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Simple and rapid determination of N-methylcarbamate pesticides in citrus fruits by electrospray ionization tandem mass spectrometry
Analytica Chimica Acta 487 (2003) 201–209
Simple and rapid determination of N-methylcarbamate pesticides in citrus fruits by electrospray ionization tandem mass spectrometry Tomomi Goto a,∗ , Yuko Ito a , Hisao Oka a , Isao Saito a , Hiroshi Matsumoto a , Hiroyuki Nakazawa b a
Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsuji-machi, Kita-ku, Nagoya 462-8576, Japan b Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan Received 28 January 2003; received in revised form 6 May 2003; accepted 6 May 2003
Abstract We developed a new analysis method for the N-methylcarbamate pesticides in citrus fruits. The pesticides were extracted from citrus fruits with cyclohexane, the extract was cleaned up by gel permeation chromatography (GPC) and determined by flow-injection electrospray ionization (ESI)–MS/MS. The new method is simple and rapid, and allows simultaneous determination of seven N-methylcarbamate pesticides in citrus fruits. The average recoveries from citrus fruits fortified at the level of 0.5 ppm ranged from 66.8 to 129.2% with the coefficients of variation ranging from 0.5 to 6.2% for intra-day (n = 5 × 3 days) and from 4.1 to 15.9% for inter-day (n = 15). The method is considered to be satisfactory for the monitoring of the carbamate pesticides residues in citrus fruits, suggesting that the present method is applicable to the other pesticide residues in foods such as organochlorine and organophosphorous pesticides. © 2003 Elsevier Science B.V. All rights reserved. Keywords: N-Methylcarbamate pesticides; Citrus fruits; Gel permeation chromatography (GPC); Flow-injection ESI–MS/MS
1. Introduction The use of pesticides in agriculture is necessary to combat a variety of pests that could destroy crops, and to improve the quality of the food produced. Agricultural use of pesticides plays a beneficial role in providing a plentiful, low-cost supply of high-quality fruits and vegetables. On the other hand, as a consequence of this use, the presence of residues in food that was critical elements of overall population health is unavoidable and pesticide residues in food is of great ∗ Corresponding author. Tel.: +81-52-910-5638; fax: +81-52-913-3641. E-mail address: tomomi 3 [email protected] (T. Goto).
importance in the evaluation of food quality. So, to evaluate whether the amounts found in food were less than the maximum residue limits is very important in the control of pesticides residue in food. N-Methylcarbamate pesticides, represented by aldicarb, carbaryl, diethofencarb, fenobucarb, methiocarb, methomyl and pirimicarb (Fig. 1) have the action on anticholinesterase, and are used all over the world. In Japan, these pesticides have been often found in citrus fruits [1–7]. It has one of the major roles as public health agencies to provide safe foods for consumers through analysis of these pesticides in citrus fruits. The most widely used method for the analysis of N-methylcarbamate pesticides in foods, is HPLC using postcolumn hydrolysis and derivatization
0003-2670/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0003-2670(03)00559-2
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Fig. 1. Chemical structures of the carbamate in pesticides.
with fluorescence detection. The typical method is as follows: the pesticides are extracted from foods with organic solvents, the extract is cleaned up by a method based on liquid–liquid or liquid–solid partition, and is determined by HPLC with postcolumn derivatization [8–10]. However, these methods are necessary for lot of time-consumed operations that require skillful techniques to confirm pesticides. In addition, co-existing substances having fluorescence from foods, especially from citrus fruits, frequently interfere with the determination of the pesticides. Therefore, it is desirable that a simple, rapid and reliable analysis method for the pesticides in citrus fruits will be developed. Tandem mass spectrometry that uses three quadrupoles in series can achieve excellent sensitivity even with a complex matrix, because it eliminates
interference prior to measurement of ions originated from target compounds. The first quadrupole functions as a mass filter that passes through only ions within a small range of masses. The second quadrupole is the collision chamber, where the isolated ions are cleaved and transferred to the third quadrupole. The third quadrupole filters the results of the cleavage so they can be scanned as the product ion spectrum. MS/MS is, therefore, effective in identifying compounds, and has proven to be as sensitive as selective detector [11]. Because the first quadrupole functions as a mass filter that passes through only ions within a small range of masses, a severe clean-up operation is not always necessary to prepare sample solution. But, complex matrices from sample may influence ion formation processes [12–14]. In order to avoid the
T. Goto et al. / Analytica Chimica Acta 487 (2003) 201–209
influence, it is effective to use a stable isotopically labeled internal standard that is not different in the physicochemical behavior from a corresponding compound expect for molecular weight. So, it is enough to eliminate roughly impurities from extracts using an appropriate clean-up method such as gel permeation chromatography (GPC), then the extract is directly injected into MS/MS using the flow-injection system [15,16]. According to the above consideration, we examined a new method using flow-injection electrospray ionization (ESI)–MS/MS for the residual N-methylcarbamate pesticides in citrus fruits.
2. Experimental 2.1. Chemicals All organic solvents (methanol, ethanol, cyclohexane, acetone and ethyl acetate) and anhydrous disodium sulfate were of pesticide grade obtained from Wako (Osaka, Japan). Regent grade formic acid was purchased from Merck (Darmstadt, Germany). The high-purity deionized water used was obtained from a PURIC-Z (Organo, Tokyo, Japan) purification system. Aldicarb, methiocarb and methomyl were obtained from Riedel-de Haen (Hanover, Germany), carbaryl, fenobucarb and pirimicarb from Wako and diethofencarb and fenobucarb-d3 (chemical purity >98.0%) from Hayashi (Osaka, Japan), respectively. Each stock standard solution and internal standard were prepared in methanol (1 mg/ml) and the working standard solutions were diluted prior to analysis.
