Anal. Chem. 2007, 79, 3794-3801
Glyphosate and AMPA Analysis in Sewage Sludge by LC-ESI-MS/MS after FMOC Derivatization on Strong Anion-Exchange Resin as Solid Support Aline Ghanem, Philippe Bados, Lucien Kerhoas, Jacqueline Dubroca, and Jacques Einhorn*
INRA, Unite´ de Phytopharmacie et Me´ diateurs Chimiques, Route de Saint-Cyr, 78026 Versailles Cedex, France
An innovative analytical method has been developed for the determination of glyphosate and aminomethylphosphonic acid (AMPA), its major metabolite, in sewage sludge. This method involved an alkaline extraction from sludge and purification on a strong anion-exchange resin. While the analytes remained fixed by ionic interactions, an “on-solid support” derivatization by FMOC-Cl was carried out. This versatile approach allowed a 10 min reaction and simple elimination of the excess of reagent. The resulting derivatives remained retained by ionic and hydrophobic interactions with the resin until being eluted by a mixed NaCl water/acetonitrile, 70/30, v/v, solution. After an appropriate dilution and adjustment of the pH, the sample was concentrated on an Oasis HLB solidphase cartridge. For quality analysis of traces in complex matrixes, LC-ESI-MS/MS in the multiple reaction monitoring positive mode was used fulfilling the European Union requirements (Decision 2002/657/CE). To overcome the matrix effects, stable isotopically labeled standards were added to the sludge extracts as internal standards and were thus derivatized during the procedure in parallel to the analytes. Mean recovery values were 70% ( 7% for glyphosate and 63% ( 3% for AMPA. Limits of detection (20 and 30 ppb dw) and limits of quantification (35 and 50 ppb dw) for glyphosate and AMPA, respectively, were sufficient to monitor samples taken from Ilede-France wastewater treatment plants where contamination currently reached 0.1-3 ppm and 2-35 ppm dw for glyphosate and AMPA, respectively. Glyphosate [N-(phosphonomethyl)glycine] is among the most widely used herbicides. Introduced by Monsanto in the early 1970s, it is a nonselective, postemergence herbicide dedicated to weed and vegetation control. Its degradation in the environment mainly occurs under biological conditions yielding aminomethylphosphonic acid (AMPA) as the major metabolite. However, AMPA can originate from the degradation of phosphonic acids in detergents.1 Glyphosate and AMPA have been frequently detected in natural waters.2,3 The presence of both contaminants was also * To whom correspondence should be addressed. E-mail: einhorn@ versailles.inra.fr. Phone: +33-1-30-83-31-20. Fax: +33-1-30-83-31-19. (1) Skark, C.; Zullei-Seibert, N.; Schottler, U.; Schlett, C. Int. J. Environ. Anal. Chem. 1998, 70, 93-104. (2) Smith, N. J.; Martin, R. C.; St Croix, R. G. Bull. Environ. Contam. Toxicol. 1996, 57, 759-765.
3794 Analytical Chemistry, Vol. 79, No. 10, May 15, 2007
shown to occur at the microgram-per-liter level in wastewaters with a significant contribution issuing from their urban use.4 They are thus suspected to be transferred to sewage sludge during wastewater treatment. Glyphosate presents a lower acute toxicity than other herbicides but appears to induce endocrine effects.5 The ecological effects of glyphosate and its metabolite still need to be documented.6,7 To assess the environmental risk of sludge used as land amendment, an in-depth knowledge of its composition is required before application. The presence of persistent pollutants such as heavy metals, PAHs, and PCBs is generally assumed, and their levels are surveyed by European regulation (Directive 86/278/CEE). Nevertheless, no data are available for pesticides, particularly for glyphosate, which is likely explained by the difficulties of analysis of such contaminants in addition to the complexity of the matrix. Highly polar and amphoteric, glyphosate often represents an analytical challenge. It has four pKa values, 0.7, 2.2, 5.9, and 10.6,8 whereas three values, 0.9, 5.6, and 10.2, characterize AMPA.9 A variety of methods have been developed for glyphosate and AMPA analysis in waters, soils, and sediments. The majority of these are tedious due to the numerous steps employed for purification and the derivatization often required for either GC10,11 or HPLC. Direct HPLC analysis is possible by ion chromatography with electrochemical or mass spectrometry detection.12-14 However, (3) Schweinsberg, F.; Abke, W.; Rieth, K.; Rohmann, U.; Zullei-Seibert, N. Toxicol. Lett. 1999, 107, 201-205. (4) Kolpin, D. W.; Thurman, E. M.; Lee, E. A.; Meyer, M. T.; Furlong, E. T.; Glassmeyer, S. T. Sci. Total Environ. 2006, 354, 191-197. (5) Richard, S.; Moslemi, S.; Sipahutar, H.; Benachour, N.; Seralini, G. E. Environ. Health Perspect. 