Screening and Quantification of Pesticides in Water Using a Dual

Dec 2, 2009 - Calculated using Advanced Chemistry Development (ACD/Labs) Software, version 8.14 for Solaris (1994−2008 ACD/Labs). There is no ...
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Anal. Chem. 2010, 82, 290–296

Screening and Quantification of Pesticides in Water Using a Dual-Function Graphitized Carbon Black Disk Nahid Amini, Mohammadreza Shariatgorji, Carlo Crescenzi, and Gunnar Thorse´n* Department of Analytical Chemistry, Stockholm University, 106 91 Stockholm, Sweden A simple platform for combining solid phase extraction (SPE) and surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) of extracted analytes, using disks prepared by embedding graphitized carbon black (GCB-4) particles in a network of polytetrafluoroethylene (PTFE), is presented. The system provides a convenient approach for rapid SALDI-MS screening of substances in aqueous samples, which can be followed by robust quantitative and/or structural analyses by liquid chromatography (LC)/MS/MS of positive samples. The extraction discs are easily transferred between gaskets where the sample extraction and desorption of selected samples is performed and the mass spectrometer. The SPE and SALDI properties of the new GCB-4 disc have been characterized for 15 pesticides with varying chemical properties, and the screening strategy has been applied to the analysis of pesticides in agricultural drainage water. Atrazine and atrazine-desethyl-2-hydroxy were detected in the sampled water by SALDI-MS screening and subsequently confirmed and quantified using LC/MS/MS. Solid phase extraction (SPE) has become a widely used technique for sample extraction and cleanup, since it provides a convenient, cost-effective alternative to liquid-liquid extraction and has been shown to be valuable for extracting, concentrating, and cleaning up molecules with a wide range of physicochemical properties, such as pesticides or pharmaceuticals. Efforts have been made to develop SPE sorbents with high capacity and selectivity, e.g., molecularly imprinted materials, which are now commercially available and have been used (inter alia) in pesticide analysis.1 However, sorbents and/or procedures with too high selectivity may fail to retain some analytes if the chemical compounds of interest have a wide range of properties, for instance, pesticides. Therefore an appropriate balance between selectivity and the ability to retain as many analytes of potential interest as possible is required. Several types of SPE sorbents have been found to be suitable for extracting pesticides from environmental water samples, for instance nonpolar to moderately polar pesticides can be readily sorbed by standard reverse-phase sorbents, such as poly(styrene-divinylbenzene) copolymer and C18* Corresponding author. (1) Chapuis, F.; Pichon, V.; Lanza, F.; Sellergren, B.; Hennion, M.-C. J. Chromatogr., B 2004, 804, 93–101.

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bonded silica.2 The extraction of polar pesticides is more challenging, but many can be adequately extracted by graphitized carbon black (GCB), which is produced by heating carbon black to 2700-3000 °C in an inert atmosphere and has been used for solid phase extraction of pesticides with varying polarities from water,3 food,4 and sediment samples.5 The use of GCB as an SPE material has the additional advantage of storage stability; herbicides stored on GCB have been shown to be as stable as those stored in water.6 In traditional SPE, short columns or cartridges packed with particles of the selected sorbent are used. Alternatively, disks can be used, consisting of a membrane loaded with smaller particles than those packed in conventional cartridges. Such disks have several advantages. Notably, the use of smaller sorbent particles increases the surface area per unit bed volume and improves the homogeneity of the packing, thereby increasing the sorption efficiency and allowing higher sampling flow rates to be applied. Void volumes, breakthrough, and channelling effects are thereby reduced.7 The possibility of using higher flow rates is a major advantage, especially when samples with large volumes, such as environmental water samples, are to be analyzed. Precise qualitative and quantitative analyses are time-consuming and costly when large numbers of samples are to be analyzed. Hence, rapid screening prior to such analyses can be very beneficial if appropriate techniques can be developed. Ideally, a screening technique should be rapid, accurate, robust, and costefficient. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is a possible option in many cases, which has attracted attention in diverse research contexts. MALDI is a soft ionization technique that provides low fragmentation, high sensitivity, and high mass accuracy and is more cost-effective and environmentally friendly than liquid chromatography (LC)/MS since it offers higher analytical throughputs and consumes much lower volumes of organic solvents. These advantages make MALDI-MS a suitable screening technique prior to use of a more laborious and/or costly quantitative method, such as LC/MS. The major drawbacks of MALDIMS are the difficulties involved in analyzing small molecules and the limitations of MALDI-MS in acquiring quantitative data. Liska, I. J. Chromatogr., A 1993, 655, 163–176. Corcia, A. D.; Marchetti, M. Anal. Chem. 1991, 63, 580–585. Torres, C. M.; Pico´, Y.; Manes, J. J. Chromatogr., A 1997, 778, 127–137. Kim, M.-S.; Kang, T. W.; Pyo, H.; Yoon, J.; Choi, K.; Hong, J. J. Chromatogr., A 2008, 1208, 25–33. (6) Sabik, H.; Jeannot, R.; Sauvard, E. Analusis 2000, 28, 835–842. (7) Thurman, E. M.; Snavely, K. TrAC, Trends Anal. Chem. 2000, 19, 18–26.

