvent and detected using GC–MS. The aim of this study was to optimize the parameters of derivatization of carbamate pesticides with xanthydrol, and thus, to sensitively determine these five compounds in water using GC–MS detection. 2. Experimental 2.1. Materials All organic solvents used were HPLC grade. Sodium chloride, potassium hydroxide, sodium bicarbonate, potassium carbonate, methylene chloride, ethyl acetate, methyl-tert-butyl ether, hexane, sodium sulfate, 9-xanthydrol (99.0%), carbaryl (99.5%), carbofuran (98%), metolcarb (99.5%), isoprocarb (99.9%), ethiofencarb (98.7%) and 4-bromo-3,5-dimethylphenyl-N-methylcarbamate (BDMC) (99.5%) as internal standard were obtained from Sigma–Aldrich (St. Louis, MO, USA).
329
standard for the injection (50 L of 10.0 mg/L in methanol) was added to each vial in order to control for injection error. 2.4. Gas chromatography–mass spectrometry All mass spectra were obtained with an Agilent 6891/5973N instrument. The ion source was operated in the electron ionization mode (EI; 70 eV). Full-scan mass spectra (m/z 45–600) were recorded for analyte identification. An HP-5MS capillary column (60 m × 0.25 mm I.D. × 0.25 m film thickness) was used. Samples were injected in the split mode (5:1). The flow rate of helium as a carrier gas was 1.0 mL/min. The injector temperature was set at 320 ◦ C. The oven temperature programs were set as follows. The initial temperature of 200 ◦ C was held for 0 min and was then increased to 260 ◦ C at a rate of 30 ◦ C/min, held for 1 min, and was then increased to 300 ◦ C at a rate of 10 ◦ C/min (held for 2 min), and was finally increased to 320 ◦ C at a rate of 5 ◦ C/min (held for 10 min). The ions selected by SIM were m/z 381, 323 and 222 for xanthyl-carbaryl, m/z 401, 344 and 222 for xanthyl-carbofuran, m/z 345, 271 and 222 for xanthyl-metolcarb, m/z 373, 237 and 222 for xanthyl-isoprocarb and m/z 405, 343, 286 for xanthyl-ethiofencarb, and m/z 439, 363, 222 for xanthyl-BDMC (internal standard). 2.5. Calibration and quantification
Surface water samples were collected from 16 basins in the Gum River without headspace in 0.5 L bottles containing 1 drop of 2 M HCl. The sampling sites were selected to uniformly represent all streams of the River.
Calibration curves for carbamates were established by extraction and derivatization after adding carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb standard solutions to the surface water. The extraction and derivatization procedures were performed in an identical manner to those described above. The ions selected for quantification were m/z 222 for xanthyl-carbaryl, m/z 401 for xanthyl-carbofuran, m/z 345 for xanthyl-metolcarb, m/z 373 for xanthyl-isoprocarb and m/z 286 for xanthyl-ethiofencarb, and m/z 222 for xanthyl-BDMC (internal standard). The ratio of the peak area of the standard to that of the internal standard was used for quantification.
