Determination of pyrethroids in vegetables by HPLC ...

Analytica Chimica Acta 447 (2001) 101–111

Determination of pyrethroids in vegetables by HPLC using continuous on-line post-elution photoirradiation with fluorescence detection T. López-López b , M.D. Gil-Garcia a , J.L. Mart´ınez-Vidal a,∗ , M. Mart´ınez-Galera a a

Department of Hydrology and Analytical Chemistry, University of Almer´ıa, La Canada de San Urbano, 04071 Almer´ıa, Spain b Laboratory of Pesticide Residues, CUAM, El Ejido, 04700 Almer´ıa, Spain Received 17 April 2001; received in revised form 23 July 2001; accepted 23 July 2001

Abstract Seven naturally non-fluorescent pyrethroids: fenpropathrin, cyfluthrin, deltamethrin, fenvalerate, acrinathrin, tau-fluvalinate and bifenthrin were determined using HPLC with a photochemically-induced post-column method, and fluorescence detection. The method was applied to the analysis of these insecticides in cucumber. Samples were extracted with dichloromethane and further cleaned up by SPE using a florisil cartridge. Matrix effect was overcame using matrix-matched standard for building up the calibration graphs and, in this way, dynamic ranges were established over more than two orders of magnitude. The limits of quantification ranged from 8 to 90 ng ml−1 (corresponding to 0.8 and 9 ␮g kg−1 in the vegetable sample), with relative standard deviations less than 8.6% using blank cucumber extract. Mean recoveries ranged from 95 to 116%. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Pyrethroid insecticides; Photochemically-induced fluorescence; HPLC; Residues; Vegetables

1. Introduction Chronologically, pyrethroids constitute the fourth major group of insecticides developed (after organochlorines, organophosphates and carbamates). They are non-systemic (in plants) contact and stomach poisons to many insects and arachnids with an additional anti-feeding action. The compounds act on the central and peripheral nervous system of target insects at very low doses [1]. Synthetic pyrethroids are effective against a wide spectrum of pest. They are widely used as pest control ∗

Corresponding author. Tel.: +34-950015429; fax: +34-950015483. E-mail address: [email protected] (J.L. Mart´ınez-Vidal).

agents in agricultural production because of their selective insecticidal activity, rapid biotransformation and excretion by the mammalian catabolic system and their non-persistence in the environment. As their range of applications has increased, the need to separate various pyrethroids has arisen, especially for multi-residue analysis. Gas chromatography (GC), using an electron capture detector (ECD) [2,3] is still the method of choice for analysis of pyrethroid residues because it is rapid, inexpensive, and convenient, despite the problems associated with instability of pyrethroids under GC conditions. GC systems with capillary columns coupled to mass spectrometers are also used for pyrethroid residues confirmation [4,5] or as a detection system per se in the quantitative GC residue analysis [6].

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 3 0 5 - 8

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T. López-López et al. / Analytica Chimica Acta 447 (2001) 101–111

However, applications of HPLC to pyrethroid residues analysis are limited. The applications include some multi-residue methods for the analysis of a variety of agrochemicals, including many pyrethroids, in fruits and vegetables [7,8] and a few applications on isolated pyrethroids [9,10]. The UV detector was used in all of them. In addition, thermospray-mass/selective ion mode (TSP-MS/SIM) detection was applied for the analysis of fenvalerate residues in fruit and vegetable extracts by HPLC [9]. Nevertheless, HPLC-UV has low sensitivity and frequently lacks selectivity, while MS detection in HPLC has made significant improvements in recent years, but it still is very complex and requires costly instruments and additional bench space. A lower cost alternative is fluorimetric detection. In general, fluorimetry is more sensitive and selective that other detection systems [11] and sensitivity can be increased using derivatization reactions [11]. Among the numerous existing derivatization procedures, photochemically-induced fluorimetry (PIF) has found extensive application in stationary media [12–17] as well as in flowing devices such as FIA [18–20] or HPLC post-column photoreaction [21–23] in the analysis of different types of compounds. Advantages of post-column approach include separation of the analytes in their original form with no necessity for complete derivatization reaction (assuming reproducibility). On the other hand, it offers the advantage of simplicity over chemical post-column reactions in that post-column pumps and another devices are not required. Previously, it has been demonstrated that some pyrethroid insecticides can be derivatized by exposure to UV irradiation, leading to the formation of strongly fluorescent photoproducts [13,19]. In this way, variables affecting the fluorescence signal were studied and methods in stationary media and FIA were developed for determining fenvalerate and deltamethrin in water and technical formulations. The aim of the present work was to develop a HPLC method for determining the insecticides fenpropathrin, cyfluthrin, deltamethrin, fenvalerate, acrinathrin, tau-fluvalinate and bifenthrin, in vegetable matrices, by continuous on-line post-column photoirradiation followed by fluorimetric detection.

