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Simultaneous determination of various pesticides in fruit juices by HPLC-DAD
Food Control 16 (2005) 87–92 www.elsevier.com/locate/foodcont
Simultaneous determination of various pesticides in fruit juices by HPLC-DAD € Sibel Topuz, G€ ul Ozhan, Buket Alpertunga * Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey Received 14 April 2003; received in revised form 25 November 2003; accepted 27 November 2003
Abstract A suitable method for the simultaneous determination of four fungicides (folpet, chlorothalonil, quinomethionat, tetradifon) and one herbicide (trifluralin) in fruit juices has been developed. The method involves the preconcentration of 25 g fruit juice samples through C18 solid-phase extraction cartridges. The pesticides were separated and quantified by reversed-phase high-performance liquid chromatography with UV-diode array detection at 220 and 260 nm. Analytical separation was performed using a concave gradient elution with acetonitrile and water on a C18 column. Recoveries from spiked apple, cherry juices and peach nectar, ranged from 93.8% to 99.5% and relative standard deviations were less than 3.4% in the concentration range of 1–16 lg/kg. The limits of detection for pesticides investigated are in the range of 0.5–1 lg/kg and the calibration curves showed good linearity >0.9988. The developed method has been tested on canned pure apple, cherry juices and peach nectar manufactured in Turkey. Results of this study showed that these samples analyzed contained no detectable residues. 2004 Elsevier Ltd. All rights reserved. Keywords: Fruit juice analysis; Pesticides; Solid-phase extraction; HPLC-DAD
1. Introduction Although the use of pesticides provides unquestionable benefits in providing a plentiful, low-cost supply of high-quality fruits and vegetables, their incorrect application may leave harmful residues, which involve possible health risk (Torres, Pic o, & Ma~ nes, 1996). As a consequence, governments and international organizations are established maximum residue limits (MRLs) in fruits and vegetables to ensure their levels (EC Council Directive, 1990). A great portion of the pesticide residues is fungicides and herbicides, which are used primarily to control spoilage of fruits and vegetables through fungal attack and to control weeds. The most frequently used methods for analysis of the fungicides and herbicides in fruits and vegetables are gas chromatography (GC) with nitrogen– phosphorous (NPD), electron-capture (ECD), a mass spectrometric (MS) detection and liquid chromatography (LC) with ultraviolet (UV) and diode-array detection (DAD) (Fillion, Sauve, & Selwyn, 2000; Hsu, *
Corresponding author. Tel.: +90-212-526-0797; fax: +90-212-5190812. E-mail address: [email protected] (B. Alpertunga). 0956-7135/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2003.11.012
Schattenberg, & Garza, 1991; Mark Lee, Papathakis, Feng, Hunter, & Carr, 1991; Martinez Vidal, Arrebola, & Mateu-Sanchez, 2002; Neidert, Trotman, & Saschenbrecker, 1994; Newsome & Collins, 1989; Pic o, Font, Molt o, & Ma~ nes, 2000). These methods are based on liquid–liquid extraction (LLE), solid-phase extraction (SPE), matrix solid-phase dispersion and supercritical fluid extraction (SFE) (Fodor-Csorba, 1992; Mattern, Liu, Louis, & Rosen, 1999; Navarro, Pic o, Marin, & Ma~ nes, 2002; Tadeo, Sanchez-Brınete, Perez, & Fernandez, 2000; Torres, Pic o, Redondo, & Ma~ nes, 1996). Fruit is often treated close to harvest or postharvest to ensure that wholesome produce reaches the consumer. The presence of pesticide residues in canned pure fruit juices can be a significant route to human exposure. Only a few procedures have been reported for the analysis of pesticide residues in fruit juices (Abad & € Montoya, 1995; Bushway, 1988; Ozhan, Topuz, & Alpertunga, 2003; Perret, Gentili, Marchese, Sergi, & D’Asceno, 2002). The development of easy analytical methods to monitor these pesticides in fruit juices is very important. In this work, we described a method based on SPE-HPLC to determine four fungicides (folpet,
88
S. Topuz et al. / Food Control 16 (2005) 87–92
centrations range of 0.025–0.4 lg/ml, were obtained by dilution with mobile phase (Acetonitrile–water, 40:60, v/ v). The standard solutions were store at 4 C and were used for 3 months.
CN Cl
O
Cl
SCCl3
N
Cl
CN Cl
O Folpet
Chlorothalonil
2.3. Analysis Cl N
S
O
O
Cl H3C
N
S
S
Cl
O Cl
Quinomethionat
Tetradifon
N(CH2CH2CH3)2 O2N
NO2
CF3
Trif luralin
Fig. 1. Structures of the studied compounds.
chlorothalonil, quinomethionat, tetradifon) and one herbicide (trifluralin), frequently used on various fruits in Turkish agriculture in fruit juices. The structures of these pesticides are illustrated in Fig. 1. The developed method has been tested on canned pure apple, cherry juices and peach nectar manufactured in Turkey.
