Journal of Chromatography A, 1355 (2014) 219–227

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Graphene based solid phase extraction combined with ultra high performance liquid chromatography–tandem mass spectrometry for carbamate pesticides analysis in environmental water samples Zhihong Shi, Junda Hu, Qi Li, Shulan Zhang, Yuhuan Liang, Hongyi Zhang ∗ College of Chemistry and Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding 071002, China

a r t i c l e

i n f o

Article history: Received 22 November 2013 Received in revised form 13 April 2014 Accepted 29 May 2014 Available online 12 June 2014 Keywords: Graphene SPE Carbamate pesticides UPLC–MS/MS Environmental water

a b s t r a c t In this paper, graphene, a new sorbent material, was synthesized and used for solid-phase extraction (SPE) of the six carbamate pesticides (pirimicarb, baygon, carbaryl, isoprocarb, baycarb and diethofencarb) in environmental water samples. The target analytes can be extracted on the graphene-packed SPE cartridge, and then eluted with acetone. The eluate was collected and dried by high purity nitrogen gas at room temperature. 1 mL of 20% (v/v) acetonitrile aqueous solution was used to redissolve the residue. The final sample solution was analyzed by ultra performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC–MS/MS) system. Under optimum conditions, good linearity was obtained for the carbamates with correlation coefficient in the range of 0.9992–0.9998. The limits of detection (S/N = 3) for the six carbamate pesticides were in the range of 0.5–6.9 ng L−1 . Relative standard deviations (RSD) for five replicate determinations were below 5.54%. RSD values for cartridge-to-cartridge precision (n = 7) were in the range of 1.27–8.13%. After proper regeneration, the graphene-packed SPE cartridge could be re-used over 100 times for standard solution without significant loss of performance. The enrichment factors for the target analytes were in the range of 34.2–51.7. The established method has been successfully applied to the determination of carbamate pesticide residues in environmental water samples such as river water, well water and lake water. © 2014 Elsevier B.V. All rights reserved.

1. Introduction As less-toxic alternatives to organophosphorus and organochlorine classes, carbamates, composed of the ester of carbamic acid with various substituents, are widely used in agricultural production as pesticides [1,2]. Although carbamates can disintegrate to some extent, they have been found frequently remaining in fruit, vegetables and crops as a result of excessive use [3]. Carbamate pesticides may also enter into the environmental water systems through various paths, including spraying, soil seepage, storage and the discharge of waste water, leading to possible contamination of the environmental water [4,5]. As inhibitors of acetylcholinesterase, carbamate pesticides could affect nerve impulse transmission, inducing dramatic toxicological effects in human beings. Moreover, carbamates and their metabolites are suspected to be carcinogens and mutagens [6]. So carbamate

∗ Corresponding author. Tel.: +86 312 5079357. E-mail address: [email protected] (H. Zhang). http://dx.doi.org/10.1016/j.chroma.2014.05.085 0021-9673/© 2014 Elsevier B.V. All rights reserved.

pesticides are included on the priority list issued by the United States Environmental Protection Agency (EPA) [7]. Therefore, the monitoring of the carbamate residue levels in various environmental water systems is of special concern to human health and environmental safety. The European Union Directive on drinking water quality (98/83/EC) established a maximum allowed concentration of 0.1 ␮g L−1 for each individual pesticide and 0.5 ␮g L−1 for total pesticides [8]. In this sense, reliable, sensitive and rapid analytical methods are urgently needed for the determination of carbamate pesticides at trace levels. Different techniques have been employed for the determination of carbamate pesticides in water and the most commonly preferred methods are liquid chromatography (LC) [9–11] and gas chromatography (GC) [12] coupled to a large number of detectors. As the thermolability of carbamate pesticides may lead to difficulty in direct GC analysis, carbamate pesticides are usually derivatized on-line [13] or off-line [14] for GC analysis. In this case, some authors do not recommend the use of GC for the analysis of carbamate pesticides and consider LC to be the most convenient technique [15]. As the carbamate pesticides are usually found