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2.2. Instruments 2.2.1. Gel permeation chromatography conditions The GPC system consisted of a Shimadzu LC-10A (Shimadzu, Tokyo) equipped with a 20× 300 ml CLNpak EV-2000 column packed with Biobees S-X3 (200–400 mesh) (Showa Denko, Tokyo, Japan); mobile phase, acetone–cyclohexane (1:4); flow rate, 4.0 ml/min. 2.2.2. Flow-injection ESI–MS/MS system The flow-injection ESI–MS/MS system consisted of an HP1100 series binary pump, and an auto sampler (Hewlett-Packard, Palo Alto, CA, USA). The MS/MS system consisted of an Quattro II triple quadrupole tandem mass spectrometer (Micromass UK, Altrincham, UK) equipped with a Z-spray (ESI) source. Polyether ether ketone (PEEK) tubing (1 m × 0.5 mm i.d.) was used for the connection of the tandem mass spectrometer with the auto sampler. 2.2.3. Mass spectrometric conditions The desolvation gas (nitrogen) temperature and flow rate were set at 200 ◦ C and 370 l/h, respectively. The ion source temperature was set at 100 ◦ C. The instrument was operated in the positive ion mode. Full scan data were collected from 50 to 300 m/z at a rate of 1 scan/s. Collision-induced dissociation was performed using argon as the collision gas at the pressure of 1.9 × 10−3 mbar in the collision cell. The other mass spectrometric parameters are summarized in Table 1. The carrier liquid was a mixture of methanol and 0.1% formic acid at a ratio of 9:1 and the flow rate of 200 l/min.
Table 1 Compound-specific ESI–MS/MS parameters for the pesticides and the internal standard Carbamate pesticides
Precursor ion (m/z)
Cone voltage (V)
Collision energy (eV)
Monitor ion (m/z)
Aldicarb Carbaryl Diethofencarb Fenobucarb Fenobucarb-d3 Methiocarb Methomyl Pirimicarb
213a 202b 268b 208b 211b 226b 163b 239b
25 15 15 25 25 23 15 30
10 10 10 15 14 20 8 25
89 145 226 95 95 121 106 72
a b
[M + Na]+ . [M + H]+ .
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2.3. Sample preparation and clean-up procedure on GPC Whole fruit sample was chopped and homogenized with a mixer. A 5 g aliquot of the homogenized sample was weighed into a 100 ml centrifuge tube and 0.1 ml of internal standard working solution (1 g/ml) was added. The mixture was blended with 100 ml of cyclohexane and 20 g of anhydrous sodium sulfate with a high-speed blender. The extracts were centrifuged at 3100 rpm for 8 min and the upper organic layer decanted into a flask. Then, place the flask in a water bath at 30 ◦ C and gently evaporated the solvent to near dryness. Allow remaining solvent to evaporate in the air. The residue was redissolved in 5 ml of GPC mobile phase. A 2 ml aliquot of the solution was loaded onto the GPC. The elution between 0 and 60 ml was discarded, and then the elution between 60 and 110 ml was collected and concentrated to dryness. A 1 ml of methanol was added to the residue and sonicated to redissolve it. A 1 l of the methanol solution was injected into the flow-injection ESI–MS/MS system. 2.4. Quantitation Calibration curves were constructed by peak-area ratios of the pesticides to the internal standard. Recoveries were calculated as the ratio of the peak-area ratio of the analyte to the internal standard from the fortified samples to the corresponding peak-area ratio of standard solutions.
3. Results and discussion 3.1. ESI–MS/MS conditions To achieve highly sensitive multiple reaction monitoring (MRM), cone voltage and collision energy should be adjusted for each pesticide. Using the selected cone voltages between 20 and 50 V, electrospray ionization mass spectra were recorded for the seven pesticides. Fenobucarb, methiocarb and pirimicarb gave clearly [M + H]+ at m/z 208, 226 and 239 under positive ion mode, respectively, and their highest intensities were obtained at the cone voltages ranging from 23 to 30 V (Table 1). However,
aldicarb, carbaryl, diethofencarb and methomyl gave only [M + Na]+ without [M + H] at m/z 213, 230, 258 and 185, respectively. In our preliminary experiments, it is suggested that use of [M + H]+ as the precursor ions gives product ion spectra with clear cleavages more easily than the case of [M + Na]+ . In order to produce [M + H]+ for aldicarb, carbaryl, diethofencarb and methomyl, we tried to measure their mass spectra at the reduced cone voltages ranging from 5 to 25 V. As a result, at the reduced cone voltage of 15 V, [M + H]+ were clearly observed with the highest intensities at m/z 202 for carbaryl, m/z 268 for diethofencarb and m/z 163 for methomyl (Table 1). However, we could not observe [M + H]+ for aldicarb. Next, we measured their fullscan ESI tandem mass spectra using the [M + H]+ as precursor ions at the cone voltage shown in Table 1, although [M + Na]+ was served as precursor ion for aldicarb. As a result, we obtained their product ion spectra with clear cleavages of the precursor ions as expected. Therefore, we chose the precursor ions and cone voltages listed in Table 1 and the following product ions as the monitor ions that showed the highest intensity for MRM: aldicarb, m/z 89; carbaryl, m/z 145; diethofencarb, m/z 226; fenobucarb, m/z 95; methiocarb, m/z 121; methomyl, m/z 106; pirimicarb, m/z 72. Each solution of seven pesticides prepared in methanol (1 g/ml) was flow-injected into the ESI–MS/MS. We investigated the intensity of each monitor ion appearing on the MRM profiles under the selected collision energies (5–40 eV). The collision energies which gave the highest intensities of the monitor ions are listed in Table 1, so we used them in the subsequent works. Calibration curves obtained under these conditions were linear over the range of 0.01–0.7 g/ml with correlation coefficients of 0.999. 