2. Materials and methods 2.1. Materials All pesticide standards were of 98–99% purity and were purchased from Riedel-de Ha€en (Seelze, Germany). All solvents (HPLC grade) were obtained from Merck (Darmstadt, Germany). A Milli-Q water purification system from Millipore (Bedford, MA, USA) was used to provide ultra-pure water in this study. C18 SPE cartridges (Bond Elut LRC-C18 , 200 mg/10 ml, Varian, CA, USA) were used in the preconcentration step. Samples were passed through the cartridges using vacuum extraction manifold system (Vac Elut 20, Varian, CA, USA). Mobile phase filtration was obtained using a 0.2 lm membrane filter (Phenomenex, CA, USA). One hundred percent apple, cherry juices and peach nectar commercially available were used. 2.2. Preparation of solutions The stock standard solutions of folpet, chlorothalonil, quinomethionat, tetradifon and trifluralin (500 lg/ ml) were prepared in acetonitrile and these were diluted with acetonitrile to give a standard solution of 5 lg/ml. Working standard solutions of each pesticide at con-
Liquid chromatographic analysis were performed with a Thermo Separation Products Liquid Chromatograph (Model Spectra System , TSP, CA, USA), equipped with HPLC pump (Spectra Series pump P4000), vacuum degasser for liquid chromatography (Solvent degasser SCM 1000), rheodyne and injection valve (Injection volume: 50 ll). System parameters were controlled with system controller (SN 4000) and chromatographic data were collected and recorded using the PC 1000 system software. The separation was carried out using a C18 , 5 lm Luna column (250 mm · 4.6 mm ID, Phenomenex, CA, USA) fitted with guard column (4 mm L · 3 mm I.D., Phenomenex, CA, USA) packed with same material. The column eluate was monitored with a UV 6000LP photo-diode array detector. The chromatographic separation was carried out using a concave gradient profile of acetonitrile and water going from 40% of acetonitrile to 90% in 20 min, this condition was held for 5 min and then back to the initial conditions. The flow rate of the mobile phase was 1 ml/min and column temperature was ambient. The DAD was set at 220 and 260 nm with a bandwidth of 4 nm. Absorbance spectra were recorded in the 200–360 nm range. 2.4. Extraction procedures In all, 10 each of apple and cherry juices and peach nectar produced by different companies were collected from the market. Fruit juice samples were stored at 4–10 C and were analyzed in 2 days. Methanol (15 ml) was added to 25 g juice sample and mixed for 10 min in a 50 ml volumetric flask. The mixture was taken to 50 ml with water. Ten milli liter aliquot of this solution was further diluted to 50 ml water in a second volumetric flask. SPE cartridges were conditioned with 5 ml of methanol and 5 ml of water. The sample was passed through the cartridges using vacuum extraction manifold system at a rate of ca. 8–10 ml/min. After washing with 2.5 ml of water, the sorbent bed was dried under vacuum for 10 min. The analytes were eluted with 3 ml of dichlormethane into a vial. The eluate was then evaporated under a gentle stream of nitrogen at 40 C. The residue dissolved in the mobile phase to a final volume of 0.2 ml and 50 ll an equivalent of 1.25 g fruit juice was injected into the HPLC system.
S. Topuz et al. / Food Control 16 (2005) 87–92
89
Fig. 2. (A) LC-DAD chromatograms of (a) unspiked apple juice sample, (b) apple juice spiked at 4 lg/kg with pesticides registered at 220 and 260 nm. Peaks (retention times, min): 1 ¼ folpet (15.65), 2 ¼ chlorothalonil (16.29), 3 ¼ quinomethionat (22.39), 4 ¼ tetradifon (24.11), 5 ¼ trifluralin (25.14). (B) LC-DAD chromatograms of (a) unspiked cherry juice sample, (b) cherry juice spiked at 4 lg/kg with pesticides registered at 220. (C) LC-DAD chromatograms of (a) unspiked peach nectar sample, (b) peach nectar spiked at 4 lg/kg with pesticides registered at 220.
90
S. Topuz et al. / Food Control 16 (2005) 87–92
2.5. Recovery studies By using the samples spiked with these pesticides the recoveries were assessed at concentrations of 1, 2, 4, 8, 16 lg/kg in fruit juices. Spiked fruit juice samples were extracted on C18 cartridges and chromatographed using the procedure outlined above for each concentration in five replicates.