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at trace concentration levels, it is necessary to carry out preconcentration and/or cleanup steps prior to instrumental analysis. Various sample preparation techniques have been employed for the extraction of carbamates from water sample, such as liquid–liquid extraction (LLE) [16], solid phase extraction (SPE) [17], solid-phase microextraction (SPME) [18], hollow fiber liquid-phase microextraction (HF-LPME) [10], single-drop microextraction (SDME) [19] and dispersive liquid–liquid microextraction (DLLME) [20]. SPE as a technique well known for its large enrichment capacity is presently the most extended method for the preconcentration of carbamate pesticide residues from water samples. In SPE procedure, the choice of appropriate adsorbent is a critical factor to obtain good recovery and high enrichment factor [21,22]. Octadecyl-bonded silica [23], polymeric sorbent [24], and mixed-mode sorbent [25] have already been used for the SPE of carbamate pesticides from water samples. In recent years, various carbon-based materials have been adopted as SPE adsorbents because of their specific properties and high stability. Graphene, a new class of carbon nanomaterial, has sparked much interest because of its remarkable mechanical, thermal and electronic properties since it was discovered by Geim in 2004 [26,27]. With a two dimensional honeycomb lattice composed of carbon atoms, graphene possesses an ultra-high specific surface area (theoretical value 2630 m2 g−1 ) [28], and both sides of the planar sheets are available for molecule adsorption [29,30]. Besides, as graphene is an electron-rich, hydrophobic nanomaterial with large specific area and ␲–␲ electrostatic stacking property [31,32], it has been served as an extraordinarily wonderful adsorbent or extraction material [33]. Nowadays, graphene and graphene-based materials have been used as adsorbents for the extraction and preconcentration of chlorophenols [34], glutathione [35], sulfonamide antibiotics [36], phthalate acid esters [37], organophosphate pesticides [38], heavy metals [39], polycyclic aromatic hydrocarbons [40], macrolides [41] and malachite green [42]. Amine modified graphene has been used to remove fatty acids and other interfering substances for the analysis of pesticides in oil crops [43]. In this paper, the performance of graphene-packed SPE cartridge for the extraction of carbamate pesticides from water samples prior to UPLC–MS/MS analysis was first demonstrated. The six targeted carbamate pesticides are pirimicarb, baygon, carbaryl, isoprocarb, baycarb and diethofencarb, their chemical structures are shown in Fig. 1. These compounds have benzyl, naphthyl or pyrimidyl in their structures, so they can exhibit strong ␲-stacking interaction with the large delocalized ␲-electron system of graphene, to be selectively adsorbed on graphene. The parameters influencing the extraction efficiency were investigated, including the type and volume of eluent solvent, the pH and volume of sample solution. For the efficient separation and quantification of the six carbamates, the UPLC and MS/MS conditions were optimized. The established method was validated and cartridge-to-cartridge precision was evaluated. The performance of graphene-packed SPE cartridge was compared with conventional sorbents such as C18 and graphitized carbon black. Finally, the proposed method was applied to the determination of the six carbamate pesticide residues in river water, lake water and well water samples.

2. Experimental 2.1. Chemicals and materials Pirimicarb (99.2%), diethofencarb (99.5%), baygon (99.5%), isoprocarb (99.2%), carbaryl (99.5%) and baycarb (99.5%) were purchased from Dikma technologies Co., Ltd. (Beijing, China). The

