Influence of complexation phenomena with multivalent cations on the analysis of glyphosate and aminomethyl phosphonic acid in water

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Available online at www.sciencedirect.com

Journal of Chromatography A, 1175 (2007) 197–206

Influence of complexation phenomena with multivalent cations on the analysis of glyphosate and aminomethyl phosphonic acid in water

Ingrid Freuze

^a^,∗

, Alain Jadas-Hecart

^a

, Alain Royer

^b

, Pierre-Yves Communal

^a^,^b

aEquipe Paysage et Biodiversit´e, UFR Science, 2 Bd Lavoisier, F-49000 Angers, France

bGIRPA, Angers Technopole, 8 rue Becquerel, F-49070 Beaucouze, France Received 14 June 2007; received in revised form 2 October 2007; accepted 5 October 2007

Available online 4 November 2007

Abstract

Experimental and theoretical influence of multivalent cations on the analysis of glyphosate and aminomethyl phosphonic acid (AMPA) was studied in pure water and in one surface water. The procedure chosen, based on derivatization with FMOC-Cl, HPLC separation, and fluorescence detection, appears highly affected at cations concentrations current in natural waters. A detailed speciation study performed with the VMINTEQ software strongly suggests that the complexes formed between analytes and cations do not dissociate during the reaction and do not react with the derivatization agent, so that only the free forms are derivatized. These results point out the necessity of a pre-treatment to prevent these interferences, even in low salinity waters. The different ways conceivable are discussed in terms of kinetic and thermodynamic considerations.

Keywords: Glyphosate; AMPA; FMOC derivatization; Metal interferences; Complexation; VMINTEQ modelling

1. Introduction

Glyphosate [(N-phosphonomethyl)glycine] is a broad- spectrum, non-selective herbicide, used for the post-emergence control of annual and perennial weeds in a variety of agricultural and non-crop applications. Its degradation in the environment is mainly due to biodegradation[1,2]leading to aminomethyl phosphonic acid (AMPA), before its ultimate mineralization[3].

Despite this degradation, glyphosate, and as a result AMPA, is regularly detected in surface water and sometimes in groundwater[4], due to its widespread use.

Many procedures have been reported for the determination of these residues in water. A summary of most of them can be found elsewhere[5,6].

To date, chromatographic methods employing different clean-up techniques and a variety of separation and detection modes are the most extensively used in routine laboratories for the analysis of these residues in water.

Gas chromatographic (GC) determinations need a previous derivatization step to convert glyphosate to a sufficiently volatile

∗Corresponding author. Tel.: +33 241 48 75 70; fax: +33 241 48 71 40.

E-mail address:ingrid.freuze@univ-angers.fr(I. Freuze).

and thermally stable derivative[7–10]. The preparation of the derivatives requires however a time-consuming reaction under anhydrous conditions, then liquid chromatographic (LC) procedures are generally preferred. A derivatization step is still often also required, due to the lack of chromophore or fluo- rophore that prevents direct detection with conventional systems such as UV–vis or fluorescence detectors. In most cases pre- or post-column derivatization with fluorescence detection are involved. The reagents used for this purpose are essentially 9- fluorenylmethylchloroformate (FMOC-Cl) for the pre-column mode, ando-phthalaldehyde (OPA) for the post-column mode.

Post-column derivatization with o-phthalaldehyde-mercap- toethanol (OPA-MERC) requires prior oxidation of glyphosate to glycine by hypochlorite solution and was initially proposed by Moye and St John[11]. Since then, numerous method adjust- ments in terms of clean-up and concentration procedures have been proposed in order to improve the detection limits (which were not sufficient for drinking water control) and to prevent the degradation of the cation-exchange column observed in the case of samples with high amount of salts[2,12–18]. This method is EPA approved but requires special equipment and needs a labo- rious extraction and clean-up procedure such as ion-exchange column chromatography, so as to reach the restrictive regulations for water in the European Union.

doi:10.1016/j.chroma.2007.10.092

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In contrast, pre-column derivatization with FMOC-Cl does not require special equipment and allows the determination of glyphosate and AMPA in natural waters at 0.1␮g/L without preconcentration[19–22]. Associated with electrospray tandem mass spectrometry detection and the use of solid phase extraction (SPE) coupled on-line with liquid chromatography (SPE-LC- ESI-MS/MS), limits of quantification as low as 50 ng/L may be obtained for both compounds, with moreover a high level of selectivity[3,23].