Table 1 Extraction recovery (%) for the assay of pesticides in apple juice (n ¼ 5) Compound
Recovery (%)
RSD (%)
1.95 ± 0.06 7.86 ± 0.14 15.92 ± 0.23
97.70 98.21 99.50
3.1 1.8 1.5
2
1.88 ± 0.04
94.20
2.1
8 16
7.82 ± 0.26 15.71 ± 0.51
97.71 98.20
3.3 3.3
2
1.90 ± 0.06
94.80
3.4
8 16
7.62 ± 0.22 15.55 ± 0.22
95.23 97.20
2.9 1.4
Tetradifon
2 8 16
1.91 ± 0.04 7.89 ± 0.11 15.89 ± 0.13
95.30 98.68 99.30
2.1 1.4 0.8
Trifluralin
2 8 16
1.88 ± 0.11 7.61 ± 0.07 15.34 ± 0.28
93.80 95.10 95.90
0.6 1.0 1.8
Folpet
Concentration (lg/kg) Added
Found (mean ± SD)
2 8 16
Chlorothalonil
3. Results and discussion The compounds investigated are very extensively applied pesticides for various fruits (apple, cherry and peach) in Turkish agriculture. Because of the fact that fruit juices are more concentrated than a single fruit, they may contain pesticide residues. Therefore intake of fruit juices may be significant route of human exposure to the pesticide. Further children drink a proportionally higher amount of fruit juices than adults, and for this reason pesticide analysis of fruit juices are especially important. The presence of natural pigments makes the analysis of fruit juices difficult. Clean up on C18 cartridges eliminated most interfering peaks and allowed good recoveries at low fortification levels. Complete separation of all compounds was achieved using a concave gradient elution with acetonitrile and water on a C18 column. This afforded good resolution in a reasonable time. A linear curve gradient did not allow satisfactory separation for folpet and chlorothalonil. The pesticides studied are highly absorbing substances in the UV region of the spectrum, with absorption maximum at 260 nm for quinomethionat and 220 nm for folpet, tetradifon, chlorothalonil and trifluralin. The procedure is illustrated in Fig. 2, which shows the chromatograms of unspiked apple, cherry juices and peach nectar and spiked samples at 4 lg/kg level with pesticides. As can be seen, the chromatograms were clean without interfering peaks in the areas of interest. SPE with C18 cartridges was used in order to achieve suitable sensitivity. The various extraction solvents were studied for the elution of the pesticides retained in the SPE cartridges. Although the elution of pesticides with different solvents produced similar recoveries, dichlormethane was considered best for extraction because a good baseline and less interfering peaks were obtained with this solvent. Juices containing no detectable amount of these pesticides were spiked at concentrations ranging from 1 to 16 lg/kg. Recoveries obtained for apple juice sample spiked with pesticides at three concentration levels are shown in Table 1. The similar recoveries were obtained for cherry juices and peach nectar in the range of 1–16 lg/kg levels of spiking. Average recoveries ranged from 93.8% to 99.5%.
Quinomethionat
Quantification of these pesticides in the sample extract was performed by use of integrated peak area with their known external standard. The linearity was observed for all of the pesticides in the range of 1–16 lg/kg (n ¼ 5) in apple juices, yielding correlation coefficients higher than 0.9988 (Table 2). The linearity values were also checked for jerry juices and peach nectar and the corresponding results were similar as reported for apple juices. The limits of detection (LODs) were calculated as the lowest concentration giving a response of three-times the average of the baseline noise defined from unspiked juices. LODs for the pesticides investigated were in the range of 0.5–1 lg/kg (Table 2). The limits of quantification (LOQs) for this method were defined as the lowest concentration of compounds in a sample that could be quantitatively determined with suitable precision and accuracy. LOQs of folpet, chlorothalonil and quinomethionat were found to be 1 lg/kg with relative standard deviation (RSD) of 0.5–1.4%. LOQs of tetradifon and trifluralin were also found to be Table 2 Calibration data and limits of detection of each pesticide (in the range of 1–16 lg/kg) in apple juice (n ¼ 5) Compound
Calibration equationa
Folpet Chlorothalonil Quinomethionat Tetradifon Trifluralin
y y y y y
a
¼ 54777x 494:48 ¼ 34829x 5720:3 ¼ 34299x þ 244:22 ¼ 24571x 1784:3 ¼ 12142x þ 2257:3
y ¼ area; x ¼ concentration (lg/kg).
r2
LODs (lg/kg)
0.9999 0.9998 1 1 0.9988
0.5 0.5 0.5 1 1
S. Topuz et al. / Food Control 16 (2005) 87–92 Table 3 Within-day and day-to-day precision and accuracy of pesticides in apple juice Compound
Concentration (lg/kg) Added
Within-day (n ¼ 5) Folpet 2 4 8
RSD (%)
RME (%)
1.95 ± 0.06 3.94 ± 0.29 7.84 ± 0.14
3.08 7.36 1.79
)2.5 )1.5 )2.0
Found (mean ± SD)
Chlorothalonil
2 4 8
1.91 ± 0.04 3.77 ± 0.01 7.88 ± 0.26
2.09 0.27 3.30
)4.5 )5.8 )1.5
Quinomethionat
2 4 8
1.86 ± 0.06 3.86 ± 0.05 7.53 ± 0.22
3.23 1.30 2.92
)7.0 )3.5 )5.9
Tetradifon
2 4 8
1.93 ± 0.04 3.81 ± 0.08 7.97 ± 0.11
2.07 2.10 1.38
)3.5 )4.8 )0.4
Trifluralin
2 4 8
1.86 ± 0.11 3.86 ± 0.02 7.53 ± 0.07
5.91 0.52 0.93
)7.0 )3.5 )5.9
Day-to-day (n ¼ 5) Folpet 2 4 8
1.95 ± 0.10 3.90 ± 0.16 7.88 ± 0.17
5.13 4.10 2.16
)2.5 )2.5 )1.5
Chlorothalonil
2 4 8
1.85 ± 0.07 3.91 ± 0.09 7.76 ± 0.13
3.78 2.30 1.68
)7.5 )2.3 )3.0
Quinomethionat
2 4 8
1.94 ± 0.07 3.72 ± 0.14 7.71 ± 0.12
3.61 3.76 1.56
)3.0 )7.0 )3.6
Tetradifon
2 4 8
1.89 ± 0.01 3.87 ± 0.12 7.81 ± 0.15
0.53 3.10 1.92
)5.5 )3.3 )2.4
Trifluralin
2 4 8
1.90 ± 0.14 3.69 ± 0.09 7.68 ± 0.15
7.37 2.44 1.95
)5.0 )7.8 )4.0
2 lg/kg with RSD of 0.6–2.2%. Differences between the matrices tested were not observed. LOQs were lower than to the MRLs set by the European Union (all >2 lg/ kg for the pesticide analyzed). The results of the assay validation study are presented in Table 3. The withinday and day-to-day reproducibilities expressed as RSD were found to be 0.27–7.36% and 0.53–7.37% respectively, indicating good precision. The accuracy of the method expressed as relative mean error (RME) between )7.8% and )0.4%, which was shown to be satisfactory. Using the improved method, folpet, chlorthalonil, quinomethionat, tetradifon and trifluralin were analyzed in canned pure apple, cherry juices and peach nectar produced by different companies in Turkey. Results of the study showed that the apple, cherry juices and peach nectar analyzed contained no detectable residues.
