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8132 J. Agric. Food Chem. 2003, 51, Assessment of Pesticide Residues in Honey Samples from Portugal and Spain CRISTINA BLASCO, MOÄ NICA FERNAÄ NDEZ, ANGELINA PENA, CELESTE LINO, M A IRENE SILVEIRA,
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8132 J. Agric. Food Chem. 2003, 51, Assessment of Pesticide Residues in Honey Samples from Portugal and Spain CRISTINA BLASCO, MOÄ NICA FERNAÄ NDEZ, ANGELINA PENA, CELESTE LINO, M A IRENE SILVEIRA, GUILLERMINA FONT, AND YOLANDA PICOÄ *, Laboratori de Bromatologia i Toxicologia, Facultat de Farmàcia, Universitat de València, Av. Vicent Andrés Estellés s/n, Burjassot, València, Spain, and Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Coimbra, 3000 Coimbra, Portugal INTRODUCTION Fifty samples of honey collected from local markets of Portugal and Spain during year 2002 were analyzed for 42 organochlorine, carbamate, and organophosphorus pesticide residues. An analytical procedure based on solid-phase extraction with octadecyl sorbent followed by gas chromatographymass spectrometry (GC-MS), for organochlorines, and by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS), for organophosphorus and carbamates, has been developed. Recoveries of spiked samples ranged from 73 to 98%, except for dimethoate (40%), with relative standard deviations from 3 to16% in terms of repeatability, and from 6 to 19% in terms of reproducibility. Limits of quantification were from to 0.1 mg kg -1. Most of the pesticides found in honey were organochlorines. Among them, γ-hch was the most frequently detected in 50% of the samples, followed by HCB in 32% of the samples and the other isomers of HCH (R-HCH and β-hch) in 28 and 26% of the samples, respectively. Residues of DDT and their metabolites were detected in 20% of the samples. Of the studied carbamates, both methiocarb and carbofuran were detected in 10% of the samples, pirimicarb in 4% and carbaryl in 2%. The only organophosphorus pesticides found were heptenophos in 16%, methidathion in 4%, and parathion methyl in 2% of honey samples. Results indicate that Portuguese honeys were more contaminated than Spanish ones. However, honey consumers of both countries should not be concerned about the amounts of pesticide residues found in honeys available on the market. Pesticides play a beneficial role in agriculture, because they help to combat the variety of pest that destroy crops, even though small amounts of pesticide residues remain in the food supply, constituting a potential risk for the human health, because of their sub-acute and chronic toxicity (1). The most widely used pesticides are organophosphorus and carbamates, which have almost completely replaced organochlorine pesticides (2). The extensive distribution of these groups of pesticides causes bees that have been fed on contaminated blossom to transfer pesticide residues into honey and finally to the consumer (1, 2). Organochlorine pesticides have been restricted or banned in agriculture since 1978 in North America and Europe because of their persistence and bioaccumulation in the environment. However, these pesticides are still frequently found in soil, from which they continue to cycle through the environment, as soil is a potential source to the atmosphere by way of volatilization and to water, plants, and animals by their movement via runoff * To whom correspondence should be addressed. Tel.: Fax: Universitat de València. University of Coimbra. (3, 4). Different studies demonstrated the bioaccumulation of organochlorine from contaminated soil to aerial and root tissues of different plants (5) and to organisms (6, 7), which can bioconcentrate these fat-soluble pesticides at times the level found in the surrounding environment. The presence of pesticide residues in honey has impelled the need for setting up monitoring programs to determine the proper assessment of human exposure to pesticides making possible to take policy decisions in the interest