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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Assessing solvent derivatization and carbon dioxide supercritical fluid simultaneous extraction/ derivatization of cyprodinil a
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Ana Aguilera , Antonio Valverde , Antonio Valverde-Monterreal , Luis Garcia-Fuentes & a
Mourad Boulaid a
Pesticide Residue Research Group, University of Almería, Almería, Spain
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Servicio Oficial de Inspección, Vigilancia y Regulación del Comercio Exterior, Almería, Spain
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Chemistry & Physics Department, Faculty of Experimental Sciences, University of Almería, Almería, Spain Published online: 05 Jun 2014.
To cite this article: Ana Aguilera, Antonio Valverde, Antonio Valverde-Monterreal, Luis Garcia-Fuentes & Mourad Boulaid (2014) Assessing solvent derivatization and carbon dioxide supercritical fluid simultaneous extraction/derivatization of cyprodinil, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 49:8, 541-549, DOI: 10.1080/03601234.2014.911553 To link to this article: http://dx.doi.org/10.1080/03601234.2014.911553
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Journal of Environmental Science and Health, Part B (2014) 49, 541–549 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2014.911553
Assessing solvent derivatization and carbon dioxide supercritical fluid simultaneous extraction/derivatization of cyprodinil ANA AGUILERA1, ANTONIO VALVERDE1, ANTONIO VALVERDE-MONTERREAL1,2, LUIS GARCIA-FUENTES3 and MOURAD BOULAID1 Pesticide Residue Research Group, University of Almerıa, Almerıa, Spain Servicio Oficial de Inspecci on, Vigilancia y Regulaci on del Comercio Exterior, Almerıa, Spain 3 Chemistry & Physics Department, Faculty of Experimental Sciences, University of Almerıa, Almerıa, Spain 1
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Derivatization of cyprodinil with different reagents and solvents has been evaluated to improve the GC/MS characterization of this fungicide. After assessing some preliminary acylation and silylation reactions, derivatization with anhydrous heptafluorobutyric anhydride (HFBA) was selected as the best derivatization option for cyprodinil. The HFBA-cyprodinil derivative was clearly identified and characterized by GC/MS (ion-trap). The spectrum of the HFBA derivative of cyprodinil was characterized by the base peak, 252 m/z ion, and two other ions with relative abundances of 5% (224 m/z ion) and 4% (420 m/z molecular ion). Conversion rates in the range of 83–92% were obtained when 0.1–1 mg cyprodinil were derivatized in vial without solvent at 25 C temperature for 120 min, with 5 mL HFBA and 5 mL pyridine. Simultaneous extraction-derivatization of cyprodinil in supercritical carbon dioxide was only achieved when no modifier was present, but conversion/recovery rates obtained in the replicate experiments carried out with 15 mL supercritical carbon dioxide at 50 C and 200 atm (n D 5), 300 atm (n D 7), and 400 atm (n D 5) were no reproducible (RSD > 50%) and ranged between 10% and 45% (related to the signal obtained for derivatization in vial). Keywords: Derivatization, GC/MS, SFE, cyprodinil.
Introduction Cyprodinil (4-cyclopropyl-6-methyl-N-phenylpyrimidine) is a systemic fungicide of the anilinopyrimidine family, a new chemical class of fungicides that are highly active against a broad range of fungi. The biological mode of action includes inhibition of methionine biosynthesis and secretion of fungal hydrolytic enzymes.[1] It was first introduced for application on cereals grains and it is currently used as a foliar fungicide in cereal grains, grapes, pome fruits, stone fruits, strawberries, vegetables, field crops, and ornamental.[2] In the southeast of Spain it is mainly used to control botrytis and sclerotinia, fungi that infect greenhouse vegetables.[3] Although the correct use of pesticides does not cause problems of public concern in health and environmental areas, if inappropriate treatments are applied and/or without respecting safety recommendations, undesiderable
Address correspondence to Ana Aguilera, Pesticide Residue Research Group, Faculty of Experimental Sciences, University of Almerıa, 04071 Almerıa, Spain; E-mail: [email protected]
residues can remain in crops. Analytical methods for determining pesticide residues include commonly gas chromatography (GC) or high-performance liquid chromatography. Analytes can be determined by GC with or without a previous derivatization step. Derivatization is employed not only to improve the thermal stability of polar compounds, but also to minimize undesiderable interactions with the column and injection liner resulting in poor peak shape or loss of mass through adsorption. Also, derivatization can be used to reposition peaks in the chromatogram from regions of extensive overlap to regions that are relatively empty of interfering peaks and to improve, if possible, the mass spectral properties of derivatives. For those analytes that present a labile hydrogen coming from different chemical function, such as secondary amine group (-NH-), primary amine group (-NH2), carboxylic group (-COOH), or hydroxyl group (-OH), several problems are usually encountered with trace level analyses; losses due to adsorption in the inlet and peak tailing due to the interaction of the analyte with active sites in the analytical column may occur.[4] Amines tend to be adsorbed by and decompose on columns resulting in tailed elution peaks, ghosting phenomena and low detector sensitivity. The
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542 derivatization for the protection of amino groups is widely employed to increase volatility and to improve the chromatographic and, if possible, mass spectral properties of derivatives.[5] Acylation (addition of –COR instead a labile -H) is one of the most widely used procedure for GC analysis of primary and secondary amines. The introduction of acyl protective groups improves the volatility, chromatographic mobility, and chemical stability of these amines.[6,7] Traditional extraction techniques require long extraction times and variable amounts of sorbents and organic solvents. Cost associated to the purchase of organic solvents and the concerns on their negative environmental impact and on human health should limit their use.[8] Supercritical fluid extraction (SFE) has been proven to be an efficient alternative for extraction of pesticide residues.[9–12] Moreover, supercritical fluids offers the possibility of developing chemical derivatization under controlled conditions of pressure and temperature, and several researchers have devised supercritical fluids derivatization and extraction (SFDE) methods, which have been revised by Field.[13] In addition of reducing sample handling and decreasing the number of preparative steps of the extracts, in situ derivatization could have the benefits that reagents can act as modifiers during the SFE and enhance the solubility of analytes in supercritical CO2. However, there is a little reference available on the application of this derivatization technique for the determination of pesticide residues.[14–16] In fact, derivatization reactions in supercritical CO2 should be chosen carefully to ensure their compatibility with the conditions used for SFE of the target analytes. The optimum reagent, reaction conditions and final analytical method must all be considered when integrating a derivatization reaction into a SFDE scheme. Certain combinations of SFE conditions might be deleterious to the formation of the derivative desired, and this should be determined initially on neat analytes before attempting derivatization in situ. The study carried out in this work had two aims. The first one was to assess the conditions required for the formation, using a simple and rapid reaction, and under mild conditions, of cyprodinil derivative to enable good GC/ MS characterization and the second one was to assay the applicability of the SFDE on this compound.
