Analytica Chimica Acta 590 (2007) 116

Review

Sample handling strategies for the determination of persistent trace organic contaminants from biota samples
Natalia Fidalgo-Used, Elisa Blanco-Gonz alez, Alfredo Sanz-Medel
Department of Physical and Analytical Chemistry, University of Oviedo, C/Julian Claver a 8, 33006 Oviedo, Spain Received 28 November 2006; received in revised form 28 February 2007; accepted 2 March 2007 Available online 12 March 2007

Abstract Even after emergence of most advanced instrumental techniques for the nal separation, detection, identication and determination of analytes, sample handling continues to play a basic role in environmental analysis of complex matrices. In fact, sample preparation steps are often the bottleneck for combined time and efciency in many overall analytical procedures. Thus, it is not surprising that, in the last two decades, a lot of effort has been devoted to the development of faster, safer, and more environment friendly techniques for sample extraction and extract clean up, prior to actual instrumental analysis. This article focuses on the state of the art in sample preparation of environmental solid biological samples dedicated to persistent organic pollutants (POPs) analysis. Extraction techniques such as Soxhlet extraction, sonication-assisted extraction, supercritical uid extraction (SFE), microwave-assisted extraction (MAE), pressurised liquid extraction (PLE) and matrix solid-phase dispersion (MSPD) are reviewed and their most recent applications to the determination of POPs in biota samples are provided. Additionally, classical as well as promising novel extraction/clean-up techniques such as solid phase microextraction (SPME) are also summarized. Finally, emerging trends in sample preparation able to integrate analytes extraction and their adequate clean-up are presented. 2007 Elsevier B.V. All rights reserved.
Keywords: Biota samples; Sample preparation; Sample handling; Extract clean-up; Extraction; Persistent organic pollutants

Contents
1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample-extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Conventional Soxhlet extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sonication-assisted extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Supercritical uid extraction (SFE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Microwave-assisted extraction (MAE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Pressurised liquid extraction (PLE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Matrix solid-phase dispersion (MSPD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extract clean-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Classical liquid adsorption chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Gel-permeation chromatography (GPC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Solid-phase microextraction (SPME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Integrated extraction and clean-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 2 3 4 5 6 7 8 8 11 12 13 14 14 14

3.

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Corresponding author. Tel.: +34 98 5103474; fax: +34 98 5103125. E-mail address: asm@correo.uniovi.es (A. Sanz-Medel).

0003-2670/$ see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2007.03.004

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1. Introduction Persistent organic pollutants (POPs) are a heterogeneous group of substances including, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs), organochlorine pesticides (OCPs) and other organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) [1,2]. POPs toxicity has been linked to observed adverse effects on human health and animals, including cancer, nervous system damage, reproductive disorders and disruption of the immune system [3,4]. Moreover, they are characterized by a high chemical and biological stability, and a high degree of lipophilicity. These characteristics make POPs prone to persist in the environment and to bioaccumulate along the food chain involving a wide range of trophic levels [5]. Because they circulate globally via the atmosphere, oceans, and other pathways, POPs released in one part of the world can travel to regions far from their source of origin [1,2]. Therefore, despite the intensive research carried out worldwide during the last three decades, it is still needed to improve our knowledge on POPs contamination, since released POPs will continue to circulate in our environment for many years to come. In addition, many of more recently recognized pollutants, such as polybrominated diphenylethers (PBDEs), polybrominated biphenyls (PBBs), polychlorinated naphthalenes (PCNs), peruorooctane sulfonate (PFOS) and related compounds, nitro musks and polycyclic musks fragrances, have distribution patterns similar to those of conventional POPs mentioned above with regard to global biospheric distribution, persistence, bioaccumulation and biomagnication [6]. Consequently, concern about the environmental fate and potential effects of these emerging POPs continues to increase [7,8]. The analytical procedures used for the determination of both conventional and emerging POPs in environmental samples (water, soil, sediment and biota) typically involve a number of equally relevant steps for sampling, sample preparation, separation and detection of target compounds, identication, quantication and data handling. The separation and detection step has been dominated by gas chromatography (GC) coupled to sensitive and specic detection systems, such as the electron capture detector (ECD) and mass spectrometry detection (MS) [9]. Nowadays, these chromatographic instrumental techniques can provide high resolution of complex mixtures on almost every matrix, while detection limits below femtograms of the desired compound are achievable. Notwithstanding the importance of such instrumental techniques, the whole analytical process can still be wasted if an unsuitable sample preparation procedure is employed before the sample reaches the detector. This is especially important in environmental analysis where the complexity of many matrices, e.g. biota, and the low concentrations at which the analytes of interest have to be identied and/or quantied (in the presence of many other closely related compounds) make prior sample treatment mandatory. Sample preparation is usually a multi-step procedure carried out off-line. This fact makes it tedious and time-consuming, prone to loss of analytes and to contamination. Therefore, today sample preparation is still the weakest link and the time-