2005, 113, 716-720. (6) Cauble, K.; Wagner, R. S. Bull. Environ. Contam. Toxicol. 2005, 75, 429435. (7) Ghanem, A.; Dubroca, J.; Chaplain, V.; Mougin, C. Environ. Chem. Lett. 2006, 4, 63-67. (8) Sprankle, P.; Meggit, W. F.; Penner, D. Weed Sci. 1975, 23, 229-234. (9) Traas, T. P.; Smith, C. E. Environmental Risk Limits for Aminomethylphosphonic Acid (AMPA). Setting Integrated Environmental Quality Standards of Netherlands Public Health and Environment National Institute, Rijksinstituut voor Volksgezondheid en Miliev (RIVM): The Netherlands, 2003, 23, 10. (10) Deyrup, C. L.; Chang, S. M.; Weintraub, R. A.; Moye, H. A. J. Agric. Food Chem. 1985, 33, 944-947. (11) Bo ¨rjesson, E.; Tortensson, L. J. Chromatogr., A 2000, 886, 207-216. (12) Zhu, Y.; Zhang, F.; Tong, C.; Liu, W. J. Chromatogr., A 1999, 850, 297301. (13) Sato, K.; Jin, J.-Y.; Takeuchi, T.; Miwa, T.; Suenami, K.; Takekoshi, Y.; Kanno, S. J. Chromatogr., A 2001, 919, 313-320. (14) Bauer, K.-H.; Knepper, T. P.; Maes, A.; Schatz, V.; Voihsel, M. J. Chromatogr., A 1999, 837, 117-128. 10.1021/ac062195k CCC: $37.00
© 2007 American Chemical Society Published on Web 04/06/2007
sensitivity and selectivity are usually reached via either precolumn15-18 or postcolumn derivatization.19,20 Precolumn derivatization of glyphosate and AMPA with 9-fluorenylmethyl chloroformate (FMOC-Cl) is advantageous since it improves the chromatographic separation of the analytes and subsequently their detection either by fluorimetry15 or mass spectrometry.17,21 Solidphase extraction methods (SPE) using various kinds of phases are also reported to allow concentration and/or purification of glyphosate prior to its analysis in waters17,20,22 or soils.21 In this study, it is proposed to perform the FMOC derivatization of glyphosate and AMPA after fixation of the analytes on a solid support conveniently chosen in the analytical strategy. Subsequently, the derivatized analytes will be detected by using LCESI-MS/MS in the multiple reaction monitoring (MRM) mode. By this approach, it was intended to gain the following advantages: (i) minimize the number of steps for sample purification and concentration, (ii) minimize the volume of “solution” to be derivatized, (iii) reduce sample handling during elimination of excess reagent and, finally, (iv) preserve the possibility of automation. The on-solid support derivatization will be the key step to satisfy all these criteria. Because sludge samples are highly complex and “dirty”, the first purification step will have to be carried out on low-pressure or atmospheric resin columns (not cartridges) using diluted extracts. In the case of a conventional approach, recovery of the free analytes should be possible only through the use of large volumes of eluent. This condition would then oblige one to carry out the liquid-phase derivatization on an aliquot to complete the reaction in a reasonable time (maximum of overnight), thus compromising the sensitivity of the overall method. In our procedure, this inconvenience is circumvented, further guarantying an easy elimination of the reagent in excess. This is the first method that determines glyphosate and AMPA residues in sewage sludge. EXPERIMENTAL SECTION To prevent the high adsorption of glyphosate and AMPA, glass materials were silanized using 5% dimethyldichloride silane (DMDCS) in hexane. After 10 min of contact, they were rinsed twice with hexane then with methanol before being dried. Chemicals and Reagents. All chemicals were of analytical grade, and 0.45 µm filtered ultrapure water (resistivity of 18.2 MΩ/ cm) was used for solutions. Glyphosate and AMPA (98% and 99% purity, respectively) were purchased from Dr. Ehrenstorfer (Augsburg, Germany). Standard solutions containing 150 µg/mL of each analyte were prepared in water and stored at 4 °C. Working standard solutions were prepared by further dilution. N-(Phosphono[14C]methyl)glycine (glyphosate; specific activity 2084 MBq/mM, purity >99%) was purchased from Dislab’System, (15) Glass, R. L. J. Agric. Food Chem. 1983, 31, 280-282. (16) Miles, C. J.; Moye, H. A. J. Agric. Food Chem. 1988, 36, 458-461. (17) Vreeken, R. J.; Speksnijder, P.; Bodeldijk-Pastorova, I.; Noij, Th. H. M. J. Chromatogr., A 1998, 794, 187-199. (18) Nedelkoska, T. V.; Low, G. K.-C. Anal. Chim. Acta 2004, 511, 145-153. (19) EPA Method 547; EPA/600/4-90/020; U.S. Environmental Protection Agency: Cincinnati, OH, 1990, 63-80. (20) Mallat, E.; Barcelo`, D. J. Chromatogr., A 1998, 823, 129-136. (21) Ibanez, M.; Pozo, O. J.; Sancho, J. V.; Lopez, F. J.; Hernandez, F. J. Chromatogr., A 2005, 1081, 145-155. (22) Patsias, J.; Papadopoulou, A.; Papadopoulou-Mourkidou, E. J. Chromatogr., A 2001, 932, 83-90.