(2) (3) (4) (5)

10.1021/ac901946b  2010 American Chemical Society Published on Web 12/02/2009

Commonly used matrixes generate cluster ions in the low molecular weight (18 MΩ/cm. Disk Setup and SPE Procedure. Graphitized carbon black (GCB) disks were made from GCB 4 particles (mesh > 400, surface area 210 m2/g; kindly provided by the Laboratori Analitici di Ricerca Associati Srl, Rome, Italy), embedded in a network of polytetrafluoroethylene (PTFE) fibrils by 3M, in the same fashion as Empore Extraction Disks.15 The sheet was cut into 13 mm diameter disks (each containing ∼23 mg GCB) and placed in Swinnex filter holders (Millipore) between two Swinnex gaskets (Millipore). The filter holders were then mounted on a SPE vacuum manifold (VacMaster-10, Biotage). Each disk was conditioned by passing 8 mL of CH2Cl2/MeOH (80/20 v/v), 2 mL of MeOH, 10 mL of 1% acetic acid, and finally 2 mL of ascorbic acid (10 g/L) through it to reduce the quinone groups present on the surface to less reactive hydroquinones.3 The SALDI characteristics of the disks were studied by collecting spectra from blank disks at a range of laser intensities as well as after 250 mL of a 10 µg/mL solution of pesticides was passed through a disk. A 200 mL volume of a 0.05 µg/mL pesticide solution was passed though the membrane to determine the limits of detection for the SALDI analysis. The intensities of the signals arising from desorption of analyte ions were compared to the background signals in regions of the spectra without any signals from either analyte or fragments of analytes. To evaluate the disks’ pesticide retention and desorption characteristics, a 1 mL portion of a solution of the 15 pesticides mentioned above in water, each at a concentration of 0.1 µg/mL, was passed through five parallel disks (conditioned as described above) at a flow rate of approximately 2 mL/min. The eluates from each disk were collected to determine the pesticide’s breakthrough values. Then, after drying the disks by passing a gentle stream of nitrogen through them for about 15 min, 1 mL of MeOH followed by 2 mL of CH2Cl2/MeOH (80/20, v/v) containing 50 mM TFA were used to desorb the analytes. The extracts were collected in Champagne vials (Agilent Technologies). A gentle stream of nitrogen was used to assist evaporation of the solvent from the eluents, which was stopped before they were completely dry. The extracts were then dissolved in 0.2 mL of MeOH/H2O 40/60, shaken and analyzed immediately by LC/ MS, as described below. To explore variations in the recoveries caused by performing SALDI analysis, a 200 mL portion of a 0.05 µg/mL pesticide solution was passed through five parallel disks, sandwiched between two gaskets. Each disk was then placed on a stainless steel plate normally used for MALDI-MS, modified in-house so that the depth of the disk plus the gaskets did not exceed that of the original plate when placed upon it. The disk was then subjected to LDI analysis, returned to its holder, and the remaining analytes were desorbed. (15) Hagen, D. F.; Markell, C. G.; Schmitt, G. A. Anal. Chim. Acta 1990, 236, 157–164.