determining step in the whole analytical procedure, accounting for about two-thirds of the total analysis time. It is also the primary source of errors and discrepancies between laboratories. Thus, the quality of this step is a key factor in the nal success of the analysis and the judicious choice of an appropriate sample preparation procedure greatly inuences the reliability and accuracy of a given environmental analysis [10,11]. The basic concept of a sample preparation is to convert a complex matrix into a sample in a format that is suitable for nal instrumental analysis. This can be achieved by a wide range of techniques, many of which have changed little over the last 100 years [12]. All those sample preparation techniques have the following common aims: to extract the analytes from the matrix, to bring them to a suitable concentration level, to remove possible interferences (clean-up step) and, when is required, to convert the analytes into a more suitable form for detection or separation. This paper reviews the main extraction and clean-up procedures, published in the last 5 years, applied to the analytical determination of both conventional and emerging persistent organic pollutants (POPs) in environmental biota (vegetal and animal) samples. Readers interested in older revisions on the topic are advised to consult previous reviews [1315]. 2. Sample-extraction techniques The analysis of POPs in biota samples requires the use of analyte extraction techniques, which allow the release of the analytes from the solid matrix, with optimum yield and selectivity, by using an adequate solvent, in such a way that as few potential interfering species as possible are carried into the analytical separation stage. The solvents may be organic liquids, supercritical uids, pressurised, or superheated liquids. The most common extraction techniques for solid and semisolid matrices include Soxhlet extraction, sonication-assisted extraction, supercritical uid extraction (SFE), microwaveassisted extraction (MAE), pressurized liquid extraction (PLE) and matrix solid-phase dispersion (MSPD). Thus, we will revise lately reported strategies and applications, using each of such approaches. 2.1. Conventional Soxhlet extraction Soxhlet extraction is a general and well-established technique developed in 1879. The technique is based on exhaustive extraction of organic compounds (analytes) in a Soxhlet system by an organic solvent, which is continuously reuxed through the sample contained in a porous thimble. The extracted analytes accumulate in a heated ask and so they must be stable in the reuxing boiling solvent. Soxhlet extraction is the oldest technique used for the isolation of non-polar and semi-polar organic pollutants from different types of solid matrices, including biota samples [10,13,16]. It has been used for decades and has been adopted by the U.S. Environmental Protection Agency (EPA) as method 3540C [2]. Although the size of the system can vary, the more common procedures use 50200 mL of organic solvent to extract the ana-

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lytes from between 1 and 100 g of biological tissue [10]. It is essential to match the solvent polarity to the solute solubility and to thoroughly wet the sample matrix with the solvent for the extraction. Typical solvents to extract POPs from animal and plant tissues are n-hexane, dichloromethane, and mixtures of toluenemethanol, nhexaneacetone and dichloromethaneacetone. Usually, animal and plant fresh tissues should be cut, shredded and then ground with sodium sulphate in order to reduce their water content and helps to open up the tissue structure which enables good penetration of solvent into the sample matrix [10]. As alternative to chemical drying with sodium sulphate, freeze-drying (water evaporation below 0 C under vacuum conditions) or liophylization of samples can be performed before extraction. The advantages of Soxhlet extraction include: it allows the use of large amount of sample (e.g. 1100 g), no ltration is required after the extraction, the technique is not matrix dependent, and many Soxhlet extractors can be set up to perform in unattended operation. Attempts to automate the technique were somewhat successful, and a few commercial systems are available in which several samples can be extracted in parallel with much shorter extraction times and less organic solvent than using conventional Soxhlet [16]. The main disadvantages of Soxhlet extraction are that it requires large amounts of solvent, the solvent must be evaporated to concentrate analytes before determination, the process takes several hours or days to complete the extraction, and it generates dirty extracts that require extensive clean-up [13,16]. Therefore, this traditional method is being replaced by other new extraction techniques such as SFE, MAE and ASE with shortened extraction times, reduced organic solvent consumption and increased pollution prevention. However, Soxhlet extraction is still an attractive option for routine analysis for its general robustness and relatively low cost. Moreover, Soxhlet extraction is widely used as a standard technique and reference for evaluating the performance of new extraction methods proposed. Soxhlet extraction was used recently to extract PAHs from whole gall bladders and liver of different sh species using dichloromethane as solvent [17]. Dichloromethane was also used to extract OCPs and PCBs from sh and bowhead whale tissue [18], OCPs, PCBs and PBDEs from several biota samples (mussel, sh, birds, mammals, lichen) [19], PCBs and PCDD/Fs from albatross muscle [20] and PBDEs from salmon [21]. Vives and Grimalt [22] reported the Soxhlet extraction of PAHs, PCBs and OCPs from sh liver with n-hexanedichloromethane (4:1, v/v). Different n-hexanedichloromethane mixtures were also used for extracting OCPs and PCBs from krill and silversh tissue [23], non-ortho PCBs congeners from sh tissue [24], OCPs, PCBs, PCDD/Fs and PCNs from several animal species (polar bear, krill, sharp-spined notothen) [25], PCBs, PCDD/Fs and PCNs from cormorants eggs and gulls [26], PBDEs from sh tissue [27,28], from sh muscle, sh liver and mussel [29] and polycyclic musk fragances from marine mammals tissue (bear, seal, sea lion, sea otters, dolphins) [30]. Manirakiza et al. [31] used a Soxhlet technique to extract OCPs and PCBs from seven sh species with n-hexaneacetone (3:1, v/v). Several n-hexaneacetone mixtures also allowed the extraction by