Saulx-les-Chartreux, France. (2-13C, 99%; 15N, 98%)-glyphosate and (13C, 99%; 15N, 98%; methylene-d2, 98%)-AMPA in water solutions at 100 µg/mL were provided from LGC Promochem, Teddington, U.K. Standard glyphosate-FMOC and AMPA-FMOC (99.5% and 96% purity, respectively) were obtained from Dr. Ehrenstorfer. FMOC-Cl (98% purity) was obtained from Sigma-Aldrich, Steinheim, Germany. Potassium dihydrogen phosphate, sodium hydroxide, sodium chloride, and acids were obtained from VWR, Fontenay-sous-Bois, France. Dimethyldichloride silane (DMDCS) was purchased from Sigma-Aldrich. Oasis HLB [poly(divinylbenzene-co-N-vinylpyrrolidone] reversedphase SPE cartridges (120 mg, 810 m2/g) were purchased from Waters, Milford, MA. LiChrolut EN [poly(styrene-co-divinylbenzene] SPE cartridges (200 mg, 1200 m2/g) and Bond-Elut octadecyl C18 SPE cartridges (200 mg, 500 m2/g) were obtained from Varian, Harbor City, CA. AG-1 × 8 [poly(styrene-co-divinylbenzene]-based trimethylammonium anion-exchange resin (100200 mesh, chloride form) was purchased from Bio-Rad Laboratories, Hercules, CA. The anion-exchange resin was converted into the HCO3- and OH- forms using 4 bed volumes of 1 N NaHCO3 and 20 bed volumes of 1 N NaOH solutions, respectively. It was then rinsed with water until pH < 9. Sewage sludge samples were collected from Plaisir and Elancourt wastewater treatment plants (WWTPs), Ile-de-France region, in the dehydrated state (20% dw). Analytical Procedure. For the radioactive tests used during development, an aqueous solution of 14C-glyphosate (9.25 kBq) was spiked on the sludge, and after 30 min, the sample was kept at 4 °C for 1 night. Radioactivity was monitored on a Wallace model 1410 (Turku, Finland) liquid scintillation counter. To evaluate effects of parameters, analyses were done by LC-UV diode array using an Aquasil C18 column (150 mm × 2 mm i.d., 3 µm, Thermo-Electron, Les Ulis, France) and linear gradient reversed chromatography with 5 mM ammonium acetate (solvent A) and acetonitrile (solvent B). Extraction. A 5 g portion of fresh sludge previously homogenized was extracted with 50 mL of 0.1 N NaOH using a horizontal shaker (300 shakes/min) for 1 h and then centrifuged 5 min at 2500g. The supernatant was filtered on 10 g of hyflosupercel silica, and 20 mL of 0.1 N NaOH was used for rinsing. Extraction was operated twice, and the supernatants were combined. A 10 mL aliquot was adjusted to pH 9 with 1 N HCl and was spiked with the internal standards (0.1 and 0.5 µg of the stable isotopically labeled glyphosate and AMPA, respectively). Fixation and Derivatization on Strong Anion-Exchange (SAX) Resin. A 300 mm × 10 mm column was packed with 1 g of AG-1 × 8 SAX resin using pH 9 ultrapure water. The 10 mL aliquot sample was subsequently percolated through the resin, and 5 mL of ultrapure water was applied onto the resin for rinsing. For derivatization, the resin was first conditioned with 2 mL of mixture A constituted of 25 mM borate buffer pH 9.2/acetonitrile (30:70 v/v), and then 2 mL of the same mixture containing 20 mg of FMOC-Cl was added. The reagent was left to react with the trapped analytes during 10 min. The resin was then rinsed and reconditioned with 15 mL of mixture B containing 25 mM borate buffer pH 9.2/acetonitrile (50:50 v/v). Another 20 mg of FMOCCl in 2 mL of mixture A was loaded to complete derivatization. Excess reagent and interferences (FMOC-OH, etc.) were eliminated by rinsing the resin, first with 15 mL of mixture B and Analytical Chemistry, Vol. 79, No. 10, May 15, 2007
