Determination of eight pesticides of varying polarity in surface waters ...

Microchim Acta (2015) 182:95–103 DOI 10.1007/s00604-014-1290-x

ORIGINAL PAPER

Determination of eight pesticides of varying polarity in surface waters using solid phase extraction with multiwalled carbon nanotubes and liquid chromatography-linear ion trap mass spectrometry Soraya Dahane & María Dolores Gil García & Ana Uclés Moreno & María Martínez Galera & María del Mar Socías Viciana & Aicha Derdour

Received: 18 March 2014 / Accepted: 13 May 2014 / Published online: 8 June 2014 # Springer-Verlag Wien 2014

Abstract We describe a MWCNT-based method for the solid-phase extraction of eight pesticides from environmental water samples. The analytes are extracted from 100 mL samples at pH 5.0 (containing 5 mmol L−1 of KCl) by passing the solution through a column filled with 20 mg of multiwalled carbon nanotubes. Following elution, the pesticides were determined by LC and electrospray ionization hybrid quadrupole linear ion trap MS. Two selected reaction monitoring transitions were monitored per compound, the most intense one being used for quantification and the second one for confirmation. In addition, an information-dependent acquisition experiment was performed for unequivocal confirmation of positive findings. Matrix effect was not found in real waters and therefore the quantitation was carried out with calibration

graphs built with solvent based standards. Except for cymoxanil, the detection and quantitation limits in surface waters are in the range from 0.3 to 9.5 ng L−1 and 1.6 to 45.2 ng L−1, respectively. Recoveries from spiked ultrapure water are ~100 %, except for the most polar pesticides methomyl and cymoxanil. The same behavior is found for real water samples (except for phosalone). The relative standard deviation is 99 % purity, were purchased from Riedel-de Haën (Seelze, Germany, www.riedeldehaen.com). Chemical structures and principal physicochemical properties of selected pesticides are included in the Electronic Supplementary Material (ESM) (See Table S1). Individual stock standard solutions of the target analytes (400 mg L−1) were prepared by weighing and dissolving the corresponding compounds in acetonitrile and stored in the dark at −20 °C, being stable for at least 3 months. Working standard solutions were prepared daily by appropriate dilution of individual stock standard solutions in acetonitrile-ultrapure water (10:90, v/v).

Determination of pesticides in environmental waters using SPE with MWCNTs and LC-QqLIT

Real water sample collection Two types of surface waters (river and natural dam) were used to evaluate the ability of MWCNTs as sorbent for preconcentration of the eight pesticides in environmental waters. Two water samples were collected from rivers located in Almería (Spain), one sample from Nacimiento River (sample NCR) and the other from Andarax River (sample AXR). Nacimiento River flows through a scarcely populated area and discharges into the Andarax River, which flows through a populated area. The sample NCR was picked up in the Nacimiento River at the top of the stream, whereas the sample AXR was picked up in the Andarax River near a wastewater treatment plant which discharges its effluent in the river flowing. In addition, two water samples were collected from two natural dams (samples BND and GND), both located in Almería (Spain). The sample BND was picked up in a dam whose water comes from the Andarax River and is used to irrigate crops, whereas the sample GND was picked from a dam whose water comes from the rainfall and is used for drinking by animals living in the area. All real water samples were collected using 1 L amber bottles, with Teflon lined caps, which were previously rinsed with the sample water on site, and were immediately carried to the laboratory. Both, river and natural dam water samples were filtered through 0.45 μm cellulose filters and stored at 4ºC in refrigerator. Solid-phase extraction The selected pesticides were extracted from environmental water samples by SPE using MWCNTs as sorbent. A Büchi Vac V-500 (Flawil, Switzerland, www.buchi.com) vacuum pump, connected to an extraction manifold of Waters (Milford, MA, USA, www.waters.com) was used in this process. MWCNTs with an average external diameter (O.D.) of 6– 13 nm, length (L) of 2.5–20 μm and 98 % carbon basic were acquired from Sigma–Aldrich (Madrid, Spain, www. sigmaaldrich.com/spain.html). The SPE cartridges packed with 20 mg of MWCNTs sorbent were prepared in the laboratory using 1 mL polypropylene body syringes and two polyethylene frits, both from Supelco (Madrid, Spain, www. sigmaaldrich.com/spain.html), to retain inside the sorbent material. To activate the sorbent, the MWCNT-SPE cartridge was firstly conditioned with 5 mL of a mixture acetone: n-hexane (50:50 v/v) and 5 mL of ultrapure water adjusted at pH 5.0±0.1 at a flow rate of 1 mL min−1. Then, 100 mL of water sample, previously filtered, was adjusted to pH 5.0±0.1 and both potassium chloride 0.005 mol L−1 and 1 % methanol (organic modifier) were added to the sample prior pre-concentration.