2. Experimental 2.1. Chemicals and solvents Analytical standards (pestanal quality) of fenpropathrin [(RS)-␣-cyano-3-phenoxybenzyl-2,2,3,tetramethylcyclopropanecarboxylate], cyfluthrin[(RS)␣ - cyano - 4- fluoro-3-phenoxybenzyl(1RS,3RS;1RS,3 SR)-3-(2,2-dichlorovinyl)-2,2,dimethylcyclopropanecarboxylate], deltamethrin [(S)-␣-cyano-3-phenoxybenzyl(1R,3R)3(2,2 dibromovinyl)2,2,dimethylcyclopropanecarboxylate], fenvalerate [(RS)-␣-cyano-3phenoxybenzyl(RS)-2(4-chlorophenyl)-3-methylbutyrate], acrinathrin [(S)-␣-cyano-3-phenoxybenzyl(Z)(1R,3S)2,2dimethyl-3[2(2,2,2trifluoro-1-trifluoromethyletoxycarbonyl) vinyl]-cyclopropanecarboxylate], tau-fluvalinate [(RS)-␣-cyano-3-phenoxybenzyl-N(2-chloro-␣,␣,␣-trifluoro-p-tolyl)-d-valinate], bifenthrin [2-methylbiphenyl-3-ylmethyl(Z)-(1RS,3RS)-3(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate] were obtained from Dr. Ehrënstorfer (Augsburg, Germany). Analytical reagent grade solvents, methanol (MeOH), acetonitrile (MeCN), dichloromethane, ethyl acetate and anhydrous sodium sulfate for pesticide residue analysis were obtained from Scharlaw (Barcelona, Spain). SPE cartridges, aminopropylbonded silica 500 mg florisil 1 g, silica 500 mg (Waters, Milford, MA, USA) were used for clean-up vegetable samples. Mobile phases were filtered through a 0.45 ␮m cellulose acetate (water) or Teflon (MeOH and MeCN) and degassed with helium prior and during use. All standards and samples were filtered through Millipore membrane Teflon filters (0.45 ␮m particle size) before injection into the chromatographic column. Distilled water, obtained from a Milli-Q water purification system from Millipore (Bedford, MA, USA), was used. 2.2. Instrumentation The HPLC was a Waters (Milford, MA, USA), composed of a Model 600 E multi-solvent delivery system, a Rheodyne 7725i manual injector valve with a 400 ␮l sample loop, a temperature control system and a Model 474 scanning fluorescence detector. Liquid chromatography (LC) separations were performed