To date, almost all these methods are used in Europe, depending upon the apparatus the laboratories are familiar with and depending upon they have the facilities required for post- column labelling. This led Monsanto Europe to carry out a round robin study to assess the capabilities of analytical laboratories to determine residues of glyphosate and AMPA in surface and groundwaters of natural origin [24]. Thirty laboratories, essentially European except one in the US, were asked to use their routine analytical method. Among the 27 laboratories who answered positively, 16 used HPLC-MS/MS or HPLC-MS after derivatization with FMOC-Cl without any clean-up, 5 used the OPA post-column derivatization with fluorescence detection, 3 of them after a Chelex 100—anion exchange clean-up/preconcentration step, and the others used GC/MS or GC/MS/MS after derivatization with a mixture of TFAA/TFE or HFB/TFA generally after a Chelex 100 extraction and anion exchange clean-up.

The results obtained showed that all these methods were suitable for the analysis of glyphosate and AMPA in surface water.

However, only those that used an ion exchange clean-up gave adequate results for groundwater. All others, including on-line SPE-LC with MS/MS detection, using labelled glyphosate as internal standard, which fulfils the requirements of excellent sensitivity and selectivity[3], did not lead to satisfactory results for groundwater. The use of spiked levels as proposed by Le Fur et al.[19]to prevent false negative interferences was neither satisfying.

In view of these results, Iba˜nez et al. [23]recently argued that one explanation was to assume the slow formation of a complex between glyphosate and some components of the matrix. This complex would be broken during the acidification used in the preconcentration/clean-up step, making glyphosate available for analysis. Moreover, its slow formation would explain why the introduction of an internal standard did not correct the matrix effect, the analyte being present in the sample longer than the internal standard. They thus re-evaluated their (SPE-LC-ESI-MS/MS) method by introducing an acidification at pH 1 followed by a posterior neutralization of the samples, previously to the FMOC-Cl derivatization step.

Using this approach, higher concentrations (between 2 and 14 times) were obtained in 80% of water samples previously analysed without any acidification. The reliability of acidification of samples as a general approach to be applied to all water samples remains however questionable due to a lack of in-depth understanding of the nature of the bias [23].

One of the reasons is that the nature of the possible interferences, and the extent at which they may affect the analysis,

were never studied in a systematic way. In agreement with the assumption of Iba˜nez and collaborators, these interferences are most likely due do the formation of complexes. At sight of the structure of the analytes (presence of amino, carboxylic and phosphonic functions), multivalent cations were the most plausible candidates. This is also in conformity with general literature where cations are usually incriminated. In order to precise this assumption, the objective of this study was to inves- tigate and quantify the influence of some alkaline (sodium, potassium), alkalino-terrous (calcium, magnesium) and metallic cations (iron, copper and zinc), in order to provide fundamental information for future studies about the development of an efficient pre-treatment step. We focused on the pre-column FMOC-Cl derivatization of glyphosate and AMPA because of its simplicity. Moreover, this method is currently in use in Europe and is even under investigation for an ISO normalization, despite the bad results obtained during the Round Robin test. Experi- ments were first performed in pure water and then extended to a real water. Results were analysed in terms of a detailed speciation of the different species present in water, thanks to a modelisation software.

2. Experimental

2.1. Reagents and glassware

Glyphosate of 99.9% purity and AMPA of 99.1% purity were obtained from Monsanto, F-69673 Bron Cedex. FMOC-Cl was purchased from Sigma–Aldrich, D-89555 Steinheim.

Potassium dihydrogen phosphate, sodium tetraborate, methanol for HPLC, sodium chloride, magnesium sulphate and iron sulphate for analysis were purchased from Merck, F-94736 Nogent sur Marne.

Acetonitrile for HPLC was bought from SDS, F-27106 Val de Reuil Cedex.

Potassium chloride, calcium chloride, copper sulphate and zinc sulphate were obtained from Sigma–Aldrich, F-38297 Saint Quentin Fallavier.

2.2. Analytical method 2.2.1. Instrumentation

The analytical system employed was composed of a Varian model 410 automatic sampler, a Varian Prostar 320 pump used in the isocratic mode, and a spectrofluorimetric detector model Jasco FP 1520. The injection volume was 100␮L and the separation was carried out on a Microsorb MV 100-5 (250 mm×4 mm) column from Varian. The mobile phase was constituted of a mixture of phosphate buffer 0.05 M (pH 5.4) and acetonitrile (70/30 v/v), with a flow rate of 1 mL/min. The fluorescent derivatives were analysed with wavelengths of excitation and emission being respectively 260 and 310 nm.