Materials and methods Reagents and materials Ethyl acetate, toluene, cyclohexane, acetone, acetonitrile, dichloromethane, and methanol were pesticide residue grade from Panreac (Barcelona, Spain) and Scharlab (Barcelona, Spain). High-purity anhydrous heptafluorobutyric anhydride (HFBA) was purchased from Supelco
Aguilera et al. (Bellefonte, PA, USA). Analytical grade dried pyridine was from Fluka (Buchs, Switzerland). Certified standard of cyprodinil (purity 99.9%) was supplied by Riedel de H€aen (Seelze, Germany). A stock standard solution of about 1000 mg L-1 of cyprodinil was prepared in acetone. Working standard solutions (0.05–10 mg L-1) were prepared in toluene and other solvents (ethyl acetate, cyclohexane, acetone, acetonitrile, dichloromethane, and methanol) from the stock standard solution by suitable dilutions. Pure standard and standard solutions were stored in dark at ¡20 C. Glass beads (3 mm diameter) were obtained from Scharlab (Barcelona, Spain), and glass microfiber filter discs from Whatman (Maidstone, England). Primary-secondary-amine (PSA) sorbent was supplied by Varian (Harbor City, CA, USA). Carbon dioxide (99.995% purity), helium and nitrogen (99.999% purity) were supplied by Air Liquide (Madrid, Spain).
GC-MS analysis Cyprodinil and cyprodinil-HFBA derivative were analysed by GC-MS by using a Varian (Walnut Creek, CA, USA) 3400 gas chromatograph-Saturn 3 ion trap mass spectrometer equipped with a model 1077 injection port, and a model 8200 CX autosampler, fitted with a DB-5MS fused-silica capillary GC column (30 m £ 0.25 mm i.d.., 0.25 mm film thickness) were used. Operating conditions for GC-MS were: 9 psi helium column head pressure; 0.75 min splitless time; 250 C injector temperature; 2 mL injection volume; 60 C initial oven temperature for 1 min, ramped to 180 C at 25 C/min, then to 280 C at 5 C/min, and held at 280 C for 15 min; 280 C transfer line temperature; and 220 C ion-trap manifold temperature. MS measurements were performed with electron impact (EI) at 70 eV in the full scan mode over the mass range of m/z 60 to 650 at 1 scan/s from 6 to 35 min. Saturn GC-MS version 5.2 software (Varian, Walnut Creek, CA, USA) was used for data collection.
SFE system An Isco SFE system, consisting of a model 260D syringe pump and controller able to operate in the pressure range of 0.6 up to 510 atm (1 atm D 101.3 kPa); a SFX 2-10 extractor with restrictor heater set at 70 C, and 2.5 stainless steel extraction cartridges with removable 2 mm frits, was used in this study. Uncoated and deactivated fused silica capillary column, 30 cm length £ 50 mm i.d., was used as restrictor, and 10 mL graduated test tubes (immersed in a 15–20 C water bath), containing 5 mL ethyl acetate, was used as collection system. In the equipment the CO2 flow is dependent of pressure and type of restrictor used. With the restrictor selected the flow rates ranged between 1– 1.2 mL min-1 with a pressure of 300 atm.
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Derivatization in organic solvents Derivatization reactions were carried out on 2 mL reaction vials, using a reactitherm dry bloc derivatization system (Pierce, Rockford, IL, USA) to heat the reaction mixtures. 0.1–1 mL of working standard solutions of cyprodinil (0.05–5 mg L-1) were placed in the reaction vials and, after mixing with the derivatization reagent (HFBA) and catalyzer (pyridine), the vial was sealed and allowed to react for 30–120 min (or overnight) in the dark at the assessed temperature (25 C and 60 C). The corresponding derivative was purified by purging with dry nitrogen followed by evaporation to dryness. The resulting residue was redissolved with 0.1–1 mL toluene and analysed by GC-MS. In addition to temperature and reaction time, the following derivatization conditions were evaluated: (a) volumes of HFBA (5–100 mL) and pyridine (0–100 mL) used to perform the acylation reaction and (b) volume (0.1 and 1 mL) and solvent type (ethyl acetate, toluene, cyclohexane, acetone, acetonitrile, dichloromethane, and methanol) of the working standard solution added to the reaction vial. Derivatization in vial without solvent was also evaluated by means of some experiments in which the solvent of the standard solution introduced in the vial was evaporated to dryness with dry nitrogen before adding the derivatization reagents. The following optimized conditions were selected to evaluate the linearity and reproducibility of derivatization process in vial, and to obtain the cyprodinil-HFBA derivative solutions used as reference in the SFE studies: derivatization in vial without solvent at 25 C temperature for 120 min, with 5 mL HFBA and 5 mL pyridine. Supercritical fluid extraction of cyprodinil and its HFBA derivative All SFE experiments were performed on glass beads packed inside 2.5 mL extraction cartridges. SFE recovery tests of cyprodinil were carried out at the spiking levels of 0.5 mg and 1 mg (100 mL of cyprodinil standard solutions in toluene of 5 or 10 mg L¡1), performing 3 and 5 replicates per level, respectively. SFE conditions were those proposed by Valverde et al.[11] for multiresidue analysis of pesticides in vegetables: dynamic extraction with 15 mL carbon dioxide at 300 atm pressure and 50 C temperature after five minutes in the static mode. Final SFE extracts were transferred to 2 mL vials, evaporated to dryness and dissolved with 1 mL toluene. Similar tests were conducted on glass beads spiked with 100 mL of the 1 mL solution of HFBA-cyprodinil derivative previously obtained by derivatization in vial of 1 mg of cyprodinil. Final SFE extracts were evaporated to dryness, dissolved with 100 mL toluene and analyzed by GC/MS, the chromatographic signal being compared with that obtained for the corresponding spiking solution, which was analyzed in the same chromatographic sequence.
Simultaneous supercritical fluid extraction and derivatization Simultaneous SFE and derivatization of cyprodinil with HFBA reagent was assessed in two steps. The first step consisted in a static period of 30 min, whereas the second one was performed under dynamic conditions just after the static period. In all cases, these experiments were carried by filling the SFE vessels with glass beads and spiking them with 100 mL of cyprodinil standard solution of 1 ppm, the derivatization reagents (5 mL HFBA and 5 mL Pyridine) being added after the solvent was evaporated under N2 stream and just before sealing the vessel. After pressurizing with CO2 at 300 atm, the SFE vessels were held statically at 50 C for 30 min. Dynamic SFE was then performed by passing through the cell 15 mL CO2 (300 atm pressure and 50 C temperature) for 10–15 min (CO2 flow rate 1–1.2 mL min-1) and using 5mL ethyl acetate as collection system. Different static modifier conditions were assessed: (a) 200 mL or 100mL of modifier (methanol, ethyl acetate, cyclohexane, acetone, toluene, and acetonitrile) and (b) no modifier. All the extractions were carried out in duplicate and the volume of final extracts was adjusted to 1 mL prior to GC-MS detection. Additional replicate experiments (n D 5) were performed without modifier at 200, 300, and 400 atm pressure, keeping constant the other SFE parameters.