Soxhlet of PBDEs from biota [32,33], from shellsh tissue, sh muscle and sh liver [34] and from cormorant liver and harbour porpoises [35]. Similarly, PBDEs, PCBs and OCPs have been extracted from guillemot liver and guillemot eggs [36], PCBs from mullet sh [37] and coots tissue [38], OCPs from salmon trout [39] and brominated ame retardants such as hexabromocyclododecane (HBCD) from biota [40]. Most recently, Holmqvist et al. [41] reported the use of n-hexane in a Soxhlet apparatus to extract PCBs and DDTs from eel tissue while Ramu et al. [42] extracted PBDEs from dolphin and porpoise tissues (blubber, liver and kidney) with diethyl ethern-hexane. Soxhlet was used to extract PCBs and PCDD/Fs from herring [43], PCBs and PCNs from pine needles [44], PCBs, PBDEs and PCNs from mussel [45] and PCBs, PCDD/Fs, PBDEs and PCNs from sh [46] using toluene. 2.2. Sonication-assisted extraction The simplest solidliquid extraction technique is to blend the solid sample with an appropriate organic solvent and ultrasonicate them. The process is carried out in discrete systems using an ultrasonic bath or a closed extractor tted with a sonic probe. Sonication involves the use of sound waves to stir the sample immersed in the organic solvent. Briey, energy in the form of acoustic sound waves in the ultrasound region above 20 kHz, is used to accelerate mass transport and mechanical removal of analytes from the solid matrix surface by a process called cavitation. This consists of the formation and implosion of vacuum bubbles trough the solvent, thus creating microenvironments with high temperatures and pressures (estimated up to 5000 C and 100 MPa) [47]. This mechanical effect of ultrasound induces a greater penetration of solvent into solid materials and improves mass transfer leading to an enhancement of sample extraction efciency. Therefore, sonication-assisted extraction is faster (530 min for sample) than the Soxhlet mode and allows extraction of large amounts of sample with a relatively low cost. Unfortunately, it still uses about as much solvent as the Soxhlet extraction, and also ltration is required after extraction. Moreover, it is labour intensive since, apart from the polarity of the solvent, the efciency of the extraction is dependent upon the nature and homogeneity of the sample matrix, the ultrasound frequency and the sonication time used [13]. Sonication-assisted extraction has been approved by EPA as method 3550B [2]. Rodr guez-Sanmart n et al. [48] reported ultrasound-assisted extraction of PAHs from mussel soft tissue using dichloromethane. PAHs were also extracted from pine needles by ultrasonic extraction with n-hexanedichloromethane (1:1, v/v) [49]. The procedure was compared with Soxhlet extraction and PLE using the same solvent. Despite similar analytical results obtained, the ultrasonic procedure was said to prevail over the other two tested in terms of availability, expeditiousness for simultaneous extractions of various samples and less instrumental cost. A fast ultrasound-assisted extraction procedure, using a mixture of methanolwater (4:1, v/v) containing 5% triethylamine (TEA) as extraction solvent, was used for the determination of 14 chlorophenols in clam tissue [50]. Smith et al. [51] studied the inuence of the extraction methodology on