with a ␮Bondapack C18 , 3.9 mm × 300 mm (10 ␮m particle size) column (Waters, Milford, MA, USA). The photochemical reaction was carried out in a post-column photochemical reactor (Softron GmbH, Gynkotek HPLC, Germering, Germany) fitted with a knitted open tube reactor coil (5 m × 1.6 mm e.d. and 0.3 mm i.d.) PTFE and a 4 W Xenon lamp. A digital Venturis FP 575 pentium personal computer using a Millennium 32 (Chromatography Manager, Waters, Milford, MA, USA) software was used for acquisition and treatment of data. A Model VV2000 LIF rotary vacuum evaporator (Heidolpf) thermostated by water circulation with a N-010 KN-18 vacuum pump (Telstar) was used to evaporate the extracts. A Model PT 2100 polytron (Kinematica AG, Luzern, Switzerland) and a Model BV-401C blender (Fagor, Guipuzcoa, Spain) were used for blending the samples. 2.3. Preparation of standards and spiked samples Standard solutions of pesticides (200 mg l−1 ) were prepared by exactly weighing and dissolving the corresponding compounds in organic solvents. These standard solutions were stable for a period of at least 3 months. Dilutions were freshly prepared for the working solutions. All solutions were protected against light with aluminum foil and were stored in a refrigerator at 4◦ C. For recovery determinations, samples (50 g) of finely chopped vegetable were spiked by addition of a standard stock solution (200 mg l−1 ), at two levels of concentration: 0.01 and 0.1 mg kg−1 (equivalent to 0.1 and 1.0 ␮g ml−1 in the final extract) for each of the pesticides. The spiked samples were allowed to stand for a few minutes before extraction to allow the spike solution to penetrate the test material. 2.4. Procedure for determining the pesticide in vegetables 2.4.1. Extraction procedure A sample (50 g) of vegetable was placed in a container glass and mixed with 105 ml of dichloromethane for 2 min with the polytron. Then, 80 g of sodium sulfate were added and the mixture was homogenized for 1 min. The extract was filtered through a 12 cm Büch-

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ner funnel and washed with two successive 25 ml portions of dichloromethane. The rinsates were added to the combined extraction fractions. The filtered liquid was collected in a 250 ml spherical flask and evaporated to nearly dryness in a rotating vacuum evaporator with a water-bath at 40 ± 1◦ C and the remaining solvent was allowed to evaporate under a slight N2 stream. 2.4.2. Clean-up procedure The residue obtained from the extract was redissolved in 5 ml hexane. A florisil (1 g) SPE column was preconditioned with 2 ml acetone and 2 ml hexane and then 1 ml of the sample extract, equivalent to 10 g cucumber, was brought onto the SPE cartridge. The collection of the eluate started directly after applying the extract. The elution began with 3 ml hexane:acetone 90:10 (v/v) and this eluate was collected in the same 25 ml spherical flask. The total eluate was concentrated to nearly dryness in a rotating vacuum evaporator with a water-bath at 40 ± 1◦ C and the remaining solvent was allowed to evaporate under a slight N2 stream. The obtained residue was redissolved in 1 ml MeCN:water 70:30 (v/v) and then filtered through 0.45 ␮m Teflon filter. The final extract contained 10 g of cucumber per 1 ml. 2.5. HPLC procedure 400 ␮l of MeCN:water 70:30 (v/v) sample solutions, equivalent to 4 g cucumber, were analyzed by HPLC with fluorimetric detection. The solvents were filtered daily before use through a 0.45 ␮m cellulose acetate (water) or Teflon (MeCN) and degassed with helium prior and during use. Samples were chromatographed by programmed gradient with MeCN:water, as mobile phase, for 22 min at a flow rate ranged from 1.0 to 1.5 ml min−1 . The fluorimetric detection was performed at an excitation wavelength (λex ) of 283 nm and at an emission wavelength (λem ) of 300 nm for all pesticides. The solvent program was as follows: initially 9 min isocratic with MeCN:water 70:30 (v/v) with a flow rate of 1.5 ml min−1 and 3 min linear gradient to MeCN:water 80:20 (v/v) with a flow rate of 1.0 ml min−1 ; then 5 min isocratic with MeCN:water 80:20 (v/v) with 1.0 ml min−1 as flow rate, followed by an additional period of 1 min linear gradient to the initial conditions and finally

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3 min in the initial conditions, time sufficient before subsequent analysis runs.