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second with 10 mL of water. The target derivatives were eluted with 30 mL of 1 N NaCl/acetonitrile (70:30 v/v) at pH 5, and recovered into 270 mL of ultrapure water at pH 3. SPE Concentration of the FMOC Analytes. Prior to the preconcentration step, the pH of the previous solution was readjusted to 3 with 1 N HCl. Cartridges of 3 mL volume packed with 120 mg Oasis HLB were used on an Autotrace SPE workstation (Tekmar, Cincinnati, OH). They were conditioned by flushing 5 mL of MeOH followed by 5 mL of water and 5 mL of phosphate buffer at pH 3. The 300 mL sample solutions obtained above were percolated at 5 mL/min. The cartridges were then rinsed with 5 mL of water and dried for 30 min under a nitrogen flow. Elution was performed with two fractions of 3.5 mL of MeOH, which were then combined and concentrated to 0.5 mL under N2 before analysis. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/ MS). A Waters HPLC pump Alliance 2695 (Milford, MA) equipped with an automatic injector model 717 was used for separation on a Discovery HS C18 column (50 mm × 2.0 mm, 5 µm) (Supelco, Bellefonte, PA), with a mobile phase consisting of 5 mM ammonium acetate buffer and acetonitrile at a flow rate of 0.15 mL/ min at 24 °C. A linear gradient from 20% to 100% of acetonitrile was used within 12 min, and the final composition was maintained 4 min. Samples of 5 µL volume were used for injection. The LC system was connected to a Quattro LC triple-quadrupole mass spectrometer (Micromass, Manchester, U.K.) with an electrospray as interface. Data acquisition and processing were performed by MassLynx NT 4.0. MRM was used with dwell times of 0.3 s/scan. Source voltages were as follows: capillary 3.2 kV, extractor 2 V. The source block and desolvation gas were heated at 120 and 400 °C, respectively. Nitrogen was used as the nebulization and desolvation gas (75 and 450 L/h, respectively). For MS/MS, collisional-induced dissociation (CID) was performed with 2.5 × 10-3 mbar of argon and 10-50 eV collision energy. Breakthrough Study. The performance of SPE for concentration of the FMOC analytes present in aqueous solutions was evaluated by means of breakthrough curves. The latter were established using a home-made automated device23 that monitored the 263 nm UV signal after percolation of 0.8 mg/L glyphosateFMOC solutions through precolumns filled with the solid phase to be tested. RESULTS AND DISCUSSION The main idea of this work was to include an “on-solid support derivatization” step in the procedure to facilitate the general treatment of the sample, particularly during the reaction itself, and the elimination of the excess of reagent. The proposed strategy consisted in the following sequence. After extraction in an aqueous medium and centrifugation, the obtained solution would be loaded on a SAX resin column. This should allow the trapping of glyphosate and its metabolite on the solid support through ionic interactions. It was then hoped that the fixed analytes could be derivatized by contact with FMOC-Cl under conditions of solvent and time to be specified. Such in situ derivatization should make it possible to easily eliminate the excess reagent by a convenient rinsing of the column. Subsequent (23) Ne´lieu, S.; Perreau, F.; Guichon, R.; Seguin, F.; Bry, C.; Einhorn, J. Anal. Bioanal. Chem. 2005, 382, 108-114.