Results and discussion GC/MS characterization of HFBA-cyprodinil derivative Cyprodinil is a secondary amine that presents one labile hydrogen and may react with HFBA to form its acylderivative. However, as far as we know, there is no reference available on the characterization of this derivatization reaction to be used for the determination of this pesticide by GC/MS analysis. The expected reaction of cyprodinil with HFBA is shown in Figure 1. This acylation reaction with HFBA was selected after assessing some preliminary acylation and silylation reactions of cyprodinil using different derivatization reagents. The HFBA derivative of cyprodinil was obtained in a number of these preliminary experiments, being identified and characterized by GC/MS. As can be seen in Figure 2, under the GC/ MS instrumental parameters above indicated, cyprodinil
Fig. 1. Course of the derivatization reaction of cyprodinil with HFBA.
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Fig. 2. m/z 224 ion chromatogram of a standard solution of cyprodinil (1 mg L-1) in toluene (a); and m/z 224 (b) and m/z 252 (c) ion chromatograms of the corresponding HFBA-cyprodinil derivative extract obtained by derivatization in vial under the optimized conditions.
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ions. The spectrum of the HFBA derivative of cyprodinil is characterized by the base peak, 252 m/z ion, and two other ions with relative abundances of 5% (224 m/z ion) and 4% (420 m/z ion). These spectral data illustrate that the molecular ion (420 m/z) was not the major ion in the spectra, corresponding the base peak 252 m/z to the fragment [M-169]. The 224 m/z [M-197] fragment is also the molecular ion observed in the spectrum of cyprodinil. These data confirm that cyprodinil derivative is obtained from the proton substitution of the NH moiety by the heptafluoro-butyric acyl group, and that the MS fragmentation pathway of the cyprodinil derivative involves the successive losses of the C3F7 and CO groups, such as it is indicated in the scheme proposed in Figure 4.
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and its HFBA derivative eluted at 15.7 and 13.2 min, respectively. Figure 2a shows the 224 m/z ion chromatogram obtained for a toluene standard solution of cyprodinil of 1 ppm, whereas Figure 2b and c include the 224 and 252 m/z ions chromatograms of the HFBA derivative obtained for 1 ppm cyprodinil standard under the optimized vial-derivatization conditions (see the section “Derivatization in vial”). Figure 3a and b show the corresponding mass spectra obtained for cyprodinil and its HFBA derivative, respectively. Examination of the mass spectrometric features of cyprodinil and the corresponding heptafluorobutiramide derivative was carried out in order to confirm the identity of the derivative by studying the molecular and fragments
Fig. 3. Mass spectra of cyprodinil (a) and HFBA-cyprodinil derivative (b).
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Fig. 4. Molecular structure of HFBA-cyprodinil derivative and the proposed MS fragmentation pathway.
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Derivatization in vial The acylation reaction of cyprodinil with HFBA could be affected supposedly by many parameters, such as reaction temperature, reaction time, amount, and type of solvent and catalyzer.[8,5] The influence of these parameters was investigated by using 2 mL vials as reactor cell. After assessing the derivatization reaction in vial with 0.1 and 1 mL standard solutions of 1 ppm cyprodinil in different solvents (ethyl acetate, toluene, cyclohexane, acetone, acetonitrile, dichloromethane, and methanol), it was found that suitable amounts of the cyprodinil derivative are only obtained when the standard solution of cyprodinil in toluene was evaporated to dryness before adding the derivatization reagents. This is the reason for which optimization experiments were performed by adding 5 mL of HFBA and 5 mL pyridine to dry residues of cyprodinil obtained by evaporation, with the aid of N2 stream, of standard solutions of cyprodinil (100 mL or 1 mL). A similar procedure has been followed by Stolker et al. for the derivatization of steroids from urine.[17] Experiments carried out to asses the influence of the catalyzer showed that the addition of pyridine is a key factor to develop the derivatization reaction because when no catalyst was added to the reaction mixture no derivatization product was observed. However, the addition of more than 5 mL of pyridine (in the range of 5–100 mL) had no effect on the product yield. It was also found that increasing both derivatization time (from 2 h up to 8 h) and temperature (from 25 C to 60 C) has no influence on the derivatization process, these results being similar to those found by Karg and others authors.[18–22] As a result, the following optimized conditions were selected to evaluate the linearity and reproducibility of the vial-derivatization process, and to obtain the cyprodinil-HFBA derivative solutions used as reference in the SFE studies: derivatization without solvent at 25 C temperature for 120 min, with 5 mL HFBA and 5 mL pyridine. Linear calibration curves were obtained from the m/z 252 ion chromatogram, at four different levels (0.1, 0.2, 0.5, and 1 mg of cyprodinil; 1 mL final volume of the GC/ MS derivatized extracts), in four different days, obtaining in all cases correlation coefficients over 0.99 with relative
standard deviations of the response factors in the range of 5–20%. Figure 5 shows an example of the regression lines obtained from the m/z 252 and 224 ions chromatograms. The slopes of the linear calibration curves (m/z 252 ion) ranged between 1.4 £ 105 and 1.8 £ 105 height units/mg L¡1, the ratio of the m/z 224 and m/252 regression lines being, in all cases, into the range of 4.5–5.5%. The amount of non-derivatized cyprodinil remaining in these experiments was determined by quantification of the cyprodinil signal, at 15.7 min, in the m/z 224 ion chromatograms, and ranged between 8% and 17%, these data representing conversion rates of cyprodinil in the range of 83–92%. As described in literature, potential problems with derivatization procedures can be, among others, the presence of unchanged derivatization reagents, which can affect chromatographic column and/or chromatographic analysis.[5,23] In order to avoid these potential problems, the addition of a sorbent material to the derivatized extracts was also evaluated as a last clean up step in the vial-derivatization procedure. 1 mL of the derivatized cyprodinil solution, previously analyzed by GC/MS, was placed in a polypropylene centrifuge tube containing 10 mg of PSA. Sample was shaken with the adsorbent for 30 s and then centrifuged at 3700 rpm (2 min), the supernatant being
Fig. 5. Regression lines obtained from the m/z 252 and 224 ions chromatograms after derivatization in vial (optimized conditions) of 0.1, 0.2, 0.5, and 1 mg of cyprodinil (1 mL final volume of GC/MS extracts).
