E L S E V I E R Journal of Chromatography A, 754 (1996) 301-331 JOURNAL OF CHROMATOGRAPHY A R e v i e w Determination of pesticide residues in fruit and vegetables C . M . T o r r e s * , Y. P i c 6 , J. M a f i e s Laboratori de Bromatologia i Toxicologia, Facultat de Farmgtcia, Universitat de Valbncia, Av. Vicent AndrOs Estell~s s/n, 4 6 1 0 0 Burjassot (Valencia), Spain Abstract A review concerning the determination of pesticide residues in fruit and vegetables is presented. The basic principles and recent developments in the extraction and quantitation of pesticides are discussed. Consideration is given to solid phase and supercritical extraction techniques, automation and robotic systems, and immunoassay procedures. K e y w o r d s : Food analysis Reviews Fruits Vegetables Extraction methods Environmental analysis: Pesticides Contents 1. Introduction ............................................................................................................................................................................ 301 2. Pesticide residues and legislation .............................................................................................................................................. 303 3. Extraction and clean-up ........................................................................................................................................................... 303 3.1, Liquid-liquid extraction (LLE) ......................................................................................................................................... 303 3.1.1. Diminution of the organic solvent toxicity ............................................................................................................... 306 3. 1.2. Elimination of the partition step .............................................................................................................................. 307 3.1.3. Elimination of the column cleanup .......................................................................................................................... 308 3.1.4. Incorporation of the newly developed pesticides ....................................................................................................... 309 3.2. Matrix solid-phase dispersion (MSPD) .............................................................................................................................. 309 3.3. Supercritical fluid extraction (SFE) ................................................................................................................................... 310 4. Instrumental analysis ............................................................................................................................................................... 314 4.1. Gas chromatography (GC) ................................................................................................................................................ 314 4.2. High-performance liquid chromatography (HPLC) ............................................................................................................. 318 4.3. Supercritical fluid chromatography (SFC) .......................................................................................................................... 324 4.4. Immunoassay (IA) ........................................................................................................................................................... 325 4.5. Other techniques .............................................................................................................................................................. 325 5. Conclusions ............................................................................................................................................................................ 328 Acknowledgments ....................................................................................................................................................................... 328 References .................................................................................................................................................................................. 328 1. Introduction The use of pesticides provides unquestionable benefits in increasing agricultural production. H o w - *Corresponding author. ever, it has the drawback of pesticide residues which remain on fruit and vegetables, constituting a po- tential risk to consumers [1]. This stimulates on one hand, the establishment of legal directives to control their levels through the M a x i m u m Residue Levels (MRLs), and on the other, a continuous look for 0021-9673/96/$15.00 �� 1996 Elsevier Science B.V. All rights reserved P I I S0021-9673 (96)00407-4