3. Results and discussion The strongest fluorescence signal for these pyrethroids occurred in binary aqueous mixtures with organic solvents, such as MeOH, ethanol and MeCN [16]. The excitation and emission spectra were established, being their λex and λem maxima between 280 to 287 nm and 319 to 338 nm, respectively. 3.1. HPLC separation and fluorescence detection after photolysis The selected pyrethroids show very close excitation and emission maxima, with emission at 330 nm after excitation at 283 nm as the better compromise wavelengths for all of them. However, at these wavelengths, a background matrix interference was found, which we were unable to eliminate with the various clean-up methods tested. Therefore, with the aim of

overcoming the interference, fluorescence was measured at 300 nm for the seven pesticides, after excitation at 283 nm, although a loss of sensitivity occurred. Mobile phase and flow rate through the C18 column and the reactor (residence times) were examined and adjusted to provide maximum responses and minimal broadening on the chromatograms. A series of gradient elution programs were evaluated ranging from 90:10 to 20:80 (v/v) MeCN:water and MeOH:water; the highest fluorescence responses and the best separation were achieved using a gradient program of MeCN:water mixtures ranged from 70:30 to 80:20 (v/v) as mobile phase. The analytical response increases as the flow rate decreases (Fig. 1) for all pyrethroids and no well-defined maximum values were reached. A flow rate ranging from 1.0 to 1.5 ml min−1 was used in the gradient program as a compromise between the residence time in the reactor and the band broadening on the chromatogram. The effect of pH on the fluorescence intensity, as well as on the separation of peaks was tested by using different buffer solutions (C T = 0.01–0.1 M) and no significant changes were found for pH values between

Fig. 1. Peak height response vs. flow-rate for: (䊉) fenpropathrin; (䉱) cyfluthrin; (䉬) deltamethrin; (䊏) fenvalerate; ( tau-fluvalinate; ( ) bifenthrin after post-column photochemical reaction and fluorescence detection.

) acrinathrin; (

)


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Fig. 2. Chromatogram corresponding to a standard of 0.4 ␮g ml−1 of: (1) fenpropathrin; (2) cyfluthrin; (3) deltamethrin; (4) fenvalerate; (5) acrinathrin; (6) tau-fluvalinate; (7) bifenthrin using the optimized chromatographic conditions.

3 and 8. Therefore, buffer was not used in the mobile phase. Fig. 2 shows the chromatogram from a mixture of the pyrethroid insecticides in the optimum conditions established. As can be seen, one chromatographic peak was obtained for each compound, except cyfluthrin, which yields two peaks corresponding to its cis and trans isomers, according to the solid standard used. In all cases, the height was used as analytical signal for quantitation, except for cyfluthrin. For this pesticide, the height or area ratios remained constant in a ±5% range for all runs. As in the technical formulation, the height or area ratios remained constant, but different than the ones obtained for the standards, all the parameters were calculated using the total area of the two peaks. 3.2. Extraction Acetone, hexane, methanol, petroleum ether, acetonitrile, dichloromethane and mixtures of them

[2–4,6,8,24] are reported to be satisfactory extractants for the studied pyrethroids. Extraction and SPE clean-up were tested with different extractants and sorbents (Table 1). Extraction with dichloromethane and clean-up with florisil were chosen because the best results were obtained. Fig. 3 shows a HPLC-fluorescence chromatogram of a cucumber blank extract, without interferents at the retention times corresponding to those of the analytes. 3.3. Analytical figures of merit and matrix effects Analytical figures of merit, when using an external standard method and pure solvent standards for calibration, are given in Table 2. The limits of quantification (LOQ) for the pyrethroid insecticides were calculated using two criteria: as the analyte concentration at which S/N = 10 [25] and as the lowest concentration where R.S.D. (%) is estimated to be less than 5% [26].

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107

Fig. 3. Chromatogram of: (a) a blank cucumber extract compared with (b) a blank cucumber extract spiked with: (1) fenpropathrin (0.03 ␮g ml−1 ); (2) cyfluthrin (0.01 ␮g ml−1 ); (3) deltamethrin (0.01 ␮g ml−1 ); (4) fenvalerate (0.02 ␮g ml−1 ); (5) acrinathrin (0.03 ␮g ml−1 ); (6) tau-fluvalinate (0.02 ␮g ml−1 ) and (7) bifenthrin (0.008 ␮g ml−1 ).