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The water sample was pre-concentrated onto the MWCNT cartridge at a flow rate of 1 mL min−1 and the sorbent was dried by successively passing air through it for 5 min and then N2 for another 5 min. The retained analytes were eluted with 3 mL of a mixture acetone:n-hexano (50:50, v/v) and the eluate was evaporated to dryness using a N2 stream. Furthermore, the obtained residue was dissolved in 1 mL of acetonitrile:water (10:90, v/v) and finally, 10 μL of the filtered extract were injected. LC-QqLIT-MS/MS procedure The chromatographic separation of the pesticides was achieved in an Agilent 1200 LC (Agilent Technologies, CA, USA www.agilent.com) provided with a binary pump. The analytical column was a ZORBAX Eclipse XDB C8 (150 mm×4.6 mm, 5 μm particle size) from Agilent. The LC mobile phase consisted of a mixture of acetonitrile (solvent A) and LC-grade water containing 0.1 % formic acid (solvent B). The elution gradient program was as follows: initially 0.5 min of 10 % A, 9 min linear gradient to 100 % A, 5 min of 100 % A, 0.1 min to return to initial conditions and finally initial conditions were reached in 10 min. The flow rate of the mobile phase was set constant at 0.4 mL min−1 during the whole process and the injection volume was 10 μL. The LC was connected to a hybrid triple quadrupole-linear ion trap (QqLIT) mass spectrometer 5500 QTRAP® (AB Sciex Instruments, CA, USA, www.absciex.com) with an electrospray interface (ESI) which was operated in positive ionization mode (PI). The Turbo Ion Spray source settings were: Ion Spray Voltage (IS) 5,500 V, Source Temperature (TEM) 500 °C, Curtain Gas (CUR) 30 psi, Collision Gas (CAD) Medium, both Ion Source Gas 1 (GS1) and Ion Source Gas 2 (GS2) 50 psi. Nitrogen was used as nebulizer gas and collision gas. For the SRM mode, the optimization was performed by direct injection of individual standard solutions of each pesticide in methanol at 0.1 mg L−1. The operational MS/MS parameters for each pesticide such as declustering potential (DP), entrance potential (EP) for precursor ions, collision energy (CE) and collision cell exit potential (CXP) for product ions, for each pesticide, are included in ESM (See Table S2). In the IDA method, only the main SRM transition for each substance is recorded and, therefore, 8 transitions were monitored, all of them in positive ion mode. This SRM experiment was recorded by using the Scheduled MRM option, setting the target scan time at 1 s with a MRM detection window of 40s. Q1 was set at low resolution and Q3 at unit. Regarding the IDA criteria, the intensity threshold was set to 500 cps and without exclusion after dynamic background subtraction of the survey scan. EPI scans were recorded at DP=100 V at one collision energy value CE=25 V, but using the collision energy spread (CES=5 V) and the LIT scanning from 80 to

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450 amu at a scan rate of 10,000 amu.s−1. The dynamic filltime option was selected on the ion trap and EPI scans were monitored in each SRM-IDA-EPI cycle, being the complete SRM-IDA-EPI cycle time of 1.58 s. The ABSciex Analyst software was used for data acquisition and processing.

S. Dahane et al.

the reference spectrum of the library and the spectrum obtained in the samples being given by the fit, reverse fit (RevFit) and purity values provided by the software. The confirmation criteria for identification of the target compounds were a fit value or purity higher than 70 %. Optimization of the SPE procedure

