Environ Sci Pollut Res DOI 10.1007/s11356-014-2876-x POTENTIALTOXICITY OF PESTICIDES IN FRESHWATER ENVIRONMENTS: PASSIVE SAMPLING, EXPOSURE AND IMPACTS ON BIOFILMS
Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry for acidic herbicides and metabolites analysis in fresh water Vincent Fauvelle & Nicolas Mazzella & Soizic Morin & Sylvia Moreira & Brigitte Delest & Hélène Budzinski
Received: 10 January 2014 / Accepted: 2 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Theoretical papers and environmental applications of hydrophilic interaction liquid chromatography (HILIC) have been published for a wide range of analytes, but to our knowledge, no study focused on acidic herbicides (e.g., triketones, phenoxy acids, sulfonylurea, and acidic metabolites of chloroacetanilides). Matrix effects are the main obstacle to natural sample analysis by liquid chromatography coupled with tandem mass spectrometry (MS) via an electrospray ionization (ESI) interface. Therefore, we paid particular attention on limiting interference by (i) adapting the emerging HILIC technique, which is generally considered more sensitive than conventional reversed phase liquid chromatography and (ii) optimizing the solid phase extraction (SPE) step using a design of experiment. A rapid and reliable off line SPE-HILIC-ESI-MS/MS method was thus developed for the quantification of acidic herbicides in fresh water, with limits of quantifications (LOQs) ranging from 5 to 22 ng L−1. Then, the analysis of freshwater samples highlighted the robustness of the method, and the importance of the chloroacetanilides metabolites among the studied analytes.
Keywords Acidic herbicides . HILIC . HPLC-ESI-MS/MS . Surface water . Matrix effects
Responsible editor: Philippe Garrigues V. Fauvelle : N. Mazzella (*) : S. Morin : S. Moreira : B. Delest Irstea, UR EABX, 33612 Cestas Cedex, France e-mail:
[email protected] V. Fauvelle (*) : H. Budzinski EPOC-LPTC, UMR 5805 CNRS, University of Bordeaux 1, 33405 Talence Cedex, France e-mail:
[email protected]
Introduction Acidic herbicides (e.g., 2,4-D, bentazon, dicamba) are widely used because they are relatively inexpensive and potent even at low application rate. Chloroacetanilides (acetochlor, Smetolachlor) are also an important class of herbicides that may be degraded in soils and water to form ethan sulfonic and oxanilic acid (ESA and OA, respectively) metabolites (Kalkhoff et al. 1998). Due to their high solubility, they are easily transferred to ground water, streams and rivers (Fung and Ikesaki 1991). Reliable analytical methods are therefore required to quantify their residues in fresh water, even at trace levels. For example, the European Directive 2009/90/EC requires a “limits of quantification (LOQs) equal or below a value of 30 % of the relevant environmental quality standard” (few ng to μg L-1). Thus, to achieve sufficiently low LOQs, a solid phase extraction (SPE) step is needed in acidic conditions in order to reach the protonated form of the analytes and thus enhance extraction recoveries on polymeric sorbents. However, several studies using reversed phase liquid chromatography (RPLC) reported strong matrix interferences under acidic SPE extraction (Tran et al. 2007; Pichon et al. 1996) due to (1) the coextraction of fulvic and humic acids present in natural samples during the SPE step, and (2) the low retention of the analytes on RPLC resulting in their coelution with interfering compounds at the start of the gradient. Indeed, matrix interferences are identified as the major limitation related to the use of electrospray ionization interface (ESI) and mass spectrometry (MS) detection for environmental samples analysis (Taylor 2005). There are several possibilities for reducing matrix effects: optimization of the sample pH, optimization of the washing step (volume and organic modifier content), reduction of the concentration factor, and the use of an appropriate internal standard (IS) calibration.