2.2.2. Calibration solutions

Glyphosate and AMPA stock calibration solutions of 1 mg/mL were separately prepared by dissolving a known amount of each analyte in water. 250␮L of each stock solu-

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Table 1

Characteristics of the surface water

Characteristic Value Unit

Calcium 128 mg/L

Zinc 0.529

Copper 0.02

Iron <0.1

Sulphate 25

Total organic carbon 5.4

TA 0 ^◦F

TAC 12.2

Glyphosate <0.1 ␮g/L

AMPA <0.1

tion was taken with precision and diluted into a volumetric flask (1 L) using ultrapure water to obtain a stock calibration solution containing both analytes at 0.25␮g/mL. Working calibration solutions from 0.1 to 5␮g/L were prepared by fortification of ultrapure water with the adequate volume of the stock solution (0.25␮g/mL), after dilution if necessary.

2.2.3. Procedure

This procedure has been adapted from the work of San- cho et al.[25]. 15 mL of sample were introduced into a 50 mL polypropylene conical tube. pH was adjusted to 9.2 by introduction of 2.5 mL of borate buffer 0.05 M. The mixture was twice extracted with 5 mL of diethyl ether (mixing 5 min, decantation 15 min) to remove most of the organic matter in the case of natural water. 7.5 mL of the aqueous phase were then derivatized (1 h, room temperature) by adjunction of 1.25 mL of a FMOC-Cl solution (1 g/L in acetonitrile) and 1.25 mL of acetonitrile. Two other washes with 5 mL of diethyl ether were realized (mixing 15 min, decantation 30 min) in order to remove the excess of derivatization reagent and the by-products formed. The aqueous phase was finally injected for analysis.

2.3. Sample preparation 2.3.1. Influence of cations

Ultrapure water samples spiked with approximately 5␮g/L of glyphosate and AMPA were fortified with each cation, independently, to produce the desired fortification levels, by the adjunction of either the solid or an intermediate solution in ultrapure water, according to the expected fortification level. The samples were then stored overnight in darkness at room temperature before analysis. It was assumed that the contact time was enough to reach the thermodynamic equilibrium.

2.3.2. Proportioned additions

These experiments were realized using a surface water (pre- senting the characteristics resumed inTable 1) as the matrix.

This water was spiked with increasing levels of glyphosate and AMPA, from 0.5 to 150␮g/L. Samples were treated and analysed.

2.4. Speciation studies

Modelisations of the glyphosate and AMPA speciation were realized with the VMINTEQ model software [26] developed by Jon Peter Gustaffsson (2006). This modified form of the MINTEQ model initially released by USEPA in 1991 is ded- icated to the calculation of equilibrium aqueous speciation, precipitation and dissolution of minerals, complexation, adsorption, solid phase saturation states, etc.

It is provided with a comprehensive database of equilibrium formation constants for numerous inorganic species but concerning organic components, data have to be incorporated. That has been done for AMPA and glyphosate.

2.4.1. Source of stability constants

Precise values of the ligand deprotonation constants are required for complexation investigations. Those reported in the literature (25^◦C) present some discrepancies: the ranges of the reported values for pK1, pK2and pK3are respectively 2.10–2.56, 5.17–5.86 and 9.75–10.86[27–33].

The best confidence interval was reported by Paschevskaya et al.[33]. Priority was given to the values used to determine the complex formation constants in order to minimize error prop- agation, after correction at infinite dilution (I= 0 M) with the Davies equation.

2.4.2. Formation constants

Most of them were selected from the IUPAC Technical Report [34]and are listed inTable 2.

For speciation in presence of copper, no significant difference was observed when calculation was performed with the data obtained for either Paschevskaya et al.[33]or the IUPAC report [34] when the associated deprotonation constants were taken into account. The data from the IUPAC report were then selected for the calculations on the real water studied, in order to use the same set of pK.

For copper and zinc, some data from the original publica- tion [35] (see Table 2) were also added, even if taking them into account lead to very similar results, due to the very low corresponding formation constants.

For iron, no data are available in case of AMPA.

The formation of mixed complexes was not taken into account due to a lack of data.

2.4.3. Temperature correction

Experiments were performed at the laboratory temperature (22^◦C) but simulations were performed at 25^◦C because the thermodynamic data needed to extrapolate from standard to experimental conditions are currently unavailable. The effect of this temperature variation on the logKvalues was thought to be negligible.