3.2. Clean-up procedure We attempted to analyse the extract from lemon without clean-up step using the above MS/MS techniques, but we could not obtain satisfactory results because of suppression effect of ionization due to coextracted substances from lemon sample as suspected [12–14]. Therefore, we decided to apply GPC to the clean-up procedure, that is used as a
T. Goto et al. / Analytica Chimica Acta 487 (2003) 201–209 Table 2 Elution profiles of pesticides from GPC column Carbamate pesticides
GPC elution volume (ml)
Aldicarb Carbaryl Diethofencarb Fenobucarb Fenobucarb-d3 Methiocarb Methomyl Pirimicarb
70–80 90–100 60–80 70–80 70–90 60–80 80–100 80–90
pre-clean-up step in the various analysis methods for the pesticides. In order to determine the optimal GPC conditions, we carried out the following experiments: a 2 ml of mixed standard solution of the N-methylcarbamate pesticides dissolved in GPC mobile phase (1 g/ml each) was injected into the GPC system. Then, the eluant was fractionated every 10 ml, concentrated to dryness and redissolved in 1 ml of methanol. The solution was analysed by the flow-injection ESI–MS/MS. As a result, all of the pesticides were eluted from the GPC under the elution volumes listed in Table 2. In GPC, methiocarb eluted first between elution volumes of 60 and 80 ml, and carbaryl was lastly eluted between 90 and 100 ml. Therefore, we discarded eluate between elution volumes of 0 and 60 ml and collected between 60 and 110 ml for the analysis of pesticides in the subsequent works. 3.3. Extraction procedure The extract should contain a minimum of matrix from citrus fruits, but extraction of the pesticides of interest must be quantitative, so extraction solvent is important in the analytical procedure. In order to select suitable extraction solvent, we evaluated various organic solvents by the following procedures: a 5 g of chopped pesticides-free lemon was weighed into a centrifuge tube, and fortified with 0.1 ml of the pesticide standard working solution (25 g/ml) and 0.1 ml of internal standard working solution (10 g/ml). Then, it was homogenized with various organic solvents and the extract was cleaned up by GPC under the above conditions.
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At first, ethyl acetate was examined. However, when ethyl acetate extract was concentrated, a vivid yellow non-volatile liquid appeared in the flask, that did not dissolve in the mobile phase for GPC and methanol, but dissolved in ethanol. The mobile phase for GPC was added into the flask containing the yellow liquid and sonicated. We applied only the mobile phase solution eliminating the yellow liquid to GPC clean up and carried out MS/MS analysis. However, recovery of pirimicarb was less than 10%. We considered that pirimicarb existed in the vivid yellow liquid that did not dissolve in the mobile phase. So, the yellow liquid was dissolved in 1 ml ethanol following addition of 4 ml of the mobile phase for GPC and the resulting solution was applied to GPC clean up. Although recovery of pirimicarb was improved more than 80%, aldicarb showed less than 20% and its MS/MS profile was asymmetrical peak. We supposed that the vivid yellow liquid causes serious problem in quantative analysis of the carbamate pesticides. In order to search extraction solvent that can extract the pesticides quantitatively and can eliminate the yellow liquid, the following organic solvents were tried: acetonitrile, acetone, acetone–cyclohexane (1:1), acetone–cyclohexane (1:4), and cyclohexane. Acetonitrile and acetone showed the same results as that of ethyl acetate. Acetone–cyclohexane (1:1) and acetone–cyclohexane (1:4) gave unsatisfactory recoveries for aldicarb, although it was observed less amount of the yellow liquid in the extraction residue. Only cyclohexane gave satisfactory result that the pesticides were able to be quantitatively extracted without the vivid yellow liquid. Resulting MS/MS profiles gave symmetrical peaks. Fig. 2 shows typical MRM profiles of the fortified lemon with the pesticides at the level of 0.5 ppm and the blank lemon according to the established analytical procedure (Fig. 3). All of the MRM profiles of the fortified samples were almost the same as the respective standards. Therefore, we conclude that the optimal extraction solvent is cyclohexane. 3.4. Validation of analytical method with fortified citrus fruits Method accuracy was determined by calculating from the calibration curves that were constructed by
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Fig. 2. Typical MRM profiles of the fortified and blank lemon samples.