Environ Sci Pollut Res
Separation of acidic herbicides is commonly performed by gas chromatography (GC) (Quintana et al. 2007) or liquid chromatography (HLPC) (Carabias-Martinez et al. 2004a; Tran et al. 2007). However, GC separation requires a time consuming derivatization step to overcome the low volatility and thermolability of the analytes (Öllers et al. 2001). Thus HPLC-ESI-MS/MS using reversed phase is the method of choice for the quantification of acidic herbicides in water (Rodil et al. 2009). Due to the poor retention of acidic herbicides on conventional RPLC, acidification of the mobile phase together with post-column base addition (Carabias-Martinez et al. 2004b; Freitas et al. 2004) or the use of an ion-pairing agent (Balinova 1996) are generally required. However, these two last solution generally imply high chromatogram baseline, and several studies demonstrated the benefits of using HILIC technique for the most polar compounds separation (Heaton et al. 2011). Thus, a zwiterrionic silica-based stationary phase column was chosen (i.e., Macherey-Nagel Nucleodur HILIC) to achieve good separation and retention of ionic compounds, together with lower IDLS (instrumental detection limits) compared to C18 or bare silica columns (Chirita et al. 2010; Vikingsson et al. 2008). HILIC is an emerging chromatographic technique for the analysis of a wide range of small polar compounds (Chirita et al. 2010; Esparza et al. 2009; Karlsson et al. 2005). It was first mentioned by Alpert et al. in the 1990s (Alpert 1990) and could be roughly represented by a partition mechanism between the organic-rich mobile phase and the pseudo-immobilized aqueous layer at the surface of the stationary phase. To our knowledge, there are no studies available on the HILIC separation of acidic herbicides, although it could be a good alternative to RPLC. Indeed, HILIC provides a theoretically improved sensitivity due to its better compatibility with the ESI interface (Nguyen and Schug 2008) and could allow reducing the SPE concentration factor or injection volume, in order to avoid matrix effects. This paper presents first the development of a HILIC-ESIMS/MS method for 19 acidic herbicides and metabolites. Then, the SPE optimization is detailed on a selected sorbent in order to find a compromise between acceptable extraction recoveries and minimized matrix effects. Afterward, the method validation parameters are listed (calibration linearity, specificity, extraction recoveries, and LOQs) and results from field application are presented.
Experimental Chemicals Formic acid was purchased from Riedel-de Haën® and ammonium acetate and triethylamine were provided by Fluka (Sigma-Aldrich, Schnelldorf, Germany). Organic solvents
(methanol (MeOH)- and acetonitrile (ACN)-HPLC grade) were obtained from Sharlau (HPLC grade, Atlantic Labo, Bruges, France); and ultrapure water (UPW) with a resistivity of 18 MΩ was produced by a Synergy UV system from Millipore (Billerica, MA, USA). All eluents were filtered through 0.45 μm regenerated cellulose filters from Whatman GmbH (Versailles, France). SPE polymeric sorbents were purchased from Waters (Oasis HLB® 30 μm, 150 and 200 mg) or offered by JT Baker (SDB-2 40 μm, 200 mg). Solid phase extractions were performed on VisiprepTM and VisidryTM from Supelco. Water samples were filtered on glass fiber filters (GF/F, 0.7 μm) provided by Whatman. Analytical standards were purchased from Dr. Erhenstorfer GmbH (Augsburg, Germany): 2,4-D, acetochlor ESA, acetochlor OA, bentazon, chlorsulfuron, dicamba, dichlorprop, diclofop, fenopop, iodosulfuron, ioxynil, MCPA, mecoprop, mesotrione, metolachlor ESA, metolachlor OA, metsulfuron-methyl, nicosulfuron, sulcotrione, bentazon-d6, dicamba-d3, MCPAd3, and metsulfuron-d3. Their purity was higher than 96.5 %. Monomolecular stock solutions were prepared in ACN (100 μg mL−1) and stored at −18 °C for 6 months. Working solutions (1.0 μg mL−1) of acidic herbicide standards, internal standards (bentazon-d6, MCPA-d3, metsulfuron-d3), and surrogate (dicamba-d3) were also prepared in ACN and stored at −6 °C for 3 months. When applying the method on natural samples, 33 other neutral pesticides were monitored using an RPLC