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Fig. 6. Full scan chromatograms obtained for a HFBA cyprodinil derivative extract before (a) and after (b) PSA clean up.
again analyzed by GC/MS. Figure 6 shows the MS-full scan chromatograms obtained for a HFAB cyprodinil derivative extract before (a) and after (b) PSA clean up. There it is evident that a cleaner chromatogram is obtained when PSA is used, and if we compare the corresponding m/z 252 ion chromatograms (see Fig. 7), it is also evident that no loss of the derivatized compound is produced during the clean up process.
Supercritical fluid extraction of cyprodinil and its HFBA derivative First SFE experiments were performed to assess the extractability of cyprodinil from glass beads by using the SFE conditions proposed by Valverde et al.[11] for multiresidue analysis of pesticides in fruits and vegetables. Results obtained in these experiments showed that cyprodinil was extracted from glass beads with mean recovery values of 61% for the 0.5 mg spikes (n D 3; RSD D 15%) and 81% for the 1 mg spikes (n D 5; RSD D 12%). Likewise, HFBA-cyprodinil derivative could be efficiently extracted from glass beads by SFE, obtaining single recovery values ranging between 110% and 120%. These results confirm
that derivatization reactions may enhance the extractability of some analytes when derivatization decreases the polarity of the analytes and increase their solubility in supercritical fluids.[24] As far as we know, no data are available in the literature on the extractability of both cyprodinil and its HFBA derivative by using SFE, but some authors have reported cyprodinil recoveries ranges between 82% to 114% on fortified vegetables and grape juice using conventional extraction methods.[25–30]
Simultaneous supercritical fluid extraction and derivatization Results obtained in the initial experiments carried out to assess the possibility of carbon dioxide supercritical fluid simultaneous extraction/derivatization of cyprodinil in glass beads showed that the addition of any modifier to the extraction cell lead to obtain null recoveries of the HBFAcyprodinil derivative. It is to say, such as it was observed in the vial-derivatization experiments, detectable amounts of HBFA-cyprodinil derivative were only obtained when no solvent was present in the reaction cell. Recovery values obtained in the replicate experiments carried out without
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Fig. 7. m/z 252 ion chromatograms obtained for a HFBA cyprodinil derivative extract before (a) and after (b) PSA clean up.
modifier with 15 mL supercritical carbon dioxide at 50 C and 200 atm (n D 5), 300 atm (n D 7), and 400 atm (n D 5) were no reproducible (RSD > 50%) and ranged between 10% and 45% (related to the signal obtained for
derivatization in vial). So that, we can conclude that the SFE technique could be used for extraction of cyprodinil but it has serious limitations to be applied to the simultaneous extraction and derivatization of this pesticide.
Simultaneous extraction/derivatization of cyprodinil Funding This work was supported by the Spanish Ministry of Education and Science and the European Commission FEDER program (project AGL2005-01418/ALI).
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References [1] Masner, P.; Muster, P.; Schmid, J. Possible methionine biosynthesis inhibition by pyrimidinamine fungicides. Pestic. Sci. 1994, 42, 163–166. [2] Waechter, F.; Weber, E.; Hertner, T.; May-Hertl, U. Chapter 89 Cyprodinil: A fungicide of the anilinopyrimidine class. In Hayes’ Handbook of Pesticide Toxicology, 3rd Ed.; Krieger, R., Ed.; Academic Press: New York, 2010; 1903–1913. [3] Fenoll, J.; Ruiz, E.; Hellın, P.; Flores, P.; Navarro, S. Heterogeneous photocatalytic oxidation of cyprodinil and fludioxonil in leaching water under solar irradiation. Chemosphere. 2011, 85, 1262–1268. [4] Mol, H.G.J.; Sunarto, S.; Steijger, O.M. Determination of endocrine disruptors in w ater after derivatization with N-methyl-N(tert. Butyldimethyltrifluoroacetamide) using gas chromatography with mass spectrometric detection. J. Chromatogr. A. 2000, 879, 97–112. [5] Kataoka, H. Gas chromatography of amines as various derivatives. In Quantitation of amonoacids and amines by chromatography; Molnar-Perl, I., Ed.; Elsevier B.V.: Amsterdam, The Netherlands, 2005; 365–404. [6] Raeppel, C.; Nief, M.; Fabritius, M.; Racault, L.; Appenzeller, B.M. Simultaneous analysis of pesticides from different chemical classes by using a derivatisation step and gas chromatographymass spectrometry. J. Chromatogr. A. 2011, 1218, 8123–8129. [7] Lee, H.B. Perfluoro and chloro amide derivatives of aniline and chloroaniline. A comparison on their formation and gas chromatographic determination by mass selective and electron capture detectors. J. Chromatogr. 1988, 457, 267–278. [8] Carro, A.M.; Gonz alez, P.; Lorenzo, R.A. Application of derivatization reactions to trace organic compounds during sample preparation based on pressurized liquid extraction. J. Chromatogr. A. 2013, 1296, 214–225. [9] Lehotay, S.J.; Valverde, A. Evaluation of different solid-phase traps for automated collection and clean-up in the analysis of multiple pesticides in fruits and vegetables after supercritical fluid extraction. J. Chromatogr. A. 1997, 765, 69–84. [10] Aguilera, A.; Brotons, M.; Rodrıguez, M.; Valverde, A. Supercritical fluid extraction of pesticides from a table-ready food composite of plant origin (Gazpacho). J. Agric. Food Chem. 2003, 51(19), 5616–5621. [11] Valverde, A.; Fernandez-Alba, R.A.; Contreras, M.; and Ag€ uera A. Supercritical fluid extraction of pesticides from vegetables using anhydrous magnesium sulfate for sample preparation. J. Agric. Food Chem. 1996, 44(7), 1780–1784. [12] Aguilera, A.; Rodrıguez, M.; Brotons, M.; Boulaid, M.; Valverde, A. Evaluation of supercritical fluid extraction/aminopropyl solidphase “In-Line” cleanup for analysis of pesticide residues in rice J. Agric. Food Chem. 2005, 53, 9374–9382. [13] Field, J.A. Coupling chemical derivatization reactions with supercritical extraction. J. Chromatogr. A. 1997, 785, 239–249.