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Table 1. List of Pesticides Used in This Study and Their Retention Times, MRM Transitions, Optimum Collision Energies, Declustering Potentials, and Entrance Potential Valuesb compound

retention time (min)

MRM transition (m/z)

collision energy (V)

declustering potential (V)

entrance potential (V)

methomyl chloridazon cyanazine carbofuran metsulfuron-methyl simazine metazachlor metalaxyl atrazine sebuthylazine (IS)a propazine parathion-methyl malathion azinphos-ethyl metolachlor diazinon

4.3 7.0 10.0 11.3 11.4 12.3 15.5 15.9 16.3 20.1 20.3 20.8 22.0 25.0 25.1 28.6

163/88 222/77 241/214 222/165 382/167 202/132 278/134 280/220 216/174 230/174 230/146 264/125 331/127 346/132 284/252 305/153

15 50 20 20 20 25 25 20 25 30 30 25 15 20 15 30

10 50 46 31 56 51 26 21 56 51 56 31 71 31 36 31

2 7 10 12 12 6 10 6 6 6 6 8 11 8 5 6

a

Internal standard. b The focusing potential was set to 300 V for all ions.

To study the effects of varying the flow rate on the breakthrough of pesticides from the disks, a 5 ng/mL solution of the pesticides in water was prepared and 20 mL portions of the solution were passed through five parallel disks, in each case at five flow rates (7-10, 15-19, 26-30, 35-40, and 46-50 mL/min). In addition, samples with equal amounts of analyte dissolved in different volumes (volumes of 100, 250, 500, 750, and 1000 mL and concentrations of 1.00, 0.40, 0.20, 0.13, and 0.10 ng/mL, respectively) were passed through five parallel disks, at a constant flow rate (30 mL/min). The eluates were collected and 500 µL of each eluate was injected manually into the LC/MS/MS system for analysis. Finally, to investigate the retention capacity of the disk, solutions of the pesticides at concentrations of 1, 5, 10 µg/ mL were prepared, 20 mL portions of each solution were passed through five parallel disks at a flow rate of 2 mL/min, and breakthrough samples were collected for LC/MS/MS analysis. SALDI Analysis. All SALDI mass spectra were obtained using a Voyager DE-STR time-of-flight mass spectrometer (Applied Biosystems) in reflector mode. A pulsed 337 nm nitrogen laser was used for desorption. The acceleration voltage was set to 20 kV, the delay time to 150 ns, and the grid voltage to 65%. All mass spectra were accumulated from five different spots, 20 shots per spot, and the resulting 100 spectra were averaged. LC/MS/MS Analysis. For LC/MS/MS, a binary liquid chromatography system (Shimadzu, Japan) consisting of two pumps (LC-10 ADvp), a degasser (DGU-14 A), a system controller (SCL10 Avp), and an autoinjector (SIL-10 ADvp) was coupled to a triple quadrupole API 2000 (Applied Biosystems/MDS Sciex, Canada) mass spectrometer. Pesticides were separated on a C-18 HPLC column (Alltima, 250 mm × 4.6 mm i.d., 5 µm particles; Alltech) as the stationary phase and a mobile phase consisting of a 30 min linear gradient of water (A) and methanol (B), both containing 1 mM of formic acid, from 50% B to 85% B. The flow rate was set to 1.0 mL/min, 0.2 mL/min of which was directed toward the mass spectrometer and the rest to waste. The retention times for all of the compounds are listed in Table 1. The analytes were ionized by positive electrospray ionization (+ESI) under the following operating parameters: curtain gas, 30 psi; collision gas, 6 psi; ion spray voltage, 4500 V; temperature, 292

