Determination of pesticide transformation products: A review of extraction and detection methods

Journal of Chromatography A, 1216 (2009) 6767–6788

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Review

Determination of pesticide transformation products: A review of extraction and detection methods ˜ J.L. Martínez Vidal, P. Plaza-Bolanos, R. Romero-González, A. Garrido Frenich ∗ Research Group “Analytical Chemistry of Contaminants”, Department of Analytical Chemistry, University of Almeria, Crta. Sacramento s/n, E-04071 Almeria, Spain

a r t i c l e

i n f o

Article history: Received 6 May 2009 Received in revised form 30 July 2009 Accepted 7 August 2009 Available online 13 August 2009 Keywords: Transformation products Metabolites Pesticides Sample extraction Liquid chromatography Mass spectrometry Known and unknown compounds Environmental samples Biological samples Food samples

a b s t r a c t Pesticides are widely applied and they can produce a variety of transformation products (TPs), through different pathways and mechanisms. Nowadays there is a growing interest related to the determination of pesticide TPs in several matrices (environmental, food and biological samples), due to these compounds can be more toxic and persistent than parent compounds, and some of them can be used as markers of exposure to different pesticides. Although solid-phase extraction (SPE) is mainly used for the extraction of TPs, alternative techniques such as solid-phase microextraction (SPME) and liquid-phase extraction (LPE) can be used. These TPs are mainly determined by liquid chromatography (LC) due to the recent developments in this technique, especially when it is coupled to mass spectrometry (MS) detectors, allowing the determination of known and/or unknown TPs. Furthermore, MS is a very valuable tool for the structural elucidation of unknown TPs. This review discusses all phases of analytical procedure, including sample treatment and analysis, indicating the main problems related to the extraction of TPs from several matrices due to their high polarity, as well as the different alternatives found for the simultaneous determination of parent compounds and TPs, using chromatographic techniques coupled to MS detection. © 2009 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6767 Pesticide transformation products: degradation mechanisms and significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6768 Extraction of TPs from several matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6773 3.1. Environmental matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6777 3.2. Biological matrices and related samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6779 3.3. Food matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6779 TPs determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6780 4.1. Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6780 4.2. Classical detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6782 4.3. MS detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6782 4.3.1. Low-resolution mass spectrometry (LRMS): analysis of target compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6782 4.3.2. High resolution mass spectrometry (HRMS): analysis of unknown compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6784 Conclusions and future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6786 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6787 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6787

1. Introduction

∗ Corresponding author. Tel.: +34 950015985; fax: +34 950015483. E-mail address: [email protected] (A. Garrido Frenich). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.08.013

Pesticides are widely present in the environment, including water and soils, and foodstuffs, as a result of the application of phytosanitary products in modern agriculture. In the last years, new pesticides, which show a more specific mode of action and have a higher polarity and lower persistence than old ones, have

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been developed. However, pesticides can be transformed in the environment, crops, animals and humans into a large number of degradation products, commonly defined as transformation products (TPs), although other terms such as metabolites or pesticide “derivatives” can be used. Currently, there is an increasing concern regarding the formation of TPs since there are evidences indicating that these products can be more toxic [1] and persistent than parent compounds [2]. Moreover, TPs can have different properties that enable them to occur in environmental areas not reached by the pesticide itself. For instance, due to their mobility in the soil–water environment, TPs can reach groundwater more easily than parent compounds [3]. In fact, they are considered relevant within the group of the so-called emerging contaminants [4]. This group includes compounds not commonly monitored in the environment but suspected of entering in it and they cause adverse effects on health [5]. In consequence, a final risk assessment should also consider the amount of TPs formed on the several matrices evaluated [6]. This concern has been expressed in the European Directive 91/414/EEC [7], which indicates the necessity for the performance of metabolism studies to identify TPs in plants, and the environmental impact assessment of relevant TPs, as well as the identification of these compounds in routine analysis. Furthermore, maximum residue limits (MRLs) have been established for several TPs of different pesticides, such as aldicarb, diuron, fipronil and malathion in a variety of agricultural products [8]. Some of these compounds can be used as markers of exposure to different pesticides, although available data is still insufficient to define biological exposure limits [9]. For instance, the presence of TPs in farmer’s human biological fluids has shown to be an indicator of occupational exposure to agrochemical compounds. In consequence, there is a need for developing analytical methodologies in order to monitor TPs in both environmental and food matrices, apart from biological fluids. Up to now, most of the developed analytical methods have been focused on the determination of pesticides in different matrices and TPs were not considered. However, they have been incorporated to the analytical methods in order to have a wider knowledge of the presence of pesticides and related compounds in several matrices, noting that some of them are amply detected [10]. Gas chromatography (GC) or liquid chromatography (LC) coupled to conventional detection systems such as flame ionisation detector (FID) [11], electron capture detector (ECD) [12], ultraviolet-visible (UV) [13,14], diode-array [15,16] or fluorescence detection (FLD) [17], have been traditionally utilised for the detection of parent compounds and certain TPs, although capillary electrophoresis (CE) can also be used [18–20]. Currently, the determination of both pesticide residues and TPs requires the use of the aforementioned chromatographic techniques hyphenated to mass spectrometry (MS) as detection system, using several analysers as quadrupoles, time of flight (TOF) or hybrid instruments [21]. In general, MS is widely applied in trace analysis laboratories due to its intrinsic characteristics, such as selectivity, sensitivity and identification–confirmation capability. In this sense, the EU indicates the use of spectrometric techniques for the unambiguous confirmation of contaminants in several types of matrices [22]. Originally, for TPs studies, GC coupled to MS (GC–MS) was mainly used [23,24] since this technique had been more extended that LC coupled to MS (LC–MS). However, many TPs showed a higher polarity than parent compounds and, thus, they required a derivatization step prior to GC analysis [25]. Nowadays, the advances in LC technology (especially in ionisation sources) have become LC–MS more popular to determine TPs because it is more adequate than GC–MS for such analysis, satisfying requirements in terms of sensitivity and selectivity [26–28]. In relation to sample treatment, the aforementioned polarity of many TPs can make difficult the extraction and pre-concentration

steps. On the other hand, the limited number of standard material is an additional problem to develop quantitative methods. Furthermore, some TPs are unknown or non-targeted compounds, and their detection and identification must be an important analytical task in order to carry out risk assessment studies. This review is focused on the study of the significant TPs found in environmental, food and biological samples, indicating the main analytical techniques used for the extraction and determination of this type of compounds, highlighting the advances in instrumentation that may facilitate the structural elucidation and identification of new TPs in these complex matrices. 2. Pesticide transformation products: degradation mechanisms and significance Pesticides are usually transformed through small changes from the parent pesticide molecule through complete mineralisation to carbon dioxide, water, chloride, phosphate, etc. [29,30]. There are a large number of TPs that have been studied in the last years, and Table 1 shows a brief summary of the most important ones, whereas Table 2 indicates the concentrations found in different samples. The transformations suffered by pesticides can be biological (metabolism) or chemical, and they can occur through reactions such as hydrolysis (reaction with water), photolysis (broken down by sunlight), oxidation and/or reduction [31,32]. For instance, in the environment, photodegradation is one of the most important factors involved in the decomposition of pesticides, and it depends on climate conditions, the presence of photosensitisers, etc. [33]. Photodegradation can occur by direct photolysis, if pesticides absorb UV light and afterwards react with substances which are present in the environment or decompose themselves; or by indirect photolysis, which is more common, and it is provoked by oxygen, hydroxy or peroxy radicals [34]. Hydrolysis is also a main degradation route in environmental matrices, and other factors such as temperature, dissolved matter and the presence of humic substances can affect the decomposition rate [11]. Despite the cited general descriptions, different situations have been described in literature related to degradation processes, synergistic effects, influence of the matrix, etc. [35], as explained below. The degradation process can be different depending on the type of pesticide and matrix. In consequence, the study of the different TPs produced in each commodity, i.e. plants, soils, food or human body, is of relevance. For instance, carbosulfan can be degraded by photolysis, hydrolysis and microbial transformation; although it has been observed that the main degradation processes in oranges are hydrolysis and oxidation [36]. Among the produced TPs (see Table 1), carbosulfan is degraded to carbofuran immediately, and then 3-hydroxycarbofuran and 3-ketocarbofuran are also detected. For instance, 3-hydroxycarbofuran and carbofuran have been found in several crops such as orange, potato and rice, at several concentration levels ranging from 0.01 to 0.08 mg/kg (see Table 2). Triazine herbicides, such as atrazine, terbutylazine, simazine and propazine, are mainly degraded by photolysis, oxidation, hydrolysis and biodegradation, leading to dealkylation of the amine groups, dechlorination, deamination and hydroxylation in water and soils [37,38], observing that dealkylation and dechlorination occur simultaneously [39]. The main degradation products in water are the dealkylated chloro TPs (desethyldesisopropylatrazine, DEDIA, and deethylatrazine, DEA). However, the main TPs in soil are 2-hydroxyatrazine (HA), 2-hydroxyterbutylazine (HTBZ), desethyl2-hydroxyatrazine (DEHA) and desisopropyl-2-hydroxyatrazine (DIHA), observing that these hydroxylated degradation products are more persistent in soils because they are adsorbed by the organic matter [40]. Basically DEA and DIHA have been mainly found in waters (see Table 2), at concentrations ranging from 0.07 to 1.3 ␮g/L, whereas other TPs have not been detected.

J.L. Martínez Vidal et al. / J. Chromatogr. A 1216 (2009) 6767–6788

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Table 1 Transformation products of pesticides. Pesticide

Transformation products

Matrices

Reference

2,4-D

2,4-Dichlorophenol

Soil

[26]

Acetochlor

Acetochlor ethane sulfonic acid (acetochlor ESA) Acetochlor oxanilic acid (acetochlor OXA)

Soil

[3]

Alachlor

Alachlor ethane sulfonic acid (alachlor ESA) 2,6-Diethylaniline (DEAN) 2-Chloro-2 ,6 -diethylacetanilide (2-Cl-DEAC) 2-Hydroxy-2 ,6 -diethylacetanilide (2-H-DEAC)

Soil Water

[2] [48]

Aldicarb

Aldicarb sulfone Aldicarb sulfoxide

Grape juices, urine

[42,97]

Amitraz

2,4-Dimethylaniline (DMA) 2,4-Dimethylformamidine (DMF) N-(2,4-dimethylphenyl)formamidine (DMPF) N,N -bisdimethylphenylformamidine (BDMPF)

Fruits

[115]

Atrazine

2-Hydroxyatrazine (HA) 2-OH terbutylazine (HTBZ) Deethylatrazine (DEA) Desisopropylatrazine (DIA) Didealkylatrazine (DDA) HTBZ DEA Desethyl-2-hydroxyatrazine (DEHA) Desethyldesisopropylatrazine (DEDIA) Desisopropyl-2-hydroxyatrazine (DIHA)

Water, soil

[31,69]

Water

[40]

Benomyl

N,N -dibutylurea

Soil

[32]

Bromoxynil

3,5-Dibromo-4-hydroxybenzamide (BrAm) 3,5-Dibromo-4-hydroxybenzoic acid (BrAC)

Soil

[122]

Carbamates

Ethylenethiourea (ETU) Propylenethiourea (PTU)

Food

[80]

Carbofuran

3-Hydroxycarbofuran 3-Ketocarbofuran 3-Hydroxycarbofuran

Soil

[29]

Urine

[97]

Carbosulfan

3-Hydroxy-7-phenol carbofuran 3-Hydroxycarbofuran 3-Ketocarbofuran 3-Keto-7-phenolcarbofuran 7-Phenolcarbofuran Carbofuran Dibutylamine