2.3. Extraction and derivatization procedure
3. Results and discussion
One hundred milliliters of water samples were transferred to a 250 mL separating funnel, and 20.0 mL of the extracting solvent was added to the solution after the addition of 25 L BDMC (10.0 mg/L in methanol). The sample was then shaken for 5 min using a mechanical shaker. The organic layer was induced into a glass-stopped tube and was concentrated to dryness using a rotary evaporator and a gentle stream of nitrogen below 40 ◦ C. The concentrated residue was dissolved in 100 L of 0.25 M xanthydrol (in methanol) and was then gently shaken using a vortex mixer after the additions of 25 L of 5.0 M HCl and 400 L distilled water. The derivatization reaction was conducted for 60 min at 60 ◦ C in darkness, and then the solution was then neutralized with 5.0 M KOH and the pH of the solution was controlled to 9.5 with about 100 mg of NaHCO3 /K2 CO3 (2:1, w/w). The solution was extracted with 4.0 mL of ethyl acetate and was then shaken for 15 min. The organic layer was concentrated with nitrogen gas. The concentrated residue was dissolved in 100 L of ethyl acetate and a 2.0 L sample of the solution was injected in the GC–MS system. Derivatization efficiencies were calculated at various temperatures (20, 30, 40, 50, 60 and 70 ◦ C), various 9-xanthydrol concentrations (10, 20, 30, 40, 50, 60 and 70 mM), heating times (10, 20, 30, 60, 90 and 120 min) and acid concentrations (0.01, 0.025, 0.05, 0.1, 0.2 and 0.5 M). The pH of each sample was controlled with 5.0 M HCl. The optimum derivatization conditions of carbamates with xanthydrol were determined by the amounts of the formed xanthyl-carbamate derivatives. Phenanthrene-d10 as an internal
3.1. Extraction of carbamates in samples
2.2. Water sampling
The selection of the extraction solvent and the pH on the extraction sample was of great importance in order to achieve satisfactory extraction efficiency for the target compounds. Based on the consideration of the solvent strength, methylene chloride, ethyl acetate, methyl-tert-butyl ether and hexane were selected as potential extraction solvents for use in this study. Extractions were performed with surface water samples spiked at a concentration of 20.0 g/L, at pH values of 3.0, 5.0, 7.0, 9.0, 11.0. As a result of extraction experimentations, methylene chloride at pH 7 gave the highest extraction efficiency (96%). 3.2. Optimization of the derivatization conditions in samples In order to overcome carbamate decomposition and to obtain thermally stable carbamate derivative compounds, the derivatization, including alkylation, acylation and silylation to block the CO–NH group is recommended. The NH of amide group is known to react with 9-xanthydrol in acidic water matrices [31,32]. The reaction between the amide group of carbamates and 9-xanthydrol is plausible, and is predicted to proceed according to a SN2 type reaction in water. In this study, the CO–NH group of carbamates underwent a substitution reaction with 9-xanthydrol under acidic conditions to produce corresponding xanthyl-carbamate derivatives, as shown
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reaction time were studied by the detection of the products. The reaction of carbamates with 9-xanthydrol showed good yields for 50.0 mM 9-xanthydrol, 0.05-M HCl and the optimal reaction temperature and time was found to be 60 min at 60 ◦ C. 3.3. Chromatography and mass spectrometry
Fig. 1. Xanthyl derivatization reaction from carbamate pesticides and 9-xanthydrol.
in Fig. 1, and it was possible to directly analyze the products by GC–MS. The optimum reaction conditions including 9-xanthydrol concentrations, HCl concentrations, reaction temperature, and
The optimum derivatization conditions were applied to the analysis of carbamates in water with 9-xanthydrol by GC–MS. Fig. 2 shows the GC–MS chromatogram of the extracted sample, and the derivatization of carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb. For GC separation of the derivative, the use of a nonpolar stationary phase was found to be efficient. The derivatives of carbamates showed a sharp peak, and the compound was quantified as an integration of the peak area. Extraneous peaks were not observed in the chromatograms near the retention times of the analytes. The mass spectra and fragmentation of xanthyl-carbaryl, xanthyl-carbofuran, xanthyl-metolcarb, xanthyl-isoprocarb and xanthyl-ethiofencarb by electron ionization at 70 eV were studied. A molecular ion peak (M+ ) was observed for each of the derivatives. Characteristic fragment ions were also observed at m/z 196 (M+ −COR), m/z 181 (M+ −NCH3 COR), and m/z 152
Fig. 2. (A) GC/MS (SIM) chromatogram of a standard solution of pesticides at 1.0 mg/L. (B) GC/MS (SIM) chromatogram corresponding to a spiked surface water sample (1.0 g/L). (1) Xanthyl-metolcarb; (2) xanthyl-isoprocarb; (3) xanthyl-carbofuran; (4) xanthyl-ethiofencarb; (5) xanthyl-BDMC(IS); (6) xanthyl-carbaryl.