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450 °C; ion source gases 1 and 2, 30 and 40 psi, respectively. To select fragments with the highest signal/noise ratios for each parent ion, the collision energy was ramped from 5 to 60 V in intervals of 5 V. Details of the MRM transitions, including the optimum collision energies, declustering potentials, and focusing potentials, are listed in Table 1. Agricultural Drainage Water Analysis. Agricultural water originating from rice fields was collected from Babolsar, a city situated in the Northern part of Iran by the Caspian Sea and stored at 4 °C awaiting analysis, when a 900 mL sample of the water was filtered though glass fiber prefilters (pore size 1.2 µm, Millipore) and passed through a GCB disk. The retained pesticides were then examined by the SALDI/MS screening and LC/MS/MS identification at a collision energy equal to 40 V and quantification procedures described in the SPE section above. RESULTS AND DISCUSSION To examine the morphology of the GCB disks, scanning electron microscope (SEM) images were taken at 90, 300, and 3000 magnifications, showing the relatively homogeneous surface of the disks, the GCB particles and pores, and single GCB particles (as illustrated in parts a, b, and c of Figure 1, respectively). Evaluation of GCB Disks as SALDI Surfaces. To assess the suitability of GCB disks as SALDI substrates, disks were probed by laser light of varying intensities in the MALDI instrument to determine their maximum tolerable laser intensity (MTLI); the highest intensity at which a SALDI substrate provides a clean background spectrum without any interference from cluster ions.14 MTLI is an important parameter, especially in postsource decay analysis of small molecules when a wide range of laser intensities is used for fragmentation. As shown in Figure 2a, a clean background spectrum was obtained in the mass range of small molecules when using a laser of the intensity applied in the SALDI mass spectrometry analysis of pesticides. The mass/ charge (m/z) differences of 12 observed in the spectrum acquired with laser intensities higher than the MTLI, Figure 2b, are assigned to carbon clusters. No peaks corresponding to the polymer used in the construction of the disk were detected, even at higher laser intensities. The performance of the disk as a SALDI

Figure 1. SEM images of a GCB disk at (a) 90×, (b) 300×, and (c) 3000× magnification.

Figure 3. SALDI spectrum of the studied pesticides retained on a GCB disk. m/z 184.9 corresponds to [methomyl + Na]+, m/z 202.1 to [simazine + H]+, m/z 216.0 to [atrazine + H]+, m/z 230.1 to [propazine + H]+, m/z 238.0 to [atrazine + Na]+, m/z 244.0 to [carbofuran + Na]+ and [chloridazon + Na]+, m/z 263.0 [cyanazine + Na]+, m/z 263.9 to [parathion - methyl + H]+, m/z 300.0 to [metazachlor + Na]+, m/z 302.1 to [metalaxyl + Na]+, m/z 306.1 to [metolachlor + Na]+, m/z 327.0 to [diazinon + Na]+, m/z 353.0 to [malathion + Na]+, and m/z 368.0 to [azinphos - ethyl + Na]+. Figure 2. Spectra of a GCB disk at (a) MTLI and (b) a higher laser intensity than MTLI showing the appearance of carbon clusters.

surface for the analysis of pesticides was also investigated. All of the studied pesticides were observed as at least one ion adduct of H+, Na+, or K+, Figure 3. Evaluation of the SPE Performance of GCB Disks. The ability of the disks to retain the pesticides present in both a relatively small volume of 1 mL and a much larger volume of 200 mL, as well as the LC/MS/MS detection limits, were evaluated and the results are summarized in Table 2. The breakthrough values listed indicate the fractions of the compounds that were not adsorbed by the disk. The obtained recoveries are comparable to previously reported results obtained using GCB cartridges.16 However, use of disks facilitates SALDI/MS analysis and greatly reduces the volume of organic solvents required for both conditioning the sorbent and desorbing the analytes. (16) Tolosa, I.; Douy, B.; Carvalho, F. P. J. Chromatogr., A 1999, 864, 121– 136.

The amounts of the analytes lost due to the SALDI screening were evaluated by determining the recoveries of the pesticides before and after the SALDI analyses. As shown in Table 2, no significant reductions in signal intensity or recovery were detected after SALDI-MS, in accordance with expectations since the area of the laser aperture of the MALDI instrument was very small (∼0.002 mm2) compared to the area of the disk (∼78.5 mm2), so losses of the analytes were negligible. The detection limits for proton, sodium, and potassium adducts of the pesticides in SALDI analyses are listed in Table 3, and the SALDI spectrum is shown in supplementary Figure 1 in the Supporting Information. At least one adduct was detected for each compound except metsulfuron-methyl. It would be possible to reduce the SALDI detection limits by using disks with smaller surface areas, but this would also reduce the flow rates that could be used and the capacity of the disks. In tests of the effects of varying the sample flow rate on the retention of analytes by the disks, parathion-methyl was completely retained at all tested flow rates and the proportions of the other Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