of health hazard (8). Different national regulations have established maximum concentrations of pesticide residues (MRLs) permitted in honey, but the lack of homogeneity causes problems in international marketing and trade. As an example, Germany, Italy, and Switzerland have set MRLs for amitraz, bromopropylate, coumaphos, cyamizole, flumetrine, and fluvalinate, which oscillate between 0.01 and 0.1 mg kg -1 in Germany, between 5 and 500 mg kg -1 in Switzerland, and are of 10 mg kg -1 in Italy (9). Up to now, maximum limits of pesticide residues in honey are not included in the Codex Alimentarius (10). The European Union (EU) legislation has regulated the MRLs for three acaricides: amitraz, coumaphos, and cyamizole, which are 0.2, 0.1, and 1 mg kg -1, respectively (11). The U. S. Environmental Protection Agency (12) has established MRLs /jf034870m CCC: $ American Chemical Society Published on Web 11/18/2003 Pesticide Residues in Honey Samples J. Agric. Food Chem., Vol. 51, No. 27, for amitraz (1 mg kg -1 ), coumaphos (0.1 mg kg -1 ), and fluvalinate (0.05 mg kg -1 ). A multiresidue method able to detect as many pesticides as possible, in a relatively short time period, is crucial for an efficient monitoring program (8, 9, 13). Generally, these methods are based on the traditional liquid-liquid extraction (LLE) or solid-phase extraction (SPE). LLE main advantage is simplicity but employs a large amount of toxic solvent and is a time-consuming procedure. Much less toxic solvents are consumed by SPE, which also offers a save in sample preparation time. However, this technique has the disadvantage of being unable to handle large sample volumes. Both, LLE (14-16) and SPE (17-19) have been selected in various multiresidue methods for extracting organochlorine, organophosphorus, carbamate, and pyrethroid pesticides in honey. The detection of pesticides is accomplished by gas chromatography (GC) or liquid chromatography (LC). Until now, GC has been the most widely used technique, because its high separation power and availability of selective detectors as electron capture (ECD), nitrogen phosphorus (NPD), and mass spectrometry (MSD) detectors. In recent years, LC has emerged as an excellent alternative technique, especially for polar and thermolabile pesticides, which are not directly determinable by GC. Mass spectrometry (MS) employing atmospheric pressure ionization (API) is becoming the detection system of choice for liquid chromatography (LC), because its versatility, high selectivity, and spectral evidence of individual solutes (20, 21). As it has been previously reviewed (13, 22), pesticide residues programs for monitoring honey are still scarce. Most studies concentrate efforts to determine residues of acaricides that are used to control Varroa jacobsoni, a parasitic mite that affects honeybee colonies (8, 23-25). Depending on the regulation of each country and beekeepers practices, the most often detected acaricides are bromopropylate, coumaphos, and fluvalinate. Only a few studies have been focussed on pesticides used for crop protection and introduced into hives by contaminated bees and wax (14, 26, 27). Most samples analyzed in Jordan during 1995 contained residues of organochlorine pesticides such as R-HCH, β-hch, and lindane, and only some of them were contaminated by organophosphorus pesticides. Pyrethroids and nitrogencontaining pesticides were not found in any sample (26). In contrast, compared to the previous report, levels and frequency of organophosphorus and carbamate pesticides were relatively higher in honey samples analyzed in India from 1993 to 1997 (27). The first aim of this study is to extend the extraction method previously proposed (19) to determine twenty eight organophosphorus and five carbamates by LC/APCI/MS and nine organochlorines by GC-MS. Validation and optimization of the SPE procedure is presented in terms of recoveries, precision, and limits of quantification. The method was applied to monitor 50 honey