2.4.4. Precipitation/adsorption

Because of the high pH (9.2) required for the derivatization some precipitates may be formed, indicated by VMINTEQ by a

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Table 2

Stability constants for glyphosate and AMPA (L: totally deprotonated species, M: metal, charges omitted in reaction for simplicity)

Cation Species Reaction logK Complexes

H⁺ AMPA L + H 10.04[34] HAMPA⁻

HL + H 5.41[34] H2AMPA

Ca²⁺ Glyphosate M + L 3.3[34] Ca(GLY)⁻

M + H + L 11.5[34] Ca(HGLY) M + 2L 5.87[34] Ca(GLY)24−

AMPA M + L 1.67[34] Ca(AMPA)

M + HL 1.06[34] Ca(HAMPA)⁺

Fe²⁺ Glyphosate M + L 6.9[34] Fe(GLY)⁻

M + H + L 12.8[34] Fe(HGLY) M + 2L 11.2[34] Fe(GLY)24−

Cu²⁺ Glyphosate M + L 12.71[33] Cu(GLY)⁻ 11.93[34]

M + 2L 18.15[33] Cu(GLY)24−

16.02[34]

M + H + L 17.54[33] Cu(HGLY) 15.85[34]

M + 2H + L 21.02[33] Cu(H2GLY)⁺ M + H + 2L 27.32[33] Cu(HGLY)(GLY)³⁻ M + 2H + 2L 34.06[33] Cu(HMPG)22−

M + 3H + 2L 37.3[33] Cu(H2GLY)HPM⁻ M + L + OH 2.06[35] Cu(GLY)(OH)²⁻

AMPA M + L 8.1[34] Cu(AMPA)

M + HL 2.6[34] Cu(HAMPA)⁺

M + 2L 14.7[34] Cu(AMPA)22−

Zn²⁺ Glyphosate M + L 8.7[34] Zn(GLY)⁻

M + H + L 7.99[35] Zn(HGLY) M + 2L 11.7[34] Zn(GLY)24−

M + L + OH −0.99[35] Zn(GLY)(OH)²⁻

AMPA M + L 5[34] Zn(AMPA)

M + HL 1.7[34] Zn(HAMPA)⁺

I= 0.1 M.

solid saturation index (SI) defined as:

SI=log IAP−logKs

where IAP is the ion activity product calculated on the basis of the reaction stoichiometry andKs is the temperature-corrected solubility constant.

This solid saturation index was checked for each experiment. When solids were allowed to form, potential adsorption of glyphosate and/or AMPA on these precipitates was not taken into account.

2.4.5. Ionic strength

Ionic strength was calculated from the ionic concentrations and pH was allowed to be calculated from mass and charge balances.

3. Results

Sodium, potassium or magnesium did not interfere in the range of concentrations experimented (up to 500 mg/L), for neither glyphosate nor AMPA. On the other hand, calcium, zinc, copper and iron appeared to bias the results obtained for glyphosate (Fig. 1). Interferences in the analysis of AMPA were

Fig. 1. Experimental influence of different metals on the analysis of glyphosate and AMPA with the FMOC method.

weaker for these levels of cations. Especially, with the incorporated concentrations of copper or zinc, none effect was observed.

As shown inFig. 1, calcium has an incidence on the results obtained with the FMOC method from concentrations as low as 100 mg/L, a concentration that can be easily encountered in natural waters. In presence of 300 mg/L of calcium, the recovery rates were about 80% for AMPA but only 50% for glyphosate.

This result is in agreement with the poor recoveries obtained on mineral waters by Le fur et al.[19].

Copper, iron and zinc interfere all at a far lower concentration than calcium. For the two first, the incidence was noteworthy as soon as 0.1 mg/L, concentration for which recovery rates lower than 25% were observed, falling to 0 for 1 mg/L of cation.

Higher but still low values were obtained in presence of zinc. We have then compared these results to the theoretical amounts of glyphosate under free form determined with the modelisation.

4. Speciation study

Simulations were performed taking the measured concentrations of glyphosate and AMPA in blank samples (i.e. without any cation) as initial concentrations.

4.1. Case of calcium

The case of calcium is particular in that a precipitate was clearly visualized during the experiments. Owing to the concentrations and pH involved, the only precipitate that could be formed was calcite (CaCO3). Because no carbonate was intentionally introduced into the reaction mixture, this led us to consider a possible contamination due to a gas exchange with the atmosphere. This reaction being rather low, it was further hypothesized that this gas exchange was insignificant during the course of the experiments (several hours) but may be higher during the storage of the concentrated borate buffer (several weeks).

The total CO2concentration (CO2T) which may be present in this buffer under the assumption of an equilibrium with the atmosphere was thus calculated at the storage temperature (20^◦C) and the resulting concentration in the reaction mixture (1.8×10⁻³M) was then taken into account in all calculations.