549 [14] Rochette, E.A.; Harsh, J.B. Supercritical fluid extraction of 2,4-D from soils using derivatization and ionic modifiers. Talanta. 1993, 40, 147–155 [15] Bakiamoh, S.B.; Maimat, R.; McGowin, A.E. Supercritical fluid extraction of chlorpyrifos and 3,5,6-trichloro-2-pyridinol from garden compost. J. Chromatogr. A. 1999, 862, 105–112. [16] King, J.W.; Zhang, Z. Derivatization reactions of carbamate pesticides in supercritical carbon dioxide. Anal. Bioanal. Chem. 2002, 374, 88–92. [17] Stolker, A.A.M.; van Ginkel, L.A.; Stephany, R.W.; Maxwell, R.J.; Prks, O.W.; Lightfield, A.R. Supercritical fluid extraction of methyltestosterone, nortestosterone and testosterone at low ppb levels from fortified bovine urine. J. Chromatogr. B. 1999, 726, 121–131. [18] Karg, F.P.M. Determination of phenylurea pesticides in water by derivatization with heptafluorobutyric anhydride and gas chromatography-mass spectrometry. J. Chromatogr. 1993, 634, 87–100. [19] Jimenez, J.J.; Nozal, M.J.; Bernal, J.L.; Santosa, M.; Mayorga, A.L. Factors affecting the extraction, hydrolysis and derivatization steps for the quantitation of total residues of amitraz in honey by gas chromatography with electron capture detection. Anal. Bioanal. Chem. 2002, 374, 300–304. [20] Worobey, B.L. Simplified heptafluorbutyrylation of linuron and its metabolite 3,4-dichloroaniline. J. Chromatogr. 1983, 262, 328–330. [21] Seiber, J.N. N-Perfluoroacyl derivatives for methylcarbamate analysis by gas chromatography. J. Agric. Food Chem. 1972, 20, 443–446. [22] Bose, J.R. Formation of the N-trifluoroacetate of carbofuran. J. Agric. Food Chem. 1977, 25, 1209–1210. [23] Herberer, T.; Stan, H. Detection of more than 50 substituted phenols as their t-butyldi-methylsilyl derivatives using gas chromatography-mass spectromethry. Analyt. Chim. Acta 1997, 341, 21–34. [24] Reche, F.; Garrig os, M.C.; S anchez, A.; Jimenez, A. Simultaneous supercritical fluid derivatization and extraction of formaldehyde by the Hantzsch reaction. J. Chromatogr. A. 2000, 896, 51–59. [25] Pose-Juan, E.; Cancho-Grande, B.; Rial-Otero, R.; Simal-Gandara, J. The dissipation rates of cyprodinil, fludioxonil, procymidone and vinclozoline during storage of grape juice. Food Control. 2005, 17, 1012–1017. [26] Munitz, M.S.; Resnik, S.L.; Montti, M.T. Method development and validation for cyprodinil and fludioxonil in blueberries by solid-phase microextraction gas chromatography, and their degradation kinetics. Food Addit. Contam. Part A. 2013, 30, 1299–1307. [27] Marin, A.; Olive, J.; Garcıa, C.; Navarro, S.; Barba, A. Dissipation rates of cyprodinil and fludioxolnil in lettuce and table grape in the field and under cold storage conditions. J. Agric. Food Chem. 2003, 51, 4708–4711. [28] Garau, V.L.; Angioni, A.; Aguilera del Real, A.; Russo, M.T.; Cabras, P. Disappearance of azoxystrobin, pyrimethanil, cyprodinil and fludioxonil on tomatoes in a greenhouse. J. Agri. Food Chem. 2002, 50, 1929–1932. [29] Giza, I.; Sztwiertnia, U.; Murawska, M. Gas chromatographic determination of cyprodinil and pyrimethanil residues in apples. Acta Chormatogr. 2001, 11, 37–41. [30] G omez-Ramos, M.M.; Ferrer C.; Malato, O.; Ag€ uera, A.; Fern andez-Alba, A.R. Liquid chromatography-high-resolution mass spectrometry for pesticide residue analysis in fruit and vegetables: screening and quantitative studies. J. Chromatogr. A. 2013, 1287, 24–37.
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Assessing solvent derivatization and carbon dioxide supercritical fluid simultaneous extraction/ derivatization of cyprodinil a
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Ana Aguilera , Antonio Valverde , Antonio Valverde-Monterreal , Luis Garcia-Fuentes & a
Mourad Boulaid a
Pesticide Residue Research Group, University of Almería, Almería, Spain
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Servicio Oficial de Inspección, Vigilancia y Regulación del Comercio Exterior, Almería, Spain
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Chemistry & Physics Department, Faculty of Experimental Sciences, University of Almería, Almería, Spain Published online: 05 Jun 2014.
To cite this article: Ana Aguilera, Antonio Valverde, Antonio Valverde-Monterreal, Luis Garcia-Fuentes & Mourad Boulaid (2014) Assessing solvent derivatization and carbon dioxide supercritical fluid simultaneous extraction/derivatization of cyprodinil, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 49:8, 541-549, DOI: 10.1080/03601234.2014.911553 To link to this article: http://dx.doi.org/10.1080/03601234.2014.911553
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Journal of Environmental Science and Health, Part B (2014) 49, 541–549 Copyright © Taylor & Francis Group, LLC ISSN: 0360-1234 (Print); 1532-4109 (Online) DOI: 10.1080/03601234.2014.911553
Assessing solvent derivatization and carbon dioxide supercritical fluid simultaneous extraction/derivatization of cyprodinil ANA AGUILERA1, ANTONIO VALVERDE1, ANTONIO VALVERDE-MONTERREAL1,2, LUIS GARCIA-FUENTES3 and MOURAD BOULAID1 Pesticide Residue Research Group, University of Almerıa, Almerıa, Spain Servicio Oficial de Inspecci on, Vigilancia y Regulaci on del Comercio Exterior, Almerıa, Spain 3 Chemistry & Physics Department, Faculty of Experimental Sciences, University of Almerıa, Almerıa, Spain 1
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Derivatization of cyprodinil with different reagents and solvents has been evaluated to improve the GC/MS characterization of this fungicide. After assessing some preliminary acylation and silylation reactions, derivatization with anhydrous heptafluorobutyric anhydride (HFBA) was selected as the best derivatization option for cyprodinil. The HFBA-cyprodinil derivative was clearly identified and characterized by GC/MS (ion-trap). The spectrum of the HFBA derivative of cyprodinil was characterized by the base peak, 252 m/z ion, and two other ions with relative abundances of 5% (224 m/z ion) and 4% (420 m/z molecular ion). Conversion rates in the range of 83–92% were obtained when 0.1–1 mg cyprodinil were derivatized in vial without solvent at 25 C temperature for 120 min, with 5 mL HFBA and 5 mL pyridine. Simultaneous extraction-derivatization of cyprodinil in supercritical carbon dioxide was only achieved when no modifier was present, but conversion/recovery rates obtained in the replicate experiments carried out with 15 mL supercritical carbon dioxide at 50 C and 200 atm (n D 5), 300 atm (n D 7), and 400 atm (n D 5) were no reproducible (RSD > 50%) and ranged between 10% and 45% (related to the signal obtained for derivatization in vial). Keywords: Derivatization, GC/MS, SFE, cyprodinil.