samples from various floral origins collected in local markets of Portugal and Spain during year MATERIALS AND METHODS Reagents and Chemicals. Pesticide standards were purchased from Sigma-Aldrich (Madrid, Spain) (see Tables 1 and 2). Methanol (HPLCgrade), petroleum ether, dichloromethane, hexane, and ethyl acetate (organic trace analysis) were obtained from Merck (Darmstadt, Germany). Stock solution of each pesticide were prepared at 1000 mg L -1 in methanol and then stored at 4 C. The carbamate and organophosphorus stock solutions were stored for 3 months, and the organochlorine solutions were stored for 1 year. Working solutions were prepared daily by appropriate dilution of aliquots obtained from stock solution in methanol. Deionized water ( 18 M cm resistivity) was Table 1. SIM Conditions for Determining Pesticides by LC APCI-MS time ion (m/z) pesticide frag/(v) dwell time (ms) monocrotophos dimethoate 272 vamidothion 284 phosphamidone carbaryl carbofuran paraoxon pirimicarb heptenophos fosmet methidathion methiocarb parathion methyl malathion fenitrothion azinphos ethyl quinalphos fenoxycarb 262 parathion ethyl 319 phenthoate fonofos diazinon 361 coumaphos 263 fenthion foxim phosalone 372 pyrazophos chlorpyriphos methyl profenofos pirimiphos ethyl chlorpyriphos ethyl 351 bromophos 451 temephos Table 2. GC-MS pesticides SIM Conditions of Organochlorine Pesticides Detected by tr(min) mol weight quantitation ion selected ions, m/z (average relative intensities, %) confirmation ion 1 deg confirmation ion 2 deg R-HCH (100) 109 (70) 219 (90) HCB (100) 282 (54) 286 (80) β-hch (100) 181 (85) 219 (70) γ-hch (100) 109 (64) 219 (90) Aldrin (100) 261 (60) 265 (70) pp -DDE (100) 318 (70) 316 (56) pp -DDD (100) 237 (64) 165 (40) op -DDT (100) 237 (66) 165 (30) pp -DDT (100) 237 (65) 165 (60) obtained from a Milli-Q SP Reagent Water System (Millipore, Bedford, MA). C 18 solid phase (particle diameters of approximately 55 µm and pore diameter 60 Å) was acquired from Análisis Vínicos (Tomelloso, Spain). Sampling. Twenty four honey samples were collected from different local markets of Coimbra (Portugal), all of them were of multi flower origin. Twenty six honey samples were taken from local markets of Valencia, those samples were from different floral origins, thyme, multi flowers, rosemary, heather, lavender, orange blossom, lemon, acorn, and eucalyptus. Four of them, V22-V25 (see Table 5), were ecological honeys. Both Portuguese and Spanish honeys were locally produced. These samples were stored in their original containers (always glass jars) at room temperature in a dark place. Extraction Procedure. Honey (5 g) was mixed with 50 ml of water and agitated by a stir bar for 10 min. At the same time, 0.5 g of C 18 sorbent was introduced into a mm ID glass chromatography column with a coarse frit No. 2 and covered with a plug of silianized 8134 J. Agric. Food Chem., Vol. 51, No. 27, 2003 Blasco et al. glass wood at the top. The solid phase was preconditioned by passing 10 ml of methanol and 10 ml of water with the aid of a vacuum pump to avoid dryness. The sample was passed through the solid phase, after that, the retained pesticides were eluted by passing first 10 ml of ethyl acetate, followed by 4 ml of methanol, and then 1 ml of dichloromethane. The eluate was evaporated to 0.5 ml, using a gentle steam of nitrogen, and transferred quantitatively with methanol into a 1-mL volumetric flask, obtaining a final extract in 100% methanol. For the analysis, 5 µl was injected into the LC-MS system, and 1 µl into the GC-MS system. Samples of honey for determining the limits of quantification (LOQs), recovery and precision were pesticide free and different from the samples studied. Recovery experiments were carried out by spiking honey samples (5 g) with volumes between 50 and 100 µl of pesticide working mixtures at appropriate concentrations in methanol. Prior to sample analysis by the proposed method, the spiked samples were let stand at room temperature for 