The results obtained in terms of calculated free forms under this hypothesis are also reported inFig. 2as solid lines together

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Fig. 2. Comparison between free and measured concentrations of glyphosate () and AMPA () in pure water fortified with approximately 5␮g/L, as related to the calcium concentrations incorporated and hypothesized CO2Tconcentra- tion.

with the experimental results (points). Theoretical concentrations of free forms under different total CO2concentrations are also figured as dashed lines to illustrate the effect of the carbonate content of the reaction mixture on the equilibrium.

From this figure, it appears that under the hypothesis for- mulated, the measured values with the FMOC method and free concentration forms are in very close agreement in the case of glyphosate, suggesting that only its free forms are derivatized.

Because the amino group in the calcium complexes is not coordinated[36], the interferences observed suggest that the basicity of the glycyl residue amino nitrogen is highly modified after complexation precluding, the initial electrophylic attack by the carbonyl group of the FMOC reagent.

In the case of AMPA, the agreement between the predictive values and the measured values is not as close as in the case of glyphosate but is still acceptable. The lowest concentration measured at the highest calcium concentration, compared to the total concentration of free forms might be due to either an imprecision of the formation constants or a partial adsorption of AMPA on the calcite precipitate. This last hypothesis is supported by the fact that the predominant calcium complex in these experimental conditions is Ca(AMPA)⁺ which is positively charged and thus more amenable to adsorption on calcite than the negatively charged complexes of glyphosate.

4.2. Case of copper

For copper, negative values for the saturation index of tenorite (CuO) were obtained, indicating that this precipitate may be formed during the course of the experiment, even if it was not observed. However this can be due to its very low concentration. Calculations were thus performed under the two hypotheses (whether solid forms or not). The results obtained in these two cases are also reported inFig. 3.

For glyphosate, values measured by the FMOC method are in close agreement with the total concentration of free forms under the two hypotheses. This again suggests that the copper complexes, like the calcium ones, are not derivatized during the course of the experiments. This hypothesis is supported by the fact that the ion is known to be bonded with N-phosphonomethylglycine through the three donor groups (amine, carboxylate, phosphonate)[33,37]. Electrophylic attack

Fig. 3. Comparison between free and measured concentrations of glyphosate () and AMPA () in pure water fortified with approximately 5␮g/L, as related to the copper concentrations incorporated and hypothesized CO2Tconcentration.

of FMOC on the amino group is thus probably consistently inferred. For AMPA the best fit between predicted and observed values was observed under the assumption that a precipitate of tenorite (CuO) is formed. This might suggest that a precipitate has been formed even if it was not experimentally observed.

Another and probably more valuable explanation is that the complex forms of AMPA are derivatized because the amino group may not be coordinated.

4.3. Case of iron

For iron(II), negative values for the saturation index of Fe(OH)2and FeCO3 were obtained, indicating that these precipitates may be formed during the course of the experiment, the last one being thermodynamically favored. This time again, calculations were performed under the two hypotheses. As shown inFig. 4, the total concentrations of free forms of glyphosate perfectly match the measured concentrations with the FMOC method when precipitates are not allowed or when a precipitate of Fe(OH)2is allowed. On the other hand, if a precipitate of FeCO3 is allowed, predictive values are much higher than those measured (curves not shown). In this case it is more difficult to conclude because the absence of precipitate was not checked except by the naked eye. The close similarity between measured and predictive values under the assumptions leading to Fig. 4, however, strongly suggests again that only the free forms are derivatized. In the case of AMPA, the decrease in the measured values together with the increase of iron(II) concentration suggest that some complex(es) is(are) formed.

However no simulation was performed because the scheme of pathway for the reaction between AMPA and iron(II) is unknown (Fig. 4).

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Fig. 4. Comparison between free and measured concentrations of glyphosate () and AMPA () in pure water fortified with approximately 5␮g/L, as related to the iron(II) concentrations incorporated and hypothesized CO2Tconcentra- tion.

4.4. Case of zinc

The case of zinc is particular in that none of the hypotheses tested (i.e. with or without precipitation of the solids susceptible to be formed) led to a good agreement between total concentrations of free forms and measured concentrations, particularly in the case of glyphosate. This result, together with the fact that

Fig. 5. Comparison between free and measured concentrations of glyphosate () and AMPA () in pure water fortified with approximately 5␮g/L, as related to the zinc(II) concentrations incorporated and hypothesized CO2Tconcentra- tion.