method developed by Lissalde et al. (2011). Bentazond6 was used as internal standard for bentazon, ioxynil and ESA/OA metabolites, MCPA-d3 corrected for all phenoxy acids and dicamba, and metsulfuron-d3 corrected for all sulfonylureas and triketones. Dicamba-d3 was chosen as extraction surrogate because it is the less retained compound during SPE step (less favorable case). As SPE was performed in acidic conditions, a lot of humic material was coextracted, implying strong signal extinction, especially for sulfonylureas and triketones. This is the reason why internal standard quantification was required. Solid phase extraction procedures About 400 mL of water sample was filtered on GF/F and the pH was adjusted to 5 with UPW/formic acid 90:10 (v/v). The optimized extraction procedure for Oasis HLB was as follows: the 150 mg cartridges were conditioned with 3 mL of methanol and 3 mL of UPW, then 200 mL of filtered, acidified, and fortified (100 μL of surrogate working solution) water sample was percolated through them. The cartridges were washed with 1.5 mL of UPW/MeOH 70:30 (v/v) (this washing fraction called F0 thereafter), and dried for 30 min under nitrogen flow. The elution step was performed with 5 mL of MeOH, the collected extract was evaporated to dryness under a nitrogen stream, and the reconstitution of the sample was obtained with
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2,940 μL of the initial HPLC eluent mixture and 60 μL of the internal standards working solution. Finally, 5 μL are injected. HPLC system and columns The HPLC system was a Dionex Ultimate 3000 (solvent rack SRD-3600 six degasser channels, DGP-3600 M pump, WPS3000 TSL Micro Autosampler, TCC-3100 HP 1xRH 2P-6P thermostated column oven). HILIC separation was carried out using a Macherey-Nagel zwitterionic Nucleodur HILIC 3 μm, 100 Å, 125 mm×2.1 mm protected by a Nucleodur HILIC 3 μm, 100 Å, 8 mm×3 mm, security guard column. Elution was performed in pseudo isocratic conditions using ammonium acetate 20 mM buffer (A), ACN (B), and MeOH (C): 0 min 5:95:0 A/B/C, 3 min 5:95:0 A/B/C, 4 min 5:0:95 A/B/ C, 5 min 5:95:0 A/B/C, and 9 min 5:95:0 A/B/C. ESI-MS/MS equipment and parameters Detection was performed with an ABSciex API 2000 tandem mass spectrometer via an ESI interface operating in negative ionization mode. The mass acquisition was performed using selected reaction monitoring (SRM); therefore, two transitions were monitored for all analytes and optimized in terms of declustering potential (DP), collision energy (CE), and cell exit potential (CXP). Transitions, DP, CE, and CXP are listed in Table 1 for acidic compounds, and are available elsewhere for neutral pesticides (Lissalde et al. 2011). Design of experiment A composite design of experiment was used to optimize the SPE step. Three parameters (extraction pH, proportion of MeOH in F0, volume of F0) with five different levels were considered. Data analysis and surface responses were performed with NemrodwTM software (LPRAI, France). Characterization of river water The river waters used as complex matrix for optimization (SPE parameters, matrix effects, validation) and for field application had the following chemical characteristics: pH between 6.5 and 8; conductivity between 150 and 700 μS cm−1; cumulated concentrations of Ca++ and Mg++ around 10 mg L−1; nitrate between 1 and 10 mg L−1; dissolved organic carbon was in the range of 5–10 mg L−1; and suspended matter was measured in the range of 3–10 mg L−1. The Ardière river is located in the Haut Beaujolais region (Eastern France) and has the following characteristics: pH of 6.8–7.0, conductivity between 130 and 180 μS cm−1; cumulated concentrations of Ca++ and Mg++ around 10–20 mg L−1; nitrate around 7 mg L−1; dissolved organic carbon was around
3 mg L−1; and suspended matter was measured in the range of 10–20 mg L−1. The Trec river is located in the Lot-et-Garonne district (South West France) and has the following characteristics: pH of 7.5–8.5, conductivity around 700 μS cm−1; cumulated concentrations of Ca++ and Mg++ around 15 mg L−1; nitrate is between 10 and 100 mg L−1; dissolved organic carbon was around 7 mg L−1; and suspended matter was around 10– 50 mg L−1.