91
From a practical point of view, the method described in this research allows a simple, rapid and sensitive determination of folpet, chlorothalonil, quinomethionat, tetradifon and trifluralin residues in fruit juices using HPLC-DAD without any derivatization. The total analysis time, including SPE and HPLC, is about 45–50 min.
References Abad, A., & Montoya, A. (1995). Application of a monoclonal antibody-based ELISA to the determination of carbaryl in apple and grape juices. Analytica Chimica Acta, 311, 365–370. Bushway, R. J. (1988). High-performance liquid chromatographic determination of carbaryl in fruit juices. Journal of Chromatography, 457, 437–441. European Community, Brussels (1990). EC Council Directive 90/642/ EEC of 27 November 1990 on fixing of maximum levels for pesticide residues in and on fruit and vegetables. Official Journal of the European Communities, L350, 0071. Fillion, J., Sauve, F., & Selwyn, J. (2000). Multiresidue method for the determination of residues of 251 pesticides in fruits and vegetables by gas chromatography/mass spectrometry and liquid chromatography with fluorescence detection. Journal of AOAC International, 83(3), 698–713. Fodor-Csorba, K. (1992). Chromatographic methods for the determination of pesticides in foods. Journal of Chromatography, 624, 353– 367. Hsu, J. P., Schattenberg, H. J., III, & Garza, M. M. (1991). Fast turnaround multiresidue screen for pesticides in produce. Journal of Association Official Analytical Chemists, 74(5), 886– 892. Mark Lee, S., Papathakis, M. L., Feng, H. C., Hunter, G. F., & Carr, J. E. (1991). Multipesticide residue method for fruits and vegetables: California Department of Food and Agriculture. Fresenius’ Journal of Analytical Chemistry, 339, 376–383. Martinez Vidal, J. L., Arrebola, F. J., & Mateu-Sanchez, M. (2002). Application of gas chromatography-tandem mass spectrometry to the analysis of pesticides in fruits and vegetables. Journal of Chromatography A, 959, 203–213. Mattern, G. C., Liu, C. H., Louis, J. B., & Rosen, J. D. (1999). GC/MS and LC/MS determination of 20 pesticides for which dietary oncogenic risk has been estimated. Journal of Agriculture and Food Chemistry, 39, 700–704. Navarro, M., Pic o, Y., Marin, R., & Ma~ nes, J. (2002). Application of matrix solid-phase dispersion to the determination of a new generation of fungicides in fruits and vegetables. Journal of Chromatography A, 968, 201–209. Neidert, E., Trotman, R. B., & Saschenbrecker, P. W. (1994). Levels and incidences of pesticide residues in selected agricultural food commodities available in Canada. Journal of AOAC International, 77(1), 18–24. Newsome, W. H., & Collins, P. (1989). Multiresidue method for fungicide residues in fruits and vegetables. Journal of Chromatography, 472, 416–421. € Ozhan, G., Topuz, S., & Alpertunga, B. (2003). A simple method for the determination of Carbaryl and 1-Naphthol in fruit juices by HPLC-DAD. Journal of Food Protection, 66(8), 1510–1513. Perret, D., Gentili, A., Marchese, S., Sergi, M., & D’Asceno, G. (2002). Validation of method for the determination of multiclass pesticide residues in fruit juices by liquid chromatography/tandem mass spectrometry after extraction by matrix solid-phase dispersion. Journal of AOAC International, 85(3), 724–730.
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Pic o, Y., Font, G., Molt o, J. C., & Ma~ nes, J. (2000). Pesticide residue determination in fruit and vegetables by liquid chromatographymass spectrometry. Journal of Chromatography A, 882, 153– 173. Tadeo, J. L., S anchez-Brınete, C., Perez, R. A., & Fernandez, M. D. (2000). Analysis of herbicide residues in cereals fruits and vegetables. Journal of Chromatography, 882, 175–191.
Torres, C. M., Pic o, Y., & Ma~ nes, J. (1996). Determination of pesticide residues in fruit and vegetables. Journal of Chromatography A, 754, 301–331. Torres, C. M., Pic o, Y., Redondo, M. J., & Ma~ nes, J. (1996). Matrix solid-phase dispersion extraction procedure for multiresidue pesticide analysis in oranges. Journal of Chromatography A, 719(1), 95–103.