Matrix effects have been reported by different authors in the determination of several classes of pesticides by GC-NPD and GC-ECD [27,28], GC-MS [29,30], HPLC-MS [31] and HPLC with photoderivatization post-column and fluorescence detection [32]. When matrix have an effect on the signal of the an-

alyte measured and this is not accounted for in the method developed, then systematic errors can affect the result and cause bias. This situation usually is described by stating that there is a lack of selectivity or that interferences occur. However, according to Massart et al. [33] selectivity and matrix effect have a more

Table 2 Analytical figures of merit for the determination of pyrethroid insecticides by using pure solvent standards Compound

Linear range (␮g ml−1 )

Regression equation

Fenpropathrin Cyfluthrin Deltamethrin Fenvalerate Acrinathrin Tau-fluvalinate Bifenthrin

0.05–3.0 0.05–3.0 0.04–3.0 0.05–3.0 0.05–3.0 0.04–3.0 0.005–3.0

Y Y Y Y Y Y Y

= 24125X − 277 = 5138972X − 1375 = 15838X + 23 = 20672X − 104 = 21313X − 122 = 28427X − 224 = 117865X + 305

R2

R.S.D.a (%)

LOQb (␮g ml−1 )

LOQc (␮g ml−1 )

0.999 0.999 0.999 0.999 0.999 0.999 0.999

4.35 6.23 2.90 3.04 1.80 2.20 1.84

0.05 0.05 0.05 0.05 0.05 0.04 0.005

0.04 0.04 0.05 0.03 0.04 0.03 0.006

Ten injections of 0.1 ␮g ml−1 . Based on the lowest concentration where the R.S.D. (%) is estimated to be less than 5%. c Based on the analyte concentration at which S/N = 10. a

b

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restricted meaning, the difference depending on the type of systematic error they cause. Matrix effect or matrix interferences lead to relative systematic errors. The factors yielding such interferences may be physical or chemical and do not lead to a response as such. Matrix interferences affect the slope of the calibration line and they can be detected by comparing the slope of the calibration line with the relationship between signal and concentration in the matrix. Therefore, during calibration matrix-matched and solvent-based standards of the same concentration were measured. In this way, a suppression effect on the analytical signal, due to the matrix, was noticed for all insecticides, except for bifenthrin, cyfluthrin and deltamethrin. Low responses of fenpropathrin, acrinathrin, tau-fluvalinate and fenvalerate (Fig. 4) in matrix extract can be attributed to incomplete photoderivatization of pesticides in the cell or to quenching processes produced by coeluting bulk plant coextracts (represented namely by pigments and cuticular waxes) which do not constitute interferent peaks because they are not fluorescent species in the

working conditions. For this reason, matrix-matched standards prepared by fortifying a blank cucumber matrix with a known concentration of insecticides were used for quantification. Table 3 shows the analytical figures of merit obtained in this way. To calculate the LOQs, samples of cucumber were fortified with decreasing levels of each pyrethroid, extracted and analyzed as described in Section 2.4. It can be seen that the LOQ calculated ranged from 8 to 90 ng ml−1 in injected solutions (corresponding to 0.8 and 9 ␮g kg−1 in the vegetable sample), according to the compound. These values are in the same order than those obtained using pure solvent standard for calibration. Fig. 3(b) shows a chromatogram of a cucumber sample spiked at concentration levels lower than LOQs. It can be seen that the signals corresponding to the analytes are clearly well defined. In all cases, the LOQs obtained in the experimental conditions are significantly lower than those reported in the literature for the determination of the same insecticides by other techniques, such as GC-ECD [34] or TLC [35] and they are in the same order

Fig. 4. Calibration curves of fenpropathrin in (䊉) pure solvent and (䉱) cucumber matrix.