Results and discussion Separation of acidic herbicides by HILIC As first detailed by Alpert et al. (1990), a high proportion of organic modifier in the mobile phase (i.e., 60 to 98 %) is required in HILIC, which is essentially a partition mechanism between the organic-rich mobile phase and the immobilized aqueous layer at the surface of the stationary phase (Chirita et al. 2011). Although HILIC interactions remain less understood than RPLC, more and more work is being done to determine the mechanisms involved (e.g., dipole-dipole, hydrogen bonding, and electrostatic interactions, partition and/or adsorption mechanisms) and to better understand their relative importance (Chirita et al. 2010; Guo and Gaiki 2005; Nguyen and Schug 2008). We chose a zwiterrionic silica-based stationary phase column (i.e., Macherey Nagel Nucleodur HILIC), which should improve the retention of ionic compounds (Chirita et al. 2010; Vikingsson et al. 2008) compared to a bare silica column. The proportion of organic modifier (ranging from 80 to 95 %), ammonium acetate concentration buffer (from 20 to 200 mM), mobile phase flow rate (from 50 to 600 μL min−1) and column oven temperature (from 20 to 50 °C) were optimized. In accordance with recent studies (Chirita et al. 2010; Guo and Gaiki 2005; Heaton et al. 2011; Li et al. 2010), we reported higher retention factors (k) with the maximum amount of ACN (Fig. 1). Depending on the analyte, the addition of MeOH to the initial isocratic elution increased sensitivity by a factor of two to 10. This gain in peak height could be explained by the protic character of MeOH and its lower surface tension (Kebarle and Tang 1993) which could facilitate ionization at the ESI interface and therefore enhance the sensitivity of detection. In agreement with the literature (Liu et al. 2008; Li et al. 2010; Chirita et al. 2010), small changes in elution order and a k decrease of about 20 % for all compounds were also observed with the addition of MeOH. No significant gain in k was observed for any compound by increasing the ammonium acetate concentration buffer (20 to 200 mM) or oven temperature (20 to 50 °C). These parameters were thus maintained at 20 mM and 25 °C, respectively, and acceptable column efficiency was found with a 200 μL min−1
Environ Sci Pollut Res Table 1 Acidic herbicides characteristics, SRM transitions and ESI-MS/MS conditions. Acquisition is performed in negation ionisation mode Herbicide
Formula
log Kow pKa
Quantitative DP (V)a CE (V)b CXP (V)c Qualitative DP (V)a CE (V)b CXP (V)c transition transition
2,4D Acetochlor ESA Acetochlor OA Bentazone
C8H6Cl2O3 C14H20NO5S C14H19NO4 C10H12N2O3S
−0.83 – – −0.46
2.87 – – 3.28
219>161 314>121 264>146 239>132
−20 −30 −30 −30
−20 −28 −12 −35
−18 −6 −6 −12
219>125 314>80 264>129 239>175
−20 −30 −30 −30
−34 −56 −32 −25
−18 −4 −6 −15
Chlorsulfuron Dicamba Dichlorprop Diclofop Fenoprop Iodosulfuron Ioxynil MCPA Mecoprop Mesotrione Metolachlor ESA Metolachlor OA Metsulfuron-Me Nicosulfuron Sulcotrione Bentazon d6 Dicamba d3 MCPA d3
C12H12ClN5O4S C8H6Cl2O3 C9H8Cl2O3 C15H12Cl2O4 C9H7Cl3O3 C13H12IN5O6S C7H3I2NO C9H9ClO3 C10H11ClO3 C14H13NO7S C15H22NO5S C15H21NO4 C14H15N5O6S C15H18N6O6S C14H13ClO5S – – –
−0.99 −1.88 2.29 1.61 3.80 1.59 2.20 −0.81 −0.19 0.11 – – −1.70 0.61 −1.70 – – –
3.40 1.87 3.00 3.43 2.84 3.22 3.10 3.73 3.11 3.12 – – 3.75 4.78 3.13 – – –
356>139 219>175 233>161 325>253 269>197 506>139 370>127 199>141 213>141 338>291 328>80 278>206 380>139 409>154 327>291 245>132 222>178 202>144
−20 −20 −20 −20 −20 −20 −20 −20 −20 −10 −35 −20 −20 −20 −20 −40 −20 −20
−46 −6 −14 −20 −18 −50 −60 −20 −20 −14 −65 −12 −20 −30 −20 −36 −6 −20
−12 −16 −16 −24 −18 −12 −10 −15 −8 −24 −6 −18 −12 −14 −26 −6 −8 −15
356>107 219>145 233>125 325>71 269>161 506>308 370>215 199>155 213>71 338>212 328>121 278>174 380>214 409>227 327>212 – – –
−20 −20 −20 −20 −20 −20 −20 −20 −20 −10 −35 −20 −20 −20 −20 – – –
−74 −6 −36 −25 −40 −40 −30 −10 −18 −42 −30 −20 −10 −18 −40 – – –
−12 −16 −16 −8 −18 −30 −20 −15 −15 −20 −6 −16 −20 −20 −20 – – –
Metsulfuron d3
–
–
–
383>142
−40
−50
−4.5
–
–
–
–
a
Declustering potential
b
Collision energy
c
Cell exit potential
mobile phase flow rate. These chromatographic conditions provided an acceptable compromise between separation efficiency (21 μm