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the determination of PAHs in pasture vegetation. The extraction efciencies of sonication and Soxhlet procedures were compared again here using dichloromethane as extraction solvent. The PAHs total amounts extracted by sonication turned out to be only 2250% of those accessible by Soxhlet. Ultrasonic-assisted extraction has been recently carried out using a dynamic extraction set-up (a ow system) which continuously supplies fresh extraction solvent to the extraction vessel. This approach may be considered as if it forces adsorbed analytes to partition continuously into new extraction solvent. A considerable reduction of extraction time, solvent consumption and sample handling, with respect to the extraction in static way, was reported [47]. Another feature of such dynamic arrangement is that the analytes are transferred out of the extraction vessel system as soon as they are extracted. This can be especially important to avoid degradation of the analytes due to sonication or if thermo-labile analytes are extracted at higher temperatures and pressures. Dome no et al. [52] used a dynamic sonicationassisted extraction procedure for extracting PAHs from lichens using hexane. The reported total extraction time was only 10 min versus 2 h in the static extraction mode and 6 h in Soxhlet extraction, while the PAHs relative recovery obtained by the three methods was similar. Similar to Soxhlet technique, only drying and homogenization is carried out before sonication-assisted extraction of biota samples. Drying the samples is performed by evaporation of water at room temperature [52] or by grinding with sodium sulphate [49]. Freeze-drying can be also used for sample drying [48,50]. 2.3. Supercritical uid extraction (SFE) SFE is an extraction technique that uses a solvent in its supercritical state. Supercritical uids have similar densities to liquids, but lower viscosities and so analytes show higher diffusion coefcients. This combination of properties results in a uid that is more penetrating has a higher solvating power and may extract solutes faster and more efciently than liquids [1214]. In addition, the density (and therefore the solvent power of the uid) may be adjusted by varying both the pressure and the temperature, affording the opportunity of theoretically performing highly selective extractions [14]. SFE utilises commercially available equipments [14] where the uid is pumped, at a pressure above its critical point, through the sample placed in an inert extraction cell (see Fig. 1). The temperature of the cell is increased to overcome the critical value of the uid. After depressurization, analytes are collected in a small volume of organic solvent or on a solid-phase lled cartridge (solid adsorbent trap). Extraction can be performed in static, dynamic or recirculating mode: performing static extraction the cell containing the sample is lled with the supercritical uid, pressurised and allowed to equilibrate; using a dynamic mode, the supercritical uid is passed through the extraction cell continuously; nally in the recirculating mode the same uid is repeatedly pumped through the sample and, after the required number of cycles, it is pumped out to the collection system.

Fig. 1. Schematic diagram of a SFE system. Reprinted with permission from [22].

Although many supercritical uids have been investigated, the most commonly used is carbon dioxide (CO2 ) because it reaches the supercritical state at a relatively low pressure (i.e. 7 MPa) and temperature (i.e. 31.3 C), it is non-toxic, nonammable, non-corrosive, chemically very inert, and affordable. Also, although CO2 is non-polar, its polarity can be adjusted with modiers such as acetone and methanol. SFE efciency is affected by a wide range of parameters such as supercritical uid nature, temperature and pressure, extraction time, the shape of the extraction cell, the sample particle size, the matrix type, the moisture content of the matrix and the analyte collection system. Due to these numerous parameters affecting the extraction efciencies, SFE affords a high degree of selectivity and the extracts are relatively quite clean (thus, they require only moderate additional clean-up). In fact, combined with solid adsorbent traps, SFE may provide a singlestep extraction and clean-up. However, the need to control so many operating parameters makes SFE optimization tedious and difcult in practice. Other disadvantages of the SFE technique include: limited sample size and high cost of the equipment. Since, the early use of SFE for extraction in the mid-1980s, numerous applications of the technique in the analysis of environmental samples has been reported [12,13,14]. In addition, SFE has been adopted by the EPA as a reference method for extracting PAHs (Method 3561) and PCBs (Method 3562) from solid environmental matrices [2]. SFE was also used recently for extracting POPs from different plant materials [5357]. The plant samples were air dried at room temperature [53,54], chemical dried with anhydrous sulphate [56] or lyophilised [57] before SFE. Ling et al. [53] reported the extraction of several OCPs from Chinese herbal medicines using SFE with CO2 at 25 MPa and 50 C (5 min static extraction time and 20 min dynamic extraction time) using Florisil as trapping sorbent. A similar procedure was used by Zuin et al. [54] for the determination of OCPs and organophosphorus pesticides in Brazils medicinal plants. Mild extraction conditions (pure CO2 ; 10 MPa and 40 C, 5 min static plus 10 min dynamic extraction time) and C18 as trapping adsorbent allowed for direct analysis of the extract by GCECD/Flame photometric detector (FPD) with no prior cleaning procedure. Quan et al. [55] reported the extraction of OCPs from ginseng by SFE using CO2 with 10 wt.% ethanolH2 O solutions as modier at 30 MPa, 60 C and C18 as