302 C.M. Torres et al. / J. Chromatogr. A 754 (1996) 301-331 pesticides less persistent and toxic for the human being. This fact has increased extraordinarily the number of pesticides registered and/or recom- mended, and analytical difficulties for their control [2,3]. To detect agricultural products that contain pes- ticide residue levels higher than the MRLs each country has available governmental agencies, which monitor pesticide residues through two different but complementary approaches: regulatory monitoring focused on raw agricultural commodities which measures the levels in individual lots for determining compliance with legal tolerances [4-11], and the Total Diet Study, in which dietary intakes of pes- ticides are determined by analysis of fruit and vegetables as consumed [11-15]. Analytical methods are needed to screen, quantify, and confirm pesticide residues in fruit and vegetables for both research and regulatory purposes. Mul- tiresidue methods (MRMs) and single residue meth- ods (SRMs) generally consist of the same basic steps, but the first ones are preferred to the second ones for the analysis of pesticides, because MRMs provide the capability of determining different pes- ticide residues in a single analysis. A review of the methods currently used to extract, isolate, and quan- tify pesticide residues in fruit and vegetables by monitoring agencies, demonstrates that they are based on classical MRMs, some developed over 30 years ago. Among the more widely used MRMs are those of Mills [16] Mills, Onley, and Gaither [17] Storherr [18] Luke [19] and Krause [20]. The method of the Association of Official Ana- lytical Chemists (AOAC), typifies the international recognized MRMs [21]. It allows the determination of many pesticide residues in fruit and vegetables, and involves an aqueous acetone extraction and laborious cleanup. Such methods, generally, applied an extraction step with a water miscible solvent, followed by a cleanup step, with an organic solvent of limited water capacity, to achieve the removal of interferences present in the sample extract and/or solid phase cleanup with silica or florisil. Finally, analyte determination is performed by gas chroma- tography (GC) or high-performance liquid chroma- tography (HPLC) with selective detectors [22,23]. These methods detect approximately 325 pesticide and pesticide-related compounds and most of them have undergone rigorous multilaboratory calibration studies, such as those needed to obtain the official acceptance by the AOAC [24]. However, their continued use still presents disadvantages, such as (i) their inefficiency as screening methods: These meth- ods are too complex, and they do not allow the generation of relevant data in time to prevent con- taminated foods from entering the marketplace, because these procedures are time consuming and labour intensive (ii) the amount of chemicals and toxic solvents that are used: it is usually by a factor of 10s-10 ~�� greater than that of the pesticide res- idues to be determined (iii) in addition, the newly developed groups of pesticides are each time more polar and/or thermodegradable and they should be incorporated into the existing MRMs. To avoid the general drawbacks of the classical methods, in recent years, significant evolution was noted in the extraction and determination of pesticide residue analysis in fruit and vegetables [25-27]. This review tries to cover the literature about the above mentioned progress published in the last 10 years for the pesticide residue analysis. The main attention is paid to simplification, miniaturization, and improve- ment of sample extraction and cleanup methods with universal microextraction procedures, solid-phase extraction (SPE) and/or solid-phase cleanup (SPC) on cartridges to replace liquid-liquid extraction (LLE), matrix solid-phase dispersion (MSPD) and selective extraction with supercritical fluid (SFE). Determination of the pesticide residues, by GC using microwave induced plasma (MIP)-atomic emission detector (AED), and tandem mass spectrometry (MS-MS), and by HPLC, for thermally labile and/ or polar pesticides and metabolites, using ion and ion-pair chromatography, additional postcolumn de- rivatization techniques and improvement of the HPLC detectors, are discussed. In addition, super- critical fluid chromatography (SFC) with different modified supercritical fluids and improved detectors for the analysis of nonpolar and polar analytes and the on-line combination SFE-SFC, are also reported. Some attention is given to the development of reliable enzyme immunoassay procedures for pes- ticides and metabolites, specially in the areas of sample preparation, validation, multiresidue capa-