Results and discussions Optimization of LC-QqLIT-MS/MS method The optimization of the chromatographic separation of the eight pesticides was performed by modification of a previously published method [27]. For all the pesticides, positive electrospray ionization (PI) mode showed higher response and, thus the protonated molecule [M + H+] was selected as the precursor ion. In order to comply with the EU requirements the two most intense transitions (SRM1 and SRM2) were selected for each pesticide [28] (See Table S2). The SRM1 was used for quantitation purposes, whereas the two SRM1 and SRM were used for identification. In addition, in order to avoid overestimations or false positive findings in quantitative analysis, other criteria were used for identification: (i) the pesticide tR in the real sample must be within ±2 % the pesticide tR in the standards and (ii) the relative abundances of the two selected SRM transitions (SRM2/SRM1) in the real samples must be between±20 % of the SRM2/SRM1 ratios in the analytical standards [29]. When working with standards, the abundances of the two selected fragments (SRM1 and SRM2) were similar for most analytes, with SRM2/SRM1 between 0.84 and 0.70, except for cymoxanil and phosalone with SRM2/SRM1 0.99 (See Table S4, ESM). On the other hand, the effect of the co-eluting residual matrix components present in the real water samples was evaluated by comparing the slopes of calibration graphs obtained using standards prepared in solvent and blank extracts of environmental water (river and dump waters) by means of a t-test [34]. Significant differences in the slopes of calibration graphs were not found for any pesticide and we stated that matrix effect was absent for all pesticides in the four environmental waters assayed. Therefore, quantitation of pesticides in real water samples was performed using external calibration curves with standards prepared in solvent. Accuracy and precision studies The recovery of the pesticides was evaluated by spiking, in triplicate, ultrapure and environmental waters at two concentration levels (0.05 and 0.10 μg L−1 for diazinon and 0.25 and 0.50 μg L−1 for the other pesticides). Recoveries were about 100 %, except for methomyl and cymoxanil (Table 1), whose low recoveries can be explained by their high water solubility (See Table S1, ESM), being only partially retained. The precision of the overall method was calculated as the relative standard deviation (RSD %) of three replicate samples giving satisfactory results (Table 1), with values ranging from 3.0 to 9.3 %. To demonstrate the applicability of SPE-MWCNT-LCQqLIT-MS/MS method, the two river waters and the two

Determination of pesticides in environmental waters using SPE with MWCNTs and LC-QqLIT

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Table 1 Mean recovery (%) and RSD (%) obtained in the pre-concentration of pesticides in ultrapure and environmental waters, using MWCNTs as sorbent Pesticides

Ultrapure water

Sample NCR

Sample ANX

Sample BNT

Sample GUA

0.25 (μg L−1)

0.50 (μg L−1)

0.25 (μg L−1)

0.50 (μg L−1)

0.25 (μg L−1)

0.50 (μg L−1)

0.25 (μg L−1)

0.50 (μg L−1)

0.25 (μg L−1)

0.50 (μg L−1)

Methomyl Cymoxanil Methidathion Malathion Penconazole Diazinona

51 (7) 80 (6) 100 (5) 110 (5) 100 (4) 106 (3)

54 (6) 59 (4) 95 (5) 122 (6) 86 (4) 121 (4)

43 (5) 86 (10) 98 (8) 86 (8) 66 (9) 91 (6)

50 (4) 57 (7) 98 (6) 97 (6) 69 (7) 58 (4)

49 (6) 79 (8) 84 (7) 77 (6) 87 (6) 111 (3)

52 (6) 71 (8) 86 (5) 107 (4) 102 (4) 96 (4)

60 (5) 71 (7) 93 (4) 89 (5) 86 (7) 94 (7)

53 (6) 68 (5) 86 (4) 107 (4) 73 (5) 70 (5)

56 (7) 74 (6) 84 (6) 109 (6) 87 (6) 71 (5)

58 (7) 68 (4) 90 (5) 76 (3) 91 (6) 72 (5)

Parathion-methyl Phosalone

89 (6) 115 (5)

81 (5) 81 (3)

81 (8) 98 (7)

83 (6) 89 (4)

79 (6) 65 (8)

72 (5) 64 (8)

95 (4) 57 (6)

79 (4) 57 (4)

84 (6) 58 (7)