Pesticides
Orange
Lemon
Intra-daya Day 1 Aldicarb 87.4 Carbaryl 99.1 Diethofencarb 94.2 Fenobucarb 97.1 Methiocarb 94.5 Methomyl 93.6 Pirimicarb 113.6
Inter-dayb Day 2
Day 3
(2.0) 98.9 (3.9) 72.7 (2.4) 87.0 (2.5) 97.2 (4.3) 86.4 (1.7) 94.2 (2.2) 102.3 (2.0) 87.7 (1.4) 94.7 (0.9) 98.6 (0.7) 95.5 (1.4) 97.1 (1.8) 97.4 (1.3) 86.5 (1.6) 92.8 (2.4) 81.2 (0.5) 73.6 (2.4) 82.8 (2.7) 123.7 (3.9) 98.3 (2.4) 111.9
Grapefruit
Intra-daya Day 1
Inter-dayb Day 2
Day 3
(12.0) 98.6 (2.6) 111.9 (2.7) 111.1 (2.9) 107.2 (6.4) 100.5 (2.2) 94.0 (1.1) 97.6 (2.6) 97.4 (6.3) 98.2 (2.4) 103.7 (2.2) 90.4 (1.3) 97.8 (1.7) 97.1 (0.9) 100.1 (1.4) 97.4 (0.7) 98.9 (4.9) 99.2 (2.0) 95.0 (1.4) 94.2 (1.5) 96.1 (8.5) 86.8 (2.9) 83.0 (2.7) 81.6 (3.7) 83.8 (10.4) 103.6 (2.6) 107.3 (2.0) 96.5 (6.1) 102.5
The values given outside the parenthesis are recovery (%) and those inside the parenthesis are C.V. (%). a Calculated from mean values (n = 5). b Calculated from mean values (n = 15).
Intra-daya Day 1
(6.7) 93.2 (3.4) 88.7 (6.3) 96.3 (1.7) 98.4 (2.8) 93.4 (3.8) 96.7 (6.0) 129.2
Inter-dayb Day 2
Day 3
(3.2) 108.4 (5.6) 66.8 (1.2) 81.6 (2.2) 83.7 (1.1) 97.5 (0.9) 94.8 (0.7) 96.6 (0.8) 96.6 (1.7) 88.7 (1.3) 87.6 (2.8) 96.1 (2.1) 95.1 (1.6) 107.3 (2.0) 113.7
(6.2) 88.8 (1.6) 84.7 (1.3) 96.2 (1.2) 97.2 (1.3) 89.9 (4.0) 96.0 (2.0) 122.9
(17.8) (3.4) (1.6) (1.3) (2.9) (3.1) (6.9)
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Table 3 Intra-day and inter-day repeatability of the present method
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Over all results indicate that the present method is sensitive, accurate and precise enough for the monitoring of the pesticide residues in citrus fruits.
4. Conclusions
Fig. 3. Analytical procedure for the pesticides.
peak-area ratios of the pesticides to internal standard, and were linear over the range of 0.01–0.7 g/ml with correlation coefficients over 0.999. Recoveries were calculated as the ratio of the peak-area of the analyte to the internal standard from the fortified samples, orange, lemon and grapefruit to the corresponding peak-area ratio of standard solutions. The recoveries from citrus fruits fortified at levels of 0.5 ppm and the coefficients of variation (C.V.) for both the intra- and inter-assay precision are listed in Table 3. As for the intra-day (n = 5 × 3 days) precision was determined by duplicate assays. The recoveries of the pesticides from orange, lemon and grapefruit ranged from 72.7 to 123.7% with C.V. less than 4.3%, from 81.6 to 111.9% with C.V. less than 3.7%, and from 66.8 to 129.2% with C.V. less than 6.2%, respectively. Inter-assay (between-day) precision was determined by duplicate assays on three successive days of pesticide-free citrus fruits sample fortified with each pesticide at 0.5 ppm. The inter-day recoveries of the pesticides ranged from 82.8 to 111.9% with C.V. less than 12.0% for orange, from 83.8 to 107.2% with C.V. less than 6.7% for lemon, and from 84.7 to 122.9% with C.V. less than 17.8% grapefruit. The limits of detection were 0.05 g/ml (S/N > 3) for each pesticide.
This paper reported the new method for the analysis of N-methylcarbamate pesticides in citrus fruits, using flow-injection ESI–MS/MS. The method involves three steps: (1) extraction with cyclohexane; (2) clean up using GPC; and (3) determination using flow-injection ESI–MS/MS. The combination use of these three steps enabled us a simple, rapid and reliable analysis of N-methylcarbamate in citrus fruits. Therefore, we strongly recommend the present method for the monitoring of the pesticides in citrus fruits. Furthermore, we suggest that the present method is applicable to the other pesticide residue in foods.
Acknowledgements The authors wish to thank Mrs. Y. Yoshimi for carrying out some of the experiments. References [1] G.J. Soleans, J. Yan, K. Hom, D.M. Goldberg, J. Chromatogr. A 205 (2000) 882. [2] R.K. Juhler, M.G. Lauridsen, M.R. Christensen, G. Hilbert, J. AOAC Int. 337 (1999) 82. [3] S.M. Dogheim, S.A.C. Alla, A.M. El-marsafy, J. AOAC Int. 978 (1999) 82. [4] B.D. Ripley, L.I. Lissemore, P.D. Leishiman, M.A. Denomme, J. AOAC Int. 196 (2000) 83. [5] US Food and Drug Administration, 1991, J. AOAC Int. 121A (1992) 74. [6] R.R. Roy, R.H. Albert, P. Wilson, R.R. Laski, J.I. Roberts, T.J. Hoffmann, R.L. Bpng, B.O. Bohannon, N.J. Yess, J. AOAC Int. 930 (1995) 78. [7] Y. Ueno, T. Ogawa, N. Okamoto, K. Taniguchi, A. Misaka, S. Nisida, H. Higuchi, Food Sanit. Res. 75 (1996) 46. [8] T. Nagayama, M. Kobayashi, H. Shioda, M. Morino, M. Ito, Y. Tamura, Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Jpn.) 470 (1994) 35. [9] Y. Tsumuraand, Y. Nakamura, K. Yoshii, Y. Tonagai, M. Higo, T. Shibata, Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Jpn.) 357 (1998) 39. [10] W. Horwitz (Ed.), Official Methods of Analysis of AOAC International, vol. 1, 17th ed., AOAC International, USA, 2000, Chapter 7, pp. 22–32.