standard stock solutions of carbamates were prepared in dark brown flask with methanol as solvent and stored in the dark at −18 ◦ C. The standard working solution was freshly prepared by diluting the stock solution with water. Graphite powder (99%) and hydrazine hydrate (50%) were purchased from J&K technology Co., Ltd. (Beijing, China). KMnO4 , P2 O5 , K2 S2 O8 , H2 O2 (30%) and concentrated H2 SO4 (95–98%) were of analytical grade and were purchased from Huaxin Chemicals Co., Ltd. (Baoding, China). Acetontrile, formic acid and methanol were of HPLC grade and were purchased from MREDA technologies Co., Ltd. (Beijing, China). Experimental water was doubly distilled de-ioned water. The empty SPE cartridges (3 mL) and SPE frits were purchased from Dikma technologies Co., Ltd. (Beijing, China). AGT Cleanert ODS C18 cartridges were purchased from Agela Techonologies INC. (Delaware, USA). VARIAN Bond Elut PRS cartridges were purchased from Varian Co. (USA). Envi-carb graphitized carbon black cartridges were purchased from Supelco Co. (USA). 2.2. Instrumentation Chromatographic separation was performed on an ACQUITYTM Ultra Performance Liquid Chromatography system (Waters, Milford, MA, USA), consisting of a binary solvent delivery system and an autosampler. MS/MS detection was performed on a Xevo® TQ tandem quadrupole mass spectrometer (Waters, USA) equipped with an electrospray ionization (ESI) source. Data were acquired and processed with MassLynx V4.1 software. JEM-100SX Transmission Electron Microscope (TEM) (Jeol Ltd, Japan), JEM-7500F Scanning Electron Microscope (SEM) (Jeol Ltd, Japan) and TU-1901 UV–vis spectrometer (Persee, China) were used to characterize the lab-produced graphene. The SPE experiments were performed on an HSE series solid-phase extraction device with a vacuum pump (Tianjin HengAo technology development Co., Ltd., Tianjin, China). MTN-2800D pressure blowing concentrator was purchased from Auto Science Co., Ltd. (Tianjin, China). 2.3. Chromatographic conditions The chromatographic separation was performed on an ACQUITY UPLC® BEH C18 column (2.1 × 100 mm i.d., 1.7 ␮m, Waters, made in Ireland) preceded by a BEH C18 VanGuardTM pre-column (2.1 × 5 mm i.d., 1.7 ␮m, Waters, made in Ireland). The mobile phase consisted of (A) 0.1% formic acid solution and (B) acetonitrile. The eluting conditions were as follows: 0–4 min, linear gradient from 30% to 40% B; 4–6 min, linear gradient from 40% to 45% B; 6–6.5 min, linear gradient from 45% to 90% B; 6.5–6.6 min, the composition of B dropped from 90% to 30%; 6.6–8.0 min, the composition of B was kept at 30%. The flow rate was 0.4 mL min−1 . The strong wash volume was 200 ␮L (90% acetonitrile, 0.1‰ formic acid) and the weak wash volume was 600 ␮L (10% acetonitrile, 0.1‰ formic acid). The column temperature and autosampler temperature were maintained at 40 ◦ C and 15 ◦ C, respectively. The injection volume was 10 ␮L. 2.4. Mass spectrometric conditions Mass spectrometry was performed on a Waters Xevo® TQ tandem quadrupole mass spectrometer equipped with electrospray ionization (ESI) source. The conditions of ESI source were as follows: Source temperature 150 ◦ C; Desolvation gas temperature, 550 ◦ C; Desolvation gas (N2 ) flow rate, 850 L h−1 ; Cone gas (N2 ) flow rate, 50 L h−1 ; Capillary voltage, 4.00 kV; Collision gas (Ar) flow rate, 0.15 mL min−1 . All the six compounds were analyzed in positive ESI mode and multiple-reaction monitoring (MRM) mode was selected for quantification.

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O CH3 O O

N H O

O O

N H

CH3

N

CH3

O

N N

N

CH3

H N

CH3

Pirimicarb

Carbaryl

Baygon

OCONHCH3

O

CH3

O

O CH(CH3)2

O

O

Baycarb

Isoprocarb

N H

O

Diethofencarb

Fig. 1. Chemical structures of the six carbamate pesticides.

2.5. Synthesis and characterization of graphene Graphite oxide was synthesized by a modified Hummers method [44,45]. Concentrated H2 SO4 (12 mL) was added into a 50 mL beaker and heated in an 80 ◦ C water bath. Then 2.5 g K2 S2 O8 and 2.5 g P2 O5 were added into the beaker. Next, 3 g Graphite powder was accurately weighed and added into the beaker. The mixture was stirred and kept at 80 ◦ C for 4.5 h. Then, it was diluted with 0.5 L of water and left overnight. After that, the mixture was filtered through a 0.22 ␮m membrane and washed with 1 L of water. The product was dried at 60 ◦ C. This pre-oxidized graphite was added into 120 mL concentrated H2 SO4 in an ice bath. After that, 15 g KMnO4 was slowly (0.5 g min−1 ) added to the mixture under stirring. The temperature must be kept below 20 ◦ C during this process. Then the ice bath was removed and the mixture was stirred at 35 ◦ C for 2 h. After that, 250 mL of water was added to the mixture in an ice bath to keep the temperature below 50 ◦ C and stirred for another 2 h. Then the mixture was diluted with 0.7 L of water again. After the addition of water, 20 mL of H2 O2 (30%, v/v) was added, causing the color turning into yellow along with bubbling. When no bubble was produced, the mixture was centrifuged for 10 min at 5000 rpm, and washed with 1 L of HCl (1:10, v/v) and 1 L of water until the pH was 7.0. The product was dark yellow when it was washed with hydrochloric acid and it turned into dark brown when it was washed with water. In the mean time, the viscosity increased with the increase of the number of washing. After washing, the product was dried at 50 ◦ C. Hydrazine was used to reduce graphene oxide to synthesize graphene. Graphite oxide (0.5 g) was dispersed in 500 mL of water, and ultrasonicated for 1 h to exfoliate graphite oxide to graphene oxide. Then 12 mL of hydrazine hydrate (50%) was added to the dispersion. The mixture was refluxed and stirred for 24 h in an oil bath at 95 ◦ C. The final product was filtered, washed with water, and dried at 50 ◦ C.