Orange, potato, rice

[138]

Chlorothalonil

1-Carbamoyl-3-cyano-4-hydroxy 2,5,6-trichlorobenzene 1,3-Dicarbamoyl-2,4,5,6-tetrachlorobenzene 2,4,5-Trichloroisophthalonitrile 2,5,6-Trichloro-4-methoxy isophthalonitrile 2,5,6-Trichloro-4-(methylthio)isophthalonitrile 4-Hydroxy-2,5,6-trichloroisophthalonitrile Isophthalonitrile

Soil, water

[60]

Chlorotoluron

3-Chloro-4-methylphenylurea (MPU)

Water

[40]

Chlorpyrifos

3,5,6-Trichloro-2-pyridinol (TCP) Diethyl phosphate (DEP) Diethyl thiophosphate (DETP) TCP

Soil Human urine

[75] [98]

Cyromazine

Melamine (1,3,5-triazine-2,4,6-triamine)

Chard

[118]



DDT

4,4 -Dichlorodiphenyldichloroethane (DDD) 4,4 -Dichlorodiphenyldichloroethylene (DDE)

Soil, water

[12,92]

Deltamethrin

3-Phenoxybenzoic acid (3-PBA)

Rat plasma

[58]

Demeton-S-methyl

Demeton-S-methyl sulfone Demeton-S-methyl sulfoxide

Grape juices

[42]

Desmediphan and phenmediphan

m-Aminophenol Aniline m-Toluidine

Soil

[51]

Diazinon

2-Isopropyl-6-methyl-4-pyrimidinol (IMP) 1-Hydroxy isopropyl diazinon 1-Hydroxy isopropyl diazoxon IMP Diazoxon Diethyl phosphate (DEP)

Soil Water

[11] [130]

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J.L. Martínez Vidal et al. / J. Chromatogr. A 1216 (2009) 6767–6788

Table 1 (Continued ) Pesticide

Transformation products

Matrices

Reference

Diethyl thiophosphate (DETP) Hydroxy diazinon Dichlobenil

2,6-Dichlorobenzamide (BAM) 2,6-Dichlorobenzoic acid (DCBA)

Groundwater

[25]

Diflubenzuron

2,6-Diflurobenzamide (DFBA) 4-Chloroacetanilide (CAA) 4-Chloroaniline (CA) 4-Chlorophenylurea (CPU) N-Methyl-4-chloroaniline (MMCA)

Forestry matrices

[59]

Endosulfan

Endosulfan sulfate Endosulfan I Endosulfan II Endosulfan diol Endosulfan lactone Endosulfan sulfate

Soil Tissue, blood

[12] [114]

Endrin

Endrin aldehyde

Soil

[12]

Fenamiphos

Fenamiphos sulfone Fenamiphos sulfoxide

Grape juices

[42]

Fenitrothion

3-Methyl-4-nitrophenol Fenitrooxon

Soil, barley, poplar leaves

[52,79,91]

Fenthion

Fenthion-sulfoxide Fenoxon Fenoxon-sulfone Fenoxon-sulfoxide Fenthion-sulfoxide

Water Orange, protection equipment

[46] [44,105]

Flonicamid

4-Trifluoromethylnicotinamide (TFNA-AM) 4-Trifluoronicotinic acid (TFNA) N-(4-trifluoronicotinoyl) glycine (TFNG)

Hops

[28]

Flubendiamide Glyphosate Heptachlor

Desiodo flubendiamide Aminomethylphosphonic acid (AMPA) Heptachlor epoxide

Food Soil, soybeans, juices Soil

[13] [20,24,109] [12]

Imidacloprid

6-Chloronicotinic acid (6-CINA) Imidacloprid-guanidine Imidacloprid-guanidine-olefin Imidacloprid-urea

Greenhouse air Soil

[15] [50]

Isoproturon

4,4-Diisopropylazobenzene (DDIPU)

Soil

[35]

Ioxynil

3,5-Diiodo-4-hydroxybenzamide (IAM) 3,5-Diiodo-4-hydroxybenzoic acid (IAC)

Soil

[122]

Malathion

Malaoxon

Soil, barley

[11,52]

Maneb

Ethylenebis (isothiocyanate) sulfide (EBIS) ETU Ethyleneurea (EU)

Tomato

[16]

MCPA

4-Chloro-2-methylphenol

Water, soil

[68]

Mesotrione

4-Methylsulfonyl-2-nitrobenzoic acid (MNBA) 2-Amino-4-methylsulfonyl-benzoic acid (AMBA) MNBA

Crops Soil, water

[17] [17]

Methiocarb

Methiocarb sulfone Methiocarb sulfoxide

Grape juices

[42]

Metolachlor

2-Ethyl-6-methylaniline (EMA) Metolachlor ESA

Soil

[48]

Metribuzin

Deaminodiketometribuzin (DA) Deaminometribuzin (DADK) Diketometribuzin (DK)

Soil

[74]

Organophosphorus pesticides

Diethyl dithiophosphate (DEDTP) Diethyl phosphate (DEP) Diethyl thiophosphate (DETP) Dimethyl dithiophosphate (DMDTP) Dimethyl phosphate (DMP) Dimethyl thiophosphate (DMTP)

Urine

[56,98]

Pyrethroids

2-(4-Chlorophenyl)-3-methylbutyric acid (CPBA) 3-PBA Cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis- and trans-DCCA) Cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-DBCA)

Urine

[23]

J.L. Martínez Vidal et al. / J. Chromatogr. A 1216 (2009) 6767–6788

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Table 1 (Continued ) Pesticide

Transformation products

Matrices

Reference

Simazine

Monodeethylsimazine

Soil

[38]

Spinosad

N-Demethylspinosyn D Spinosyn B Spinosyn K

Food samples

[76]

Terbutylazine Terbutryne

Deethylterbutylazine (DETBZ) 2-Hydroxyterbutylazine (HTBZ)

Soil Water

[38] [40]

Thiophanate methyl

2-Aminobenzimidazole Carbendazim

Water

[83]

In food samples, the degradation of some pesticides, such as carbamates and organophosphorus pesticides (OPPs), takes place through oxidative mechanisms [41] producing their oxidative sulfoxide and sulfone. In this case, the degradation rate also depends on the pesticide and type of food. For instance, aldicarb (carbamate) is degraded to aldicarb sulfoxide and then, both compounds are degraded to aldicarb sulfone, whereas demeton-S-methyl (OPP) is simultaneously converted into demeton-S-methyl sulfone and demeton-S-methyl sulfoxide [42], detecting both TPs at concentrations higher than 0.7 ␮g/L in juices samples (see Table 2). In relation to the type of sample, it can be observed that the oxidation of parent compounds to degradation products can become lower in antioxidant-rich foods such as juices, and the pesticide resistance can increase in these types of matrices [43]. Fig. 1 shows the degradation pathway of fenthion in oranges, detecting five TPs in the selected varieties of oranges [44]. It can be observed that the main TP of the oxidative process is fenthion-sulfoxide, with a decay rate lower than the corresponding to the active compound. On the other hand, fenthion-sulfone kept almost constant during the experiment, indicating that its formation and degradation rates are of the same order of magnitude. Finally, whereas these two TPs are mainly formed due to an oxidation process, fenoxon, fenoxon-sulfoxide and fenoxon-sulfone are more related to hydrolytic processes, taking into account that they are detected after rain events. Fenthion is also applied to olive crops to combat olive fly [45], and several TPs can be detected in different matrices depending on their lipophilic properties. Fenthion is detected in olive oil, which is a lipophilic matrix, since it is a lipophilic compound (log Ko/w 4.8). On the contrary, more polar compounds such as fenthionsulfoxide (TP showing a log Ko/w 1.9) are not detected in this type of matrix. However, fenthion-sulfoxide, fenthion-O-analogue, and a small amount of fenthion-sulfone have been detected in olive fruit pulp, and only the most polar TP, fenthion-sulfoxide, has been found in environmental water near to olive crops [46]. A similar pattern can be observed for endosulfan TPs, which are distributed as a function of their lipophilicity. For example, in the environment, there is a continuous degradation of endosulfan II to endosulfan I, whereas in mammals or other biological matrices (e.g. placenta), endosulfan I and II are converted into more hydrosoluble TPs such as endosulfan sulfate, endosulfan lactone or endosulfan diol [47]. Endosulfan I, II and sulfate have been detected in rat tissues (see Table 2) at concentrations ranging from 0.02 to 3.11 mg/kg, although they have not been detected in plasma samples. It is important to notice that whereas the information on sorption and degradation of pesticides in soils is large, the information on these processes for their TPs is still scarce [48]. For example, imidacloprid, which is used as a crop pest insecticide, seed and flea-control treatment, produces several TPs (see Table 1) with similar structure than the parent compound, but showing different sorption capabilities. One of these TPs, imidacloprid-urea, is less adsorbed on soil than imidacloprid, whereas imidacloprid-

guanidine is adsorbed to a much greater extent than the original compound [49]. Another question is the fact that some TPs show stronger insecticidal activity than the parent compound [50], or they can be more toxic and persistent than the original compound, as it happens with carbamates [51] or certain OPPs such as malathion and fenitrothion. In this case, their TPs, malaoxon and fenitrooxon are responsible for the insecticidal activity of the parent compounds [52]. Besides, malathion is usually degraded by hydrolysis, obtaining other products, such as diethyl thiosuccinate, O,O-dimethyl phosphorothionic acid and other unexpected products in natural waters [34]. As aforementioned, the presence of pesticide TPs in biological matrices, such as plasma, urine and tissue, can indicate human exposure to agrochemical products [53]. Dialkyl(thio)phosphates have been analysed in urine as a marker of exposure to various OPPs [54], although the determination of alkyl phosphates can provide overall information about exposure [55–57]. For instance, some TPs products have been detected in human urine at concentrations lower than 1404 ng/L (Table 2). On the other hand, the determination of some pyrethroids and related compounds, such as deltamethrin and 3-phenoxybenzoic acid (which is a TP that has little or non-demonstrable toxicity), can be used to provide a basis for understanding the susceptibilities to these compounds on different ages and species of mammals [58]. This compound has been found in rat plasma at concentrations lower than 0.318 mg/L (see Table 2). In consequence, the initial TPs of most pesticides should also be considered as residues of concern in food and drinking water, and should be included within the risk assessment processes, due to the fact that some of these TPs are more persistent or possess higher toxicity than parent compounds (e.g. DDE is more persistent than DDT), as previously commented. Apart from the examples given above, it must be noticed that, for instance, diflubenzuron is quickly degraded in the environment (mainly by hydrolysis and photolysis), producing TPs such as 4-chloroacetanilide, 4-chloroaniline and N-methyl-4-chloroaniline, which have been classified as mutagens by the National Cancer Institute and the Cancer Assessment Group of the US Environmental Protection Agency (US EPA) [59]. The same pattern has been observed for chlorotalonil and its main TP, 4-hydroxy-2,5,6-trichloroisophthalonitrile, which appears to be more persistent, mobile and toxic than the parent compound in soil and water [60]. Occasionally, the presence of two or more pesticides and TPs can provoke some dramatic consequences due to synergistic effects. Malathion (OPP) shows a low mammalian toxicity, but if it is administered together with a small dose of parathion (another OPP), its toxicity increases considerably because parathion is degraded to paraoxon. This TP inhibits carboxylesterases which are in charge of transforming malathion into a harmless substance, malathion acid [61]. In relation to legislation, the current documents [7] indicate the need for metabolism studies which permit the identification of metabolic and degradation products in some matrices. Consid-