Fig. 6. Same asFig. 5but with data from Kobylecka et al.[38].

formation constants of zinc complexes are classified as provisional in the IUPAC report[34], due to high uncertainty in the reported data, led us to suspect a possible error in these constants. This hypothesis was partly upheld by the fact that using the data reported by Kobylecka et al.[38], a better agreement was obtained (Fig. 6).

The similarity between the shape of the observed values and those predicted while assuming that no solid is formed suggests that this hypothesis is valid and that the discrepancy between the two sets of data most probably results from an overestimation of the formation constants. Adjusting the value of the one of Zn(GLY)⁻to obtain the best fit curve led to a value of logK= 7 at a 0.1 M ionic strength instead of the reported values of 8.7 [34]and 7.61[38].

4.5. Proportioned additions

According to these results, obtained in pure water, the reliability of the FMOC method for the analysis of a real water appeared questionable, that is why we realized proportioned additions on a surface water whose composition is given inTable 1. This water, that initially contained neither glyphosate nor AMPA, was fortified with increasing levels of these compounds. The concentrations detected with the FMOC method were highly under-estimated (Fig. 7). The recovery rates were on average 70% for AMPA and 30% only for glyphosate. The levels of fortification ranged from 0.5 to 150␮g/L. All the results are not represented on the graph for purposes of clarity but the ten- dency was always the same. Corresponding simulations were performed for the different concentrations of glyphosate and AMPA experimented and for calculated concentrations taking into account the concentrations of the major ions of the water (Table 1) together with the dilution induced by the introduction of the borate buffer. The total carbonate content was recalcu-

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Fig. 7. Detected and predicted (free forms) concentrations of glyphosate () and AMPA () in surface water fortified with increasing levels of the two compounds, as related to the introduced concentrations.

lated on the basis of a 1.8×10⁻³M concentration in the borate buffer.

As no precipitate was observed during the experiments in pure water with any of the ions studied, except in the case of calcium, and because good fits were observed under this assumption, only calcite was allowed to precipitate. The total concentrations of free forms were calculated and compared with the detected concentrations observed.

In an initial step, the possible interactions between the DOC and metals were not taken into account. This procedure led to predicted values far below the detected concentrations, especially for glyphosate. However, setting the copper concentration to zero led the predictive values for glyphosate to perfectly match the measured concentrations when the adjusted formation constant (logK= 7) for the formation of Zn(GLY)⁻was used for calculation.

From this observation, it was hypothesized that copper could be complexed on the DOC.

A second set of simulations was thus performed, considering possible cation binding to DOC. This was performed using the NICA-Donnan model available in the VMINTEQ software and briefly described in the VMINTEQ help file. A more thorough description may be found in Kinniburgh et al.[39]. This model was preferred among the three models included in VMINTEQ because it has the largest database for calculating cation binding to humic substances. Default parameters were used for the simulation (i.e. 70% of the DOC is made of fulvic acid, has a C content of 50%) and parameters for “generic” fulvic and humic acids (Milne et al., 2003, quoted by Gustafsson[26]) are applicable.

The results of these simulations are reported in Fig. 7 as plain traits together with the experimental results (points) and the results obtained using the provisional logKvalue reported in Popov et al.[34]for the Zn(GLY)⁻formation (dashed line).

As might be seen, the glyphosate concentrations measured perfectly match the total concentrations of free forms when using the adjusted value for the non-protonated zinc complex. Clearly zinc is the main metal which interferes in the analysis. Cal- cium also interferes but to a lesser extent. Copper, on the other hand, despite its strong complexation ability with glyphosate,

does not interfere because it is preferably bound to humic substances.

The measured concentrations of AMPA are lower than the total concentration of free forms. This trend was observed in experiments performed with calcium (seeFig. 2). One possible explanation is that the dissociation constant of Ca(HAMPA)⁺ which is the dominant form of the calcium complexes in our experimental conditions might be a little under-estimated. How- ever, using an adjusted value for this constant – so that total concentration of free forms of AMPA perfectly matches the measured concentrations, in experiments performed in pure water fortified with calcium – does not lead to a real improvement of the fit. It is thus most probable that the observed discrepancies are due to either some metal interactions which were not taken into account or some adsorption of the free forms on the calcite precipitate which appears at the highest calcium concentration.

These hypotheses were not further investigated because it was beyond the scope of this paper.

4.6. Other cations

The interferences that could be due to some other cations were checked by computing the total concentration of free forms for hypothesized concentrations of the cations for which values of the stability constants were available (i.e. Co²⁺, Mn²⁺, Cd²⁺, Al³⁺, Ni²⁺, La³⁺).

The results of these simulations in pure water at pH 9.2 are shown in Fig. 8, for cation concentrations ranging from 0 to 15␮M.