2.6. Solid-phase extraction procedures Graphene (30 mg) was placed in a 3 mL empty SPE cartridge using an upper frit and a lower frit to avoid adsorbent loss. Prior to extraction, the SPE cartridge was preconditioned with 3 mL methanol, 3 mL acetone, 3 mL acetonitrile and 9 mL doubly distilled

water. The sample solution (50 mL) was passed through the cartridge at a flow rate of 1 mL min−1 . Then 5 mL acetone was used to elute the analytes retained on the cartridge. The eluent was collected and dried at room temperature under the nitrogen protection. 1 mL of 20% (v/v) acetonitrile aqueous solution was used to redissolve the residue. The final solution was filtered through a 0.22 ␮m membrane, and 10 ␮L of the solution was injected into the UPLC–MS/MS system for analysis. 3. Results and discussion 3.1. Characterization of graphene Characterization of the lab-produced graphene was carried out by using TEM, SEM and UV–vis scanning. From the TEM image (Fig. 2A), we can see clearly the transparent laminate structure with intrinsic wrinkles, which is a characteristic feature of the singlelayer graphene sheet. The SEM image (Fig. 2B) indicates that the graphene agglomerates consist of randomly aggregated and crumpled nanosheets. In addition to this, UV–vis spectra were used to confirm the graphene oxide and graphene. As shown in UV–vis spectra (Fig. 2C), graphene oxide dispersion has an absorption peak at 230 nm, and graphene dispersion has a wide absorption peak at 260 nm. This phenomenon is in accordance with the description in the literature [34]. 3.2. Optimization of UPLC–MS/MS conditions In order to realize good separation of the six carbamates, the composition of mobile phase and elution program were optimized. Experimental results showed that complete separation of the six carbamates could be achieved within 5.5 min by using the chromatographic conditions described in Section 2.3. For the recondition of the column, the total run time was 8 min. The mass spectrometric detection was performed by using positive ion electrospray MS/MS in MRM mode. The qualitative ion pairs were selected based on their full scan mass spectra obtained by directly infusing standard solutions into the mass spectrometer ESI source. Two transitions between the parent ion and the most abundant daughter ions were monitored for the identification of

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Fig. 2. (A) TEM image of graphene. (B) SEM image of graphene. (C) UV–vis spectra of graphene and graphene oxide dispersion.

each compound, and the ion pair with relatively higher intensity was selected for quantification. For each analyte, the optimized parameters including cone voltage, collision voltage, qualitative ion pairs and quantitative ion pair are listed in Table 1.

3.3. Optimization of SPE procedures Several factors affecting SPE performance were investigated in this paper. These parameters included the type and volume of eluent solvent, the pH of sample solution, as well as the volume of the

sample. Extraction recovery (R) was used to evaluate the extraction efficiency and R is expressed as follows: R=

CV × 100% Co Vo

where C is the analyte concentration (ng mL−1 ) in the reconstituted solvent, Co is the initial concentration of analyte in water sample. V and Vo are the volumes of the reconstituted solvent and water sample, respectively. Three replicates were performed for these studies. Statistical analysis of the data (significance level) was carried out by using statistical software IBM SPSS statistics 19.

Table 1 Optimized multiple reaction monitoring (MRM) parameters for the detection of the carbamate pesticides. Compound Isoprocarb Carbaryl Baycarb Baygon Pirimicarb Diethofencarb

Cone voltage (V)

Collision voltage (V)

Qualitative ion pair (m/z)

22 22 20 20 20 20 15 15 28 28 16 16

14 8 25 12 15 8 15 8 20 16 30 10

194.20 → 95.00 194.20 → 137.10 202.05 → 127.05 202.05 → 145.00 208.09 → 95.00 208.09 → 152.00 210.15 → 111.00 210.15 → 168.10 239.17 → 72.00 239.17 → 182.10 268.18 → 124.03 268.18 → 226.15

Quantitative ion pair (m/z) 194.20 → 95.00 202.05 → 145.00 208.09 → 95.00 210.15 → 111.00 239.17 → 72.00 268.18 → 226.15

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Fig. 3. Effect of the type of eluent on the extraction recoveries of six carbamate pesticides. Other conditions: sample, 1 mL of sample solution containing 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb; volume of eluent, 5 mL.