Introduction Cyprodinil (4-cyclopropyl-6-methyl-N-phenylpyrimidine) is a systemic fungicide of the anilinopyrimidine family, a new chemical class of fungicides that are highly active against a broad range of fungi. The biological mode of action includes inhibition of methionine biosynthesis and secretion of fungal hydrolytic enzymes.[1] It was first introduced for application on cereals grains and it is currently used as a foliar fungicide in cereal grains, grapes, pome fruits, stone fruits, strawberries, vegetables, field crops, and ornamental.[2] In the southeast of Spain it is mainly used to control botrytis and sclerotinia, fungi that infect greenhouse vegetables.[3] Although the correct use of pesticides does not cause problems of public concern in health and environmental areas, if inappropriate treatments are applied and/or without respecting safety recommendations, undesiderable
Address correspondence to Ana Aguilera, Pesticide Residue Research Group, Faculty of Experimental Sciences, University of Almerıa, 04071 Almerıa, Spain; E-mail: [email protected]
residues can remain in crops. Analytical methods for determining pesticide residues include commonly gas chromatography (GC) or high-performance liquid chromatography. Analytes can be determined by GC with or without a previous derivatization step. Derivatization is employed not only to improve the thermal stability of polar compounds, but also to minimize undesiderable interactions with the column and injection liner resulting in poor peak shape or loss of mass through adsorption. Also, derivatization can be used to reposition peaks in the chromatogram from regions of extensive overlap to regions that are relatively empty of interfering peaks and to improve, if possible, the mass spectral properties of derivatives. For those analytes that present a labile hydrogen coming from different chemical function, such as secondary amine group (-NH-), primary amine group (-NH2), carboxylic group (-COOH), or hydroxyl group (-OH), several problems are usually encountered with trace level analyses; losses due to adsorption in the inlet and peak tailing due to the interaction of the analyte with active sites in the analytical column may occur.[4] Amines tend to be adsorbed by and decompose on columns resulting in tailed elution peaks, ghosting phenomena and low detector sensitivity. The
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542 derivatization for the protection of amino groups is widely employed to increase volatility and to improve the chromatographic and, if possible, mass spectral properties of derivatives.[5] Acylation (addition of –COR instead a labile -H) is one of the most widely used procedure for GC analysis of primary and secondary amines. The introduction of acyl protective groups improves the volatility, chromatographic mobility, and chemical stability of these amines.[6,7] Traditional extraction techniques require long extraction times and variable amounts of sorbents and organic solvents. Cost associated to the purchase of organic solvents and the concerns on their negative environmental impact and on human health should limit their use.[8] Supercritical fluid extraction (SFE) has been proven to be an efficient alternative for extraction of pesticide residues.[9–12] Moreover, supercritical fluids offers the possibility of developing chemical derivatization under controlled conditions of pressure and temperature, and several researchers have devised supercritical fluids derivatization and extraction (SFDE) methods, which have been revised by Field.[13] In addition of reducing sample handling and decreasing the number of preparative steps of the extracts, in situ derivatization could have the benefits that reagents can act as modifiers during the SFE and enhance the solubility of analytes in supercritical CO2. However, there is a little reference available on the application of this derivatization technique for the determination of pesticide residues.[14–16] In fact, derivatization reactions in supercritical CO2 should be chosen carefully to ensure their compatibility with the conditions used for SFE of the target analytes. The optimum reagent, reaction conditions and final analytical method must all be considered when integrating a derivatization reaction into a SFDE scheme. Certain combinations of SFE conditions might be deleterious to the formation of the derivative desired, and this should be determined initially on neat analytes before attempting derivatization in situ. The study carried out in this work had two aims. The first one was to assess the conditions required for the formation, using a simple and rapid reaction, and under mild conditions, of cyprodinil derivative to enable good GC/ MS characterization and the second one was to assay the applicability of the SFDE on this compound.
Materials and methods Reagents and materials Ethyl acetate, toluene, cyclohexane, acetone, acetonitrile, dichloromethane, and methanol were pesticide residue grade from Panreac (Barcelona, Spain) and Scharlab (Barcelona, Spain). High-purity anhydrous heptafluorobutyric anhydride (HFBA) was purchased from Supelco
Aguilera et al. (Bellefonte, PA, USA). Analytical grade dried pyridine was from Fluka (Buchs, Switzerland). Certified standard of cyprodinil (purity 99.9%) was supplied by Riedel de H€aen (Seelze, Germany). A stock standard solution of about 1000 mg L-1 of cyprodinil was prepared in acetone. Working standard solutions (0.05–10 mg L-1) were prepared in toluene and other solvents (ethyl acetate, cyclohexane, acetone, acetonitrile, dichloromethane, and methanol) from the stock standard solution by suitable dilutions. Pure standard and standard solutions were stored in dark at ¡20 C. Glass beads (3 mm diameter) were obtained from Scharlab (Barcelona, Spain), and glass microfiber filter discs from Whatman (Maidstone, England). Primary-secondary-amine (PSA) sorbent was supplied by Varian (Harbor City, CA, USA). Carbon dioxide (99.995% purity), helium and nitrogen (99.999% purity) were supplied by Air Liquide (Madrid, Spain).
GC-MS analysis Cyprodinil and cyprodinil-HFBA derivative were analysed by GC-MS by using a Varian (Walnut Creek, CA, USA) 3400 gas chromatograph-Saturn 3 ion trap mass spectrometer equipped with a model 1077 injection port, and a model 8200 CX autosampler, fitted with a DB-5MS fused-silica capillary GC column (30 m £ 0.25 mm i.d.., 0.25 mm film thickness) were used. Operating conditions for GC-MS were: 9 psi helium column head pressure; 0.75 min splitless time; 250 C injector temperature; 2 mL injection volume; 60 C initial oven temperature for 1 min, ramped to 180 C at 25 C/min, then to 280 C at 5 C/min, and held at 280 C for 15 min; 280 C transfer line temperature; and 220 C ion-trap manifold temperature. MS measurements were performed with electron impact (EI) at 70 eV in the full scan mode over the mass range of m/z 60 to 650 at 1 scan/s from 6 to 35 min. Saturn GC-MS version 5.2 software (Varian, Walnut Creek, CA, USA) was used for data collection.
SFE system An Isco SFE system, consisting of a model 260D syringe pump and controller able to operate in the pressure range of 0.6 up to 510 atm (1 atm D 101.3 kPa); a SFX 2-10 extractor with restrictor heater set at 70 C, and 2.5 stainless steel extraction cartridges with removable 2 mm frits, was used in this study. Uncoated and deactivated fused silica capillary column, 30 cm length £ 50 mm i.d., was used as restrictor, and 10 mL graduated test tubes (immersed in a 15–20 C water bath), containing 5 mL ethyl acetate, was used as collection system. In the equipment the CO2 flow is dependent of pressure and type of restrictor used. With the restrictor selected the flow rates ranged between 1– 1.2 mL min-1 with a pressure of 300 atm.