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Table 1 Calibration curves and detection limits of carbamate pesticides in water. Compound
Linear range (g/L)
Carbaryl Carbofuran Metolcarb Isoprocarb Ethiofencarb
0.2–20 0.2–20 0.2–20 0.2–20 0.2–20
Linear curve
r
Detection limits (g/L)
y = 0.0611x − 0.0117 y = 0.1285x + 0.0252 y = 0.2765x − 0.0496 y = 0.1854x − 0.0205 y = 1.3918x − 0.2033
0.9993 0.9994 0.9993 1.000 0.9996
LOD
LOQ
0.007 0.002 0.007 0.006 0.009
0.022 0.007 0.021 0.019 0.028
Table 2 Intra- and inter-day laboratory precision and accuracy results for the analysis of carbamate pesticides in surface water (n = 5). Compounds
Spiked Conc (g/L)
Intra-day
Inter-day
Mean ± SD (g/L)
Accuracy (%)
Precision (%)
Mean ± SD (g/L)
Accuracy (%)
Precision (%)
Carbaryl
0.1 1.0 10.0
0.12 ± 0.01 0.96 ± 0.11 11.12 ± 0.39
115.0 95.7 111.2
14.7 11.8 3.5
0.12 ± 0.02 1.01 ± 0.09 11.23 ± 1.58
114.8 101.1 112.3
15.0 9.0 14.0
Carbofuran
0.1 1.0 10.0
0.11 ± 0.01 1.10 ± 0.13 9.30 ± 0.64
113.2 109.9 93.0
13.2 12.2 6.9
0.12 ± 0.02 1.17 ± 0.11 11.43 ± 1.59
114.3 117.3 114.3
14.8 8.98 13.8
Metolcarb
0.1 1.0 10.0
0.12 ± 0.02 1.12 ± 0.07 10.16 ± 1.49
115.0 112.1 101.6
13.8 6.6 14.7
0.12 ± 0.02 0.92 ± 0.12 10.22 ± 0.27
114.8 92.2 102.2
13.0 13.0 2.61
Isoprocarb
0.1 1.0 10.0
0.09 ± 0.01 0.91 ± 0.10 8.98 ± 0.79
92.2 90.7 89.9
14.9 10.6 8.8
0.09 ± 0.01 0.98 ± 0.06 7.92 ± 1.06
94.5 98.2 79.2
10.5 6.12 13.4
Ethiofencarb
0.1 1.0 10.0
0.10 ± 0.01 0.90 ± 0.13 9.61 ± 1.44
98.3 90.0 96.1
14.3 14.4 15.0
0.10 ± 0.01 1.07 ± 0.15 10.7 ± 0.71
102.3 106.5 107.0
(M+ −NCH3 CORCHO), where R is the remaining moiety besides amide group (–NHCO) of carbamates. 3.4. Validation of the assay The method was validated, establishing the limit of detection (LOD), the limit of quantification (LOQ), linearity, precision and accuracy. LOD and LOQ were defined as the analyte concentration corresponding to a signal/noise ratio of 3 and 10, respectively in surface water samples which did not contain any of the analytes. The LODs of carbamates in this study were in the concentration range of 0.002–0.009 g/L and the LOQs were in the concentration range of 0.007–0.028 g/L (Table 1). The calibration curves of carbamates were constructed by the extraction and analysis of spiked samples. Examination of the standard curve by calculation of a regression line of the peak area ratios for xanthyl-carbamate derivatives to the internal standard on concentrations, using a least-squares fit, demonstrated a linear relationship, with correlation coefficients being greater than 0.999 (Table 1). The accuracy can be assessed by determining the recovery of spiked samples: Intra-day accuracy was evaluated using five spiked samples at concentrations of 0.1, 1.0 and 10.0 g/L for carbamates. The inter-day accuracy was determined using the sample recovery on three different days. The accuracy was in range of approximately 79–113% and the precision of the assay was less than 15%, as shown in Table 2. 3.5. Method application This paper was designed to describe a method for the determination of carbamates in surface water samples, using GC–MS. When the proposed method was applied to water samples, interfering peaks were not observed in the chromatograms.
12.8 14.0 6.6
Using the proposed method, the levels of carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb were analyzed in sixteen surface water. None of the carbamate pesticides were detected in any of the samples. Although carbamate pesticides were not detected in these samples, they must be continuously monitored for surface water samples, as they can arise from a variety of sources, and pose a considerable toxicological risk. 4. Conclusions In this paper, a simple and sensitive method to detect carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb in water samples is presented, based on a new derivatization method with 9-xanthydrol. The major advantages of this method are as follows: (1) The derivatization reaction is simple and selective. (2) The proposed method has a high sensitivity of 0.007–0.028 g/L (LOQ) for surface water. (3) This method requires relatively inexpensive instrumentation. The proposed GC–MS method permits the reliable analysis of trace carbaryl, carbofuran, metolcarb, isoprocarb and ethiofencarb in environmental water samples. Acknowledgment This work was supported by Korea Ministry of Environment (MOE) as “the Environmental Health Action Program”. References [1] R.T. Goad, J.T. Goad, B.H. Atieh, R.C. Gupta, Toxicology Mechanisms and Methods 14 (2004) 233. [2] R.C. Gupta, Toxicology Mechanisms and Methods 14 (2004) 103. [3] A.K. Ray, M.C. Ghosh, Aquatic toxicity of carbamates and organophosphates, in: R.C. Gupta (Ed.), Toxicology of Organophosphate and Carbamate Compounds, Academic Press, San Diego, CA, 2005.