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Table 3 Analytical figures of merit for the determination of pyrethroid insecticides by using blank cucumber matrix as solvent standards Compound

Linear range (␮g ml−1 )

Regression equation

Fenpropathrin Cyfluthrin Deltamethrin Fenvalerate Acrinathrin Tau-fluvalinate Bifenthrin

0.05–3.0 0.05–3.0 0.05–3.0 0.05–3.0 0.05–3.0 0.04–3.0 0.005–3.0

Y Y Y Y Y Y Y

= 20129X − 31 = 7148521X − 4537 = 16657X + 364 = 18239X + 456 = 18925X + 19 = 23817X + 508 = 124540X + 2

R2

R.S.D.a (%)

LOQb (␮g ml−1 )

LOQc (␮g ml−1 )

0.998 0.998 0.998 0.999 0.999 0.999 0.998

6.67 8.57 3.26 3.57 2.6 2.42 1.96

0.08 0.08 0.06 0.08 0.09 0.07 0.01

0.09 0.08 0.06 0.08 0.09 0.06 0.008

Ten injections of 0.1 ␮g ml−1 . Based on the lowest concentration where the R.S.D. (%) is estimated to be less than 5%. c Based on the analyte concentration at which S/N = 10. a

b

that those obtained by HPLC/UV [8] or PIF [14,15]. LOQ are lower than the maximum residue limit (MRL) established in European Union [36] for these pesticides.

matrix-matched standards, the best recoveries were obtained, ranging from 95 to 116%.

3.4. Spike recoveries

The influence of other pesticides on the determination of 0.1 ␮g ml−1 of pyrethroids was studied by first testing 0.1 ␮g ml−1 of each interferent pesticide and if interference occurred, reducing the concentration progressively until interference ceased. The criterion for interference was a error in quantification of pyrethroids higher than 5%. The results obtained are summarized in Table 5. It can be observed that permethrin, cypermethrin, cyhalothrin and tralomethrin, among 36 compounds, interfere, at a concentration level higher than 0.05 ␮g ml−1 .

3.5. Selectivity

Samples of cucumber were spiked with 10 and 100 ␮g kg−1 of each pyrethroid, extracted and analyzed as described in Section 2.4, to determine recoveries. The recoveries obtained using both matrix-matched calibration and solvent calibration can be observed in Table 4. It can be seen that errors were obtained in some cases when calibration graphs built with standards in pure solvent were used; the errors agree with the slope of the calibration graph, according to the compound. Nevertheless, when using

Table 4 Mean recoveries and standard deviations (n = 6) from cucumber samples spiked with 0.01 and 0.1 mg kg−1 of pyrethroid insecticides calculated using for quantitation the calibration graphs in pure solvent and in blank cucumber matrixa Spiked samples, 0.1 ␮g ml−1

Compound

Solvent Fenpropathrin Cyfluthrin Deltamethrin Fenvalerate Acrinathrin Tau-fluvalinate Bifenthrin a

95 89 116 110 97 108 102

(5.6) (6.4) (3.3) (6.2) (2.9) (3.7) (2.4)

R.S.D. (%) in parentheses (n = 6).

Matrix 102 95 110 107 102 116 99

(8.6) (4.0) (3.1) (6.8) (3.2) (4.4) (2.7)

Spiked samples, 1.0 ␮g ml−1 Solvent 87 97 121 83 85 77 105

(3.1) (2.5) (1.8) (1.9) (1.0) (2.1) (1.3)

Matrix 105 101 114 102 103 103 99

(3.4) (2.6) (1.8) (2.0) (1.0) (2.1) (1.3)

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Table 5 Study of interferences at λex/em of 283/300 nm Name

NFa

IFb

ILc

Fuberidazole Carbendazim Thiabendazole Fenitrothion Pirimiphos-methyl Chlorpyriphos Imidacloprid 2-Aminobencimidazole Bendiocarb Aminocarb Butocarboxim Butocarboxim-sulfoxide Aldicarb Aldicarb-sulfoxide Aldicarb-sulfone Oxamyl Methomyl Formetanate Propoxur Carbofuran Carbaryl Methiocarb Methiocarb-sulfone 3-Hydroxycarbofuran 1-Naphthol Permethrin Cypermethrin Cyhalothrin Tralomethrin Tetramethrin Tefluthrin Diflubenzuron Triflumuron Hexaflumuron Lufenuron Flufenoxuron