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elution of the FMOC analytes and SPE concentration would finally yield a methanolic (or acetonitrile) sample solution to subject to LC-ESI-MS/MS analysis. Other alternatives were tested preliminarily but were not successful. They included the use of (i) a cation-exchange column instead of the anionic one to fix the analytes or (ii) a hydrophobic phase to trap the reagent and allow its reaction with a percolating solution of the analytes. However, the strategy based on the use of an anion-exchange support to undergo the derivatization with FMOC-Cl was successfully accomplished. The development and optimization of this strategy are described in Figure 1. Extraction. Analyte extraction was attempted in alkaline medium (0.1 N NaOH) following the example of soil studies.16 The efficiency of this medium was tested using sludge samples fortified with radiolabeled [14C] and unlabeled glyphosate (9.25 KBq, 5 ppm fresh weight). High recoveries (>80%) were obtained for the extraction of [14C]-glyphosate from sludges after 0-72 h of contamination. The alkaline aqueous medium was thus found strongly desorptive for glyphosate even from highly organic matrixes. No further improvement was obtained using a more concentrated alkaline solution or adding sonication. Interactions between the SAX Resin and the FMOC Analytes. According to our strategy, it was essential to ensure the fixation of the analytes on the SAX resin in order to purify the sample and subsequently perform the FMOC-Cl derivatization. Since counterions with the lowest selectivity for anionexchange chromatography are most readily exchanged by target anions, we converted the commercial chloride resin into the hydroxide or hydrogen carbonate form (the selectivity order being as follows: OH- < HCO3- < Cl-). The trapping efficiency for our analytes was examined by means of radioactivity experiments on both types of resin. A 10 mL aqueous solution (pH 10-13) containing [14C]-glyphosate was applied onto a glass column filled with 1 g of SAX support. No breakthrough was detected in the effluents. Experiments were also carried out to check if FMOC derivatives could be fixed on the solid support under the alkaline conditions and how they could be eluted further. The fixation of the FMOC products on the SAX resin was studied using unlabeled standards. Trapping was found efficient (0% breakthrough) on both OH- and HCO3- forms of resin, with the sample pH ranging from 9 to 13. In contrast, some breakthrough was observed when using Cl- (>23% of glyphosate-FMOC and >43% of AMPA-FMOC). Aqueous solutions of NaCl are often used for the elution of linked ionic analytes because of the high affinity of Cl- ions for this type of resin.15 However, this eluent (1-2 N solutions) was not strong enough since it yielded only 7% and 13% recovery of glyphosate-FMOC from OH- and HCO3- resins, respectively. By contrast, the introduction of an organic modifier like acetonitrile in the saline medium strongly improved the elution efficiency, indicating that both ionic and hydrophobic interactions had to be involved. With 10-30% of acetonitrile, recoveries reached from 30% to 51% (OH-) and to 75% (HCO3-). However, the difference observed between both phases led us to suspect a possible degradation of the analyte during the elution process. This question will be addressed next. Higher percentages of acetonitrile were not tested to limit the extent of dilution necessary before operating the SPE concentration. The influence of the pH on
Figure 1. (a) Analytical strategy involving the FMOC-Cl derivatization onto the SAX resin. (b) Chemical principle of the derivatization approach (e.g., glyphosate).
elution was also studied, and pH 5 was found optimal. Stability of FMOC Derivatives on the SAX Resin during Derivatization. The behavior of glyphosate- and AMPA-FMOC on the SAX resin was studied in conditions simulating the derivatization process. Aqueous solutions of these compounds were applied at alkaline pH (9 and 10) for fixation. The solid support was conditioned and treated by FMOC-Cl as described in the Experimental Section (see final procedure). Recoveries through elution were evaluated by LC-UV. A significant loss of FMOC analytes was observed particularly for AMPA-FMOC. Recoveries (n ) 3) were 90% ( 5% and 63% ( 6% (HCO3-) versus 62% ( 5% and 20% ( 3% (OH-) for glyphosate-FMOC and the metabolite-FMOC derivative, respectively. In the presence of OH- ions, the resin environment becomes more alkaline, probably promoting the hydrolysis of the linked derivatives. AMPA-FMOC thus appears more sensitive to such reaction. This competitive effect was reduced when operating with a resin conditioned under the less alkaline HCO3- form. The influence of the pH of the solution to be percolated before “derivatization” was also noted. Hydrolysis was partly reduced when loading the sample at a lower alkaline pH (9 instead of 10). Recoveries became 87% ( 6% and
78% ( 4% (HCO3-) versus 71% ( 3% and 32% ( 4% (OH-) for the glyphosate-FMOC and metabolite-FMOC derivatives, respectively. Requirements of the “On-Solid Support Derivatization”. The FMOC derivatization of glyphosate and AMPA is usually performed in solution (cf., in waters, soil, or sediment extracts). The reaction requires (1) an alkaline medium (often pH 9) for the reactivity of the amine function and (2) the presence of acetonitrile for reagent solubility. The following experiments were performed to check whether resin-bound glyphosate and AMPA could react with FMOC-Cl and in which optimal conditions (Figure 2). pH Conditions. Samples (10 mL) spiked with analytes (150 µg), simulating sludge extracts, were applied on the resin at pH values ranging from 7 to 10. In these preliminary experiments, solutions were made from ultrapure water in order to avoid matrix effects. The following sequence was tested: (i) sample percolation on the SAX resin (HCO3-) followed by conditioning with mixture A (see the Experimental Section), (ii) loading of FMOC-Cl (10 mg) in mixture A for derivatization and 5 min of contact, (iii) support rinsing with mixture B followed by water only, and (iv) elution Analytical Chemistry, Vol. 79, No. 10, May 15, 2007
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Figure 2. Influence of various parameters on the yields of FMOC derivatization for glyphosate and AMPA fixed on a HCO3- resin (ultrapure water solutions spiked with 150 µg of each analyte, two replicates, yields evaluated by HPLC-UV; b glyphosate, 2 AMPA).