3.3.1. Effect of the type of eluent solvent Selection of the type of eluent solvent is of vital importance for the extraction efficiency of the analytes. In this work, four kinds of eluent solvents (acetone, acetonitrile, methanol and ethanol) were tested. 1 mL of sample solution containing 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb was loaded onto the graphene-packed SPE cartridge, and eluted with 5 mL of the potential eluent solvent. Other experimental conditions were carried out as described in Section 2.6 and the results are shown in Fig. 3. Among the potential eluent solvents, methanol showed relatively low recovery for most of the carbamates, while acetone exhibited the highest extraction recoveries for carbaryl, baycarb and diethofencarb, showing significant difference from the other three solvents (p < 0.01). For baygon and isoprocarb, acetone demonstrated similar recovery with acetonitrile and ethanol (p > 0.05). For the elution of pirimicarb, acetone behaved similar with acetonitrile (p > 0.05). In this study, acetone was proved to be more effective compared with other tested solvents, and it was chosen as eluent solvent in the following experiments.

Fig. 4. Effect of volume of eluent on the extraction recoveries of six carbamate pesticides. Other conditions: sample, 1 mL of sample solution containing 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb; eluent solvent, acetone.

affects the existing state of targeted analytes but also influences the charge species and density on the sorbent surface [46]. In this work, the effect of pH on the extraction efficiency was evaluated at pH 3.00, 5.00, 6.80, 8.20, 10.00 and 12.00, and phosphate buffer solutions were used to adjust the pH. It should be mentioned that pH 8.20 was the original pH of the solution which was prepared by diluting the mixed standard solution with water. As shown in Fig. 5, when the pH was increased from 3 to 6.80, the extraction recoveries increased significantly for pirimicarb, baycarb (p < 0.001), diethofencarb (p < 0.01) and carbaryl (p < 0.05), while for baygon and isoprocarb, no significant increase was observed (p > 0.05). From pH 6.8 to 8.2, the extraction recoveries for all the six carbamates didn’t change much (p > 0.05). Then from pH 8.2 to 10.0, the extraction recoveries of baygon, carbaryl, isoprocarb and baycarb decreased sharply (p < 0.001 for baygon, carbaryl and baycarb; p < 0.01 for isoprocarb), and the extraction recoveries for pirimicarb

3.3.2. Effect of the volume of eluent The volume of eluent is an important parameter for the efficient elution of the analytes. Therefore, the effect of the volume of acetone on the extraction efficiency of the analytes was investigated. 1 mL of sample solution containing 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb was loaded onto the graphene-packed SPE cartridge, and then eluted with 1.0–7.0 mL of acetone. Other experimental conditions were carried out as described in Section 2.6. As shown in Fig. 4, the extraction recoveries for all the six carbamates increased with the increase of the volume of acetone, and reached the maximum value with 5 mL of acetone as eluent solvent. Then with the further increase of the volume of acetone, different behaviors were observed for the carbamates: the extraction recoveries for pirimicarb, carbaryl and diethofencarb kept almost constant (p > 0.05), and the recoveries of baygon, baycarb and isoprocarb showed a decreasing trend (p < 0.01). Based on an overall consideration, 5 mL of acetone was employed in the following experiments. 3.3.3. Effect of pH of the sample The pH of the sample solution is another important factor influencing the extraction efficiency for the reason that pH not only

Fig. 5. Effect of sample pH on the extraction recoveries of six carbamate pesticides. Other conditions: sample, 1 mL of sample solution containing 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb; eluent, acetone; eluent volume, 5 mL.

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Table 2 Linearity, limit of detection and limit of quantification for the six carbamate pesticides. Compound

Linear range (␮g L−1 )

Regression equation

R

LOD (ng L−1 )

LOQ (ng L−1 )

Pirimicarb Baygon Carbaryl Isoprocarb Baycarb Diethofencarb

0.005–5 0.025–100 0.010–200 0.025–100 0.030–60 0.025–50

Y = 22,794.25 + 604,919.40X Y = 56,511.95 + 69,356.45X Y = 66,523.57 + 30,251.73X Y = −56,730.70 + 74,346.90X Y = −21,980.45 + 62,965.53X Y = 38,362.25 + 125,428.78X