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trapping adsorbent. Pure CO2 was used by Zhu and Lee [56] to extract PCBs from pine needles and exhibited good extraction efciencies and recoveries (90%). The extract was collected using 3 mL of hexane and used directly in the GCMS analysis. Crespo and Yusty [57] compared SFE with Soxhlet for extracting PCBs from algae samples. Athough both methods yield comparable results, SFE offered the advantage of detecting all PCBs studied at lower concentrations, while extraction time and amount of solvents needed were reduced. Several applications of SFE for extracting POPs from different animal tissues have also been reported [5861]. Efcient SFE extraction of PBDEs from whale tissues was obtained with CO2 at 40 C and 28 MPa, ow rate 2 mL min1 and trapping into C18 [58]. The extract was analysed directly without additional cleanup by GCMS. The same SFE procedure was applied to extract PCBs, chlordane, toxaphenes and PBDEs from seal tissue [59]. Ner n et al. [60] made a comparison between Soxhlet and SFE extraction for the determination of OCPs and some metabolites in frog tissues The study highlighted the main advantages of SFE versus Soxhlet procedures, including efciency (higher recoveries), time consumption, cost and more environment friendly. It must be kept in mind, however, that SFE requires an optimization in depth since the extraction behaviour is strongly affected by the type of sample. Similar to plant materials, in all the procedures reported above [5860] animal tissue samples were desiccated with sodium sulphate before the extraction step to make the sample matrix more accessible to the supercritical uid. In fact, Antunes et al. [61] applied SFE to extract PCBs and OCPs from sh muscle using three types of raw materials: fresh llet, fresh llet grounded with anhydrous sodium sulphate and freeze-dried llet. They found that supercritical CO2 could extract organochlorine compounds from freeze-dried sh llets, but they were not extracted efciently from fresh sh. The pressure had a signicant effect on extraction, while temperature did not signicantly affect the efciency of the extraction. 2.4. Microwave-assisted extraction (MAE) MAE uses microwave radiation (0.3300 GHz) as the source of heating a solid samplesolvent mixture [62]. Due to the particular effects of microwaves on matter (namely dipole rotation and ionic conductance) heating with microwaves is instantaneous and occurs in the heart of the sample, leading to very fast extraction. Heat generation in the sample by the microwaves eld requires the presence of a dielectric compound. The greater the dielectric constant, the more thermal energy is released and the more rapid is the heating for a given frequency. Consequently, the effect of microwave energy is strongly dependent on the nature of both the solvent and the solid matrix. Usually, the extraction solvent has a high dielectric constant, so that it strongly absorbs the microwave energy. However, in some cases (for thermolabile compounds), the microwaves may be absorbed only by the matrix, resulting in heating of the sample and release of the solutes into the cold solvent. Therefore, the nature of the solvent is of great importance in MAE: it should selectively and efciently solubilize the analytes in the sample but, at the same time, it should absorb the microwaves without leading to a

strong heating (so as to avoid eventual degradation of the analyte compounds). Thus, it is common practice to use a binary mixture (e.g. hexaneacetone, 1:1) where only one of the solvent is absorbing microwaves. Other important parameters affecting the extraction process are the applied power, the temperature and the extraction time. Moreover, the water content of the sample needs to be carefully controlled to avoid excessive heating, allowing reproducible results. The application of microwave energy to the samples may be performed either in closed vessels with pressure and temperature control (pressurised MAE) or in open vessels at atmospheric pressure (focused MAE) [14,15]. Whereas in focused MAE, the temperature is limited by the boiling point of the solvent at atmospheric pressure, in pressurised MAE the temperature may be elevated by simply applying adequate pressures [14]. Today MAE is considered a good alternative to traditional Soxhlet extraction for POPs in environmental solid samples because it reduces extraction time (e.g. 2030 min per batch of as many as 12 samples), uses small amounts of solvents (30 mL in MAE versus 300 mL in Soxhlet extraction) and improves extraction yields. Consequently, several applications are reported [63] and an ofcial EPA method 3546 (Microwave Extraction) has been approved for the extraction of organic compounds from solid environmental samples [2]. However, MAE has also several drawbacks. They include that the extract must be ltered after extraction, polar solvents are needed, clean-up of extracts is almost always needed (because MAE is very efcient) and the equipment is moderately expensive. Several recent studies have reported the use of MAE for extracting different POPs from plants [6467] and animal tissues [6872]. Since the water content of the sample has a great effect on the extraction process, usually samples were air dried at room temperature [6567], lyophilised [65,66], freeze-dried [68] or chemical dried with anhydrous sulphate [70,71] before MAE. Cai et al. [64] used MAE to extract OCPs from Chinese teas before solid-phase microextraction (SPME)GCECD analysis. The recoveries of MAE were compared with those of ultrasonic extraction and results showed that MAE provided better recoveries (efciencies) and shorter extraction times than ultrasonic extraction. Barriada-Pereira et al. [65] carried out a comparative study between MAE and Soxhlet extraction of 21 OCPs from plants using n-hexaneacetone (1:1, v/v) as solvent in both cases. Both techniques showed similar recoveries but Soxhlet extraction was more laborious and required higher solvent consumption and longer extraction times than MAE. The developed MAE procedure was applied to the determination of the same OCPs in tree leaves [66] and ve species of plants [67]. Carro et al. [68] reported a reliable and simple procedure to extract PCBs from freeze-dried mussels using pressurised MAE with n-pentanesodium hydroxide (5%) or dichloromethanenpentane (1:1) at different temperature (7090 C) during 10 min. Wittmann et al. [69] compared a combination of saponication and liquidliquid extraction (S-LLE) with MAE for the GCECD determination of trichlorobenzenes (TCBs) in cod. In both cases, n-pentane was used as the extraction solvent. Both methods were appropriate for the detection of TCBs at concentration levels typically observed in marine biota. However,