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Table 2 TPs concentrations found in samplesa . Transformation products Environmental samples 2-Cl-DEAC; 2-H-DEAC; DEAN

Samples

Concentration

Observations

Reference

Water

0.8–2.7 ␮g/L

Leaching properties

[48]

3-Methyl-4-nitrophenol; fenitrooxon

Poplar leaves

3-Methyl-4-nitrophenol: <1.2 mg/kg Fenitrooxon: 0.4 mg/kg

Study in treated poplar leaves

[79]

Acetochlor ESA; acetochlor OXA

Soil

Acetochlor ESA: 1–60 ␮g/kg OXA: 2–130 ␮g/kg

Soils spread with acetochlor

[3]

Alachlor ESA; metolachlor ESA

Soil

Alachlor ESA < 210 ␮g/kg Metolachlor ESA < 90 ␮g/kg

Persistence and mobility studies

[2]

Alachlor ESA; alachlor OXA; metolachlor ESA; metolachlor OXA

Soil

Alachlor ESA: ND-0.18 mg/kg Alachlor OXA: ND-0.54 mg/kg Metolachor ESA: ND-0.01 mg/kg Metolachlor OXA: ND-0.07 mg/kg

Field application and persistence studies

[14]

AMPA

Soils

0.003–0.382 mg/kg

Different depths

[24]

BAM; DCBA

Groundwater

BAM: 0.060–3.100 mg/L DCBA: 0.039–0.049 mg/L

17 samples

[25]

BrAC; BrAm; IAC; IAM

Soil

BrAM: <0.5 ␮M BrAC, IAC, IAM: ND

Degradation studies

[122]

DDD; DDE

Soil, water

DDD < 0.07 ␮g/L DDE < 0.05 ␮g/L

5 types of water

[92]

DEA; DEDIA; DEHA; DETBZ; DIA; DIHA; HA; HTBZ

Water

DEA: 70–120 ng/L DIHA: <5 ng/L

Rest of TPs not detected

[37]

DEA; DEDIA; DEHA; DIHA; HTBZ

Water

DEA: 0.3–1.3 ␮g/L

54 samples. Only DEA was detected

[40]

DEA; DIA

Soil

DEA: <0.05–2.02 ␮g/kg DIA: <0.05–1.32 ␮g/kg

47 samples

[84]

DEP; O,O -diethyl phosphorothioate; O,O -dimethyl phosphorothioate

Surface water

DEP: 97.7 ␮g/L O,O -Diethyl phosphorothioate: 2.2 ␮g/L O,O -Dimethyl phosphorothioate: 67.3 ␮g/L

Fruit growing area

[72]

DETBZ; monodeethylsimazine

Soil

DETBZ: 0.25–1.78 mg/kg Monodeethylsimazine: 0.29–0.91 mg/kg

Degradation studies

[38]

EMA; metolachlor ESA

Soil

EMA: 0.6 ␮g/L

Leaching properties

[48]

IMP

Soil Water

1–4 mg/kg 2–3.5 mg/L

Degradation studies

[11]

Malaoxon N,N -Dibutylurea TCP

Water Soil Soil

<1 mg/L ND-0.41 mg/kg 5.42–24.40 ␮g/kg

Degradation studies Degradation studies 3 samples

[11] [32] [75]

Urine

ND (<10 ␮g/L)

Sample: pest control operator

[97]

Rat plasma

>0.318 mg/L

Toxicokinetic study

[58]

Human urine

3-PBA: ND-1404 ng/L cis-DBCA: ND-69 ng/L CPBA: ND-58 ng/L cis-DCCA: ND-519 ng/L trans-DCCA: ND-823 ng/L

Pest control operators (6) and non-occupationally people (2)

[23]

Biological samples and related matrices 3-Hydroxycarbofuran; aldicarb sulfone; aldicarb sulfoxide 3-PBA 3-PBA; cis-DBCA; cis-DCCA; CPBA; trans-DCCA

6-CINA

Greenhouse air

ND

Dissipation process

[15]

DEDTP; DEP; DETP; DMDTP; DMP; DMTP

Human urine

DEDTP: ND-28 mg/kg DEP: ND-129 mg/kg DETP: ND-116 mg/kg DMDTP: ND-425 mg/kg DMP: ND-1096 mg/kg DMTP: ND-1801 mg/kg

1146 samples. Data provided as mg/kg on creatinine basis

[55]

DMP; DMTP

Human urine

DMP: ND-52 ␮g/L DMTP: 10–47 ␮g/L

Exposed (1) and unexposed (11) people

[56]

Endosulfan I; endosulfan II; endosulfan diol; endosulfan lactone; endosulfan sulfate

Rat tissue, plasma

Endosulfan I: ND-3.11 mg/kg Endosulfan II: ND-1.19 Endosulfan sulfate: 0.02–0.22 mg/kg

Not detected in plasma samples

[114]

J.L. Martínez Vidal et al. / J. Chromatogr. A 1216 (2009) 6767–6788

6773

Table 2 (Continued ) Transformation products Endosulfan ether; endosulfan lactone; endosulfan sulfate Food samples 3-Hydroxycarbofuran; carbofuran; dibutylamine

a

Samples

Concentration

Observations

Reference

Human serum

ND

5 samples

[125]

Orange, potato, rice

3-Hydroxycarbofuran: 0.01–0.05 mg/kg Carbofuran: 0.04–0.08 mg/kg Dibutylamine: 0.06–0.09 mg/kg

Real samples

[138]

Aldicarb sulfone; aldicarb sulfoxide

Grape juices

ND (<4.6 ␮g/L)

100 samples

[42]

BDMPF; DMA; DMF; DMPF

Fruits

BDMPF: 0.005–0.05 mg/kg DMA: 0.025 mg/kg DMF: 0.03–0.05 mg/kg DMPF: 0.05–0.1 ␮g/kg

30 samples (2 positives)

[115]

Demeton-S-methyl sulfone; demeton-S-methyl sulfoxide

Grape juices

Demeton-S-methyl sulfone: 1.2–2.1 ␮g/L Demeton-S-methyl sulfoxide: 0.7–3.1 ␮g/L.

100 samples (3 positives)

[42]

ETU; PTU

Food

ETU: 0.32 mg/kg PTU: 0.19 mg/kg.

90 samples (2 positive rice samples)

[80]

Fenamiphos sulfone; fenamiphos sulfoxide

Grape juices

Fenamiphos sulfone: ND (<1 ␮g/L) Fenamiphos sulfoxide: 3.2–10.1 ␮g/L

100 samples (5 positives)

[42]

Fenitrooxon

Barley

0.015–0.040 mg/kg

Degradation studies

[52]

Fenthion-sulfone; fenthion-sulfoxide; malaoxon; omethoate

Olive oil

Fenthion-sulfone: ND (<0.030 mg/kg) Fenthion-sulfoxide: (0.055–0.13 mg/kg) Malaoxon: 0.022 mg/kg Omethoate 0.022–0.044 mg/kg

30 samples (2 positives)

[108]

Fipronil; fipronil desulfinyl; fipronil sulfone; fipronil sulfide Malaoxon Melamine

Pollen

Fipronil desulfinyl <0.1 ␮g/kg

[106]

Barley Chard

ND-0.155 mg/kg 0.37–2.76 mg/kg

60 samples (2 positives). Only fipronil desulfinyl was detected Degradation studies 20 treated samples

Methiocarb sulfone; methiocarb sulfoxide

Grape juices

Methiocarb sulfone: 0.5–0.8 ␮g/L Methiocarb sulfoxide: 0.2–2.4 ␮g/L

100 samples (5 positives)

[42]

TFNA; TFNA-AM; TFNG

Hops

TFNA: 0.302–0.470 mg/kg TFNA-AM: 0.038–0.177 mg/kg TFNG: 0.098–0.204 mg/kg

12 treated hops

[28]

[52] [118]

Abbreviations. ND: not detectable. For other abbreviations see Table 1.

ering this information, the most abundant and/or frequent TPs with toxicological relevance should be included in the definition of “residue”, and they should be determined in routine analysis [62], as it was indicated for dichlobenil and related TPs in water [25]. However, it is difficult to decide which compounds should or might be included in monitoring programs and routine analysis, since there are thousands of potential TPs present in plants, animals and in the environment. In this sense, metabolism studies gain importance since they can provide adequate information and data for future trials, and they may indicate which compounds could be included in following tests. Normally, these studies have been mainly focused on food analysis, and the most relevant TPs have been included in the definition of residue, being determined simultaneously with the parent compound, such as dimethoate, fenamiphos, etc. [63]. According to the new situations described in other kind of matrices, especially in environmental matrices, more work is demanded as it was indicated by REACH (registration, evaluation and authorisation of chemicals) legislation [64] established by EU, which highlighted the need for characterisation of possible degradation, transformation or reaction processes and an estimation of the environmental distribution and fate.

3. Extraction of TPs from several matrices Nowadays, many of the available analytical methods are focused on the determination of parent compounds, whereas few methods are available for the analysis of TPs [65]. Furthermore, sample treatment is the most tedious step in the analytical procedure, bearing in mind that it can take over 60% of the analyst’s time [66]. Currently, there is a trend towards the simplification of this stage, trying to find simple methods which allow the determination of a wide range of compounds. Another important difficulty related to the determination of TPs is that these compounds are usually detected at very low levels (␮g/kg or lower), and thus pre-concentration step is a pre-requisite in analysis. An additional problem involved in TP determination is that these analytes show higher polarity than parent pesticides, which can make difficult the simultaneous determination of both types of compounds. According to the revised literature, a variety of sample preparation techniques have been used for the extraction of TPs from different matrices, although solid-phase extraction (SPE) is mainly applied [67–70], as it can be observed in Table 3, where recoveries have also been included. However, other extraction techniques that can be found in literature are liquid–liquid extraction (LLE)

6774

Table 3 Extraction techniques used for the isolation of transformation productsa . Transformation products

Extraction technique

Observations

Recovery (%)

Reference

Soil

LLE

Extraction with dichloromethane/hexane Clean-up with SPE: OASIS cartridges

54–130

[60]

2,6-Diflurobenzamide;4-chloroacetanilide; 4-chloroaniline; 4-chlorophenylurea; N-methyl-4-chloroaniline

Pine groves

SLE

>90

[59]

2-Aminobenzimidazole; carbendazim

Water

SLM

Not provided

[83]

2-Isopropoxyphenol; 2,2-dimethyl-2-dihydrobenzofuran-7-ol; 2,3,5-trimethylphenol; ␣-naphtol 3,5,6-Trichloro-2-pyridinol; IMP

Surface water

SPE

Extraction with acetonitrile SPE clean-up: aminopropyl cartridges DHE and 15% of TOPO as organic solvent. Acceptor: 0.015 M H2 SO4 C18 cartridges

>73

[70]

Sludge

PLE and SPE

>92

[75]

3,5-Dibromo-4-hydroxybenzamide; 3,5-dibromo-4-hydroxybenzoic acid; 3,5-diiodo-4-hydroxybenzamide; 3,5-diiodo-4-hydroxybenzoic acid 3-Hydroxy carbosulfan; aldicarb sulfone; aldicarb sulfoxide 3-Methyl-4-nitrophenol; 4-nitrophenol; fenitrooxon; paraoxon ethyl; paroxon methyl; S-methylisomer of fenitrothion 3-Methyl-4-nitrophenol; fenitrooxon 3-Methyl-4-nitrophenol; fenitrooxon

Soil

SLE

Extraction with CH2 Cl2 /acetone (50:50, v/v). SPE Florisil cartridges Water

Not provided

[122]

Groundwater

SPE

77–97

[19]

Water

SPE

Graphite carbon cartridges LiChrolut EN cartridges

79–105

[67]