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Derivatization in organic solvents Derivatization reactions were carried out on 2 mL reaction vials, using a reactitherm dry bloc derivatization system (Pierce, Rockford, IL, USA) to heat the reaction mixtures. 0.1–1 mL of working standard solutions of cyprodinil (0.05–5 mg L-1) were placed in the reaction vials and, after mixing with the derivatization reagent (HFBA) and catalyzer (pyridine), the vial was sealed and allowed to react for 30–120 min (or overnight) in the dark at the assessed temperature (25 C and 60 C). The corresponding derivative was purified by purging with dry nitrogen followed by evaporation to dryness. The resulting residue was redissolved with 0.1–1 mL toluene and analysed by GC-MS. In addition to temperature and reaction time, the following derivatization conditions were evaluated: (a) volumes of HFBA (5–100 mL) and pyridine (0–100 mL) used to perform the acylation reaction and (b) volume (0.1 and 1 mL) and solvent type (ethyl acetate, toluene, cyclohexane, acetone, acetonitrile, dichloromethane, and methanol) of the working standard solution added to the reaction vial. Derivatization in vial without solvent was also evaluated by means of some experiments in which the solvent of the standard solution introduced in the vial was evaporated to dryness with dry nitrogen before adding the derivatization reagents. The following optimized conditions were selected to evaluate the linearity and reproducibility of derivatization process in vial, and to obtain the cyprodinil-HFBA derivative solutions used as reference in the SFE studies: derivatization in vial without solvent at 25 C temperature for 120 min, with 5 mL HFBA and 5 mL pyridine. Supercritical fluid extraction of cyprodinil and its HFBA derivative All SFE experiments were performed on glass beads packed inside 2.5 mL extraction cartridges. SFE recovery tests of cyprodinil were carried out at the spiking levels of 0.5 mg and 1 mg (100 mL of cyprodinil standard solutions in toluene of 5 or 10 mg L¡1), performing 3 and 5 replicates per level, respectively. SFE conditions were those proposed by Valverde et al.[11] for multiresidue analysis of pesticides in vegetables: dynamic extraction with 15 mL carbon dioxide at 300 atm pressure and 50 C temperature after five minutes in the static mode. Final SFE extracts were transferred to 2 mL vials, evaporated to dryness and dissolved with 1 mL toluene. Similar tests were conducted on glass beads spiked with 100 mL of the 1 mL solution of HFBA-cyprodinil derivative previously obtained by derivatization in vial of 1 mg of cyprodinil. Final SFE extracts were evaporated to dryness, dissolved with 100 mL toluene and analyzed by GC/MS, the chromatographic signal being compared with that obtained for the corresponding spiking solution, which was analyzed in the same chromatographic sequence.
Simultaneous supercritical fluid extraction and derivatization Simultaneous SFE and derivatization of cyprodinil with HFBA reagent was assessed in two steps. The first step consisted in a static period of 30 min, whereas the second one was performed under dynamic conditions just after the static period. In all cases, these experiments were carried by filling the SFE vessels with glass beads and spiking them with 100 mL of cyprodinil standard solution of 1 ppm, the derivatization reagents (5 mL HFBA and 5 mL Pyridine) being added after the solvent was evaporated under N2 stream and just before sealing the vessel. After pressurizing with CO2 at 300 atm, the SFE vessels were held statically at 50 C for 30 min. Dynamic SFE was then performed by passing through the cell 15 mL CO2 (300 atm pressure and 50 C temperature) for 10–15 min (CO2 flow rate 1–1.2 mL min-1) and using 5mL ethyl acetate as collection system. Different static modifier conditions were assessed: (a) 200 mL or 100mL of modifier (methanol, ethyl acetate, cyclohexane, acetone, toluene, and acetonitrile) and (b) no modifier. All the extractions were carried out in duplicate and the volume of final extracts was adjusted to 1 mL prior to GC-MS detection. Additional replicate experiments (n D 5) were performed without modifier at 200, 300, and 400 atm pressure, keeping constant the other SFE parameters.
Results and discussion GC/MS characterization of HFBA-cyprodinil derivative Cyprodinil is a secondary amine that presents one labile hydrogen and may react with HFBA to form its acylderivative. However, as far as we know, there is no reference available on the characterization of this derivatization reaction to be used for the determination of this pesticide by GC/MS analysis. The expected reaction of cyprodinil with HFBA is shown in Figure 1. This acylation reaction with HFBA was selected after assessing some preliminary acylation and silylation reactions of cyprodinil using different derivatization reagents. The HFBA derivative of cyprodinil was obtained in a number of these preliminary experiments, being identified and characterized by GC/MS. As can be seen in Figure 2, under the GC/ MS instrumental parameters above indicated, cyprodinil
Fig. 1. Course of the derivatization reaction of cyprodinil with HFBA.
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Fig. 2. m/z 224 ion chromatogram of a standard solution of cyprodinil (1 mg L-1) in toluene (a); and m/z 224 (b) and m/z 252 (c) ion chromatograms of the corresponding HFBA-cyprodinil derivative extract obtained by derivatization in vial under the optimized conditions.
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ions. The spectrum of the HFBA derivative of cyprodinil is characterized by the base peak, 252 m/z ion, and two other ions with relative abundances of 5% (224 m/z ion) and 4% (420 m/z ion). These spectral data illustrate that the molecular ion (420 m/z) was not the major ion in the spectra, corresponding the base peak 252 m/z to the fragment [M-169]. The 224 m/z [M-197] fragment is also the molecular ion observed in the spectrum of cyprodinil. These data confirm that cyprodinil derivative is obtained from the proton substitution of the NH moiety by the heptafluoro-butyric acyl group, and that the MS fragmentation pathway of the cyprodinil derivative involves the successive losses of the C3F7 and CO groups, such as it is indicated in the scheme proposed in Figure 4.
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and its HFBA derivative eluted at 15.7 and 13.2 min, respectively. Figure 2a shows the 224 m/z ion chromatogram obtained for a toluene standard solution of cyprodinil of 1 ppm, whereas Figure 2b and c include the 224 and 252 m/z ions chromatograms of the HFBA derivative obtained for 1 ppm cyprodinil standard under the optimized vial-derivatization conditions (see the section “Derivatization in vial”). Figure 3a and b show the corresponding mass spectra obtained for cyprodinil and its HFBA derivative, respectively. Examination of the mass spectrometric features of cyprodinil and the corresponding heptafluorobutiramide derivative was carried out in order to confirm the identity of the derivative by studying the molecular and fragments
Fig. 3. Mass spectra of cyprodinil (a) and HFBA-cyprodinil derivative (b).