T. Goto et al. / Analytica Chimica Acta 487 (2003) 201–209 [11] R.S. Sheridan, J.R. Meola, J. AOAC Int. 982 (1999) 82. [12] S. Takatsuki, S. Nemoto, R. Matsuda, K. Sasaki, M. Toyoda, Shokuhin Eiseigaku Zasshi (J. Food Hyg. Soc. Jpn.) 314 (1999) 40. [13] D.M. Holstege, D.L. Scharberg, E.R. Tor, L.C. Hart, F.D. Galey, J. AOAC Int. 1263 (1994) 77.
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[14] J. Henion, E. Brewer, G. Rule, Anal. Chem. 650A (1998) 70. [15] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Anal. Chem. 882 (1998) 70. [16] J.J. Stry, J.S. Amoo, S.W. George, T. Hamilton-Johnson, E. Stetser, J. AOAC Int. 651 (2000) 83.
Simple and rapid determination of N-methylcarbamate pesticides in citrus fruits by electrospray ionization tandem mass spectrometry Tomomi Goto a,∗ , Yuko Ito a , Hisao Oka a , Isao Saito a , Hiroshi Matsumoto a , Hiroyuki Nakazawa b a
Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsuji-machi, Kita-ku, Nagoya 462-8576, Japan b Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan Received 28 January 2003; received in revised form 6 May 2003; accepted 6 May 2003
Abstract We developed a new analysis method for the N-methylcarbamate pesticides in citrus fruits. The pesticides were extracted from citrus fruits with cyclohexane, the extract was cleaned up by gel permeation chromatography (GPC) and determined by flow-injection electrospray ionization (ESI)–MS/MS. The new method is simple and rapid, and allows simultaneous determination of seven N-methylcarbamate pesticides in citrus fruits. The average recoveries from citrus fruits fortified at the level of 0.5 ppm ranged from 66.8 to 129.2% with the coefficients of variation ranging from 0.5 to 6.2% for intra-day (n = 5 × 3 days) and from 4.1 to 15.9% for inter-day (n = 15). The method is considered to be satisfactory for the monitoring of the carbamate pesticides residues in citrus fruits, suggesting that the present method is applicable to the other pesticide residues in foods such as organochlorine and organophosphorous pesticides. © 2003 Elsevier Science B.V. All rights reserved. Keywords: N-Methylcarbamate pesticides; Citrus fruits; Gel permeation chromatography (GPC); Flow-injection ESI–MS/MS
1. Introduction The use of pesticides in agriculture is necessary to combat a variety of pests that could destroy crops, and to improve the quality of the food produced. Agricultural use of pesticides plays a beneficial role in providing a plentiful, low-cost supply of high-quality fruits and vegetables. On the other hand, as a consequence of this use, the presence of residues in food that was critical elements of overall population health is unavoidable and pesticide residues in food is of great ∗ Corresponding author. Tel.: +81-52-910-5638; fax: +81-52-913-3641. E-mail address: tomomi 3 [email protected] (T. Goto).
importance in the evaluation of food quality. So, to evaluate whether the amounts found in food were less than the maximum residue limits is very important in the control of pesticides residue in food. N-Methylcarbamate pesticides, represented by aldicarb, carbaryl, diethofencarb, fenobucarb, methiocarb, methomyl and pirimicarb (Fig. 1) have the action on anticholinesterase, and are used all over the world. In Japan, these pesticides have been often found in citrus fruits [1–7]. It has one of the major roles as public health agencies to provide safe foods for consumers through analysis of these pesticides in citrus fruits. The most widely used method for the analysis of N-methylcarbamate pesticides in foods, is HPLC using postcolumn hydrolysis and derivatization
0003-2670/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0003-2670(03)00559-2
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Fig. 1. Chemical structures of the carbamate in pesticides.
with fluorescence detection. The typical method is as follows: the pesticides are extracted from foods with organic solvents, the extract is cleaned up by a method based on liquid–liquid or liquid–solid partition, and is determined by HPLC with postcolumn derivatization [8–10]. However, these methods are necessary for lot of time-consumed operations that require skillful techniques to confirm pesticides. In addition, co-existing substances having fluorescence from foods, especially from citrus fruits, frequently interfere with the determination of the pesticides. Therefore, it is desirable that a simple, rapid and reliable analysis method for the pesticides in citrus fruits will be developed. Tandem mass spectrometry that uses three quadrupoles in series can achieve excellent sensitivity even with a complex matrix, because it eliminates
interference prior to measurement of ions originated from target compounds. The first quadrupole functions as a mass filter that passes through only ions within a small range of masses. The second quadrupole is the collision chamber, where the isolated ions are cleaved and transferred to the third quadrupole. The third quadrupole filters the results of the cleavage so they can be scanned as the product ion spectrum. MS/MS is, therefore, effective in identifying compounds, and has proven to be as sensitive as selective detector [11]. Because the first quadrupole functions as a mass filter that passes through only ions within a small range of masses, a severe clean-up operation is not always necessary to prepare sample solution. But, complex matrices from sample may influence ion formation processes [12–14]. In order to avoid the
T. Goto et al. / Analytica Chimica Acta 487 (2003) 201–209
influence, it is effective to use a stable isotopically labeled internal standard that is not different in the physicochemical behavior from a corresponding compound expect for molecular weight. So, it is enough to eliminate roughly impurities from extracts using an appropriate clean-up method such as gel permeation chromatography (GPC), then the extract is directly injected into MS/MS using the flow-injection system [15,16]. According to the above consideration, we examined a new method using flow-injection electrospray ionization (ESI)–MS/MS for the residual N-methylcarbamate pesticides in citrus fruits.