Environmental water Poplar leaves

SPME SPME (PDMS-DVB)

Not provided Not provided

[91] [79]

4,4 -DDD; 2,4 -DDD; DDE

Seawater, groundwater

SPME

>85

[77]

4-Chloro-2-methylphenol

Water, soil

SPE

80

[68]

4-Nitrophenol; aminoparathion; malaoxon; O,O-dimethyldithiophosphoric acid; O,O-dimethylthiophosphoric acid; paraoxon Alachlor ESA; alachlor OXA; metolachlor ESA; metolachlor OXA

River water

LLE

PDMS-DVB SLE extraction with hexane/acetone (1:1) prior SPME Carbowax/TPR 100 and PDMS/DVB C18 cartridges. Soil extracted in alkaline media prior SPE Hexane

Not provided

[33]

Soil

MAE

Extraction with methanol/water (50:50, v/v) Clean-up step with C18 cartridges

>71

[14]

J.L. Martínez Vidal et al. / J. Chromatogr. A 1216 (2009) 6767–6788

Sample

Environmental samples 1-Carbamoyl-3-cyano-4-hydroxy 2,5,6-trichlorobenzene; 1,3-dicarbamoyl2,4,5,6-tetrachlorobenzene; 2,4,5-trichloroisophthalonitrile; 2,5,6-trichloro-4-methoxy isophthalonitrile; 2,5,6-trichloro-4(methylthio)isophthalonitrile; 4-hydroxy-2,5,6trichloroisophthalonitrile; isophthalonitrile

Crops, soil, water

SLE

BAM; DCBA DA, DADK; DK

Groundwater Soil

SPE PLE

DDA; DEA; DIA;

Ground, surface water

SPE

DDD; DDE DDE; DDD

Soil Water

HS-SPME HS-LPME

DEA; DEDIA; DEHA; DETBZ; DIA; DIHA; HA; HTBZ DEA; DEDIA; DEHA; DIHA; HA DEA; DIA

Water

SPE

Groundwater Soil

SPE MIP

DEA; DIA; HA DEDIA; DETBZ; deethyl-2-hidroxyterbutylazine; desethylterbumeton; DIA; DIHA; 2-hidroxysimazine; HTBZ DEP; O,O -diethyl phosphorothioate; O,O -dimethyl phosphorothioate Fenthion-sulfoxide

Water Water

On-line SPE Direct injection

Surface water

SAX

Acetonitrile/water (60:40, v/v) OASIS cartridges Methanol/water (75:25, v/v) C18 and cation exchange cartridges PDMS Room temperature ionic liquids LiChrolut EN cartridges

>70

[17]

52–90 73–97

[25] [74]

95–100

[69]

68–87 Not provided

[12] [92]

94–125

[37]

68–110 116–121

[40] [84]

28–104 Not provided

[89] [139]

Elution with acetonitrile Chloroform

79–96

[72]

94–104

[46]

Extraction with acetonitrile 2 clean-up steps: LLE (n-hexane) and SPE (Florisil) Extraction with ethyl acetate Methanol

74–104

[106]

Not provided

[11]

86–89

[118]

LiChrolut EN cartridges PLE using acetone as extractant prior MIP PRP-1 cartridges Centrifugation prior analysis

LLE

Pollen

SLE

IMP; malaoxon

Water, soil

LLE and SLE

Melamine

Chard

SLE

Greenhouse air Personal protection equipment

SPE SLE

Amberlite XAD-2 Acetone: CH2 Cl2 (50:50, v/v)

70–75 >66

[15] [105]

Water, rat plasma

Direct injection

Protein precipitation with acetonitrile for rat plasma

Not provided

[130]

Human urine

LLE

52–107

[98]

3-Hydroxy carbofuran 3-Hydroxy carbofuran; aldicarb sulfone; aldicarb sulfoxide; 3-PBA

Human urine Human urine

SPE SPE

84 81–115

[96] [97]

94

[58]

3-PBA; cis-DCCA; trans-DCCA

Human urine

SPE

Ethyl acetate/acetonitrile (70:30, v/v) OASIS NH2 or graphite carbon cartridges Protein precipitation with acetonitrile C18 cartridges

93–101

[100]

3-PBA; CPBA; cis-DCCA; trans-DCCA; cis-DBCA

Human urine

SPE

87–121

[23]

DEDTP; DETP; DEP; DMDTP; DMP; DMTP DEDTP; DETP; DMDTP; DMTP

Human urine Human urine

SPE LLE

C18 cartridges Clean-up by LLE (n-hexane) C18 cartridges Acetonitrile

98–105 60–107

[57] [71]

Endosulfan I; endosulfan II; endosulfan diol; endosulfan lactone; endosulfan sulfate

Rat tissues

Soxhlet

Extraction with dichloromethane Clean-up with Florisil

73–132

[114]

Biological samples and related matrices 6-CINA Fenoxon; fenoxon-sulfone; fenoxon-sulfoxide; fenthion-sulfone; fenthion-sulfoxide; 1-Hydroxy isopropyl diazinon; 1-hydroxy isopropyl diazoxon; 2-isopropyl-6-methyl-4-pyrimidinol; diazoxon; diethyl phosphate; diethyl thiophosphate; hydroxy diazinon; 3,5,6-Trichloro-2-pyridinol; diethyl phosphate; diethyl thiophosphate

Rat plasma

6775

River water

Fipronil; fipronil desulfinyl; fipronil sulfone; fipronil sulfide

J.L. Martínez Vidal et al. / J. Chromatogr. A 1216 (2009) 6767–6788

AMBA; MNBA

6776

Table 3 (Continued ) Transformation products Endosulfan ether; endosulfan lactone; endosulfan sulfate

Extraction technique

Observations

Recovery (%)

Reference

Human serum

LLE

Extraction with n-hexane:diethylether (1:1, v/v) Clean-up with n-hexane

60–65

[125]

Pear

SLE

Ethyl acetate

83–101

[115]

Orange, potato, rice

PLE

Dichloromethane

55–94

[138]

Honeybees

MSPD

C18 as sorbent and elution with dichloromethane/methanol (85:15, v/v) C18 cartridges

63–98

[81]

>80

[42]

Aldicarb sulfone; aldicarb sulfoxide; demeton-S-methyl sulfone; demeton-S-methyl sulfoxide; fenamiphos sulfone; fenamiphos sulfoxide; methiocarb sulfone; methiocarb sulfoxide AMPA

Grape juices

SPE

Soybean

SLE

Desiodo flubendiamide

Vegetables, soil

SLE

EBIS; ETU; EU

Tomato

SLE

ETU; PTU Fenthion-sulfone; fenthion-sulfoxide; malaoxon; omethoate Fenoxon; fenoxon-sulfone; fenoxon-sulfoxide; fenthion-sulfoxide N-Demethylspinosyn D; Spinosyn B; Spinosyn K

Fruits and vegetables Olive oil

MSPD HS-SPME

Extraction with water Not provided Addition of acetonitrile to precipitate proteins Extraction with 85–99 acetonitrile Clean-up step: LLE with chloroform and 500 mg of activated charcoal powder >70 Acetonitrile/dichloromethane/chloroform (1:1:1, v/v/v) Carbon as sorbent 63–67 PDMS 80–106

Orange

SLE

Ethyl acetate

70–94

[44]

Crops, soil, water, beef, poultry, milk, eggs

SLE

>70

[76]

TFNA-AM; TFNA; TFNG

Hops

SLE

Extraction with acetonitrile/water (80:20, v/v) Clean-up with C18 cartridges Extraction with acetonitrile/water (1:1) 2 clean-up steps: SPE (C18 and Nexus) and LLE (ethyl acetate)

66–119

[28]

[20]

[13]

[16] [80] [108]

a Abbreviations. DHE: dihexylether; HS-LPME: head-space liquid-phase microextraction; LLE: liquid–liquid extraction; MAE: microwave assisted extraction; MIP: molecularly imprinted polymer; MSPD: matrix solid-phase dispersion; PDMS/DVB: polydimethylsiloxane/divinylbenzene; SAX: strong anion-exchange disk; SLE: solid–liquid extraction; SLM: supported liquid membrane; SPE: solid-phase extraction; TOPO: tri-n-octylphosphineoxide. For other abbreviations see Table 1.

J.L. Martínez Vidal et al. / J. Chromatogr. A 1216 (2009) 6767–6788

Food samples 2,4-Dimethylaniline; 2,4-dimethylformamidine; N,N -bisdimethylphenylformamidine; N-(2,4-dimethylphenyl)formamidine 3-Hydroxycarbofuran; 3-keto-7-phenolcarbofuran; 3-ketocarbofuran; 3-hydroxy-7-phenol carbofuran; 7-phenolcarbofuran; carbofuran; dibutylamine 6-Chloronicotinic acid; aldicarb sulfone; aldicarb sulfoxide

Sample

J.L. Martínez Vidal et al. / J. Chromatogr. A 1216 (2009) 6767–6788

6777

Fig. 1. Degradation pathways of fenthion and persistence curves for fenthion and transformation products in (A) Valencia Navel variety and (B) Navel Late variety. Reprinted from [44] copyright 2007, with permission from American Chemical Society.

[46,71], anion-exchange disk [72], thin layer chromatography (TLC) [73], pressurised liquid extraction (PLE, also known as accelerated solvent extraction, ASE) [74,75], solid–liquid extraction (SLE) [76], solid-phase microextraction (SPME) [77–79], matrix solid-phase dispersion (MSPD) [80–82], supported liquid membrane (SLM) [83] and molecularly imprinted polymers (MIPs) [84]. In relation to the matrices under study, the current literature can be classified into three main groups: environmental, biological and food matrices. Water and soil are the most common matrices studied in environmental monitoring; whereas urine is widely analysed as biological sample. Finally, in the food group, a wide variety of crops and matrices have been studied. 3.1. Environmental matrices The analysis of TPs in environmental matrices is mainly focused on the study of waters (including surface waters, wastewater) and soils. Considering the techniques mentioned above, SPE is the preferred procedure for water analysis and it is used as either extraction or clean-up technique. LLE is additionally used but to

a lesser extent since it can result less selective than SPE; besides the need for the utilisation of non-polar solvents (e.g. chloroform, hexane) to perform the extraction can reduce the scope of this extraction technique to less polar TPs, depending on the corresponding log Ko/w . In comparison with LLE, reported recoveries are comparable, although bearing in mind solvent consumption and sample-handling, SPE seems more convenient. A variety of SPE sorbents have been used for the analysis of TPs in water samples. For instance, SPE has been applied for the extraction of TPs from water [4] using hydrophilic and lipophilic sorbents such as OASIS HLB cartridges (see Table 3), which are widely used in pesticide residue analysis. Other sorbents such as LiChrolut cartridges and graphitised carbon black (GBC) disks have also been used for the isolation of herbicides (triazines and ureas) and their TPs from water samples [85]. Cyclohexyl sorbents have been applied for the determination of diethyl phosphates, although certain TPs from this group, such as dimethylphosphate, cannot be extracted with this type of cartridge [57]. Taking into account that TPs are relatively polar, the utilisation of C18-based cartridges is unusual, but surprisingly, they have been applied for the determination of MCPA [68] and atrazine TPs

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Fig. 2. Extraction flow chart for the TPs products of chlorothalonil of water samples. Group I compounds: chlorothalonil, pentachloronitrobenzene, 4-hydroxy2,5,6-trichloroisophthalonitrile, 2,5,6-trichloro-4-methoxyisophthalonitrile, 1-carbamoyl-3-cyano-4-hydroxy-2,5,6-trichlorobenzene, 2,4,5-trichloroisophthalonitrile, 2,5,6-trichloro-4-methylthioisophthalonitrile and isophthalonitrile. Group II compounds: 1,3-dicarbamoyl-2,4,5,6-tetrachlorobenzene. Abbreviations. SPE: solid-phase extraction; MeOH: methanol; ACN: acetonitrile; EtOAC: ethyl acetate. Reprinted from [60] copyright 2008 with permission from Elsevier.