+ − + − + − − − − − − − − − − − − − − − − − − − + − − − − − − − − − − −

+ + + − + − − − − − − − − − − − − − − − − − − − + + + + + − + + + + + +

− − − − − − − − − − − − − − − − − − − − − − − − − 0.1 0.05 0.05 0.1 − − − − − − −

a

Native fluorescence. UV-induced fluorescence. c Interference level (␮g ml−1 ). b

4. Conclusions An adequately sensitive and specific HPLC method, using post-column photochemical reaction and fluorescence detection, has been developed and analytical figures of merit were determined for seven pyrethroid insecticides in cucumber. Quantification based on standards prepared in blank matrix extract was carried out to compensate for the matrix-induced effects

and to obtain more accurate results. Quantification limits compare favourably with the ones obtained by GC-ECD and TLC and are in the same order as those obtained by HPLC UV and PIF with FIA, or in stationary media and lower than the MRLs established in European countries. Acknowledgements The authors are grateful to INIA (Project CAL00-063) for financial support. References [1] T. Roberts, D. Hutson, Metabolic Pathways of Agrochemicals. Part 2. Insecticides and Fungicides, The Royal Society of Chemistry, Cornwall, UK, 1999. [2] G.F. Pang, C.L. Fan, Y.Z. Chao, T.S. Zhao, J. Chromatogr. A 667 (1994) 348. [3] G.F. Pang, C.L. Fan, Y.Z. Chao, C.L. Fan, J.J. Zhang, X.M. Li, J. AOAC Int. 82 (1999) 186. [4] A.R. Fernández-Alba, A. Valverde, A. Aguera, M. Contreras, J. Chromatogr. A 686 (1994) 263. [5] G.F. Pang, Y.Z. Can, C.L. Fan, J.J. Zhang, X.M. Li, J. Mu, D.N. Wang, S.M. Liu, W.B. Song, H.P. Li, S.S. Wong, R. Kubinec, J. Tekel, S. Tahotna, J. Chromatogr. A 882 (2000) 231. [6] Y. Nakamura, Y. Tonogai, Y. Tsumura, Y. Ito, J. AOAC Int. 76 (1993) 1348. [7] E.R. Brouwer, E.A. Struys, J.J. Vreuls, U.A.T. Brinkman, Fresenius J. Anal. Chem. 350 (1994) 487. [8] G.F. Pang, Y.Z. Chao, C.L. Fan, J.J. Zhang, X.M. Li, T.S. Zhao, J. AOAC Int. 78 (1995) 1481. [9] C. Liu, G.C. Matter, X. Yu, R.T. Rosen, J.D. Rosen, J. Agric. Food Chem. 39 (1991) 718. [10] E. Papadopoulou-Mourkidou, Chromatographia 20 (1985) 376. [11] A. Coly, J.J. Aaron, Talanta 46 (1998) 815. [12] A. Coly, J.J. Aaron, Talanta 41 (1994) 1475. [13] A. Coly, J.J. Aaron, Analyst 119 (1994) 1205. [14] J.J. Aaron, A. Coly, Analyst 121 (1996) 1545. [15] A. Coly, J.J. Aaron, Anal. Chim. Acta 360 (1998) 129. [16] A. Coly, J.J. Aaron, Talanta 49 (1999) 107. [17] M. Maafi, M.C. Mahedero, J.J. Aaron, Talanta 44 (1997) 2193. [18] L.F. Garc´ıa, S. Eremin, J.J. Aaron, Anal. Lett. 29 (1996) 1447. [19] A. Coly, J.J. Aaron, Analusis 24 (1996) 107. [20] L.F. Garc´ıa, J.J. Aaron, Mikrochim. Acta 126 (1997) 289. [21] C.J. Miles, H.A. Moye, Chromatographia 24 (1987) 628. [22] C.J. Miles, H.A. Moye, Anal. Chem. 60 (1988) 220. [23] B.M. Patel, H.A. Moye, R. Weinberger, Talanta 38 (1991) 913. [24] R.J. Maguire, J.H. Carey, J.H. Hart, R.J. Tkacz, H. Lee, J. Agric. Food Chem. 37 (1989) 1153.

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