C.M. Torres et al. I J. Chromatogr. A 754 (1996) 301-331 303 bility and commercial kits and biosensors, which generally involve an immobilized enzyme or anti- body as the basis of selectivity. 2. Pesticide residues and legislation As has been previously commented, since 1960's fresh fruits and vegetables have been checked for pesticide residues [4,11]. Nowadays the number of pesticides that could be detected number over 380. About 99 of them are actually found [9,11]. These pesticides present a wide variety of uses and physico-chemical properties. In this way it is com- mon to equate pesticides with insecticides. This is erroneous since the term pesticide is a general classification and includes mainly insecticides, her- bicides and fungicides. Each group of compounds includes different chemical families and types of action, and also, one compound may present a diversity of uses. Table 1 listed the pesticide residues found in fresh fruits an vegetables by the official agencies during 1992-1993 in pesticide residues, their uses and chemical class. The levels of pesticide residues are controlled by the MRLs, which are established by each country and sometimes cause conflicts because residue levels acceptable in one country could be unacceptable in other. This problem has revealed the need to har- monize the different MRLs, which has mainly been dealt with by two international organizations: the European Union (EU) at European level and the Codes Alimentarius Commission of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) [2,28]. The first EU directive was promulgated in 1976 (Directive 76/895/EEC) and it fixed the MRLs of certain pesticides in/on fruits and vegetables. This directive was modified and extended in later direc- tives, published in 1980 (80/428/EEC), 1981 (81/ 36/EEC), 1982 (82/528/EEC), 1988 (88/298/ EEC) and 1989 (89/186/EEC). The main disadvan- tage associated with these directives is that they only give partial harmonization because they do not cover all the pesticides traded, since they only reach to 64 active ingredients. National legislations cover more: about 380 in Spain, 360 in Germany, 400 in Nether- lands, etc. At world level, there are more than 600 active ingredients in the market [29]. In 1990, the EU promulgated a new directive (90/642/EEC) relating to the MRLs in selected plant products, including fruits and vegetables. Its object is to avoid the diversity of MRLs in order to facilitate the future European one-trade system. It fixes the MRLs for all the EU countries, eliminating the possibility that some countries approve higher MRLs. As a result, the products with residue con- tents higher than MRLs established by the EU can not be moved between the member countries [2,28]. Moreover, there are directives relating to the ban of marketing and use of some OCPs. They started with directive 79/119/EEC and included 83/131 / EU, 85/298/UE, 86/355/EU, 87/181/EU, 87/477/ EU, 89/365/EU, 90/533/EU. The directive 79/100/ EEC establishes sampling methods ~br the official control of pesticide residues in fruits and vegetables, and 85/591/EEC treats the introduction of sampling and analysis methods to control products for human consumption. Recently, a directive 91/414/EU about the marketing of pesticide products was published. It demands a large number of studies on residues before an active ingredient can be authorized at European level [281. 3. Extraction and clean-up 3. I. Liquid-liquid extraction (LLE) The existing multiresidue methodology makes possible the determination of OCPs, OPPs, MCs, triazine and thiocarbamate herbicides, Dithiocarbam- ates, and other contaminants in crops. These MRMs are continuously being revised to reduce their dis- advantages. It is possible to diminish the following drawbacks: 1. Toxicity of solvents used 2. Partition step 3. Column cleanup 4. Incorporation of the newly developed pesticides