[69] and despite the polar nature of such compounds, the reported recoveries are consistent (Table 3). Due to the wide range of different TPs determined by SPE, it is not possible to establish a general rule or which one suits better. However, it is important to notice that adequate recoveries are normally obtained regardless the SPE cartridge selected, which means that there is a suitable SPE sorbent for each group of TPs, according to the revised references. Nevertheless, if one is looking for a versatile sorbent for the extraction of a wide range of pesticides and TPs, OASIS cartridges are probably the most suitable because of their hydrophilic and lipophilic characteristics allowing a more generic extraction. On the other hand, there are certain TP families that are considerably difficult to extract simultaneously. Thus, Chaves et al. [60] developed several procedures for the extraction of different TPs from the same parent compound: chlorothalonil. The analytes were extracted from waters and soils (Fig. 2) using different procedures based on SPE, changing the pH of the sample and the elution solvent. In another application, C18 cartridges have been used for the separation of acetochlor, alachlor and metolachlor from their corresponding oxanilic and sulfonic acids [86], using ethyl acetate and methanol as eluent agents. Apart from using selective elution, an additional strategy based on the combination of different sorbents (including cartridges and disks) is also described. The application of anion-exchange can be very interesting for the determination of very polar TPs. For instance, strong anion-exchanger (SAX) disks have been used for the determination of glyphosate and its main TP, aminophosphoric acid (AMPA) in natural waters [87], providing good recoveries. In a

similar way, triazine herbicides and TPs can be isolated from water and soils using SAX and C18. The first device is a disk that retains the TPs, whereas the second one is a cartridge that retains the parent compounds [88]. Furthermore, on-line SPE has been successfully applied for the determination of polar pesticides and TPs in surface water [89,90]. This is an interesting option that can diminish sample pre-treatment and increase sample-throughput. However, the application of this automated extraction (and clean-up) is not widespread, probably because of the equipment cost and simplicity of normal SPE. SPME is another technique that has been applied for TP analysis in water. The use of SPME for the determination of pesticide residues in this kind of matrix is well-known, and therefore it was obvious that this technique could be applied for this aim. SPME is normally utilised as an automated extraction device coupled to GC and MS. However, the applications described in literature employ SPME and subsequent analysis by LC coupled to classical UV detection. Besides, this approach has been used for the analysis of TPs from typical non-polar pesticides such as fenitrothion (OPP) [91] or DDT [77]. Due to the polarity of these analytes, typical SPME fibres have been applied, such as polydimethylsiloxane/divinylbenzene (PDMS-DVB) fibres, which are the usual choice in SPME-GC applications for pesticide residue analysis (Table 3). In spite of the use of SPME reduces samplehandling and minimises solvent consumption, its application in water analysis is reduced, probably due to certain performance drawbacks of SPME, such as lack of reproducibility, short lifetime of the fibres and possible carry-over problems with certain analytes. Nevertheless, the optimised methods reported in literature show an interesting sensitivity for trace analysis [12]. In relation to the use of other microextraction techniques, head-space liquid-phase microextraction (HS-LPME) has been applied for the determination of DDT TPs in water samples [92]. This type of LPME performs the extraction by heating the sample and retaining the analytes in a suspended drop which is then directly injected in the LC system. This device is not a common LPME procedure since a room temperature ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate) instead of an organic solvent was used to form the drop. Very low limits of detection (LODs) were obtained (<1 ␮g/L), but recovery results were not reported. Soils represent another important environmental matrix for the monitoring of TPs. In literature, there is a variety of extraction techniques that have been applied, but the common procedure involves a SLE followed by an SPE step. The SLE stage can be carried out by mechanical shaking or employing PLE [74,93] or MAE apparatus [14]. The recoveries obtained by PLE and MAE are quite similar to those obtained by SLE, although it is difficult to compare them since the studied analytes are different. Almaric et al. [84] described the use of PLE to extract atrazine TPs; the raw extract was then purified by SPE using a MIP-based sorbent. The MIP procedure was used for the selective extraction of atrazine and TPs taking advantage of the common structure of the parent compound and some of the TPs, which can provide a new approach for the selective determination of these types of compounds. In this case, it can be observed that the obtained recoveries are overall high (around 120%) in comparison to other SLE/SPE procedures. Bearing in mind desirable characteristics such as high sample-throughput or minimised sample-handling, PLE seems the most suitable option for routine analysis of TPs in soils, in the same way as it is applied for the monitoring of other organic residues and contaminants in these samples. Alternatively, the utilisation of SPME is also reported for the analysis of soil samples [12]; the target compounds are DDT TPs, the same analytes determined in water samples by this extraction technique. Another environmental matrix of interest is air. The number of publications related to the determination of organic contaminants

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such as polycyclic aromatic hydrocarbons is very extensive. However, the literature focused on the monitoring of pesticide TPs is quite scarce, despite this information can result very valuable for exposure studies. Garrido-Frenich et al. [15] described the analysis of imidachloprid and one of its TPs in greenhouse air by using special sampling pumps equipped with different solid sorbents, which were then extracted by Soxhlet. 3.2. Biological matrices and related samples The group of biological samples normally includes matrices such as urine, serum or blood. In the revised references, the majority of studies are related to the determination of pesticide TPs in urine, although there are also several studies focused on serum and plasma samples. These are liquid matrices, and for this reason, SPE and LLE have been applied [94–96], although some differences in their use can be remarked. Firstly, reported recoveries are slightly higher in the SPE-based methods, regardless the type of TP. The SPE procedures involve a pre-concentration of the sample in the cartridge, and it is possible to improve recovery rates. This is an important characteristic in comparison to LLE since the available amount (or volume) of biological samples is normally reduced. Therefore, the intrinsic features of SPE permit the analysis of low volumes of urine [23,97]. On the other hand, it has been noted that the LODs obtained using SPE [57] for OPP TPs in urine were much lower than the corresponding LODs achieved by using LLE [98] in spite of using MS/MS detection in both studies. In other studies, SPE and LLE have been combined providing a more efficient sample treatment. Thus, ethylenethiourea, which is the main TP of ethylenebisdithiocarbamates (e.g. as mancozeb, zineb and mentiram) can be isolated from human urine, applying SPE, using florisil as sorbent, and LLE with dichloromethane [99]. Urine can be considered as a “clean” matrix; however, in some applications, a clean-up step is mandatory in order to determine the analytes. This was observed in the monitoring of aldicarb TPs. Fig. 3 compares the LC-UV chromatograms ( = 210 nm) corresponding to a human urine control sample spiked with aldicarb sulfoxide, aldicarb sulfone, hydroxycaroburan, aldicarb and carbofuran, without clean-up and changing the type of sorbent used in this step (graphite carbon with florisil or NH2 cartridges). The clean-up is necessary to determine these compounds [97]. Another approach is based on the performance of an on-line SPE stage, which has also been utilised in other liquid samples (e.g. water) [100]. The difficulty of analysing TPs and parent compounds simultaneously has been pointed out in previous sections. Thus, Bielawski et al. [101] analysed carbamates, pyrethroids, OPPs, organochlorine pesticides (OCPs) and TPs in meconium by using different SPE and LLE steps. First, a SPE method to isolate parent pesticides was optimised and afterwards, a methanolic/hydrochloric acid methyl ester derivatization with LLE was also carried out for the analysis of the TPs. On the contrary, in other biological samples, such as urine, it is possible to find common approaches for the simultaneous determination of TPs. These compounds can be released by an enzymatic (or acid) hydrolysis procedure [102] that can be used for the determination of TPs from OPPs, such as diazinon, malathion, pirimiphos, and azinphos, pyrethroids and herbicides [65,103]. Alternatively, ion exchange has been used for the analysis of TPs, but high LODs have been reported, observing that lyophilisation can produce better results for the determination of OPP TPs in human urine [57]. The application of extraction techniques that have been utilised in other liquid matrices, such as water, has not been described. For instance, SPME is widely used in water analysis, but up to our knowledge, determination of TPs in urine samples has not been

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reported. The same situation can be remarked for LPME-based methodologies applied in biological samples. On the other hand, an interesting recent approach related to the automation of the extraction/clean-up stage based on the use of turbulent flow chromatography (TFC) is available. This technology performs an on-line treatment by utilising special columns packed with particles showing pores on their surface and large interstitial spaces. Under these conditions, low-size molecules (e.g. pesticide TPs) diffuse faster than high-size molecules (e.g. matrix compounds) and these small molecules diffuse into the pores and chemically interact with them depending on their affinity. Therefore, this system can be useful for the separation of the analytes from complex matrices, such as biological fluids [104]. It is important to notice that further studies related to a thorough monitorization of TPs in biological fluids (especially in human fluids) are required in order to increase the available information, which is somehow separated. The current studies are focused on specific groups of TPs and in certain matrices. It would be very interesting to perform studies in a wider scope, for instance, monitoring a variety of groups of TPs, although this could imply the development of different extraction procedures. In this way, it may achieve a general overview of the levels of these compounds in humans. In relation to the situation described above, there are several studies focused on the analysis of pesticide TPs in greenhouse air [15] and in personal protection equipment [105] in areas using pesticides in farming practices. 3.3. Food matrices Very different commodities are included within this group, and general trends are complicated to establish; besides, there is a variety of TPs studied which complicates this aim. Despite one can find two main categories, crops (i.e. vegetables, fruits, oils) and animal products (i.e. meat, egg), the extraction methods are selected considering solid and liquid matrices. For solid matrices, SLE is the preferred technique, regardless the combination TPs/matrix. In vegetables and fruits, further clean-up is rarely applied, whereas in other matrices, such as meat products, an SPE stage has been used. Additionally, parent compounds and TPs can be extracted by SPE applying selective elution. Thus, fipronil and its TPs can be extracted from pollen [106], using florisil during the clean-up step after LLE. Firstly, the use of dichloromethane allowed the elution of fipronil TPs, whereas fipronil was eluted using dichloromethane:methanol. MSPD has also been used in the determination of TPs in food products. This technique is based on the dispersion of the sample on a sorbent, allowing the simultaneous disruption and homogenisation of solid and semi-solid samples, as well as the extraction, fractionation, and clean-up of analytes in a single step. MSPD has been used for the extraction of some carbamate pesticides and two of their TPs (ethylenethiourea and propylenethiourea) from different matrices (avocado, lemon, nuts, orange) [80], aldicarb sulfone and sulfoxide from honeybees [81], and for the determination of some organochlorine TPs in egg [82]. In comparison with SLE, MSPD provided lower recovery values for the analysis of ethylenethiourea in vegetables (Table 3), probably due to the fact that sorbent and matrix are manually mixed, whereas in SLE, solvent and matrix are thoroughly mixed, normally with homogenisers or shakers, increasing recovery. In liquid samples, SPE has obviously been applied [107], although SPME-based methodologies have been proposed as an alternative. The use of head-space SPME (HS-SPME) has been reported for the determination of TPs of OPPs in olive oil, allowing the simultaneous assessment of a wide range of compounds, minimising sample manipulation and avoiding the use organic solvents

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Fig. 3. LC-UV chromatograms of urine samples containing aldicarb sulfoxide, aldicarb sulfone, 3-hydroxycarbofuran, aldicarb and carbofuran ( = 210 nm) without clean-up (a), after passage the extract through graphite carbon and florisil cartridge (b) and after passage the extract through graphite carbon and NH2 cartridge. Peak number: (1) aldicarb sulfoxide; (2) aldicarb sulfone; (3) 3-hydroxycarbofuran; (4) aldicarb; (5) carbofuran. Reprinted from [97] copyright 2000 with permission from Elsevier.