As might be seen from this figure, nickel, cadmium and to a lesser extent manganese might be potential interferents. These interferences are particularly significant in the case of nickel for which concentration as low as 1␮M, i.e. 58.71␮g/L leads to a free fraction of glyphosate lower than 10%.

4.7. Ability of the existing clean-up/extraction treatment steps to prevent complexation

In view of these results, the FMOC method appears to be powerful to assess the concentrations of free forms of glyphosate and AMPA, and this may be of value in toxicological studies.

However, as the aim of the analysis is before all to obtain the total

Fig. 8. Predicted (free forms) rates of glyphosate in pure water as related to the concentrations of different cations.

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concentrations of these analytes, a reliable clean-up/extraction treatment step seems absolutely necessary.

Some procedures have already been proposed in the literature including acidification [23], extraction on ion exchange resins either to extract glyphosate and AMPA or to eliminate metal interferences. In all cases good results were obtained in terms of recovery rate for various studied waters. The problem is that the ionic composition of waters, especially in cations, was never specified, making it difficult to extrapolate the results to the case of other waters. However, the results obtained in this study allow to grasp the capacities of these methods to eliminate interferences.

The acidification at pH 1, recently proposed by Iba˜nez et al.[23], seems at first sight a good solution because it facilitates the dissociation of complexes in favor of free forms. Simulations performed with the VMINTEQ software showed that, among the cations investigated, on a thermodynamical basis, only copper is able to form some complexes, all the other complexes being dissociated at this low pH. High concentrations are furthermore necessary to generate significant reduction of the free forms for glyphosate (80% for a concentration of copper of 2 mg/L to 50%

for a concentration of 10 mg/L). By the fact, this method appears thermodynamically able to prevent most of the interferences.

However the kinetic aspects should also be considered. It is assumed in this method (1) that acidification during 1 min is enough to dissociate all the complexes and (2) that the required neutralization to pH 9.2 before the FMOC derivatization does not shift the equilibrium towards the formation of complexes.

As mentioned by Iba˜nez et al. [23], this will require further investigations.

Other researchers have chosen anion-exchange resins for extraction of AMPA and glyphosate before derivatization [2,12,40–42]. In this case, the only way to have a significant negative fraction for both compounds, which is a prerequisite to extract them, is to adjust the pH at a value exceeding 7.

Unfortunately, calcite precipitation may appear in this pH range and greatly affect the resin retention capacity, especially in the case of hydroxide regenerated resin, for which calcite precipitation is favored due to the release of hydroxide ions in the percolated sample. Moreover, in this pH range, the complexes of glyphosate and AMPA are negatively charged and so most probably extracted together with free forms. In this context, it is doubtful that the interferences due to cations will be eliminated.

It is thus surprising that high recoveries were obtained with spiked natural water samples. In fact, a thorough examination of the above mentioned papers shows that high recoveries were only obtained when elution was performed with citrate buffer [2,12,42]. Corbera et al.[40]who used HCl or NaCl solution as eluent, obtained for their part poor recoveries for high salinity waters, despite high recoveries in low salinity waters. Taking into account that citrate is a cation complexing agent, this suggests that the differences observed might be due to the citrate decomplexation of glyphosate and AMPA prior to the derivatization step, rather than to a better eluting power of citrate as suggested by these authors. It should be noted however that citrate is unable to disrupt the strong interactions between copper and glyphosate or AMPA, which means that false results might be obtained in

presence of this cation, especially in cases of waters with low organic content. So, even if this kind of extraction was reported to process successfully for some waters, thermodynamic and kinetic considerations strongly suggest that this will not be the case for all waters.

Clean-up of water based initially on iron loaded Chelex 100 resin (ligand-exchange) and then AG1-X8 (anion-exchange) resin columns was also reported to perform well[13]. Samples are adjusted to pH 2 before elution on the iron loaded Chelex column. For most waters, this pH is adequate in that it favors the free forms, authorizing their retention on the Chelex iron resin.

However, speciation calculations show that some iron complexes (Fe(GLY) and Fe(HGLY)⁺) are still present at this pH and will thus pass through the resin because they can be extracted neither by anion nor ligand exchange. Some copper complexes do not dissociate as well and might also lead to some interferences, depending upon the ability of the iron Chelex resin to displace copper from those complexes. Unfortunately no data exists in the literature to confirm or infirm these potential interferences, methods being always validated on waters for which the ionic composition is rarely specified in terms of metal concentrations.