304 C.M. Torres et al. / J. Chromatogr. A 754 (1996) 301-331 Table 1 Pesticides, isomers and breakdown products that have been detected in fresh fruits and vegetables Compound Use Chemical class References Acephate Insecticide OPPs" [9,11 ] Aldicarb Insecticide MCs h [ 11 ] Aldicarb sulfone Insecticide MCs h [11] Aldrin Insecticide OCPs c [ 11 ] Anilazine Herbicide Triazine [ 11 ] Azinphos-ethyl Insecticide OPPs [9] Azinphos-methyl Insecticide OPPs [9,11 ] Bitertanol Fungicide Nitrogen Heterocyclic [9] Bromide (inorganic) - [9] Bromopropylate Acaricide Bromo Benzylate [9] Bupirimate Fungicide Nitrogen Heterocyclic [9] Captafol Fungicide Dicarboximide [9, I 1 ] Captan Fungicide Dicarboximide [9,11 ] Carbaryl Insecticide MCs [9,1 I ] Carbendazim Insecticide MCs [9,11 ] Carbofuran Insecticide MCs [ I 1 ] Carbophenothion Insecticide OPPs [ I l ] Chlordane Insecticide OCPs [ I 1 ] Chlordimeform insecticide Formamide [ 11 ] Chlorfenvinphos Insecticide OPPs [9,11 ] Chlorobenzilate Acaride Chloro Benzylate [9] Chlorothalonil Fungicide Nitrogen heterocyclic [9,11] Chlorpropham Herbicide Carbamate [9] Chlorpyrifos Insecticide OPPs [9,11 ] Chlorpyrifos-methyl Insecticide OPPs [9, I 1 ] Chlozolinate Fungicide Nitrogen Heterocyclic [9] Cypermerthrin Fungicide Pyrethrine [9,11 ] Daminozide Grow regulator Hydrazide [9] DCPA Herbicide Chlorophenoxy [ 11 ] p,p'-DDE Insecticide OCPs [9] DDT Insecticide OCPs [ 11 ] Dehamethrin Fungicide Pyrethrine [9] Demeton Insecticide OPPs [ 11 ] Diazinon Insecticide OPPs [9,11 ] Dichlobenil Herbicide Nitriles [ 11 ] Dichlofluanid Fungicide Nitrogen Heterocyclic [9] Dichlorvos Insecticide OPPs [9,11 ] Dicloran Fungicide Sustituted Aromatic [9,11 ] Dicofol Insecticide OCPs [9,11 ] Dicrotophos Insecticide OPPs [ 11 ] Dieldrin Insecticide OCPs [9,11 ] Dimethoate Insecticide OPPs [9,11 ] Diphenylamine Other treatments - [9,11 ] Diquat Herbicide Bipyridyl [9] Dithianon Fungicide Nitrogen Heterocyclic [9] Disulphoton Insecticide OPPs [ 11 ] Endosulfan-alfa Insecticide OCPs [9,11 ] Endosulfan-beta Insecticide OCPs [9,11 ] Endosulfan sulphate Insecticide OCPs [9,11 ] Endrin Insecticide OCPs [9,11 ] EPN Insecticide OPPs [ 11 ] Esfenvalerate Insecticide Pyrethrine [ 11 ]

C.M. Torres et al. / J. Chromatogr. A 754 (1996) 301-331 305 Table 1 (continued) Compound Use Chemical class References Ethion Insecticide OPPs [9] Ethoprop Insecticide OPPs 111 ] Etrimfos Insecticide OPPs [91 Fenarimol Fungicide Nitrogen Heterocyclic 19] Fenitrothion Insecticide OPPs [9,11 ] Fenthion Insecticide OPPs [9,11 ] Fenthion sulphone Insecticide OPPs 19] Fenthion sulphoxide Insecticide OPPs [9] Fenvalerate Insecticide Pyrethrine [91 Folpet Fungicide Dicarboximide 19, I 1 ] Fonofos Insecticide OPPs [ 11 I Heptachlor Insecticide OCPs [ 11 ] Heptachlorobenzene Insecticide OCPs I I 1 ] "y-HCH Insecticide OCPs 19, l 1 ] lmazalil Fungicide Nitrogen Heterocylic [9,11 ] Iprodione Fungicide Nitrogen Heterocyclic [9.1 I ] Linuron Herbicide substituted Ureas I I 11 Malathion Insecticide OPPs [9,11 ] Mancozeb Fungicide Dithiocarbamates 191 Maneb Fungicide Dithiocarbamates 19] Mecarbam Insecticide MCs 19,11 ] Metalaxyl Fungicide Nitrogen Heterocyclic [91 Methamidophos Insecticide OPPs [9,11 ] Methidathion Insecticide OPPs [9,11 ] Methiocarb Insecticide MCs [9, I 1 ] Methomyl Herbicide Carbamate [ 11 I Metribuzin Herbicide Triazine 191 Mevinphos Insecticide OPPs [9,11 ] Mirex Insecticide OCPs [ I 1 ] Monocrotophos Insecticide OPPs 19, I 1 ] Myclobutanil Fungicide Nitrogen Heterocyclic [ 11 ] 1 -Naphtol 191 Omethoate Insecticide OPPs 19, I 1 ] Ortophenylphenol Fungicide Substituted aromatics 19] Oxadiazon Insecticide OPPs [ 11 ] Oxamyl Herbicide Carbamate [ I 1 ] Parathion Insecticide OPPs [9, t 1 ] Parathion-methyl Insecticide OPPs [9, I 1 ] Penconazole Fungicide Nitrogen Heterocyclic [91 Pentachloroanisole Fungicide Substituted aromatics [9] Permethrin Insecticide Pyrethrine [9, I I ] Phentoate Insecticide OPPs [ 9 ] Phorate Insecticide OPPs [ I I ] Phosalone Insecticide OPPs [9,11 ] Phosmet Insecticide OPPs 19, I 1 ] Phosphamidon Insecticide OPPs 19,11 ] Pirimicarb Insecticide MCs 19 I Pirimiphos-methyl Insecticide OPPs [9, I 1 ] Prochloraz Fungicide Nitrogen Heterocyclic [9] Procymidone Fungicide Dicarboximide [9,11 ] Profenofos Insecticide OPPs I 11 ] Pronamide Herbicide Amide [ 1 I ] Propargite Acaricide Sulphite 19,11 ] (Continued on p. 306)