0.9996 0.9998 0.9992 0.9992 0.9996 0.9995

0.5 5.6 1.0 3.0 6.9 5.0

1.5 18.6 4.9 12.7 23.3 16.9

Table 3 Results for precision test. Repeatability (n = 5)

Compound

Pirimicarb Baygon Carbaryl Isoprocarb Baycarb Diethofencarb

Cartridge-to-cartridge precision RSD% (n = 7)

C1 (ng mL−1 )

RSD%

C2 (ng mL−1 )

RSD%

C3 (ng mL−1 )

RSD%

0.005 0.025 0.010 0.025 0.030 0.025

3.09 4.19 5.54 3.33 1.57 0.89

2 10 20 10 12 10

0.97 0.97 4.22 4.01 3.13 1.03

5 25 50 25 30 25

2.91 2.56 3.05 1.07 4.45 2.42

and diethofencarb kept almost constant (p > 0.05). It was reported that carbamate pesticides are likely to decompose in strong acidic and strong alkaline conditions, especially in strong alkaline solution at pH > 10 [6]. In our experiment, the original pH values of the environmental water samples are as follows: river water, pH = 7.01; lake water, pH = 6.99; well water, pH = 6.64. They are all in the pH range with relatively constant and high extraction efficiency. Therefore, to facilitate the extraction process, no adjustment of sample pH was performed in the following experiments. 3.3.4. Effect of sample volume In this part, the influence of sample volume was investigated. The graphene-packed SPE cartridge was loaded with 1–100 mL aqueous standard solutions containing 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb in all cases. Other experimental conditions were carried out as described in Section 2.6. The results are illustrated in Fig. 6. It could be seen that extraction recoveries did not change significantly for pirimicarb, baygon, carbaryl, baycarb and diethofencarb (p > 0.05) when the sample volume increased from 25 mL to 50 mL.

Fig. 6. Effect of sample volume on the recoveries of six carbamate pesticides. Other conditions: amount of carbamates, 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb; eluent, acetone; eluent volume, 5 mL.

1.88 1.27 8.13 1.62 1.46 3.11

But when sample volume increased up to 100 mL, the extraction recoveries significantly reduced for all of the carbamates (p < 0.001). Therefore, 50 mL was employed as the loading volume. 3.4. Comparison with other sorbent materials To evaluate the validity of graphene adsorbent, the extraction efficiency of graphene was compared with several commonly used commercialy available sorbent materials including VARIAN Bond Elut PRS, AGT CleanertTM ODS C18 and ENVI-Carb graphitized carbon black (GCB). The same amounts (30 mg) of different adsorbents were accurately weighed and packed in 3 mL SPE cartridges. 50 mL of sample solutions containing 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb were loaded onto the cartridges and eluted under the optimized experimental conditions. Three replicates were carried out for each sorbent material. As shown in Fig. 7, graphene has highest recoveries among those studied adsorbents except for

Fig. 7. Comparison of performance of graphene with several other adsorbents (PRS, C18 and graphitized carbon black (GCB)) for the SPE of six carbamate pesticides. Other conditions: sample, 50 mL of aqueous solution containing 25 ng of baygon, diethofencarb and isoprocarb, 50 ng of carbaryl, 30 ng of baycarb, and 5 ng of pirimicarb; eluent, acetone; eluent volume, 5 mL.

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Table 4 Recoveries of carbamate pesticides from river water, lake water and well water. Compound

Pirimicarb

Baygon

Carbaryl

Isoprocarb

Baycarb

Diethofencarb

Added (ng mL−1 )

River water recovery (%)

Lake water recovery (%)

Well water recovery (%)

0.005 1 5 0.025 5 25 0.01 10 50 0.025 5 25 0.03 6 30 0.025 5 25

85.0 103.1 83.6 96.1 97.4 86.9 110.4 100.6 92.4 103.2 106.0 94.9 97.6 110.3 100.7 101.9 100.8 88.2

111.0 95.1 81.1 85.3 93.7 85.4 106.4 81.8 81.2 101.5 107.9 87.7 84.3 110.7 93.6 100.9 90.7 84.1

91.6 104.7 97.8 96.1 101.2 87.9 97.4 106.8 110.3 97.1 104.9 85.0 108.9 105.5 97.3 103.0 100.3 102.9

carbaryl. This is probably because that carbaryl has a naphthyl in its structure and the ␲–␲ interaction between carbaryl and graphene is stronger compared with other sorbents. The results proved the merits of graphene as SPE adsorbent. Furthermore, compared with commercially available SPE cartridges, the reusability of graphene-packed SPE cartridge is excellent. After proper regeneration, the graphene-packed SPE cartridge could be reused. The regeneration procedures are as follows: the cartridge was eluted with 9 mL of acetone, 3 mL of methanol, 3 mL of acetonitrile and 9 mL of water in series. From the results of

our experiment, the graphene-packed SPE cartridge could be used at least 100 times for the adsorption of standard solution without significant loss of performance.