2. Experimental 2.1. Chemicals All organic solvents (methanol, ethanol, cyclohexane, acetone and ethyl acetate) and anhydrous disodium sulfate were of pesticide grade obtained from Wako (Osaka, Japan). Regent grade formic acid was purchased from Merck (Darmstadt, Germany). The high-purity deionized water used was obtained from a PURIC-Z (Organo, Tokyo, Japan) purification system. Aldicarb, methiocarb and methomyl were obtained from Riedel-de Haen (Hanover, Germany), carbaryl, fenobucarb and pirimicarb from Wako and diethofencarb and fenobucarb-d3 (chemical purity >98.0%) from Hayashi (Osaka, Japan), respectively. Each stock standard solution and internal standard were prepared in methanol (1 mg/ml) and the working standard solutions were diluted prior to analysis.
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2.2. Instruments 2.2.1. Gel permeation chromatography conditions The GPC system consisted of a Shimadzu LC-10A (Shimadzu, Tokyo) equipped with a 20× 300 ml CLNpak EV-2000 column packed with Biobees S-X3 (200–400 mesh) (Showa Denko, Tokyo, Japan); mobile phase, acetone–cyclohexane (1:4); flow rate, 4.0 ml/min. 2.2.2. Flow-injection ESI–MS/MS system The flow-injection ESI–MS/MS system consisted of an HP1100 series binary pump, and an auto sampler (Hewlett-Packard, Palo Alto, CA, USA). The MS/MS system consisted of an Quattro II triple quadrupole tandem mass spectrometer (Micromass UK, Altrincham, UK) equipped with a Z-spray (ESI) source. Polyether ether ketone (PEEK) tubing (1 m × 0.5 mm i.d.) was used for the connection of the tandem mass spectrometer with the auto sampler. 2.2.3. Mass spectrometric conditions The desolvation gas (nitrogen) temperature and flow rate were set at 200 ◦ C and 370 l/h, respectively. The ion source temperature was set at 100 ◦ C. The instrument was operated in the positive ion mode. Full scan data were collected from 50 to 300 m/z at a rate of 1 scan/s. Collision-induced dissociation was performed using argon as the collision gas at the pressure of 1.9 × 10−3 mbar in the collision cell. The other mass spectrometric parameters are summarized in Table 1. The carrier liquid was a mixture of methanol and 0.1% formic acid at a ratio of 9:1 and the flow rate of 200 l/min.
Table 1 Compound-specific ESI–MS/MS parameters for the pesticides and the internal standard Carbamate pesticides
Precursor ion (m/z)
Cone voltage (V)
Collision energy (eV)
Monitor ion (m/z)
Aldicarb Carbaryl Diethofencarb Fenobucarb Fenobucarb-d3 Methiocarb Methomyl Pirimicarb
213a 202b 268b 208b 211b 226b 163b 239b
25 15 15 25 25 23 15 30
10 10 10 15 14 20 8 25
89 145 226 95 95 121 106 72
a b
[M + Na]+ . [M + H]+ .
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2.3. Sample preparation and clean-up procedure on GPC Whole fruit sample was chopped and homogenized with a mixer. A 5 g aliquot of the homogenized sample was weighed into a 100 ml centrifuge tube and 0.1 ml of internal standard working solution (1 g/ml) was added. The mixture was blended with 100 ml of cyclohexane and 20 g of anhydrous sodium sulfate with a high-speed blender. The extracts were centrifuged at 3100 rpm for 8 min and the upper organic layer decanted into a flask. Then, place the flask in a water bath at 30 ◦ C and gently evaporated the solvent to near dryness. Allow remaining solvent to evaporate in the air. The residue was redissolved in 5 ml of GPC mobile phase. A 2 ml aliquot of the solution was loaded onto the GPC. The elution between 0 and 60 ml was discarded, and then the elution between 60 and 110 ml was collected and concentrated to dryness. A 1 ml of methanol was added to the residue and sonicated to redissolve it. A 1 l of the methanol solution was injected into the flow-injection ESI–MS/MS system. 2.4. Quantitation Calibration curves were constructed by peak-area ratios of the pesticides to the internal standard. Recoveries were calculated as the ratio of the peak-area ratio of the analyte to the internal standard from the fortified samples to the corresponding peak-area ratio of standard solutions.