72 (4) 60 (7)

a

0.05 and 0.1 μg L−1 RSD, relative standard deviation (n=3) between parentheses

dump waters, spiked at the same concentration levels that ultrapure water, were analyzed in triplicate. Firstly, retention times of the eight pesticides in the four real water samples were within ±2 % of the corresponding tR in the standards and the relative abundances of the two selected transitions were within ±20 % of the two SRM ratios in the analytical standards (See Table S2, ESM). In addition, the EPI spectra acquired during the analysis of real water samples were identified by the spectral library with a match quality of 70 %. As for recoveries, the target pesticides kept in real waters the same behaviour as in ultrapure water but, a significant decrease in the recoveries of the less polar pesticide phosalone was found in three of the four real water samples, which can be due to the matrix components existing in these water samples, according to the sampling sites. Humic acids (HAs), with 26.3 % of aromatic carbon (more than fulvic acids), can interact with electron-rich sites of MWCNTs surfaces via π-π interactions [35] and compete with organic molecules by direct site competition and pore blockage on MWCNTs. In addition, due to its high aromatic carbon percentage, HAs can interact with the more hydrophobic compounds, which would reduce their adsorption to MWCNTs. In the light of these results, we propose this last mechanism to explain the low recoveries obtained for phosalone in the more complex environmental water. Although it seems a contradiction between the low recoveries obtained for phosalone and the absence of matrix effect, this behaviour can be explained because most organic matter was removed in the SPE procedure (by interaction with electron-rich sites of MWCNTs surfaces via π-π interactions) and, therefore, it was absent in the blank extracts when the matrix matched calibration graphs were built. However, when the pesticides were added to the real water samples, they were preferably adsorbed onto the MWCNTs whereas the organic matter present in these samples interacted with the most apolar

pesticide phosalone reducing its adsorption to MWCNTs. This demonstrates that comparing the slopes of calibration graphs built in solvent and in blank extract of real samples is not always a safe procedure to check the effect of matrix components and the standard addition method should be used when there is a risk for any analyte. In this way, a deep study about the physico-chemical properties of analytes should be always undertaken as the first step when developing an analytical method.

Comparative study of the MWCNTs-LC-QqLIT method According to the literature, the pre-concentration of the target pesticides in aqueous samples was carried out with different conventional SPE sorbents, the most frequently used being Oasis HLB although another sorbents such as Chroma bond or even Sep-Pak C18 cartridges in tandem have been used (See Table S5, ESM). As can be seen the LODs obtained with our method (using 20 mg of MWCNTs) were in the same order or lower than those obtained using the above mentioned sorbents. The recoveries were also in the same order, except for the most polar pesticides methomyl and cymoxanil. In fact, our previous studies showed recoveries near 90 % for these two pesticides when using 50 mg of sorbent, but phosalone was not recoveried at all (0 % recovery). Therefore, a compromise between both situations was chosen. As for methods using MWCNTs, the high sensitivity provided by the QqLIT detection allowed to use less quantity of sorbent, unlike the methods using this sorbent and less sensitive detection, such as UV (300 mg) or MS (100 mg). Also, the highly sensitive QqLIT detection allowed working with low sample volumes, thus allowing low pre-concentration factors (100 in our work v.s. 250–1,000 in the other cases). An additional advantage provided by the QqLIT detection is

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the absence of matrix effect, which is present in the methods using LC-MS. However, in addition to the analytical performance, it is also interesting to take into account the cost of the adsorbent. In this sense, the price of one Oasis HLB cartridge was about 7.5 euros, whereas the price of one MWCNTs cartridge used in this work was 4.9 euros, including the polypropylene syringe and frits, which involves a cost reduction of 30 %. Moreover, MWCNTs cartridges used to pre-concentrate solvent or clean water samples can be re-used up to ten times, after washing with 5 mL methanol, whereas conventional cartridges are usually discarded.

Conclusions In the present paper, SPE cartridges packed in the laboratory with 20 mg of MWCNTs were successfully used to pre-concentrate eight pesticides in surface water samples. High efficiency was obtained except for the most polar ones methomyl and cymoxanyl, whose recoveries were lowered to get acceptable recoveries for the most apolar phosalone. We can conclude that differences in the polarity of the analytes are not compatible with good recoveries using MWCNTs. The pesticides were analyzed by liquid chromatography coupled to a hybrid triple quadrupole-linear ion trap-mass spectrometer at trace levels working in SRM and IDA modes. The developed methodology was applied to the analysis of two river waters and two natural dam water samples, spiked with the target pesticides, and the two criteria to avoid overestimations (tR and SRM2/SRM1 in real samples) were accomplished. The IDA mode provided additional EPI spectra at low concentration levels, thus allowing reliable identification at the quantitation level for those pesticides with SRM2/SRM1