3.5. Method validation The proposed method was validated in the following aspects: linearity, limit of detection (LOD), limit of quantification (LOQ), precision and recovery.

Fig. 8. The TIC chromatograms of the well water sample (top one) and the spiked well water sample (bottom one). Pirimicarb, tR = 1.00 min; Baygon, tR = 2.39 min; Carbaryl, tR = 2.78 min; Isoprocarb, tR = 3.54 min; Baycarb, tR = 4.88 min; Diethofencarb, tR = 5.15 min.

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3.5.1. Linearity, LOD and LOQ To establish the calibration curves, 50 mL aqueous solutions containing seven concentration levels of carbamate pesticide standards were extracted and analyzed under the optimized experimental conditions. The results are summarized in Table 2. It could be seen from Table 2 that excellent linear relationships were obtained for all the six carbamate pesticides with the correlation coefficients (R) ranging between 0.9992 and 0.9998. LODs (S/N = 3) and LOQs (S/N = 10) varied from 0.5 to 6.9 ng L−1 and 1.5 to 23.3 ng L−1 , respectively. These data indicated that the proposed method has excellent sensitivity. 3.5.2. Precision Precision was evaluated by carrying out repeatability test at three different concentration levels. For each concentration level, five replicate samples were processed with the established method. The results are presented in Table 3. The relative standard deviations (RSD) for repeatability test were below 5.54%, which demonstrates good precision of the established method. To evaluate the cartridge-to-cartridge precision, seven replicate graphene-packed SPE cartridges were prepared, and used for the sample extraction according to the experimental procedures described in Section 2.6. The RSD values for the seven tested cartridges are as follows: pirimicarb 1.88%, baygon 1.27%, carbaryl 8.13%, isoprocarb 1.62%, baycarb 1.46% and diethofencarb 3.11%. 3.5.3. Recovery Relative recovery experiment was carried out by spiking carbamate pesticide standards at low, medium and high concentration levels into river water sample, lake water sample and well water sample containing known amount of carbamate pesticides. Then the spiked samples were processed by the established method. Three replicates were carried out for each concentration level, and the results of relative recovery are summarized in Table 4. The recoveries for the carbamate pesticides in river water, lake water and well water (40 m-deep) samples were in the range from 81.1% to 111.0%. 3.6. Application of the established method to the analysis of environmental water samples Under the optimized conditions, the established method was applied to the determination of the six carbamate pesticide residues in various environmental water samples. Water samples were collected from different regions in China, including river water (Tangshan, China), lake water (Taihu, China), forty-meterdeep well water (Qinhuangdao, China). The experimental results showed that baygon and carbaryl were not found in the water samples. Isoprocarb was not detected in river water and well water, and it was found in lake water, but the concentration was less than LOQ. Baycarb, diethofencarb and pirimicarb were detected in all the water samples, but the concentration levels were less than LOQ except that 0.0062 ␮g L−1 of pirimicarb was detected in well water. The TIC chromatograms of the well water sample and the spiked well water sample are shown in Fig. 8. The enrichment factors for the carbamate pesticides were in the range of 34.2–51.7. 4. Conclusion In this paper, graphene-based SPE was combined with UPLC–MS/MS to determine the carbamate pesticides for the first time. Possessing large surface area and strong adsorption ability, graphene proved to be a good adsorbent for the SPE enrichment and purification of the six carbamate pesticides. At the same time,