with a 1 N NaCl solution in acetonitrile/water, 30/70, v/v, at pH 5. The FMOC derivatization was found effective under these conditions: the obtained yields of the derivatives were 78% ( 7% and 27% ( 6% for glyphosate and AMPA, respectively. They did not differ significantly (t test, four degrees of freedom, P < 0.05) at pH values between 7 and 9 (three points, two replicates, n ) 6), In contrast, at pH 10 the yield for glyphosate was significantly lower with 62% as a mean value (t test, five degrees of freedom, P < 0.05). The pH 9 value to fix the analytes was kept for the rest of the study, and various other parameters were tested in order to improve the yields especially for the metabolite. Several compositions of borate buffer-acetonitrile (pH and borate buffer concentration, acetonitrile percentage) were tested 3798 Analytical Chemistry, Vol. 79, No. 10, May 15, 2007
for both resin conditioning and FMOC-Cl loading. In the 8.210 interval, the pH of the buffer was found optimal at 9.2 and in each case more favorable to glyphosate. The best results were further obtained at the 25 mM concentration. The derivatization was effective even in the absence of borate, probably because of a residual alkalinity within the resin after percolation. At 100 mM, derivatization was insufficient since borate ions can compete with the analytes on the resin thus providing some breakthrough. Excess of FMOC-Cl and Time of Reaction. Since during normal use the reagent can be rapidly hydrolyzed into inactive FMOCOH, an excess of the reagent is generally recommended. Suspecting the same in our conditions, FMOC-Cl was applied at different doses. The optimal dose was found to be 20 mg, a higher excess
Figure 3. Positive and negative ESI-MS responses of glyphosate-FMOC (m/z 392 or 390) and AMPA-FMOC (m/z 334 or 332) for different mobile phase compositions: A, B, C (2.5 mM NH4OAc), D, E, F (5 mM NH4OAc), and G, H, I (10 mM NH4OAc) with 25%, 50%, and 75% of acetonitrile, respectively.
giving rise to precipitates in the column. Surprisingly, it was also observed that the time of contact between the reagent and the fixed analytes (even with sonication assistance) had no significant influence (in the 5-30 min interval) on the final yields of derivatization for both analytes. It was thus concluded that the reaction is quite fast and probably blocked rapidly. Further, the difference in the conversion yields for glyphosate and AMPA might be due to the deeper migration of the metabolite during percolation, protecting it to some extent from the subsequent introduction of the reagent. This hypothesis was supported by the presence of the single anionic function (phosphonate) in AMPA versus a double one (phosphonate and carboxylate) in the glyphosate molecule. This factor and a possible intrinsic difference of reactivity between both amines (secondary vs primary) should be considered together. Reagent Volume and Number of “Derivatizations”. In an effort to get a better distribution of FMOC-Cl within the resin, we tested several reagent volumes (from 1 to 9V0, V0 being the volume of the resin employed). The derivatization yields remained considerably lower for AMPA than for glyphosate, and no significant effect of the reagent volume was observed. A 2V0 loading of the reagent solution was chosen as a compromise. These results suggest that the reaction could be limited by a rapid fixation of FMOC-Cl by interaction with the polymeric backbone of the resin, hampering the reagent to reach the linked AMPA molecules. To overcome this problem, we tested a “double derivatization” each time using the same parameters (10 min of contact, 20 mg of FMOC-Cl loaded in 2V0 of mixture A). The solid support was rinsed and reconditioned intermediately in order to release new active sites before carrying out the second reagent application. With the latter, the reagent could be distributed more deeply within the phase, allowing reaction with the underivatized resin-bound analytes. Indeed, the yields increased from 60% to 82% by operating twice
for glyphosate and doubled for AMPA in ultrapure water. A third application of the reagent did not enhance the yields further. For sludge samples in which analytes may compete with other amino acids, the double-derivatization process gave even better yields (88% and 49% for glyphosate and AMPA, respectively). Excess Reagent Removing. FMOC-Cl in excess as well as FMOC-OH, its hydrolysis product, can compete for trapping with target derivatives during the SPE concentration, which may result in a lower efficiency of this step. Furthermore, they can interfere with analytes during the chromatographic separation used for analysis. To selectively remove these interferences we tested various mixtures of aqueous borate buffer (pH 9.2)-acetonitrile, with ratios ranging from 30/70, v/v, to 70/30, v/v, and volumes from V0 to 15V0. The efficiency of these compositions was evaluated by comparing peak areas of residual reagent in the LCUV chromatograms of the washes. The best results, i.e., allowing a maximum elimination of FMOC-Cl and maintaining the fixation of the FMOC analytes, were obtained using 15V0 of borateacetonitrile 50/50, v/v. Optimization of the SPE Preconcentration. The performance of three reversed phases toward the fixation of FMOCglyphosate was evaluated by comparing their breakthrough volumes (VB; see the Experimental Section). At pH 3 and 3% of acetonitrile in the percolated solutions, recorded performances were as follows: C18 (VB e 50 mL) < LiChrolut EN (VB e 220 mL) < Oasis HLB (VB e 500 mL). The EN polymeric phase is often more efficient than C18 due to the active aromatic sites that allow π-π interactions.24 Such interactions likely occur with the aromatic backbone of the FMOC derivatizing group of the analyte. Furthermore, the occurrence of the pyrrolidone group, a hydrogen acceptor, in the Oasis HLB support makes it even more efficient (24) Thurman, E. M.; Mills, M. S. Solid Phase Extraction: Principles and Practice; Wiley: New York, 1998; Vol. 147.