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S-LLE was more time consuming and required larger organic solvents volumes. Bayen et al. [70] reported the rst validated method for the quantication of major PBDE congeners (47, 99 and 100) in marine biological tissues using MAE with 25 mL of n-pentanedichlorometane (1:1, v/v) as extraction solvent. MAE was compared here to Soxhlet extraction for extracting PBDEs from standard reference materials (SRM 2978 and SRM 1588a) with comparable analytical results (<15% variation). However, using MAE the extraction solvent volume (25 mL) was substantially lower than that required for conventional Soxhlet extraction and the extraction time was reduced from several hours to 25 min. The method was applied to the determination of POPs including PBDEs in a wide range of mangrove biota organisms [71]. Pena et al. [72] developed a MAE procedure for the extraction of six regulated PAHs from polluted sh muscle using n-hexane. Two types of sh samples representatives from low and high fat content sh (turbot and salmon) were considered for experimental optimization. Other samples of low and high fat content (mussel and lamprey) were also analysed to verify the applicability of the developed procedure. Accuracy validation using NIST SRM 2977 reference material was carried out and recoveries around 90% for the studied compounds were obtained. 2.5. Pressurised liquid extraction (PLE) This technique, also named pressurized uid extraction (PFE), was originally launched by Dionex Inc. in 1995 under the name accelerated solvent extraction (ASETM ) [73,74]. PLE is a solid-liquid extraction process performed in closed-vessels at relatively elevated temperatures, usually between 80 and 200 C, and elevated pressures, between 10 and 20 MPa. Therefore, PLE is quite similar to SFE but CO2 is replaced by organic solvents to mitigate potential polarity troubles [74]. Extraction is carried out under pressure to maintain the conventional organic solvents in its liquid state, but extracting at temperatures well above their atmospheric boiling points. Therefore, the solvent is still below its critical conditions during PLE but has enhanced solvation power and lower viscosities and hence allows higher diffusion rates for analytes. In this way the extraction efciency increases, minimizing solvent needed and expediting the extraction process. Both static and ow-through extraction systems can be used (Fig. 2) [14,75]: in the static extraction mode, the sample is loaded in an inert cell and pressurized with solvent heated above its boiling point during some time (then, the extract is automatically removed and transferred to a vial). The ow-through extraction mode uses fresh solvent continuously introduced to the sample. This improves the extraction efciency but, of course, diluting the extract [75]. In PLE, the pressure is of comparatively minor importance because its role is just to maintain the solvent in its liquid state. This reduces the number of parameters that need to be optimized to achieve efcient extractions compared with SFE. The main parameters to consider now are temperature and time and so the time devoted to development and optimization of the extraction procedure can be reduced. Moreover, method set-up is gener-

Fig. 2. Schematic diagram of a PLE system. Reprinted with permission from [10].