Simultaneous determination of various pesticides in fruit juices by HPLC-DAD € Sibel Topuz, G€ ul Ozhan, Buket Alpertunga * Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey Received 14 April 2003; received in revised form 25 November 2003; accepted 27 November 2003
Abstract A suitable method for the simultaneous determination of four fungicides (folpet, chlorothalonil, quinomethionat, tetradifon) and one herbicide (trifluralin) in fruit juices has been developed. The method involves the preconcentration of 25 g fruit juice samples through C18 solid-phase extraction cartridges. The pesticides were separated and quantified by reversed-phase high-performance liquid chromatography with UV-diode array detection at 220 and 260 nm. Analytical separation was performed using a concave gradient elution with acetonitrile and water on a C18 column. Recoveries from spiked apple, cherry juices and peach nectar, ranged from 93.8% to 99.5% and relative standard deviations were less than 3.4% in the concentration range of 1–16 lg/kg. The limits of detection for pesticides investigated are in the range of 0.5–1 lg/kg and the calibration curves showed good linearity >0.9988. The developed method has been tested on canned pure apple, cherry juices and peach nectar manufactured in Turkey. Results of this study showed that these samples analyzed contained no detectable residues. 2004 Elsevier Ltd. All rights reserved. Keywords: Fruit juice analysis; Pesticides; Solid-phase extraction; HPLC-DAD
1. Introduction Although the use of pesticides provides unquestionable benefits in providing a plentiful, low-cost supply of high-quality fruits and vegetables, their incorrect application may leave harmful residues, which involve possible health risk (Torres, Pic o, & Ma~ nes, 1996). As a consequence, governments and international organizations are established maximum residue limits (MRLs) in fruits and vegetables to ensure their levels (EC Council Directive, 1990). A great portion of the pesticide residues is fungicides and herbicides, which are used primarily to control spoilage of fruits and vegetables through fungal attack and to control weeds. The most frequently used methods for analysis of the fungicides and herbicides in fruits and vegetables are gas chromatography (GC) with nitrogen– phosphorous (NPD), electron-capture (ECD), a mass spectrometric (MS) detection and liquid chromatography (LC) with ultraviolet (UV) and diode-array detection (DAD) (Fillion, Sauve, & Selwyn, 2000; Hsu, *
Corresponding author. Tel.: +90-212-526-0797; fax: +90-212-5190812. E-mail address: [email protected] (B. Alpertunga). 0956-7135/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2003.11.012
Schattenberg, & Garza, 1991; Mark Lee, Papathakis, Feng, Hunter, & Carr, 1991; Martinez Vidal, Arrebola, & Mateu-Sanchez, 2002; Neidert, Trotman, & Saschenbrecker, 1994; Newsome & Collins, 1989; Pic o, Font, Molt o, & Ma~ nes, 2000). These methods are based on liquid–liquid extraction (LLE), solid-phase extraction (SPE), matrix solid-phase dispersion and supercritical fluid extraction (SFE) (Fodor-Csorba, 1992; Mattern, Liu, Louis, & Rosen, 1999; Navarro, Pic o, Marin, & Ma~ nes, 2002; Tadeo, Sanchez-Brınete, Perez, & Fernandez, 2000; Torres, Pic o, Redondo, & Ma~ nes, 1996). Fruit is often treated close to harvest or postharvest to ensure that wholesome produce reaches the consumer. The presence of pesticide residues in canned pure fruit juices can be a significant route to human exposure. Only a few procedures have been reported for the analysis of pesticide residues in fruit juices (Abad & € Montoya, 1995; Bushway, 1988; Ozhan, Topuz, & Alpertunga, 2003; Perret, Gentili, Marchese, Sergi, & D’Asceno, 2002). The development of easy analytical methods to monitor these pesticides in fruit juices is very important. In this work, we described a method based on SPE-HPLC to determine four fungicides (folpet,
88
S. Topuz et al. / Food Control 16 (2005) 87–92
centrations range of 0.025–0.4 lg/ml, were obtained by dilution with mobile phase (Acetonitrile–water, 40:60, v/ v). The standard solutions were store at 4 C and were used for 3 months.
CN Cl
O
Cl
SCCl3
N
Cl
CN Cl
O Folpet
Chlorothalonil
2.3. Analysis Cl N
S
O
O
Cl H3C
N
S
S
Cl
O Cl
Quinomethionat
Tetradifon
N(CH2CH2CH3)2 O2N
NO2
CF3
Trif luralin
Fig. 1. Structures of the studied compounds.
chlorothalonil, quinomethionat, tetradifon) and one herbicide (trifluralin), frequently used on various fruits in Turkish agriculture in fruit juices. The structures of these pesticides are illustrated in Fig. 1. The developed method has been tested on canned pure apple, cherry juices and peach nectar manufactured in Turkey.