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Table 1. MS/MS Conditions and Performances of the MRM Method (ESI Positive vs Negative) compd
Tr (min)
ESI
cone voltage (V)
glyphosate-FMOC
3.2
positive
AMPA-FMOC
a
8.9
transition
collision energy (eV)
C/Q % (RSD)a
LOD (pg)
LOQ (pg)
linearity (r2)
19
392 f 214 (Q) 392 f 170 (C)
9.5 10
62(4)
30
50
0.9995
negative
20
390 f 168 (Q) 390 f 150 (C)
10 25
39(2)
80
140
0.9968
positive
19
334 f 156 (Q) 334 f 112 (C)
8.5 10
36(4)
50
80
0.9997
negative
11
332 f 110 (Q) 332 f 136 (C)
10 15
38(9)
50
125
0.9972
Average value calculated from 12 injections of standard solutions (three replicates, four concentration levels).
Figure 4. MRM chromatograms in the positive mode for quantitation of (a) glyphosate-FMOC and (b) AMPA-FMOC in a sewage sludge sample (Plaisir, January 2005). Concentrations found in ppm dw: 1.8 (glyphosate) and 33.3 (AMPA). Analytes are confirmed by C/Q × 100 being within (20% for glyphosate and (25% for AMPA of the expected values (see Table 1; cf., European Commission Decision 2002/657/ CE). IS: isotopically labeled standard.
toward polar solutes, i.e., glyphosate-FMOC.24,25 The influence of pH and acetonitrile percentage on VB was thus studied for optimization in the case of Oasis HLB. The VB decreased dramatically when the pH was changed to 5 or 7 (ca. 10 mL in both cases). Acidic conditions appeared necessary to maintain a zero net charge form (according to pKa values) of glyphosateFMOC, the most favorable for retention. A low percentage of acetonitrile could be admitted, e.g., 3% or 5% yielding with our system VB values of 500 or 235 mL, respectively, but not 7% (VB < 2 mL). With the use of 120 mg Oasis HLB cartridges instead of precolumns (cf., VB measurements, Experimental Section) 99% ( 2% and 102% ( 3% recoveries were obtained for glyphosateFMOC and AMPA-FMOC, respectively, using 300 mL spiked solutions (3% acetonitrile/water pH 3). On the basis of these results, the eluates originating from the SAX resin (pH > 5, 30% acetonitrile) should be acidified to pH 3 and 10-fold diluted before SPE on Oasis cartridges with no risk of breakthrough. LC-ESI-MS/MS Conditions. Glyphosate-FMOC and AMPAFMOC were analyzed and compared in both electrospray (ESI)
positive and negative modes. The MS procedure was first investigated using direct infusion of methanolic solutions of standard FMOC derivatives. The cone voltage was optimized, and the variations of current ion intensities were evaluated for both [M + H]+ (positive ionization) and [M - H]- ions (negative ionization). The results showed optimum values at 19 V for both compounds in the positive mode. Concerning the [M - H]- ions, optimal values were found at 20 and 11 V for glyphosate-FMOC and AMPA-FMOC, respectively. The effects of ammonium acetate and organic modifier percentage in the mobile phase were studied, these factors being known to influence the ionization process.26 The analyte response increased with increasing acetonitrile percentage and was always higher for the glyphosate derivative. The 5 mM concentration of ammonium acetate was found optimal for the [M + H]+ ions. In the negative mode, all intensities were lower than in the positive mode (Figure 3). For MS/MS optimization, the fragmentation of both [M + H]+ and [M - H]- ions was investigated as a function of collision energy. Two ion transitions (parent ion f product ion) for
(25) Hennion, M.-C. J. Chromatogr., A 1999, 856, 3-54.