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Table 2. Recoveries and Breakthroughs of the Test Pesticides Determined after Passing 1 mL of a 1.0 µg/mL Solution and 200 mL of a 0.05 µg/mL Solution through GCB Disks, and Recoveries after SALDI Screening Analysisa

compound

1 mL breakthrough % (RSD %)

recovery % (RSD %)

200 mL breakthrough % (RSD %)

recovery % before SALDI analysis (RSD %)

recovery % after SALDI analysis (RSD %)

LC/MS/MS LOD (pg/mL)

methomyl chloridazon cyanazine carbofuran metsulfuron- methyl simazine metazachlor metalaxyl atrazine propazine parathion-methyl malathion azinphos-ethyl metolachlor diazinon

0.3 (88) n.d.b n.d.b 0.2 (88) n.d.b 0.1 (93) 0.1 (98) 0.1 (87) 0.1 (97) 22 (2) n.d.b n.d.b n.d.b n.d.b 0.1 (75)

98 (7) 96 (6) 109 (14) 101 (7) 99 (11) 104 (6) 97 (6) 100 (6) 97 (8) 74 (9) 78 (6) 90 (8) 90 (9) 95 (8) 69 (10)

38 (3) 19 (7) 25 (4) 31 (12) 20 (5) 22 (3) 28 (3) 20 (6) 21 (2) 27 (7) n.d.b 19 (6) 15 (3) 22 (8) 17 (5)

62 (3) 85 (5) 77 (6) 69 (8) 76 (6) 80 (2) 77 (4) 79 (8) 75 (6) 70 (6) 76 (8) 78 (3) 80 (7) 72 (6) 57 (8)

60 (5) 86 (3) 75 (7) 67 (9) 76 (4) 80 (6) 76 (2) 77 (5) 75 (4) 71 (5) 75 (4) 78 (2) 79 (6) 72 (3) 55 (6)

19 81 280 5 4 9 50 11 10 7 201 6 125 84 86

a The values are presented as percentages of the initial amounts of each compound present in the samples (averages of five replicates). LC/ MS/MS method LOD values were defined as 3 times the noise observed in analyses of compounds eluted from the disks after passage of 200 mL of a solution containing 0.05 µg/mL of each pesticide. b n.d., not detected.

Table 3. LOD Values, Defined As 3 Times the Observed Noise, for SALDI Analysis of Pesticides Retained on the GCB Membrane after 200 mL of a 0.05 µg/mL Solution Had Passed through a 78.5 mm2 Membrane LOD (ng/mL) compound methomyl chloridazon cyanazine carbofuran metsulfuron-methyl simazine metazachlor metalaxyl atrazine propazine parathion-methyl malathion azinphos-ethyl metolachlor diazinon a

+

[M + H] a

n.d. 10 21 8 n.d.a 4 25 n.d.a 2 2 14 25 n.d.a 12 4

[M + Na]+

[M + K]+

37 50 21 48 n.d.a n.d.a 37 11 n.d.a 12 n.d.a 25 7 21 n.d.a

21 37 37 45 n.d.a 30 30 11 n.d.a 37 11 n.d.a n.d.a n.d.a 37

n.d., not detected.

analytes retained with the highest tested rate (∼50 mL/min) ranged from 50% for methomyl to 90% for azinphos-ethyl, which have octanol-water partition coefficients (log P values) of 0.6 and 3.6, respectively (Figure 4a). The particularly strong retention of parathion-methyl is probably due to the relatively lower electron density on the benzene ring of this compound, which increases the π-π interactions with the electron-rich aromatic structure of the GCB surface. These observations are in agreement with previous findings that benzene derivatives with electron-withdrawing substitutes were retained more strongly than expected on a GCB analytical column.17 The retention profile for all of the compounds, except parathion-methyl, was almost identical across the range of flow (17) Tanaka, N.; Kimata, K.; Hosoya, K.; Miyanishi, H.; Araki, T. J. Chromatogr., A 1993, 656, 265–287. (18) Calculated using Advanced Chemistry Development (ACD/Labs) Software, version 8.14 for Solaris (1994-2008 ACD/Labs).