2. Materials and methods 2.1. Materials All pesticide standards were of 98–99% purity and were purchased from Riedel-de Ha€en (Seelze, Germany). All solvents (HPLC grade) were obtained from Merck (Darmstadt, Germany). A Milli-Q water purification system from Millipore (Bedford, MA, USA) was used to provide ultra-pure water in this study. C18 SPE cartridges (Bond Elut LRC-C18 , 200 mg/10 ml, Varian, CA, USA) were used in the preconcentration step. Samples were passed through the cartridges using vacuum extraction manifold system (Vac Elut 20, Varian, CA, USA). Mobile phase filtration was obtained using a 0.2 lm membrane filter (Phenomenex, CA, USA). One hundred percent apple, cherry juices and peach nectar commercially available were used. 2.2. Preparation of solutions The stock standard solutions of folpet, chlorothalonil, quinomethionat, tetradifon and trifluralin (500 lg/ ml) were prepared in acetonitrile and these were diluted with acetonitrile to give a standard solution of 5 lg/ml. Working standard solutions of each pesticide at con-
Liquid chromatographic analysis were performed with a Thermo Separation Products Liquid Chromatograph (Model Spectra System , TSP, CA, USA), equipped with HPLC pump (Spectra Series pump P4000), vacuum degasser for liquid chromatography (Solvent degasser SCM 1000), rheodyne and injection valve (Injection volume: 50 ll). System parameters were controlled with system controller (SN 4000) and chromatographic data were collected and recorded using the PC 1000 system software. The separation was carried out using a C18 , 5 lm Luna column (250 mm · 4.6 mm ID, Phenomenex, CA, USA) fitted with guard column (4 mm L · 3 mm I.D., Phenomenex, CA, USA) packed with same material. The column eluate was monitored with a UV 6000LP photo-diode array detector. The chromatographic separation was carried out using a concave gradient profile of acetonitrile and water going from 40% of acetonitrile to 90% in 20 min, this condition was held for 5 min and then back to the initial conditions. The flow rate of the mobile phase was 1 ml/min and column temperature was ambient. The DAD was set at 220 and 260 nm with a bandwidth of 4 nm. Absorbance spectra were recorded in the 200–360 nm range. 2.4. Extraction procedures In all, 10 each of apple and cherry juices and peach nectar produced by different companies were collected from the market. Fruit juice samples were stored at 4–10 C and were analyzed in 2 days. Methanol (15 ml) was added to 25 g juice sample and mixed for 10 min in a 50 ml volumetric flask. The mixture was taken to 50 ml with water. Ten milli liter aliquot of this solution was further diluted to 50 ml water in a second volumetric flask. SPE cartridges were conditioned with 5 ml of methanol and 5 ml of water. The sample was passed through the cartridges using vacuum extraction manifold system at a rate of ca. 8–10 ml/min. After washing with 2.5 ml of water, the sorbent bed was dried under vacuum for 10 min. The analytes were eluted with 3 ml of dichlormethane into a vial. The eluate was then evaporated under a gentle stream of nitrogen at 40 C. The residue dissolved in the mobile phase to a final volume of 0.2 ml and 50 ll an equivalent of 1.25 g fruit juice was injected into the HPLC system.
S. Topuz et al. / Food Control 16 (2005) 87–92
89
Fig. 2. (A) LC-DAD chromatograms of (a) unspiked apple juice sample, (b) apple juice spiked at 4 lg/kg with pesticides registered at 220 and 260 nm. Peaks (retention times, min): 1 ¼ folpet (15.65), 2 ¼ chlorothalonil (16.29), 3 ¼ quinomethionat (22.39), 4 ¼ tetradifon (24.11), 5 ¼ trifluralin (25.14). (B) LC-DAD chromatograms of (a) unspiked cherry juice sample, (b) cherry juice spiked at 4 lg/kg with pesticides registered at 220. (C) LC-DAD chromatograms of (a) unspiked peach nectar sample, (b) peach nectar spiked at 4 lg/kg with pesticides registered at 220.
90
S. Topuz et al. / Food Control 16 (2005) 87–92
2.5. Recovery studies By using the samples spiked with these pesticides the recoveries were assessed at concentrations of 1, 2, 4, 8, 16 lg/kg in fruit juices. Spiked fruit juice samples were extracted on C18 cartridges and chromatographed using the procedure outlined above for each concentration in five replicates.