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[4] Q.X. Li, B.D. Hammock, J.N. Seiber, Journal of Agricultural and Food Chemistry 39 (1991) 1537. [5] T.K. Lau, W. Chu, N. Graham, Water Science and Technology 55 (2007) 275. [6] Ethiofencarb Powered by Atlassian Confluence and Zen Foundation, http://toxipedia.org/display/toxipedia/Ethiofencarb, 2012. [7] Canadian Federal Environment Minister, Canadian Environmental Quality Guidelines, http://ceqg-rcqe.ccme.ca/ [8] Q. Wu, Q. Chang, C. Wu, Journal of Chromatography A 1217 (2010) 1773. [9] L.j. He, C.J. Wang, Y.J. Sun, International Journal of Environmental Analytical Chemistry 89 (2009) 439. [10] Q.H. Wu, X. Zhou, Y.M. Li, Analytical and Bioanalytical Chemistry 393 (2009) 1755. [11] K. Saeid, H. Mohammadreza, Analytica Chimica Acta 699 (2011) 113. [12] S.M. Goulart, R.D. Alves, A.A. Neves, Analytica Chimica Acta 671 (2010) 41. [13] P. Morrica, P. Fidente, S. Seccia, Biomedical Chromatography 19 (2005) 107. [14] M.G. Cahill, G. Caprioli, M. Stack, S. Vittori, K.J. James, Analytical and Bioanalytical Chemistry 400 (2011) 587. [15] C.Y. Hao, B. Nguyen, X.M. Zhiao, Journal of AOAC International 93 (2010) 400. [16] F. Boujelbane, F. Oueslati, N.B. Hamida, Desalination 250 (2010) 473. [17] K. Nagasawa, H. Uchiyama, A. Ogamo, Journal of Chromatography 144 (1977) 77.
[18] E. Crespo-Corral, M.J. Santos-Delgado, L.M. Polo-Diez, Journal of Chromatography A 1209 (2008) 22. [19] E. Ballesteros, M. Gallego, M. Valcarcel, Journal of Chromatography 633 (1993) 169. [20] Y.S. Oh-Shin, M. Ko, H.S. Shin, Journal of Chromatography A 769 (1997) 285. [21] E. Crespo-Corral, M.J. Santos-Delgado, L.M. Polo-Díez, J. Sanz-Perucha, Journal of Chromatography A 1132 (2006) 241. [22] T. Okumura, K. Imamura, Y. Nishikawa, Analyst 120 (1995) 2675. [23] M. Saraji, N. Esteki, Analytical and Bioanalytical Chemistry 391 (2008) 1091. [24] J.W. King, Z. Zhang, Analytical and Bioanalytical Chemistry 374 (2002) 88. [25] J. Zhang, H.K. Lee, Journal of Chromatography A 1117 (2006) 31. [26] J.Y. Lee, H.K. Lee, Analytical Chemistry 83 (2011) 6856. [27] M.J. Santos-Delgado, B.S. Rubio, G.T. Fernandez-Tostado, L.M. Polo-Diez, Journal of Separation Science 28 (2005) 2130. [28] R. Carabias-Martínez, C. García-Hermida, E. Rodríguez-Gonzalo, L. RuanoMiguel, Journal of Chromatography A 921 (2001) 287. [29] A.M. Filho, F.N. dos Santos, P.A. de P. Pereira, Microchemical Journal 96 (2010) 139. [30] H. Chen, R. Chen, S. Li, Journal of Chromatography A 1217 (2010) 1244. [31] P. Herbert, L. Santos, M. Bastos, M. Barros, A. Alves, Journal of Food Science 67 (2002) 1616. [32] H.S. Shin, E.Y. Yang, Chemistry Central Journal 6 (2012) 157.