3. Results and discussion 3.1. ESI–MS/MS conditions To achieve highly sensitive multiple reaction monitoring (MRM), cone voltage and collision energy should be adjusted for each pesticide. Using the selected cone voltages between 20 and 50 V, electrospray ionization mass spectra were recorded for the seven pesticides. Fenobucarb, methiocarb and pirimicarb gave clearly [M + H]+ at m/z 208, 226 and 239 under positive ion mode, respectively, and their highest intensities were obtained at the cone voltages ranging from 23 to 30 V (Table 1). However,
aldicarb, carbaryl, diethofencarb and methomyl gave only [M + Na]+ without [M + H] at m/z 213, 230, 258 and 185, respectively. In our preliminary experiments, it is suggested that use of [M + H]+ as the precursor ions gives product ion spectra with clear cleavages more easily than the case of [M + Na]+ . In order to produce [M + H]+ for aldicarb, carbaryl, diethofencarb and methomyl, we tried to measure their mass spectra at the reduced cone voltages ranging from 5 to 25 V. As a result, at the reduced cone voltage of 15 V, [M + H]+ were clearly observed with the highest intensities at m/z 202 for carbaryl, m/z 268 for diethofencarb and m/z 163 for methomyl (Table 1). However, we could not observe [M + H]+ for aldicarb. Next, we measured their fullscan ESI tandem mass spectra using the [M + H]+ as precursor ions at the cone voltage shown in Table 1, although [M + Na]+ was served as precursor ion for aldicarb. As a result, we obtained their product ion spectra with clear cleavages of the precursor ions as expected. Therefore, we chose the precursor ions and cone voltages listed in Table 1 and the following product ions as the monitor ions that showed the highest intensity for MRM: aldicarb, m/z 89; carbaryl, m/z 145; diethofencarb, m/z 226; fenobucarb, m/z 95; methiocarb, m/z 121; methomyl, m/z 106; pirimicarb, m/z 72. Each solution of seven pesticides prepared in methanol (1 g/ml) was flow-injected into the ESI–MS/MS. We investigated the intensity of each monitor ion appearing on the MRM profiles under the selected collision energies (5–40 eV). The collision energies which gave the highest intensities of the monitor ions are listed in Table 1, so we used them in the subsequent works. Calibration curves obtained under these conditions were linear over the range of 0.01–0.7 g/ml with correlation coefficients of 0.999. 3.2. Clean-up procedure We attempted to analyse the extract from lemon without clean-up step using the above MS/MS techniques, but we could not obtain satisfactory results because of suppression effect of ionization due to coextracted substances from lemon sample as suspected [12–14]. Therefore, we decided to apply GPC to the clean-up procedure, that is used as a
T. Goto et al. / Analytica Chimica Acta 487 (2003) 201–209 Table 2 Elution profiles of pesticides from GPC column Carbamate pesticides
GPC elution volume (ml)
Aldicarb Carbaryl Diethofencarb Fenobucarb Fenobucarb-d3 Methiocarb Methomyl Pirimicarb
70–80 90–100 60–80 70–80 70–90 60–80 80–100 80–90
pre-clean-up step in the various analysis methods for the pesticides. In order to determine the optimal GPC conditions, we carried out the following experiments: a 2 ml of mixed standard solution of the N-methylcarbamate pesticides dissolved in GPC mobile phase (1 g/ml each) was injected into the GPC system. Then, the eluant was fractionated every 10 ml, concentrated to dryness and redissolved in 1 ml of methanol. The solution was analysed by the flow-injection ESI–MS/MS. As a result, all of the pesticides were eluted from the GPC under the elution volumes listed in Table 2. In GPC, methiocarb eluted first between elution volumes of 60 and 80 ml, and carbaryl was lastly eluted between 90 and 100 ml. Therefore, we discarded eluate between elution volumes of 0 and 60 ml and collected between 60 and 110 ml for the analysis of pesticides in the subsequent works. 3.3. Extraction procedure The extract should contain a minimum of matrix from citrus fruits, but extraction of the pesticides of interest must be quantitative, so extraction solvent is important in the analytical procedure. In order to select suitable extraction solvent, we evaluated various organic solvents by the following procedures: a 5 g of chopped pesticides-free lemon was weighed into a centrifuge tube, and fortified with 0.1 ml of the pesticide standard working solution (25 g/ml) and 0.1 ml of internal standard working solution (10 g/ml). Then, it was homogenized with various organic solvents and the extract was cleaned up by GPC under the above conditions.
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At first, ethyl acetate was examined. However, when ethyl acetate extract was concentrated, a vivid yellow non-volatile liquid appeared in the flask, that did not dissolve in the mobile phase for GPC and methanol, but dissolved in ethanol. The mobile phase for GPC was added into the flask containing the yellow liquid and sonicated. We applied only the mobile phase solution eliminating the yellow liquid to GPC clean up and carried out MS/MS analysis. However, recovery of pirimicarb was less than 10%. We considered that pirimicarb existed in the vivid yellow liquid that did not dissolve in the mobile phase. So, the yellow liquid was dissolved in 1 ml ethanol following addition of 4 ml of the mobile phase for GPC and the resulting solution was applied to GPC clean up. Although recovery of pirimicarb was improved more than 80%, aldicarb showed less than 20% and its MS/MS profile was asymmetrical peak. We supposed that the vivid yellow liquid causes serious problem in quantative analysis of the carbamate pesticides. In order to search extraction solvent that can extract the pesticides quantitatively and can eliminate the yellow liquid, the following organic solvents were tried: acetonitrile, acetone, acetone–cyclohexane (1:1), acetone–cyclohexane (1:4), and cyclohexane. Acetonitrile and acetone showed the same results as that of ethyl acetate. Acetone–cyclohexane (1:1) and acetone–cyclohexane (1:4) gave unsatisfactory recoveries for aldicarb, although it was observed less amount of the yellow liquid in the extraction residue. Only cyclohexane gave satisfactory result that the pesticides were able to be quantitatively extracted without the vivid yellow liquid. Resulting MS/MS profiles gave symmetrical peaks. Fig. 2 shows typical MRM profiles of the fortified lemon with the pesticides at the level of 0.5 ppm and the blank lemon according to the established analytical procedure (Fig. 3). All of the MRM profiles of the fortified samples were almost the same as the respective standards. Therefore, we conclude that the optimal extraction solvent is cyclohexane. 3.4. Validation of analytical method with fortified citrus fruits Method accuracy was determined by calculating from the calibration curves that were constructed by
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Fig. 2. Typical MRM profiles of the fortified and blank lemon samples.