Cation-exchange resin in the H⁺form might also be a choice for extraction of cations before derivatization. In this case, the exchange of cations with the proton fixed on the resin might lead to both an acidification of water and a shift in the equilibrium towards the free forms during the sample percolation.

Ideally, these free forms should thus percolate through the resin, the cations being retained, and derivatization of the percolate should give accurate results, assuming that interactions between free forms and the resin polymer are low. This development is however only suitable on a thermodynamical basis. On a kinetic basis, there is no evidence that dissociation will proceed during the percolation, the kinetics of ion-exchange being somewhat lower than the kinetics of other types of interactions[2]. How- ever, even in the worst case (none of the complexes dissociated during the percolation), the retention of cations on the resin induces a shift in favor of free forms in the eluate which is in favor of good derivatization yields. In this case, one might be acutely aware that enough time should be left before derivatization for the shift in equilibrium to proceed. In numerous papers, no information is given on the rate at which the samples were percolated and on the time allowed for the equilibrium to proceed before derivatization. This surely may lead to inaccurate results. To conclude, even if this analysis of the ability of resins to be used as pre-treatment steps before derivatization depends of course on the water quality, it highlights that numerous questions remain unanswered concerning these pre-treatment steps.

Besides these frequently used pre-treatments, another way to prevent metal interferences is to use a complexing agent prior to the derivatization step. This kind of pre-treatment is currently in use in the OPA method where EDTA is added to shift the equilibrium towards the free forms of glyphosate and AMPA.

In the case of FMOC derivatization, this reagent was proved to lead to significant interferences in fluorimetric detection[19]. To our knowledge, no other reagent was experimented but this way should be considered, because it might be a solution of choice in terms of reliability, price and facility of use.

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5. Conclusion

The results obtained in this study clearly indicate that the FMOC method, yet one of the most employed in France and under investigations for an ISO normalization, may lead in some cases to an under estimation of the concentrations of glyphosate and AMPA, in the presence of multivalent cations. It is demonstrated here that only the free forms of AMPA and glyphosate are able to react with the FMOC reagent. With some metals, as copper, the complexes formed at pH 9.2 are extremely stable, implying that the reaction is nearly stoichiometric. For example, for 1␮g/L of glyphosate (6 nM) in presence of an equivalent molar concentration of free copper (0.4␮g/L Cu²⁺), the fraction under free form is only 10%. For information, the maximum levels authorized in drinking water in France are 0.2 mg/L for iron, 1 mg/L for copper and 5 mg/L for zinc. This means that this kind of interference could occur in some waters, particularly those with a low organic content (essentially groundwater). For waters with higher DOC, some of these interferences will be reduced due to a partial complexation of interfering cations on the organic matter. This was demonstrated in this work in the case of copper. This may be the reason why most of the problems described in the literature were observed for groundwaters and not in surface waters.

One should note that this kind of interferences is probably not specific to the FMOC method, as proved by the results obtained during the round robin study. All the methods without decomplexation step led to inaccurate results in groundwater, whatever the derivatization agent or the detection mode. The direct consequence, from an analytical point of view, is that AMPA and glyphosate have to be released before derivatization. Thermodynamically, an acidification at pH 1 should allow the decomplexation of most complexes. Yet, part of them, especially those with high formation constants, is susceptible to be reformed during the reversion to pH 9.2, required for the derivatization. To date, no kinetic data being available, this method cannot be extended with a sufficient confidence to all kind of waters. The results obtained with methods based on extraction on anionic resins, or based on a prior elimination of metals thanks to cationic resins, are also difficult to generalize. Some of the thermodynamical considerations presented here let us suppose that these techniques would probably not be fitted for use in all types of water. Moreover, numerous questions remain about the kinetic of the reactions, deciding for the quality of the results obtained. Eventually, there is a lack of data about the potential efficiency of the addition of a complexing agent such as EDTA.

Simulations realized thanks to the VMINTEQ software indicate that EDTA is thermodynamically able to dissociate complexes of glyphosate and AMPA at pH 9.2. The addition of this reagent might so allow the elimination of the potential ionic interferents in the case of a MS/MS detection. Care should however be taken about a possible kinetic limitation which can be a limiting fac- tor. In the case of a fluorimetric detection, this addition is in any case a solution due to the formation of fluorescent interfering compounds.

To conclude, numerous questions still remain about the choice of an adequate clean-up, which could be used with con-

fidence in all types of water. Researches should now be carried out in this way, on the basis of the fundamental phenomena explained in this work. This is actually under investigation in our laboratory.

Acknowledgement

This work was developed under financial support of Mon- santo, B-1150 Brussels.

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