3 h to achieve the solvent evaporation and the pesticide distribution in the honey. Liquid Chromatography-Mass Spectrometry. The equipment used was a Hewlett-Packard (Palo Alto, CA) HP-1100 Series LC-MSD system equipped with a binary solvent pump, an autosampler, and a mass selective detector (MSD) consisting of a standard API source that can be configured as APCI. An HP Chemstation software version A was used for LC-MS control and signal acquisition. The chromatographic separation was carried out on a Luna C 18 column ( mm I.D., particle size 5 µm) protected by a Securityguard cartridge C 18 (4 2 mm I.D.), both from Phenomenex (Madrid, Spain). The methanol/water gradient selected to separate compounds at a flow rate of 0.8 ml min -1 was 65% of methanol, which was increased linearly to 70% of methanol in 30 min, then increased to 80% of methanol in 20 min, and held at 80% of methanol for 10 min. Return to the initial conditions was carried out in 10 min. The APCI interface in negative ionization mode was operated at 400 C vaporized temperature, 6 bar pressure of nebulizer gas, 8 L min -1 drying gas flow-rate, 350 C drying gas temperature, 4000 V capillary voltage, and 25 µa corona current discharged. Full-scan LC- MS chromatograms were obtained by scanning from m/z 100 to 400 with a scan time of 0.75s. Time-scheduled selected-ion monitoring (SIM) of the most abundant ion of each compound was used for quantification as it is shown in Table 1. Gas Chromatography-Mass Spectometry. GC analysis was carried out on a Trace GC-MS 2000 (Thermo Finnigan, Manchester, UK) system with Xcalibur-software-based data acquisition. The injector temperature was 220 C, and the detector one was 280 C. Sample was injected in the splitless mode, and the splitless was opened after 60 s. A fused silica capillary column (30 m 0.25 mm I.D., 0.25 µm) with chemically bonded phases DB-5 was used. The oven temperature was as follows: initial temperature of 150 C, held for 1 min, increased to 230 C at3 C min -1, held for 5 min, and then increased to 250 C at 3 C min -1 and held for 15 min. The MS ionization potential was 70 ev, and the temperatures were as follows: ion source 250 C, transfer line 200 C, and analyzer 230 C. Analysis was performed in SIM mode monitoring specific ions of each analyte as it is shown in Table 2. The most intense ion was used for quantification and the second and third ion for confirmation. Identification criteria was based on (a) the chromatographic retention data, and (b) the relative peak heights of the three characteristic masses in the sample peak that must be within (20% of the relative intensity of these masses in the mass spectrum of the standard analyzed in the GC/MS system. RESULTS AND DISCUSSION Organophosphorus and Carbamates Analysis. A multiresidue method previously reported to analyze twenty two organophosphorus pesticides (19) in honey was adapted for the analysis of thirty-three pesticides, five of which are carbamates, and the others organophosphorus. As reported previously, the organophosphorus and carbamates gave intense mass spectra under negative ionization mode conditions (19, 28). The calibration curves constructed were linear over the range of interest. The correlation coefficient were Table 3. Limits of Quantifications (LOQs) and Mean Recovery with Relative Standard Deviations (RSDs) of the Studied Pesticides by LC APCI-MS and GC MS peak number pesticide mean recovery, % ± RSDs, % (n ) 5) under repeatability conditions under reproducibility between days conditions LOQ mg kg -1 LOQ 5 LOQ LOQ 5 LOQ 1 vamidothion ± 9 93± ± ± 15 2 dimethoate ± ± 9 42 ± ± 12 3 phosphamidone ± 6 95± 8 91 ± ± 11 4 carbofuran ± 8 89± 9 88 ± ± 12 5 monocrotophos ± 9 92± ± ± 11 6 carbaryl ± 7 93± ± ± 11 7 pirimicarb ± ± ± ± 15 8 paraoxon ± 9 83± ± ± 14 9 heptenophos ± 7 89± 9 92 ± ± methidathion ± 9 92± ± ± fosmet ± ± ± ± parathion methyl ± 9 83± ± ± methiocarb ± 8 85± 9 80 ± ± 9 14 malathion ± ± ± ± fenitrothion ± ± 9 85 ± ± 9 16 azinphos