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Figure 4. Breakthrough values for pesticides when (a) a constant volume of a solution was passed through GCB disks at different flow rates, and (b) samples containing the same amounts of pesticides, but in different volumes, were passed through GCB disks.

rates tested. The fact that parathion-methyl was almost completely retained by the disks, even at a flow rate of 50 mL/ min, indicates that no channels were created as a result of the higher flow rate. The volume of sample passed through a disk can also influence the retention of analytes, so the effect of this variable was studied (19) Kuster, M.; Lo´pez de Alda, M. J.; Barata, C.; Raldu´a, D.; Barcelo´, D. Talanta 2008, 75, 390–401.

by passing varying volumes of water, up to 1 L, spiked with a constant amount of pesticides through the disks, Figure 4b. As expected, based on log P values of the pesticides, the same profile was observed when the sample volume was increased as when the flow rate was increased. Again, no breakthrough was observed for parathion-methyl even when 1 L of sample was passed through a disk. For the pesticides with relatively high log P values, the breakthrough was almost constant with increasing sample volumes, but for compounds with lower log P values, which are retained more weakly by GCB, the breakthrough values increased with increases in the sample volume. A number of spiked water samples containing different concentrations of the pesticides, at a constant volume, were also passed through the disks. At 1 µg/mL, the breakthrough values were less than 6% for all of the analytes except propazin and methomyl, for which the values were ∼22%. The breakthrough values for most of the pesticides increased quite sharply with increases in concentration from 1 to 10 µg/mL. Almost all of the applied methomyl and carbofuran appeared in the breakthrough solution when the solution containing 10 µg/mL of the pesticides was passed through the disks, whereas no significant changes were observed in the breakthrough values of azinphos-ethyl and parathion-methyl. The retention of these analytes is based on reversed phase and π-π interactions, rather than the anion exchange properties of GCB, since all of the studied analytes, except metsulfuron-methyl, are neutral at the working pH.18 The ability to use such high flow rates and large sample volumes are major advantages when handling environmental samples, which are normally available in large volumes but contain low concentrations of analytes.

Figure 5. SALDI mass spectrum of agricultural drainage water showing atrazine (m/z 216.1) and one of its degradation products, atrazine-desethyl-2-hydroxy (m/z 170.0).

Agricultural Drainage Water Analysis. The applicability of this method for screening and analyzing pesticides in real, complex matrixes was investigated in tests with water collected from the drainage channels of a rice field. High amounts of pesticides are generally used in rice cultivation, and their presence in drainage water collected in such areas has been previously reported.19 Duplicate samples of water collected for this study were passed through disks and analyzed by LDI. Ions with m/z ratios of 216.1 and 170.0 were detected in the screening (Figure 5), potentially corresponding to atrazine and one of its major degradation products, atrazine-desethyl-2-hydroxy. Further identification and quantification by LC/MS/MS confirmed the presence of atrazine, at a concentration of 110.7 ± 0.3 µg/L (Figure 6a,b). The presence of atrazine-desethyl-2-hydroxy was confirmed by comparing its retention time and fragmentation pattern in analyses of the samples with those of the corresponding standard, Figure 6c,d.

Figure 6. LC/MS/MS spectra of (a) atrazine present in agricultural drainage water, (b) a standard solution of atrazine, (c) atrazine-desethyl2-hydroxy present in agricultural drainage water, and (d) a standard solution of atrazine-desethyl-2-hydroxy. All of the spectra were collected at a collision energy of 40 V. Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

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CONCLUSIONS The potential of using GCB disks for both SALDI and SPE, at least for pesticide analyses, has been demonstrated in this work. The disk format offers the possibility of automating both SALDI and SPE steps by placing disks in 96-well plates, thereby providing a high-throughput analytical technique with substantial time and cost savings for analyses of environmental samples.

Ehsan Jalilian at the Department of Inorganic Chemistry, Stockholm University, for performing the SEM analysis.

ACKNOWLEDGMENT N.A. and M.S. contributed equally to this work. The authors would like to acknowledge Saied Ghods for his assistance and

Received for review August 28, 2009. Accepted November 6, 2009.

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SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AC901946B