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Fig. 4. Molecular structure of HFBA-cyprodinil derivative and the proposed MS fragmentation pathway.
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Derivatization in vial The acylation reaction of cyprodinil with HFBA could be affected supposedly by many parameters, such as reaction temperature, reaction time, amount, and type of solvent and catalyzer.[8,5] The influence of these parameters was investigated by using 2 mL vials as reactor cell. After assessing the derivatization reaction in vial with 0.1 and 1 mL standard solutions of 1 ppm cyprodinil in different solvents (ethyl acetate, toluene, cyclohexane, acetone, acetonitrile, dichloromethane, and methanol), it was found that suitable amounts of the cyprodinil derivative are only obtained when the standard solution of cyprodinil in toluene was evaporated to dryness before adding the derivatization reagents. This is the reason for which optimization experiments were performed by adding 5 mL of HFBA and 5 mL pyridine to dry residues of cyprodinil obtained by evaporation, with the aid of N2 stream, of standard solutions of cyprodinil (100 mL or 1 mL). A similar procedure has been followed by Stolker et al. for the derivatization of steroids from urine.[17] Experiments carried out to asses the influence of the catalyzer showed that the addition of pyridine is a key factor to develop the derivatization reaction because when no catalyst was added to the reaction mixture no derivatization product was observed. However, the addition of more than 5 mL of pyridine (in the range of 5–100 mL) had no effect on the product yield. It was also found that increasing both derivatization time (from 2 h up to 8 h) and temperature (from 25 C to 60 C) has no influence on the derivatization process, these results being similar to those found by Karg and others authors.[18–22] As a result, the following optimized conditions were selected to evaluate the linearity and reproducibility of the vial-derivatization process, and to obtain the cyprodinil-HFBA derivative solutions used as reference in the SFE studies: derivatization without solvent at 25 C temperature for 120 min, with 5 mL HFBA and 5 mL pyridine. Linear calibration curves were obtained from the m/z 252 ion chromatogram, at four different levels (0.1, 0.2, 0.5, and 1 mg of cyprodinil; 1 mL final volume of the GC/ MS derivatized extracts), in four different days, obtaining in all cases correlation coefficients over 0.99 with relative
standard deviations of the response factors in the range of 5–20%. Figure 5 shows an example of the regression lines obtained from the m/z 252 and 224 ions chromatograms. The slopes of the linear calibration curves (m/z 252 ion) ranged between 1.4 £ 105 and 1.8 £ 105 height units/mg L¡1, the ratio of the m/z 224 and m/252 regression lines being, in all cases, into the range of 4.5–5.5%. The amount of non-derivatized cyprodinil remaining in these experiments was determined by quantification of the cyprodinil signal, at 15.7 min, in the m/z 224 ion chromatograms, and ranged between 8% and 17%, these data representing conversion rates of cyprodinil in the range of 83–92%. As described in literature, potential problems with derivatization procedures can be, among others, the presence of unchanged derivatization reagents, which can affect chromatographic column and/or chromatographic analysis.[5,23] In order to avoid these potential problems, the addition of a sorbent material to the derivatized extracts was also evaluated as a last clean up step in the vial-derivatization procedure. 1 mL of the derivatized cyprodinil solution, previously analyzed by GC/MS, was placed in a polypropylene centrifuge tube containing 10 mg of PSA. Sample was shaken with the adsorbent for 30 s and then centrifuged at 3700 rpm (2 min), the supernatant being
Fig. 5. Regression lines obtained from the m/z 252 and 224 ions chromatograms after derivatization in vial (optimized conditions) of 0.1, 0.2, 0.5, and 1 mg of cyprodinil (1 mL final volume of GC/MS extracts).
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Fig. 6. Full scan chromatograms obtained for a HFBA cyprodinil derivative extract before (a) and after (b) PSA clean up.
again analyzed by GC/MS. Figure 6 shows the MS-full scan chromatograms obtained for a HFAB cyprodinil derivative extract before (a) and after (b) PSA clean up. There it is evident that a cleaner chromatogram is obtained when PSA is used, and if we compare the corresponding m/z 252 ion chromatograms (see Fig. 7), it is also evident that no loss of the derivatized compound is produced during the clean up process.
Supercritical fluid extraction of cyprodinil and its HFBA derivative First SFE experiments were performed to assess the extractability of cyprodinil from glass beads by using the SFE conditions proposed by Valverde et al.[11] for multiresidue analysis of pesticides in fruits and vegetables. Results obtained in these experiments showed that cyprodinil was extracted from glass beads with mean recovery values of 61% for the 0.5 mg spikes (n D 3; RSD D 15%) and 81% for the 1 mg spikes (n D 5; RSD D 12%). Likewise, HFBA-cyprodinil derivative could be efficiently extracted from glass beads by SFE, obtaining single recovery values ranging between 110% and 120%. These results confirm
that derivatization reactions may enhance the extractability of some analytes when derivatization decreases the polarity of the analytes and increase their solubility in supercritical fluids.[24] As far as we know, no data are available in the literature on the extractability of both cyprodinil and its HFBA derivative by using SFE, but some authors have reported cyprodinil recoveries ranges between 82% to 114% on fortified vegetables and grape juice using conventional extraction methods.[25–30]
Simultaneous supercritical fluid extraction and derivatization Results obtained in the initial experiments carried out to assess the possibility of carbon dioxide supercritical fluid simultaneous extraction/derivatization of cyprodinil in glass beads showed that the addition of any modifier to the extraction cell lead to obtain null recoveries of the HBFAcyprodinil derivative. It is to say, such as it was observed in the vial-derivatization experiments, detectable amounts of HBFA-cyprodinil derivative were only obtained when no solvent was present in the reaction cell. Recovery values obtained in the replicate experiments carried out without
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548
Fig. 7. m/z 252 ion chromatograms obtained for a HFBA cyprodinil derivative extract before (a) and after (b) PSA clean up.
modifier with 15 mL supercritical carbon dioxide at 50 C and 200 atm (n D 5), 300 atm (n D 7), and 400 atm (n D 5) were no reproducible (RSD > 50%) and ranged between 10% and 45% (related to the signal obtained for
derivatization in vial). So that, we can conclude that the SFE technique could be used for extraction of cyprodinil but it has serious limitations to be applied to the simultaneous extraction and derivatization of this pesticide.
Simultaneous extraction/derivatization of cyprodinil Funding This work was supported by the Spanish Ministry of Education and Science and the European Commission FEDER program (project AGL2005-01418/ALI).
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