(26) Mathis, J. A.; McCord, B. R. Forensic Int. 2005, 154, 159-166.
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quantitation (Q) and for confirmation (C) were chosen in each case according to intensity and specificity criteria for application of the MRM mode. In agreement with the MS results, a better sensitivity in the positive mode than in the negative one was observed for both analytes (Table 1).21 The possible matrix effects were then investigated. The calibration curves obtained from real sludge extracts with standard addition (R1) were compared with those from external methanolic standards (R2) (seven concentration levels e6 to 5000 pg/µL g 5 µL injected). A satisfactory linearity for both curves (R2 > 0.999) was noticed. But slope ratios of 3 (R1/R2) for glyphosate-FMOC and of 2 for AMPA-FMOC were observed, proving the existence of positive matrix effects. Therefore, isotopically labeled standards were used for quantitation instead of the laborious standard addition method. These internal standards (IS) were added to the extract before purification to undergo FMOC derivatization like the analytes. Analytical Performances. To determine recovery rates, sludge extracts were spiked at two concentration levels, 10 and 100 ppm (dw), and the entire analytical procedure was applied. Recoveries determined at 10 ppm (n ) 3), after subtracting sludge initial contamination and using standard addition calibration, were 70% ( 7% for glyphosate and 63% ( 3% for AMPA. The results obtained at 100 ppm were very similar. The method developed with internal calibration was then validated, by evaluating repeatability and limits of detection (LODs) and quantification (LOQs). The repeatability is expressed by the RSD of the analyte quantification in sludge samples. The sample used in the validation study was found to contain (n ) 5) both analytes at 1.5 ppm dw (glyphosate) and 16.6 ppm dw (AMPA), with RSDs of 8% and 2%, respectively. The LODs and LOQs were estimated as the analyte concentrations producing signal/noise ratios of 3:1 and 5:1, respectively, in the MRM chromatograms. Their values were extrapolated from signals exhibited by unspiked sludge samples since they were initially contaminated at significant levels and found lower than 50 ppb dw for both analytes (LOD 20 and 30 ppb, LOQ 35 and 50 ppb for glyphosate and AMPA, respectively). These limits were close to those reported for soils,21 although this matrix is less complex than sludge. (27) Williams, G. M.; Kroes, R; Munro, I. C. Regul. Toxicol. Pharmacol. 2000, 31, 117-165. (28) Steber, J.; Wierich, P. Chemosphere 1987, 16, 1323-1337.
Application to WWTP Samples. Our method was applied to monitor sewage sludge contamination during 1 year (July 2004June 2005) in two WWTPs from Ile-de-France region. All samples were found to contain both compounds. Glyphosate concentrations ranged between 0.2 and 2.9 ppm dw at Elancourt and between 0.5 and 1.8 ppm dw at Plaisir. Concentrations of the metabolite were always higher (2.3-20.9 ppm dw and 9.6-33.3 ppm dw at Elancourt and Plaisir, respectively) than those of glyphosate reflecting its various possible origins.27,28 Figure 4 shows an example of the LC-ESI-MS/MS quantitation and characterization of glyphosate and AMPA in one of those samples (Plaisir, January 2005). CONCLUSIONS In this work, we developed, optimized, and validated a method for glyphosate and AMPA analysis in sewage sludge. We proved herein the possibility of FMOC-Cl derivatization on a SAX resin for bound glyphosate and AMPA. This approach has the advantage of allowing a rapid reaction and easy sample handling since purification, derivatization, and elimination of excess reagent occur on the same support. After elution of the FMOC derivatives, samples are concentrated on Oasis HLB cartridges via appropriate aqueous dilution and pH adjustment. Followed by LC-ESI-MS/ MS in the positive MRM mode, our procedure appears, despite the complexity of the matrix, sufficiently sensitive and precise to quantify glyphosate and AMPA residues in real sludge samples. Monitoring of these analytes in two WWTPs of Ile-de-France region led us to observe a contamination of glyphosate which rarely exceeded 2 ppm dw, whereas AMPA corresponding levels were 10-40-fold higher over all the year. According to the LODs of our method (20 and 30 ppb, respectively), much lower levels of the contaminants could be detected in sludges. ACKNOWLEDGMENT The authors are grateful to the French Agency for Environment and Energy Management (ADEME) and to the National Institute for Agricultural Research (INRA) for the financial support of Project 0275048 and the Ph.D. fellowship of A.G. They also thank the WWTPs of Elancourt and Plaisir for providing sludge samples. F. Perreau is acknowledged for helpful discussions. Received for review November 21, 2006. Accepted March 1, 2007. AC062195K
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