ethyl ± 9 74± ± ± fenoxycarb ± 8 85± ± ± phenthoate ± 9 77± ± ± parathion ethyl ± ± ± ± quinalphos ± ± 7 75 ± ± 7 21 fenthion ± ± ± ± fonofos ± ± ± ± diazinon ± ± 9 92 ± ± 9 24 coumaphos ± ± ± ± foxim ± ± ± ± phosalone ± 9 90± 9 93 ± ± pyrazophos ± 8 89± 7 89 ± ± 8 28 chlorpyriphos methyl ± ± 9 87± ± profenofos ± ± ± ± pirimiphos ethyl ± ± ± ± bromophos ± ± ± ± temephos ± ± ± ± chlorpyriphos ethyl ± ± 9 83± ± R-HCH ± 6 90± 7 93± 9 87± HCB ± 3 85± 9 90± ± β-hch ± 8 93± 5 92± ± 9 37 γ-hch ± 9 91± 7 87± ± aldrin ± 3 83± 7 82± 6 82± pp DDE ± 8 93± 4 92± ± 8 40 pp DDD ± 7 89± 6 87± ± 9 41 op DDT ± 9 98± 8 96± ± pp DDT ± 8 93± 9 90± ± 12 The precision and accuracy of the procedure obtained by analysis of five spiked honey samples at two concentration levels (the limits of quantification (LOQs) and 5 times the LOQ) are summarized in Table 3. Recoveries ranged from 73 to 95% with RSDs from 6 to 16% in terms of repeatability (intraday precision), and from 9 and 19% in terms of reproducibility (interday precision). Only dimethoate recovery was lower than 50%. The LOQs, also listed in Table 3, varied from to 0.1 mg kg -1. These values correspond to the lowest concentration of compound that gives a response that can be quantified with an interassay RSD of less than 20%. Sensitivity was good enough to ensure a reliable determination. An example of a typical LC-MS chromatogram of a sample spiked at LOQs levels of the thirty-three studied pesticides is shown in Figure 1A. Some pesticides coeluted at the same retention time and various peaks from the matrix are observed in the initial part of the chromatogram as it is shown in the chromatogram of an unspiked sample (Figure 1B), consequently, the use of individual ion chromatogram of each pesticide enabled the selective identification and quantification of doubtful peaks. Organochlorine Analysis. The extraction method was also extended to determine nine organochlorine pesticides. Preliminary experiments were carried out to find the best eluent for Pesticide Residues in Honey Samples J. Agric. Food Chem., Vol. 51, No. 27, Table 4. Recoveries (%) a and Repeatability (RSD) of Organochlorine Pesticides from Honey Samples Spiked at 0.1 mg kg -1 of Each Pesticide Using Different Solvents b pesticides hexane petroleum ether MeOH 10 ml EA + 4 ml MeOH + 1mLDCM R-HCH 68 ± 8 97± 8 98± 8 97± 6 HCB 79 ± ± 7 90± 8 92± 3 β-hch 72 ± ± ± 7 95± 8 γ-hch 79 ± ± ± ± 9 Aldrin 58 ± 6 68± ± 6 79± 3 pp -DDE 84 ± 8 78± 7 74± 7 90± 8 pp -DDD 82 ± 7 99± 7 92± 7 91± 7 op -DDT 84 ± 8 77± 8 85± 8 98± 9 pp -DDT 98 ± 7 77± 7 88± 7 89± 8 Figure 1. LC APCI-MS chromatograms of (A) untreated honey sample spiked at 5 times the LOQ (Peak identification as Table 3) and (B) a non spiked honey. organochlorine pesticides from solid phase. Methanol, hexane, petroleum ether, and the previously tested eluent (ethyl acetate, methanol, and dichloromethane) were evaluated as elution solvents. As it is summarized in Table 4, satisfactory results were obtained with most of the solvents tested. These results are in accordance with a previous published paper that uses SPE with C 18 and hexane for the extraction of organochochlorines in honey (18). However, the selected elution (ethyl acetate, methanol, and dichloromethane) was preferred because of the high recoveries obtained without extracting large quantities of interferences and the possibility to perform a simultaneous extraction of organochlorine, organophosphorus, and carbamate pesticides. The detector response was linear in the concentration range between LOQ and 100 times the LOQ and correlations were better than Table 3 gives recoveries of honey samples obtained by quintuplicate analysis of spiked honeys at two concentration levels (LOQs, and 5 times LOQs). The mean a Each value is the mean of five determinations. b DMC, dichloromethane; EA, ethyl acetate; MeOH, methanol. recoveries for GC determined pesticides were from 79 to 98% with within-day R
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