[108]. This application results interesting since olive oil is a very complex matrix and the analysis of compounds at trace levels in this fatty commodity usually requires an extensive labour, including several extraction and clean-up stages. Another microextraction technique that can be found in literature is SLM; in particular, SLM has been used for the simultaneous extraction of glyphosate and aminomethylphosphonic acid (AMPA) from orange, apple and juices [104], minimising the volume of organic solvent used during the extraction procedure. Finally, certain methodologies can be applied for the simultaneous extraction of a high number of pesticides and their TPs in food analysis. A worldwide methodology is based on extraction with ethyl acetate, and it has been used for the extraction of fenthion and TPs from oranges [44]. Recently, the QuEChERS (quick, easy, cheap, effective, robust and safe) procedure has become very popular since it has been shown to be a powerful procedure in pesticide residue analysis in foodstuffs [110,111]. This methodology allows the simultaneous extraction of polar and non-polar compounds with adequate recoveries, and thus it can be suitable for the extraction of a wide range of compounds, including parent compounds and TPs. This approach has been successfully applied for the simultaneous analysis of pesticide residues and their toxic TPs in fruits and vegetables [112,113]. It is important to notice that these multi-residue methodologies have been mainly found in food applications. Furthermore, the comprehensive monitoring of TPs is not commonly performed and normally the TPs studies are those included in legislation (and obviously with available standards).

4. TPs determination 4.1. Separation In general, most TPs are more polar and less volatile than parent compounds, and they are also thermolabile. In this sense, LC is a suitable technique for the determination and identification of polar TPs, because GC may require a previous derivatization step [57], as it can be observed in Table 4. For instance, alkylphosphates must be converted into volatile compounds with derivatizing agents, such as diazomethane, diazopentane, triazenes or pentafluorobenzyl bromide (PFBBr) [9] prior to GC analysis. Nevertheless, it is important to notice that many TPs are GC-amenable (e.g. DDT, OPP and OCP TPs) or even GC and LC-amenable (e.g. some OPP TPs). In GC, most studies utilised a typical non-polar column with a phase composition equivalent to 5% phenyl–95% polydimethylsiloxane (PDMS). This is the typical GC column for pesticide trace analysis and, thus it is also applied in TPs determination. The use of columns showing a higher polarity (e.g. 50% PDMS or polyethyleneglycol-based phase) improving separation (e.g. better peak shape) for more polar TPs has been also reported [67,72]. Less common is the use of highly non-polar columns with a phase composition equivalent to 100% PDMS [11,105,114]. In general, splitless injection was the preferred injection technique, with injection volumes ranging from 1 to 4 ␮L. Furthermore, programmed-temperature vaporisation (PTV) was also applied [23] in the analysis of pyrethroid TPs in urine samples by GC–MS/MS.

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Table 4 Analytical determination techniques for the determination of transformation products using conventional detections and limits of detection (LOD)a . Transformation products

Sample

Technique

Detection

LOD

Reference

Non-chromatographic techniques Fenthion-sulfoxide

River water

Voltamperometry

Square-wave AdSV

0.44 ␮g/kg

[46]

Transformation products

Sample

Derivatization

Detection

LOD

Reference

GC analysis DDD; DDE DEDTP; DETP; DMDTP; DMTP

Soil Human urine

Not necessary Derivatization with PFBBr Derivatization with methyl iodide Not necessary

ECD (T = 280 ◦ C) FPD

<0.15 ng/g 1–5 ␮g/L

[12] [71]

FPD (T = 260 ◦ C)

0.02–0.12 ␮g/L

[72]

0.03–0.04 ␮g/L

[125]

0.05–0.10 ␮g/kg

[105]

DEP; O,O -diethyl phosphorothioate; O,O -dimethyl phosphorothioate Endosulfan ether; endosulfan lactone; endosulfan sulfate Fenoxon; fenoxon-sulfone; fenoxon-sulfoxide; fenthion-sulfone; fenthion-sulfoxide; Fenthion-sulfone; fenthion-sulfoxide; malaoxon; omethoate IMP; malaoxon Transformation products LC analysis 2,6-Diflurobenzamide; 4-chloroacetanilide; 4-chloroaniline; 4-chlorophenylurea; N-methyl-4-chloroaniline

Surface water

Personal protection equipment

Not necessary

ECD (T = 300 ◦ C) confirmation by GC–IT-MS/MS NPD (T = 300 ◦ C)

Olive oil

Not necessary

FTD (T = 250 ◦ C)

<0.01 mg/kg

[108]

Water, soil, chicory

Not necessary

FID (T = 250 ◦ C)

Not provided

[11]

Human serum

Sample

Mobile phase

Detection

LOD

Reference

Forestry matrices

Acetonitrile–methanol– citrate disodium hydrogenphosphate, pH 7

DAD ( = 245 and 260 nm) ED (1.35 V)

DAD: 2.0–25.2 ␮g/L

[59]

Methanol–water, 0.6% ammonia Acetonitrile–water

UV ( = 270 nm)

∼0.1 ␮g/L

[83]

UV ( = 210 nm)

8–10 ␮g/L

[97]

River water

Water–acetonitrile, 1% acetic acid

DAD ( = 220 nm). Confirmation by single quadrupole

0.05–0.1 ␮g/L

[67]

ED: 0.07–1.8 ␮g/L

2-Aminobenzimidazole; carbendazim

Water

3-Hydroxy carbofuran; aldicarb sulfone; aldicarb sulfoxide 3-Methyl-4-nitrophenol; 4-nitrophenol; fenitrooxon; paraoxon ethyl; paroxon methyl; S-methylisomer of fenitrothion 3-Methyl-4-nitrophenol; Fenitrooxon

Human urine

River water

Acetonitrile–citric acid–Na2 HPO4 buffer, pH 6.5

DAD ( = 220 and 275 nm) DCAD (1.2 V)

1.2–11.8 ␮g/L

[91]

3-PBA

Rat plasma

UV ( = 210 nm)

<0.03 mg/L

[58]

4,4 -DDD; 2,4 -DDD; DDE

Seawater, groundwater Greenhouse air

Acetonitrile–water, pH 2.4 with H3 PO4 Methanol–water, 83:17, v/v Acetonitrile–0.01 M phosphate buffer, pH 3 Acetonitrile–K2 HPO4 , pH 7 Acetonitrile/water, ammonium acetate 9 mM Methanol–water, 90:10, v/v Acetonitrile–phosphate buffer, pH 7.2 Acetonitrile–KH2 PO4 , pH 7 Acetonitrile–water (60:40, v/v) Acetonitrile–100 mM SDS Methanol–acetonitrile–2% ammonium acetate

UV ( = 238 nm)

0.6–1.2 ␮g/L

[77]

DAD ( = 220 nm)

250 ␮g/L

[15]

UV ( = 210 nm)

5–10 ␮g/kg

[14]

FLD (exc = 227 nm; em = 424 nm)

<0.010 mg/kg

[17]

UV ( = 238 nm)

0.05–0.08 ␮g/L

[92]

DAD ( = 220 nm)

0.02–0.10 ␮g/L

[40]

DAD ( = 220 nm)

0.1 ␮g/L

[89]

UV ( = 230 nm)

<0.01 mg/kg

[13]

DAD ( = 232 and 280 nm) UV ( = 250 nm)

<0.35 mg/kg

[16]

<0.010 mg/kg

[76]

6-CINA Alachlor ESA; alachlor OXA; metolachlor ESA; metolachlor OXA AMBA; MNBA

Soil Crops, soil, water

DDD; DDE

Waters

DEA; DEDIA; DEHA; DIHA; HA

River and ground water Surface water

DEA; DIA; HA Desiodo flubendiamide EBIS; ETU; EU N-Demethylspinosyn D; spinosyn B; spinosyn K

Vegetables and soils Tomato Food samples

a Abbreviations. AdSV: adsorptive-stripping voltammetry; DAD: diode-array detection; DCAD: direct current amperometrical detection; ECD: electron capture detection; ED: electrochemical detection; FID: flame ionisation detection; FLD: fluorescence detection; FPD: flame photometric detection; FTD: flame thermoionic detector; NPD: nitrogen phosphorus determination; PFBBr: pentafluorobenzyl bromide; SDS: sodium dodecylsufate. For other abbreviations see Table 1.

This technique can be useful in order to reduce degradation of certain analytes in the injection port since the evaporation is performed at low temperature in the first stages (i.e. 70 ◦ C). However, PTV is not usually applied in TP analysis by GC, although it is often used for the analysis of parent compounds.

In relation to LC, C18-based stationary phases (reversed-phase) have been commonly applied in the revised literature. Hybrid particles (silica and polymer) for the analysis of TPs from herbicides and metribuzin, have been applied [98], utilising a non-commercial stationary phase based on a reverse-phase/weak anion-exchanger for

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the analysis of chlorpyrifos TPs in urine samples. As a consequence of the widespread use of columns with particle size ≤2 ␮m in trace analysis (i.e. pesticide or veterinary drug residues analysis), the utilisation of ultra-high performance liquid chromatography (UHPLC) has been also reported in TP analysis, for instance for the analysis of amitraz TPs in fruits [115]. The main advantages of UHPLC are well-known (e.g. reduction of running time or narrower peaks than conventional LC) and the utilisation of this type of technology is rapidly increasing. Apart from typical LC or UHPLC, other LC modes have been applied for the separation of TPs. The use of multidimensional LC (LC × LC) has been described for the determination of six TPs from OPPs in urine samples [56]. In this case, considering the matrix nature (liquid and not specially complicated) the authors use a first C18-based column followed by a second column with an alkylamide-based stationary phase, and perform an on-line cleanup so that the urine samples are directly injected. The result is an interesting approach related to the reduction of sample pretreatment. This kind of strategy has been also applied in other fields (e.g. veterinary drug residue analysis) and in other clean matrices (i.e. water [116]), but also in more complex matrices, such as egg [117]. As certain TPs are small ionic compounds, their separation can result very difficult using conventional reversed-phase LC. The use of ionic or ion pair chromatography is a suitable option for the separation of these ionic compounds, which are poorly separated by usual LC (e.g. dialkylphosphates [54]), obtaining a more efficient separation [118]. Recently, hydrophilic interaction chromatography (HILIC) has been used as an alternative to ion pair LC for the separation of very polar compounds (e.g. certain antibiotics) that are not retained in typical reversed-phase columns [119,120]. This modality is similar to normal-phase LC, but it uses polar mobile phases that are compatible with MS detection. Bearing in mind that TPs normally are polar compounds and that some of them are highly polar, HILIC could be an interesting alternative to common LC separation. However, up to our knowledge, the use of this modality has not been reported yet in these applications. Further information (i.e. mobile phase) about the separation of TPs described in the revised literature is shown in Tables 4 and 5. 4.2. Classical detection In relation to the detection techniques, a significant number of studies included in the present review have applied classical detectors coupled to GC or LC. In GC, there is a variety of detectors that have been used (Table 4), such as ECD [12], FID [11], nitrogen phosphorus detector (NPD) [105] and flame thermoionic detector (FTD) [108]. In some cases, a derivatization procedure is used in order to make TPs more volatile and decreased the detection limit of the analytical method. For that purposed, several reagents can be used such as methyl iodide [72] or PFBBr [71], which has the advantage of forming only one reaction product with each TP. On the other hand, UV detection and diode-array detection (DAD) have been widely used as classical detectors in LC (Table 4), although FLD was also applied for the analysis of mesotrione TPs [17]. In general, detection wavelengths range from 210 to 280 nm. In all the cases it can be observed that low detection limits were obtained for a wide range of pesticides. For instance, carbamates TPs can be determined in urine by UV detection using a detection wavelength of 210 nm [97]. Because at this wavelength, most of the organic and inorganic compounds can absorb, some interfering compounds can also be detected and efficient extraction and cleanup techniques should be used in order to minimise interferences. At the same wavelength a TPs of deltamethrin (3-phenoxybenzoic acid, 3-PBA) [58] was detected, although it also presented signifi-