ally straightforward because the same solvent recommended in the ofcial and routine Soxhlet methods can be used. Therefore, PLE is an attractive technique because it is fast (e.g. extraction time is approximately 15 min per sample), uses less solvent volume (1540 mL), no ltration is required after extraction, the instrumentation allows extraction in unattended operation (at least 24 samples can be processed sequentially) and different sample sizes can be accommodated (e.g. 11, 22 and 33 mL vessels are available). The main two disadvantages of PLE include limited selectivity (so, it usually requires further clean-up of the extract obtained) and higher capital cost than SFE and MAE systems. The acceptance of PLE as an ofcial EPA method (method 3545) for the determination of POPs in a variety of environmental solid samples [2] has convinced many authors to use this technique for sample preparation in environmental analysis [5,12,76]. Therefore, numerous applications of PLE for the extraction of POPs from biota samples (vegetal and animal) have been reported in the last 6 years [7793]. In all the reported procedures PLE was applied to dry samples since, as in the case of Soxhlet extraction, the absence of water in the samples makes the sample matrix more accessible to organic solvents. Therefore, samples were dried by grinding with sodium sulphate [78,88] or with hydromatrix [80,82,90], air-dried [79], freeze-dried [83,85,86,89,90,93] or lyophilised [87] before PLE. Weichbrodt et al. [77] used PLE for extracting organochlorine compounds from cod liver and sh llets. Extraction was performed with ethyl acetatecyclohexane (1:1, v/v), allowing for direct use of gel-permeation chromatography as clean-up step without solvent exchange. Suchan et al. [78] compared PLE with conventional Soxhlet extraction for extracting OCPs and PCBs from sh llets using two different solvent mixtures (hexanedichloromethane (1:1, v/v) and hexaneacetone (4:1, v/v). It was found that PLE, with both solvents tested, was comparable to Soxhlet. Tao et al. [79] applied PLE for extracting DDT and its metabolites from wheat with hexane/acetone (1:1, v/v) (pressure 101 MPa, temperature 120 C). Moreno et al. [80] investigated the extraction of 65 pesticides including OCPs from greasy vegetable matrices such as avocado using PLE with ethyl acetatecyclohexane (1/1, v/v) at 120 C and a pressure of 12 MPa. Adou et al. [81] reported an analytical procedure based on PLE before GCECD or GCFPD for the determination of

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different pesticides in fruits and vegetables. The recoveries were around 70% for almost all the compounds assayed. PLE was also used by Haib et al. [82] for the extraction of OCPs from tobacco samples with acetone at 100 C and 10 MPa of pressure. Focant et al. [83] used PLE to extract PCBs and PCDD/Fs from highly fatty biological matrices (poultry, eggs, mackerel and sperm whale blubber).The optimal extraction conditions were obtained using hexane as extration solvent and a pressure of 10 MPa. Similarly, Bj orklund et al. [84] reported the use of PLE to extract PCBs from fat-containing matrices such as sh meal and a certied reference material (CRM 349, cod liver oil) using hexane as extraction solvent. G omez Ariza et al. [85] used a mixture of dichloromethanepentane (15/85, v/v) to extract PCBs from biota samples (clams, oysters, eggs of spoonbill, mussels and sh) using PLE. Quantitative recoveries (from 90 to 106%) were reported for both native and spiked PCB congeners. The method was validated using the standard reference material SRM 2974 (mussel tissue) and compared to Soxhlet and matrix solid-phase dispersion extraction. Kitamura et al. [86] developed a method based on the combination of PLE with DMSOacetonitrile (1:9, v/v) as the extraction solvent (14 MPa and 180 C) and DMSOacetonitrilehexane partitioning for the determination of PCBs and Dioxin congener (PCDDs/Fs) in lipid-rich biological matrices (beef, pork, chicken, and sh). Dioxin congener levels extracted by this method were almost identical to those obtained by conventional solvent extraction methods, such as those employing acetone/hexane or toluene, but PLE was advantageous in shortening analysis time. Martinez et al. [87] reported the extraction of PAHs from mussels by PLE with recoveries between 65 and 150% using hexanedichloromethane (1:1, v/v) at a pressure of 10 MPa and a temperature of 150 C. J ansk a et al. [88] selected hexaneacetone (1:1, v/v) as the most suitable extraction solvent for extracting PAHs from sh tissue and spruce needles samples using PLE. Relatively good recoveries (not signicantly different from those obtained by employing classic techniques such as Soxhlet extraction and extraction enhanced by sonication) were obtained. Liguori et al. [89] also extracted several PAHs from blue mussel, salmon llet, sh oil and sh by means of PLE using dichloromethane as solvent (pressure 10 MPa, temperature 100 C). Recoveries between 99 and 105% were obtained for spiked samples. The method was validated by analysing two certied reference materials (CRM 2977, mussel tissue and T0621, olive oil). PLE with dichloromethane as solvent (14 MPa and 100 C) was also used for extracting PBDEs from blue mussel [90] and PBDEs, hexabromocyclododecane (HBCD) and methoxylated polybrominated diphenyl ether congeners (MeO-PBDEs) from sea lion blubber [91]. In a recent investigation by Saito et al. [92] PLE was applied with success to extract a broader spectrum of POPs (PCBs, PBDEs, OCPs, etc.) from biological tissues. Dichloromethaneacetone (1:1, v/v) was used as the extraction solvent at a temperature of 100 C and a pressure of 10 MPa. Jiang et al. [93] reported a PLE procedure to extract also several OPPs (PCBs, PCDDs/DFs and PCNs) from freeze-dried seafood samples (sh, bivalves, shrimp, crab, and cephalopods).