Table 1 Extraction recovery (%) for the assay of pesticides in apple juice (n ¼ 5) Compound
Recovery (%)
RSD (%)
1.95 ± 0.06 7.86 ± 0.14 15.92 ± 0.23
97.70 98.21 99.50
3.1 1.8 1.5
2
1.88 ± 0.04
94.20
2.1
8 16
7.82 ± 0.26 15.71 ± 0.51
97.71 98.20
3.3 3.3
2
1.90 ± 0.06
94.80
3.4
8 16
7.62 ± 0.22 15.55 ± 0.22
95.23 97.20
2.9 1.4
Tetradifon
2 8 16
1.91 ± 0.04 7.89 ± 0.11 15.89 ± 0.13
95.30 98.68 99.30
2.1 1.4 0.8
Trifluralin
2 8 16
1.88 ± 0.11 7.61 ± 0.07 15.34 ± 0.28
93.80 95.10 95.90
0.6 1.0 1.8
Folpet
Concentration (lg/kg) Added
Found (mean ± SD)
2 8 16
Chlorothalonil
3. Results and discussion The compounds investigated are very extensively applied pesticides for various fruits (apple, cherry and peach) in Turkish agriculture. Because of the fact that fruit juices are more concentrated than a single fruit, they may contain pesticide residues. Therefore intake of fruit juices may be significant route of human exposure to the pesticide. Further children drink a proportionally higher amount of fruit juices than adults, and for this reason pesticide analysis of fruit juices are especially important. The presence of natural pigments makes the analysis of fruit juices difficult. Clean up on C18 cartridges eliminated most interfering peaks and allowed good recoveries at low fortification levels. Complete separation of all compounds was achieved using a concave gradient elution with acetonitrile and water on a C18 column. This afforded good resolution in a reasonable time. A linear curve gradient did not allow satisfactory separation for folpet and chlorothalonil. The pesticides studied are highly absorbing substances in the UV region of the spectrum, with absorption maximum at 260 nm for quinomethionat and 220 nm for folpet, tetradifon, chlorothalonil and trifluralin. The procedure is illustrated in Fig. 2, which shows the chromatograms of unspiked apple, cherry juices and peach nectar and spiked samples at 4 lg/kg level with pesticides. As can be seen, the chromatograms were clean without interfering peaks in the areas of interest. SPE with C18 cartridges was used in order to achieve suitable sensitivity. The various extraction solvents were studied for the elution of the pesticides retained in the SPE cartridges. Although the elution of pesticides with different solvents produced similar recoveries, dichlormethane was considered best for extraction because a good baseline and less interfering peaks were obtained with this solvent. Juices containing no detectable amount of these pesticides were spiked at concentrations ranging from 1 to 16 lg/kg. Recoveries obtained for apple juice sample spiked with pesticides at three concentration levels are shown in Table 1. The similar recoveries were obtained for cherry juices and peach nectar in the range of 1–16 lg/kg levels of spiking. Average recoveries ranged from 93.8% to 99.5%.
Quinomethionat
Quantification of these pesticides in the sample extract was performed by use of integrated peak area with their known external standard. The linearity was observed for all of the pesticides in the range of 1–16 lg/kg (n ¼ 5) in apple juices, yielding correlation coefficients higher than 0.9988 (Table 2). The linearity values were also checked for jerry juices and peach nectar and the corresponding results were similar as reported for apple juices. The limits of detection (LODs) were calculated as the lowest concentration giving a response of three-times the average of the baseline noise defined from unspiked juices. LODs for the pesticides investigated were in the range of 0.5–1 lg/kg (Table 2). The limits of quantification (LOQs) for this method were defined as the lowest concentration of compounds in a sample that could be quantitatively determined with suitable precision and accuracy. LOQs of folpet, chlorothalonil and quinomethionat were found to be 1 lg/kg with relative standard deviation (RSD) of 0.5–1.4%. LOQs of tetradifon and trifluralin were also found to be Table 2 Calibration data and limits of detection of each pesticide (in the range of 1–16 lg/kg) in apple juice (n ¼ 5) Compound
Calibration equationa
Folpet Chlorothalonil Quinomethionat Tetradifon Trifluralin
y y y y y
a
¼ 54777x 494:48 ¼ 34829x 5720:3 ¼ 34299x þ 244:22 ¼ 24571x 1784:3 ¼ 12142x þ 2257:3
y ¼ area; x ¼ concentration (lg/kg).
r2
LODs (lg/kg)
0.9999 0.9998 1 1 0.9988
0.5 0.5 0.5 1 1
S. Topuz et al. / Food Control 16 (2005) 87–92 Table 3 Within-day and day-to-day precision and accuracy of pesticides in apple juice Compound
Concentration (lg/kg) Added
Within-day (n ¼ 5) Folpet 2 4 8
RSD (%)
RME (%)
1.95 ± 0.06 3.94 ± 0.29 7.84 ± 0.14
3.08 7.36 1.79
)2.5 )1.5 )2.0
Found (mean ± SD)
Chlorothalonil
2 4 8
1.91 ± 0.04 3.77 ± 0.01 7.88 ± 0.26
2.09 0.27 3.30
)4.5 )5.8 )1.5
Quinomethionat
2 4 8
1.86 ± 0.06 3.86 ± 0.05 7.53 ± 0.22
3.23 1.30 2.92
)7.0 )3.5 )5.9
Tetradifon
2 4 8
1.93 ± 0.04 3.81 ± 0.08 7.97 ± 0.11
2.07 2.10 1.38
)3.5 )4.8 )0.4
Trifluralin
2 4 8
1.86 ± 0.11 3.86 ± 0.02 7.53 ± 0.07
5.91 0.52 0.93
)7.0 )3.5 )5.9
Day-to-day (n ¼ 5) Folpet 2 4 8
1.95 ± 0.10 3.90 ± 0.16 7.88 ± 0.17
5.13 4.10 2.16
)2.5 )2.5 )1.5
Chlorothalonil
2 4 8
1.85 ± 0.07 3.91 ± 0.09 7.76 ± 0.13
3.78 2.30 1.68
)7.5 )2.3 )3.0
Quinomethionat
2 4 8
1.94 ± 0.07 3.72 ± 0.14 7.71 ± 0.12
3.61 3.76 1.56
)3.0 )7.0 )3.6
Tetradifon
2 4 8
1.89 ± 0.01 3.87 ± 0.12 7.81 ± 0.15
0.53 3.10 1.92
)5.5 )3.3 )2.4
Trifluralin
2 4 8
1.90 ± 0.14 3.69 ± 0.09 7.68 ± 0.15
7.37 2.44 1.95
)5.0 )7.8 )4.0
2 lg/kg with RSD of 0.6–2.2%. Differences between the matrices tested were not observed. LOQs were lower than to the MRLs set by the European Union (all >2 lg/ kg for the pesticide analyzed). The results of the assay validation study are presented in Table 3. The withinday and day-to-day reproducibilities expressed as RSD were found to be 0.27–7.36% and 0.53–7.37% respectively, indicating good precision. The accuracy of the method expressed as relative mean error (RME) between )7.8% and )0.4%, which was shown to be satisfactory. Using the improved method, folpet, chlorthalonil, quinomethionat, tetradifon and trifluralin were analyzed in canned pure apple, cherry juices and peach nectar produced by different companies in Turkey. Results of the study showed that the apple, cherry juices and peach nectar analyzed contained no detectable residues.