Pesticides
Orange
Lemon
Intra-daya Day 1 Aldicarb 87.4 Carbaryl 99.1 Diethofencarb 94.2 Fenobucarb 97.1 Methiocarb 94.5 Methomyl 93.6 Pirimicarb 113.6
Inter-dayb Day 2
Day 3
(2.0) 98.9 (3.9) 72.7 (2.4) 87.0 (2.5) 97.2 (4.3) 86.4 (1.7) 94.2 (2.2) 102.3 (2.0) 87.7 (1.4) 94.7 (0.9) 98.6 (0.7) 95.5 (1.4) 97.1 (1.8) 97.4 (1.3) 86.5 (1.6) 92.8 (2.4) 81.2 (0.5) 73.6 (2.4) 82.8 (2.7) 123.7 (3.9) 98.3 (2.4) 111.9
Grapefruit
Intra-daya Day 1
Inter-dayb Day 2
Day 3
(12.0) 98.6 (2.6) 111.9 (2.7) 111.1 (2.9) 107.2 (6.4) 100.5 (2.2) 94.0 (1.1) 97.6 (2.6) 97.4 (6.3) 98.2 (2.4) 103.7 (2.2) 90.4 (1.3) 97.8 (1.7) 97.1 (0.9) 100.1 (1.4) 97.4 (0.7) 98.9 (4.9) 99.2 (2.0) 95.0 (1.4) 94.2 (1.5) 96.1 (8.5) 86.8 (2.9) 83.0 (2.7) 81.6 (3.7) 83.8 (10.4) 103.6 (2.6) 107.3 (2.0) 96.5 (6.1) 102.5
The values given outside the parenthesis are recovery (%) and those inside the parenthesis are C.V. (%). a Calculated from mean values (n = 5). b Calculated from mean values (n = 15).
Intra-daya Day 1
(6.7) 93.2 (3.4) 88.7 (6.3) 96.3 (1.7) 98.4 (2.8) 93.4 (3.8) 96.7 (6.0) 129.2
Inter-dayb Day 2
Day 3
(3.2) 108.4 (5.6) 66.8 (1.2) 81.6 (2.2) 83.7 (1.1) 97.5 (0.9) 94.8 (0.7) 96.6 (0.8) 96.6 (1.7) 88.7 (1.3) 87.6 (2.8) 96.1 (2.1) 95.1 (1.6) 107.3 (2.0) 113.7
(6.2) 88.8 (1.6) 84.7 (1.3) 96.2 (1.2) 97.2 (1.3) 89.9 (4.0) 96.0 (2.0) 122.9
(17.8) (3.4) (1.6) (1.3) (2.9) (3.1) (6.9)
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Table 3 Intra-day and inter-day repeatability of the present method
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Over all results indicate that the present method is sensitive, accurate and precise enough for the monitoring of the pesticide residues in citrus fruits.
4. Conclusions
Fig. 3. Analytical procedure for the pesticides.
peak-area ratios of the pesticides to internal standard, and were linear over the range of 0.01–0.7 g/ml with correlation coefficients over 0.999. Recoveries were calculated as the ratio of the peak-area of the analyte to the internal standard from the fortified samples, orange, lemon and grapefruit to the corresponding peak-area ratio of standard solutions. The recoveries from citrus fruits fortified at levels of 0.5 ppm and the coefficients of variation (C.V.) for both the intra- and inter-assay precision are listed in Table 3. As for the intra-day (n = 5 × 3 days) precision was determined by duplicate assays. The recoveries of the pesticides from orange, lemon and grapefruit ranged from 72.7 to 123.7% with C.V. less than 4.3%, from 81.6 to 111.9% with C.V. less than 3.7%, and from 66.8 to 129.2% with C.V. less than 6.2%, respectively. Inter-assay (between-day) precision was determined by duplicate assays on three successive days of pesticide-free citrus fruits sample fortified with each pesticide at 0.5 ppm. The inter-day recoveries of the pesticides ranged from 82.8 to 111.9% with C.V. less than 12.0% for orange, from 83.8 to 107.2% with C.V. less than 6.7% for lemon, and from 84.7 to 122.9% with C.V. less than 17.8% grapefruit. The limits of detection were 0.05 g/ml (S/N > 3) for each pesticide.
This paper reported the new method for the analysis of N-methylcarbamate pesticides in citrus fruits, using flow-injection ESI–MS/MS. The method involves three steps: (1) extraction with cyclohexane; (2) clean up using GPC; and (3) determination using flow-injection ESI–MS/MS. The combination use of these three steps enabled us a simple, rapid and reliable analysis of N-methylcarbamate in citrus fruits. Therefore, we strongly recommend the present method for the monitoring of the pesticides in citrus fruits. Furthermore, we suggest that the present method is applicable to the other pesticide residue in foods.
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