cant absorbance at 230 nm. The sensitivity was four times higher at 210 nm and although there was a considerable increase in the background absorbance with respect to 230 nm, 210 nm was selected in order to carry out toxicokinetic studies. Furthermore, acetonitrile and phosphoric acid was selected for the mobile phase in order to reduce the background. However, for the determination of other TPs, (N-demethylspinosyn, 2-aminobenzimidazole), higher detection wavelengths can be used, increasing the selectivity of the detection procedure [76,83]. Another way of increasing selectivity is the use of DAD. The application of DAD allows the determination of herbicides and TPs at concentration levels of 0.1 ␮g/L, as demanded by current legislation, indicating the suitability of this detection technique [40], eliminating false positives because of the spectral capability of this type of detector [15]. In some cases, the use of different detection wavelengths allows the detection of co-eluted compounds such as maneb and one of its TPs (ethyleneurea) [16]. Both compounds present the same retention time, but due to the different spectra, the individual quantification at 232 nm (ethyleneurea) and 280 nm (maneb) is possible. Besides, DAD can be combined with other type of detectors, such as electrochemical for the determination of TPs of diflubenzuron [59] and fenitrothion [91]. The combination of both detection techniques improves the sensitivity and selectivity of the analytical method. 4.3. MS detection 4.3.1. Low-resolution mass spectrometry (LRMS): analysis of target compounds Although the aforementioned conventional detectors have been utilised in pesticide residue and TPs analysis, MS detection has become the most popular methodology for the analysis of such compounds as it can be observed in Table 5 . MS offers high sensitivity and selectivity, no derivatization is required when coupled to LC and identification and confirmation can be carried out in a single step. Fig. 4 shows the chromatograms of postharvest ophenylphenol, diphenyl, thiabendazole, imazalil and its major TP R14821 in citrus fruits [121]. It can be noticed that target compounds were not distinguished by UV detection and they appeared as minor peaks. However, when MS detection was used, the occurrence of interferences was not so critical in the detection of the analytes since LC–MS provided higher selectivity. Structural identification of TPs is important in any study of the fate on any toxic contaminant in biotic or abiotic systems, being significant for human and environmental risk assessment. In this sense, MS detection is an important tool since it provides useful qualitative information, and thus it has been mainly used for these purposes. For the MS analysis of TPs, several strategies can be used: targeted or non-targeted compound analysis. In targeted compound analysis, TPs are known and included within a defined MS method, and they can also be monitored in routine analysis. In this case, adequate results have been obtained for the analysis of pesticides working with low-resolution mass spectrometry (LRMS) instruments, such as triple quadrupole (QqQ) analysers [122–124] and ion trap analysers (IT) [23,125], mainly operating in tandem MS (MS/MS). The QqQ analyser is the most utilised one and it permits the application of the four existing MS/MS modes: product ion scan, precursor-ion scan, neutral loss scan and selected-reaction monitoring (SRM). For the determination of atrazines and TPs, it can be observed that TPs are structurally similar to the parent pesticide, with the central ring and the attached –NH– groups intact. In consequence, they can produce the same product ions and neutral losses, which can be used to detect and quantify these compounds. Thus, the precursor-ion m/z 68 ([N C–NH–C N–H]+ )

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Table 5 Analytical techniques for the determination of transformation products using MS detection and limits of detection (LOD)a . Transformation products GC analysis 1-Carbamoyl-3-cyano-4-hydroxy 2,5,6-trichlorobenzene; 1,3-dicarbamoyl2,4,5,6-tetrachlorobenzene; 2,4,5-trichloroisophthalonitrile; 2,5,6-trichloro-4-methoxy isophthalonitrile; 2,5,6-trichloro-4(methylthio)isophthalonitrile; 4-hydroxy-2,5,6-trichloroisophthalonitrile; Isophthalonitrile 3,5,6-Trichloro-2-pyridinol; IMP 3-methyl-4-nitrophenol; fenitrooxon 4-nitrophenol; aminoparathion; malaoxon; O,O-dimethyldithiophosphoric acid; O,O-dimethylthiophosphoric acid; paraoxon BAM; DCBA

Sample

Derivatization

Detection

LOD

Reference

Soil, water

Not necessary

MS (Q)

15 ␮g/kg

[60]

Sewage sludge, agricultural soil Poplar leaves River water

Derivatization with MTBSTFA Not necessary Derivatization with PFBBr

MS (Q)

4–62 ng/g

[75]

MS (Q) MS (Q)

0.6–2.1 ␮g/kg Not provided

[79] [33]

Groundwater

Derivatization with BSTFA Not necessary Not necessary Not necessary

MS (Q)

<0.1 ␮g/L

[25]

2–19 ng/L <0.1 ␮g/L GC–MS: <5 ng/L

[23] [69] [37]

MEKC–UV: 0.1–0.25 ␮g/L 0.05–0.17 ␮g/L

[57]

0.03–0.30 mg/kg

[114]

CPBA; 3-PBA; cis-DCCA; trans-DCCA; cis-DBCA DDA; DEA; DIA; DEA; DEDIA; DEHA; DETBZ; DIA; DIHA; HA; HTBZ

Human urine Water Tap, ground and river water

DEDTP; DETP; DEP; DMDTP; DMP; DMTP

Human urine

Not necessary

Endosulfan I; endosulfan II; endosulfan diol; endosulfan lactone; endosulfan sulfate

Rat tissues

Not necessary

MS/MS (IT) MS (Q) MS (electric–magnetic–electric sector) UV ( = 210 nm) MS/MS (QqQ) Chemical ionisation MS (Q)

Sample

Mobile phase

Detection

LOD

Reference

Soil, water

Acetonitrile–water, 0.05% acetic acid

MS (Q)

5 ␮g/kg

[60]

Water

Methanol–water, 0.01% formic acid

MS/MS (Q-TOF)

Not provided

[130]

Pear

Methanol–ammonium formate, 10 mM (UHPLC)

MS/MS (Q-TOF)

2–7 ␮g/kg

[115]

Surface water

Acetonitrile–water

MS/MS (QqQ)

0.2–0.5 ␮g/L

[70]

Human urine

Acetonitrile–water, 20 mM acetic acid Methanol–water, 0.1% acetic acid

MS/MS (QqQ)

<0.25 mg/L

[98]

MS/MS (QqQ)

0.4–1.0 ␮g/L

[122]

Orange, potato, rice

Acetonitrile–methanol–water, 1 mM ammonium acetate

MS/MS (Q-TOF)

10–70 ␮g/kg

[138]

Water and soil

Acetonitrile–water, 0.01% formic acid Methanol–water, TFA pH 3

MS/MS (Q-hexapole-Q)

1 ␮g/L

[68]

MS (Q)

8–90 ␮g/kg

[81]

Grape juices

Methanol–water, 10 mM ammonium formate

MS/MS (QqQ)

0.03–1.20 ␮g/L

[42]

Soil

Methanol–water

MS/MS (QqQ)

1.25–12.5 ␮g/kg

[74]

Transformation products LC analysis 1-Carbamoyl-3-cyano-4-hydroxy 2,5,6-trichlorobenzene; 1,3-dicarbamoyl2,4,5,6-tetrachlorobenzene; 2,4,5-trichloroisophthalonitrile; 2,5,6-trichloro-4-methoxy isophthalonitrile; 2,5,6-trichloro-4(methylthio)isophthalonitrile; 4-hydroxy-2,5,6-trichloroisophthalonitrile; isophthalonitrile 1-Hydroxy isopropyl diazinon; 1-hydroxy isopropyl diazoxon; 2-Isopropyl-6-methyl-4-pyrimidinol; diazoxon; diethyl phosphate; diethyl thiophosphate; hydroxy diazinon; 2,4-Dimethylaniline; 2,4-dimethylformamidine; N,N -bisdimethylphenylformamidine; N-(2,4-dimethylphenyl)formamidine 2-Isopropoxyphenol; 2,2-dimethyl-2-dihydrobenzofuran-7-ol; 2,3,5-trimethylphenol; ␣-naphtol 3,5,6-Trichloro-2-pyridinol; DEP; DETP 3,5-Dibromo-4-hydroxybenzamide; 3,5-dibromo-4-hydroxybenzoic acid; 3,5-diiodo-4-hydroxybenzamide; 3,5-diiodo-4-hydroxybenzoic acid 3-Hydroxycarbofuran; 3-keto-7-phenolcarbofuran; 3-ketocarbofuran; 3-hydroxy-7-phenol carbofuran; 7-phenolcarbofuran; carbofuran; dibutylamine 4-Chloro-2-methylphenol

Soil

6-Chloronicotinic acid; aldicarb sulfone; aldicarb sulfoxide Aldicarb sulfone; aldicarb sulfoxide; demeton-S-methyl sulfone; demeton-S-methyl sulfoxide; fenamiphos sulfone; fenamiphos sulfoxide; methiocarb sulfone; methiocarb sulfoxide DA, DADK; DK

Honeybees

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Table 5 (Continued ) Transformation products

Sample

Mobile phase

Detection

LOD

Reference

DEDIA; DETBZ; deethyl-2-hidroxyterbutylazine; desethylterbumeton; DIA; DIHA; 2-hidroxysimazine; HTBZ DEA; DIA ETU; PTU Fenoxon; fenoxon-sulfone; fenoxon-sulfoxide; fenthion-sulfoxide Fipronil; fipronil desulfinyl; fipronil sulfone; fipronil sulfide Melamine

Water

Methanol–water, 0.01 formic acid

MS/MS (Q-TOF)

Not provided

[139]

Soil Food Orange

Methanol–water Methanol–water Methanol–10 mM ammonium formate Methanol–water

MS/MS (IT) MS (Q) MS/MS (Q-TOF and IT-MS)

<0.05 ␮g/kg <0.25 mg/kg 0.4–5.6 ␮g/kg

[84] [80] [44]

MS/MS (QqQ)

<0.05 ␮g/kg

[106]

MS/MS (QqQ)

<0.01 mg/kg

[118]

TFNA-AM; TFNA; TFNG

Hops

Methanol–0.5 mM TFHA (ion pair separation) Acetonitrile–water, 0.2% formic acid

MS/MS (QqQ)

<2.5 ␮g/kg

[28]

Pollen Chard

a Abbreviations. BSTFA: N,O-bis(trimethylsilyl)-trifluoroacetamide; HRMS: high resolution mass spectrometry; IT: ion trap; MEKC: micellar electrokinetic chromatography; MTBSTFA: N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide; PFBBr: pentafluorobenzyl bromide; Q: single quadrupole; QqQ: triple quadrupole; SDS: sodium dodecyl sulfate; TFA: trifluoroacetic acid; TFHA: tridecafluoroheptanoic acid. For other abbreviations see Table 1.

and constant neutral loss m/z 42 (CH3 –CH CH2 ) can be used to monitor these compounds [126], which indicates that TPs can be selectively detected without having previous knowledge regarding their identity. In the same way, neutral-scan mode has been used for the identification of transformation products of furathiocarb, using a neutral loss corresponding to m/z 131 [126]. Moreover, neutral loss strategy has been also used to confirm the identity of nitro-degradation product of isoproturon [127]. When QqQ analyser works in SRM mode, it shows several advantages and interesting characteristics for target analysis such as the increase of selectivity, reduction of number of interferences and high sensitivity. Another important point is the possibility of diminishing the analysis time, including extraction and instrumental determination [128] because of the aforementioned increase in selectivity and sensitivity. These properties can reduce the need for exhaustive sample pre-treatment and the high speed acquisition allows the simultaneous monitoring of up to 70–80 transitions if multiple reaction monitoring (MRM) is applied [113,129]. One aspect that can facilitate the determination of this type of compounds by MS is the presence of acidic or basic centres in these molecules, which favours efficient ionisation, and low detection limits can be obtained [102].