graphene-packed SPE cartridge has fine reusability (a graphenepacked SPE cartridge could be used at least 100 times based on the experience in our lab). The cartridge-to-cartridge precision was good. Satisfactory linearity, repeatability and recovery were achieved with graphene-packed SPE coupled to UPLC–MS/MS for the analysis of six carbamate pesticides. LOD and LOQ are lower than other existing methods, demonstrating that the proposed method is highly sensitive. The results indicated that the proposed method could be used efficiently for the determination of trace carbamates in various water samples. Acknowledgments Financial support from the National Natural Science Foundation of China (20875020, 20575016) and the Natural Science Foundation of Hebei Province China (B2013201234) are gratefully acknowledged. The authors also thank the Human Resources and Social Security Department of Hebei Province for the financial support from the Scientific and Technological Foundation for Selected Overseas Chinese Scholars (2011). Special thanks are given to the project sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry of China. References [1] S. Moinfar, M.R.M. Hosseini, Development of dispersive liquid-liquid microextraction method for the analysis of organophosphorus pesticides in tea, J. Hazard. Mater. 169 (2009) 907–911. [2] C. Przybylski, V. Bonnet, Combination of H-1 nuclear magnetic resonance spectroscopy and mass spectrometry as tools for investigation of the thermolytic and solvolytic effects Case of carbamates analysis, J. Chromatogr. A 1216 (2009) 4787–4797. [3] X.H. Wang, J. Cheng, H.B. Zhou, X.H. Wang, M. Cheng, Development of a simple combining apparatus to perform a magnetic stirring-assisted dispersive liquidliquid microextraction and its application for the analysis of carbamate and organophosphorus pesticides in tea drinks, Anal. Chim. Acta 787 (2013) 71–77. [4] E. Ballesteros, M.J. Parrado, Continuous solid-phase extraction and gas chromatographic determination of organophosphorus pesticides in natural and drinking waters, J. Chromatogr. A 1029 (2004) 267–273. [5] J. Cheng, Y.T. Xia, Y.W. Zhou, F. Guo, G. Chen, Application of an ultrasoundassisted surfactant-enhanced emulsification microextraction method for the analysis of diethofencarb and pyrimethanil fungicides in water and fruit juice samples, Anal. Chim. Acta 701 (2011) 86–91. [6] A. Santalad, L. Zhou, F.J. Shang, D. Fitzpatrick, R. Burakham, S. Srijaranai, J.D. Glennon, J.H.T. Luong, Micellar electrokinetic chromatography with amperometric detection and off-line solid-phase extraction for analysis of carbamate insecticides, J. Chromatogr. A 1217 (2010) 5288–5297. [7] US Environmental Protection Agency, National Survey of Pesticides in Drinking Water Wells, Phase II Report, EPA 570/9-91-020, National Technical Information Service, Springfield, VA, 1992. [8] EU Council, EU Council Directive on the Quality of Water Intended for Human Consumption, 98/83/CE, 1998. [9] C.Y. Hao, B. Nguyen, X.M. Zhao, E. Chen, P. Yang, Determination of residual carbamate, organophosphate, and phenyl urea pesticides in drinking and surface water by high-performance liquid chromatography/tandem mass spectrometry, J. AOAC Int. 93 (2010) 400–410. [10] G.Y. Zhao, C. Wang, Q.H. Wu, Z. Wang, Determination of carbamate pesticides in water and fruit samples using carbon nanotube reinforced hollow fiber liquidphase microextraction followed by high performance liquid chromatography, Anal. Methods 3 (2011) 1410–1417. [11] N. Makihata, T. Kawamoto, K. Teranishi, Simultaneous analysis of carbamate pesticides in tap and raw water by LC/ESI/MS, Anal. Sci. 19 (2003) 543–549. [12] H. Chen, R.W. Chen, S.Q. Li, Low-density extraction solvent-based solvent terminated dispersive liquid-liquid microextraction combined with gas chromatography-tandem mass spectrometry for the determination of carbamate pesticides in water samples, J. Chromatogr. A 1217 (2010) 1244–1248. [13] L. Guo, H.K. Lee, Low-density solvent based ultrasound-assisted emulsification microextraction and on-column derivatization combined with gas chromatography-mass spectrometry for the determination of carbamate pesticides in environmental water samples, J. Chromatogr. A 1235 (2012) 1–9. [14] E.Y. Yang, H.S. Shin, Trace level determinations of carbamate pesticides in surface water by gas chromatography-mass spectrometry after derivatization with 9-xanthydrol, J. Chromatogr. A 1305 (2013) 328–332. [15] J.M. Soriano, B. Jiménez, G. Font, J.C. Moltó, Analysis of carbamate pesticides and their metabolites in water by solid phase extraction and liquid chromatography: a review, Crit. Rev. Anal. Chem. 31 (2001) 19–52. [16] S.M. Goulart, R.D. Alves, A.A. Neves, J. Humberto de Queiroz, T. Condé de Assis, M.E.L.R. de Queiroz, Optimization and validation of liquid-liquid extraction

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