91
From a practical point of view, the method described in this research allows a simple, rapid and sensitive determination of folpet, chlorothalonil, quinomethionat, tetradifon and trifluralin residues in fruit juices using HPLC-DAD without any derivatization. The total analysis time, including SPE and HPLC, is about 45–50 min.
References Abad, A., & Montoya, A. (1995). Application of a monoclonal antibody-based ELISA to the determination of carbaryl in apple and grape juices. Analytica Chimica Acta, 311, 365–370. Bushway, R. J. (1988). High-performance liquid chromatographic determination of carbaryl in fruit juices. Journal of Chromatography, 457, 437–441. European Community, Brussels (1990). EC Council Directive 90/642/ EEC of 27 November 1990 on fixing of maximum levels for pesticide residues in and on fruit and vegetables. Official Journal of the European Communities, L350, 0071. Fillion, J., Sauve, F., & Selwyn, J. (2000). Multiresidue method for the determination of residues of 251 pesticides in fruits and vegetables by gas chromatography/mass spectrometry and liquid chromatography with fluorescence detection. Journal of AOAC International, 83(3), 698–713. Fodor-Csorba, K. (1992). Chromatographic methods for the determination of pesticides in foods. Journal of Chromatography, 624, 353– 367. Hsu, J. P., Schattenberg, H. J., III, & Garza, M. M. (1991). Fast turnaround multiresidue screen for pesticides in produce. Journal of Association Official Analytical Chemists, 74(5), 886– 892. Mark Lee, S., Papathakis, M. L., Feng, H. C., Hunter, G. F., & Carr, J. E. (1991). Multipesticide residue method for fruits and vegetables: California Department of Food and Agriculture. Fresenius’ Journal of Analytical Chemistry, 339, 376–383. Martinez Vidal, J. L., Arrebola, F. J., & Mateu-Sanchez, M. (2002). Application of gas chromatography-tandem mass spectrometry to the analysis of pesticides in fruits and vegetables. Journal of Chromatography A, 959, 203–213. Mattern, G. C., Liu, C. H., Louis, J. B., & Rosen, J. D. (1999). GC/MS and LC/MS determination of 20 pesticides for which dietary oncogenic risk has been estimated. Journal of Agriculture and Food Chemistry, 39, 700–704. Navarro, M., Pic o, Y., Marin, R., & Ma~ nes, J. (2002). Application of matrix solid-phase dispersion to the determination of a new generation of fungicides in fruits and vegetables. Journal of Chromatography A, 968, 201–209. Neidert, E., Trotman, R. B., & Saschenbrecker, P. W. (1994). Levels and incidences of pesticide residues in selected agricultural food commodities available in Canada. Journal of AOAC International, 77(1), 18–24. Newsome, W. H., & Collins, P. (1989). Multiresidue method for fungicide residues in fruits and vegetables. Journal of Chromatography, 472, 416–421. € Ozhan, G., Topuz, S., & Alpertunga, B. (2003). A simple method for the determination of Carbaryl and 1-Naphthol in fruit juices by HPLC-DAD. Journal of Food Protection, 66(8), 1510–1513. Perret, D., Gentili, A., Marchese, S., Sergi, M., & D’Asceno, G. (2002). Validation of method for the determination of multiclass pesticide residues in fruit juices by liquid chromatography/tandem mass spectrometry after extraction by matrix solid-phase dispersion. Journal of AOAC International, 85(3), 724–730.
92
S. Topuz et al. / Food Control 16 (2005) 87–92
Pic o, Y., Font, G., Molt o, J. C., & Ma~ nes, J. (2000). Pesticide residue determination in fruit and vegetables by liquid chromatographymass spectrometry. Journal of Chromatography A, 882, 153– 173. Tadeo, J. L., S anchez-Brınete, C., Perez, R. A., & Fernandez, M. D. (2000). Analysis of herbicide residues in cereals fruits and vegetables. Journal of Chromatography, 882, 175–191.
Torres, C. M., Pic o, Y., & Ma~ nes, J. (1996). Determination of pesticide residues in fruit and vegetables. Journal of Chromatography A, 754, 301–331. Torres, C. M., Pic o, Y., Redondo, M. J., & Ma~ nes, J. (1996). Matrix solid-phase dispersion extraction procedure for multiresidue pesticide analysis in oranges. Journal of Chromatography A, 719(1), 95–103.