Fig. 4. Comparison of chromatograms of orange sample fortified with ophenylphenol (OPP), diphenyl (DP), thiabendazole (TBZ), imazalil (IMZ) and its metabolite R14821 (IMZ-M). Detection MS-SIM and UV ( = 230 nm). Reprinted from [121] copyright 2003, with permission from Elsevier.

In relation to quantification process in targeted compound analysis, matrix effect can be an important source of errors, and several strategies can be used. For instance, it was observed that during the determination of some carboxylic acid TPs in urine [65], a signal enhancement was noted. The use of isotope labelled internal standards (IS) is the best choice to compensate matrix effects, but the lack of standards for most of TPs and their high cost make necessary the use of alternative procedures, such as matrix-matched standard calibration. Additionally, if the analytes are present at low concentrations, other alternatives such as efficient clean-up procedures are necessary to obtain reliable results. In biological samples, ionic TPs can be excreted directly, whereas the less polar ones have to be conjugated to increase their polarities prior to excretion. LC–MS gives the possibility of measuring the conjugates, providing useful information on the metabolism of pesticides in exposed people [130], and most of conjugates involves the fragmentation of the groups [M−176]− and ions at m/z 113 and 175, characteristics of glucoronides, and [M−80]− , characteristic of sulfates. However, due to the presence of high amounts of other conjugates, some interfering peaks can be found, and an efficient clean-up procedure is demanded again.

4.3.2. High resolution mass spectrometry (HRMS): analysis of unknown compounds The use of a QqQ analyser requires the previous selection of the appropriate SRM transitions of the TPs prior to analysis by using the corresponding standards. However, the information related to nontarget and/or unknown TPs not included in the study will be missed. For this purpose, the MS analysis can be carried out using time of flight (TOF) analysers, which are high resolution mass spectrometry (HRMS) instruments permitting the performance of accurate mass measurements. These measurements are greatly useful in structural elucidation and identification of compounds. Indeed, TOF-MS has been the selected technique for the determination of TPs in environmental, biological and food matrices [131,132], and the study of the structure of degradation products [133]. The utilisation of TOF analysers offers several advantages for the analysis of TPs, such as high resolution (more than 5000 FWHM), mass accuracy, and full scan spectrum acquisition. A significant characteristic of TOF instruments is a consequence of the recording of full scan spectra. Thanks to this information, it is possible to detect compounds (known or unknown) after analysis. This is an important advantage since it is possible to review and to look for additional compounds, not included in the initial analysis. One of the problems is that there are not commercial reference standards available for many of them. In this sense, the performance of accurate mass measurements by TOF and the utilisation of accu-

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Fig. 5. Extracted ion chromatograms obtained form the LC/TOF-MS analysis of pear extract: (a) m/z 294.19 ± 0.02 Da and (b) m/z 163.12 ± 0.02 Da; (c) accurate mass spectrum of the peak at 12.16 min, which corresponds to amitraz transformation product 1 (N-2,4-dimethylphenyl-N-methylformamidine). Reprinted from [132] copyright 2007, with permission from American Chemical Society.

rate mass databases can be applied for the elucidation and/or identification of these compounds [134]. Thus, secondary amide ethanesulfonic acid TPs of acetochlor, alachlor, and metolachlor were identified by TOF-MS due to their characteristic fragmentation and use of diagnostic ions [135]. Fig. 5 shows the extracted chromatogram of m/z 294.195 and 163.12, which correspond to amitraz and a fragment ion. The peak at 28.78 min is due to amitraz (Fig. 5a), but an additional peak was observed at 12.16 min (Fig. 5b), when m/z 163.12 was monitored. This peak was due to an amitraz TP that had the same structure that the fragment ion observed at 28.78 min [132], observing that fragmentation patterns of parent compound can be useful for the identification of TPs. An interesting advantage of the use of TOF for the elucidation of TPs is the combination of accurate mass measurements with the isotopic pattern of the TPs, as it has been shown by García-Reyes et al. [136], who elucidated several chlorinated pesticide TPs using the isotopic pattern as a filter. In the same way, imazalil and prochloraz

TPs were detected in citrus, without routine monitoring methods, based on the same isotopic patterns of the identified parent compounds [137]. Thus, the presence and the number of chlorine atoms present in the suspected species can easily be found by taking into account the relative intensity of 37 Cl/35 Cl. One of the main problems associated with the TOF instrument is that it is not possible to isolate a precursor-ion to obtain clean MS/MS spectra and only fragmentation can be enhanced if higher “fragmentator” voltages are used [138]. Bearing in mind that this analyser cannot provide structural information from fragmentation, hybrid instruments such as the quadrupole TOF analyser (Q-TOF) have been introduced trying to reach a more accurate identification. These hybrid instruments have facilitated the elucidation of pesticide TPs in degradation studies [115,139,140], as well as they have permitted the differentiation between structural isomers. For instance, two TPs of diazinon with the same nominal mass, m/z 305, can be distinguished when hybrid Q-TOF analyser is

6786

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Fig. 6. UPLC–QqTOF-MS chromatogram of a treated pear extract 7 days after the treatment. Peaks filled in black are those whose identity cannot be confirmed by any type of standards. Peaks unfilled are those whose identity was confirmed by the use of an analytical standard. Time scale (min). Reprinted from [115] copyright 2008, with permission from Elsevier.

used [130], due to the product ion spectra obtained, and they can allow the differentiation between isomeric analytes. In the same way, the use of Q-TOF can help to distinguish between two possible TPs of diuron (C8 H9 N2 OCl2 and C9 H13 N2 Cl2 ) with the same nominal mass of m/z 219 [4]. Another example is shown in Fig. 6, where Q-TOF was used for the determination of several amitraz TPs [115], where preliminary identification of TPs was accomplished using the experimentally determined m/z values to compute the empirical formula. In order to assure the reliability of the detected compound, Hernández et al. [131] proposed that in order to consider a peak as potential TP, it should appear in several of the treated samples collected at different times, with a reproducible retention time (±0.1 min) and m/z value (±20 ppm). Despite the increasing use of TOF instrument in the determination of unknown compounds, MS/MS can also be applied for this purpose and several and interesting approaches can be used. First, the identification can be performed by obtaining the full scan mass spectrum, but a limited m/z range can be examined simultaneously to increase sensitivity. In certain cases, matrix components can interfere in the determination of TPs, and thus it is necessary to use several MS–MS modes, such as constant neutral loss and precursorion scan modes to increase the selectivity of the detection [126]. This strategy has been used for the identification of TPs. In the study of phenylureas TPs, the precursor-ion scan was used to monitor the common product ion corresponding to m/z 72 ([(CH3 )2 NCO]+ ), which is specific of this type of compounds [141]. However, this approach has some limitations because some TPs can have a different structure in the side of the molecule from which the diagnostic ion comes, and negative results can be provided. 5. Conclusions and future trends Pesticides can be degraded and they can produce new analytes which can be more toxic than parent compounds or they can also have insecticidal properties, as well as high persistence in the environment. Taking into account these TP properties, the development of analytical methods that include these compounds are highly demanded, especially regarding the high toxicity of some of them. In this field, the recent approaches in LC–MS/MS may contribute enormously to the study of biotransformation products or risk assessment. Some TP structures suggested in the past have been unambiguously identified by the application of analysers such as Q-TOF. Due to the fact that pesticide metabolism is really complex, it is necessary to develop and validate new analytical methods that allow the accurate determination of new TPs in food, envi-

ronmental and biological matrices. Moreover, some of these new compounds should be proposed as biomarkers which could address toxicological questions. Although the use of Q-TOF can overcome some shortcomings related to the elucidation of TPs, some problems are still present. For instance, it is not possible to obtain an unequivocal elucidation of the structure of some TPs, and due to the lack of commercial standards, the combination of this technique with others such as nuclear magnetic resonance (NMR) may help to avoid problems related to the structure of these compounds. Bearing in mind that no single instrument is able to provide all the information required in metabolism studies, the utilisation of several instruments providing complementary information seems to be necessary. In this sense, general protocols to identify TPs must be proposed. New analysers, such as the Orbitrap, allow accurate mass measurement in MSn experiments for small molecule research, and additional information can be obtained [21]. In the same way, the IT analyser has been also used for the determination of some TPs in fruits [44], providing qualitative information that could be used to ascertain whether these compounds or any other substances were present or not in the sample, although the main limitation is its low dynamic range, and only a limited number of ions can be fragmented simultaneously [26]. Furthermore, new analysers such as quadrupole linear IT (Q-LIT) and IT-TOF can be used in these studies. The hybrid IT-TOF analyser permits the performance of multiple MS stages (as the Orbitrap), and the possibility of obtaining mass accuracy measurements for different ions obtained by MSn , increasing the structural data available. This instrument has been already used in the detection of non-targeted compounds in herbal preparations [142]. Furthermore, the use of multidimensional LC can be an excellent way of performing efficient and automated clean-up in complex matrices in order to detect several classes of TPs [56]. In relation to automated clean-up systems, TFC-based technology can be interesting as a potential tool in the pre-treatment sample, as reported in other fields of trace analysis such as veterinary drug analysis [104]. In the same way, although GC-TOF has already been used for the determination of TPs in food [143], GC × GC-TOF can be applied for qualitative and quantitative determination of pesticide residues and TPs in complex matrices such as animal feed [144]. Regardless of the analytical technique, some further drawbacks must be addressed. The availability of TP standards is a key factor, since the determination of such compounds in environmental, food or biological matrices is only possible whenever the commercial standard is in the market. Otherwise, the study of TPs is limited to a qualitative stage. Moreover, TPs legislation is focused on wellknown compounds, and this knowledge is always preceded by the corresponding quantitative study in a variety of matrices. In this sense, considering the revised literature, further research should be performed in order to achieve a more comprehensive overall view of the occurrence of TPs (e.g. biological fluids) by developing multi-residue methods (including screening methodologies) for the monitoring of a variety of TPs and not only certain groups. Furthermore, the data related to the levels found in real samples is still insufficient (Table 2). Another problem is the stability of analytes in the samples before and during the whole analytical procedure. Full validation of a method must include stability experiments for long-term stability, freeze and thaw stability. Processed sample stability must be assessed for any procedure. Bearing in mind all these aspects, more efforts must be done in the analysis of TP pesticides in several matrices, paying attention in the structural elucidation of these compounds and fully validation of the proposed methods, taking into account stability of the analytes.

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Acknowledgements The authors gratefully acknowledge Spanish Ministry of Science and Innovation (MICINN-FEDER) for financial support (Project Ref. AGL2006-12127-C02-01). PPB acknowledges her grant (F.P.U.) from the Spanish Ministry of Science and Innovation (Ref. AP20053800). RRG is also grateful for personal funding through the Ramón y Cajal Program (Spanish Ministry of Science and Innovation-EFS).

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