When water is employed as the extraction solvent in PLE the authors tend to use a different name to highlight the fact that water is an environment-friendly solvent. Thus, terms such as pressurized hot water extraction, subcritical water extraction (SWE), superheated water extraction and high temperature water extraction can be found in the literature [12,76,94]. Because the polarity of water decreases markedly as the temperature is increased, superheated water at 100200 C, under a relatively low pressure, can act as a medium to non-polar solvent (ethanol or acetone) and is an efcient extraction solvent for many analytes [12,76,94]. A limitation in extracting with hot water is that it fails to recover compounds that are hydrophobic, thermolabile, or easily hydrolysable. Therefore, until now, only a few examples of subcritical water extraction of POPs from biota samples have been published [76]. Recently, Morales et al. [95] used water modied with sodium dodecyl sulfate (SDS) as extractant for extracting PAHs from environmental solid samples (soil, sediment, trout and sardine). A comparison between three operational modes (static, dynamic and staticdynamic mode) was carried out and efciencies close to 100% were obtained for the three modes tested. Wennrich et al. [96] also applied subcritical water extraction (SWE) to extract OCPs and chlorobenzenes from fruit and vegetables. 2.6. Matrix solid-phase dispersion (MSPD) MSPD is a process for the disruption and extraction of solid samples introduced in 1989 [97]. MSPD combines aspects of several analytical techniques, performing sample disruption while dispersing the components of the sample on and into a solid support. In this way a chromatographic material is generated that possesses unique character for the extraction of compounds from the dispersed sample [98]. In MSPD the sample is mixed (for liquid and semi-solid samples) or blended (for solid samples) with an appropiate sorbent until a homogeneous mixture is obtained (complete disruption and dispersion of the sample on the solid support); this mixture is packed into an empty column, from which the analytes of interest are eluted with a suitable organic solvent while interfering matrix compounds are selectively retained on the column [99]. Another possibility is the elution of interfering compounds in a washing step while, the target analytes are next eluted by a different solvent. Finally, additional clean-up is performed or the sample is directly analysed. Sometimes, the MSPD column is coupled on-line with a solid phase extraction (SPE) column or, as in several applications, the SPE sorbent is packed in the bottom part of the MSPD column to remove interfering matrix components [97,100]. MSPD can be regarded as a valid sample preparation technique, alternative to more classical methods, especially for solid and semisolid samples. It is simple, requires a small sample size, has a short extraction time, uses less solvent than conventional techniques, does not require preparation and maintenance of equipment and offers the possibility of simultaneously performing extraction and cleanup. However, the negative aspect is that MSPD is fairly labour intensive, requiring the sample to be ground up with the solid matrix and packed into a column

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for extraction, and quite a number of applications still use large volumes of solvents for extraction and clean-up. The selectivity of a MSPD procedure depends on the sorbent/solvent combination used. Most methods reported to date use reverse-phase materials, such as C8 - and C18 -bonded silica as the solid support; silica, Florisil and chemically-modied sorbents are used less frequently. For analyte extraction from animal tissues, C18 -bonded silica is by far the most popular sorbent while, for plant samples, both C8 - and C18 -bonded silica and also Florisil are used extensively [98]. The nature of the elution solvent is also important since the target analytes should be efciently desorbed while the bulk of the remaining matrix components should be retained in the column. Most sorbents have been tested in combination with a large variety of solvents, ranging from alkanes through toluene, dichloromethane and alcohols to water at elevated temperatures. Most environmental applications of MSPD deal with extracting pesticides from fruit, vegetables and animal tissue [98]. However, there are also papers reporting the extraction of other POPs such as PAHs from sh tissue [101], PCBs from shellsh and sh [85], PCBs from animal fatty samples [102], PCBs and PBDEs from different biota material [103]; and pesticides, PCBs, PBDEs and polybrominated biphenyl (PBBs) from several marine species [104]. Dry samples are most effectively homogenised and dispersed with the solid support used in MSPD. Therefore, samples are usually dried with anhydrous sodium sulphate [101103] or freeze-dried [85] before blending with the MSPD sorbent. Pensado et al. [101] described the performance of MSPD for the extraction of PAHs from sh tissue (salmon and turbot). The suitability of different solid supports (Florisil, C18 , and acidic silica gel) was tested as well as the inuence on the extraction efciency of the natural fat content in samples. Under optimal conditions, tissue samples were dispersed with C18 and anhydrous sodium sulphate and transferred to a SPE cartridge containing orisil and C18 . Cartridges were eluted with acetonitrile and the extract directly analysed. GomezAriza et al. [85] compared MSPD to PLE and Soxhlet for extracting PCBs from shellsh and sh. In MSPD, the samples (freeze-dried) were blended with Florisil, and the mixture was placed in a glass column containing Florisil and eluted with dichloromethanepentane (15:85, v/v). The obtained extract was clean enough for direct analysis by GCMS and GCECD. Similar advantages were obtained using both MSPD and PLE methods, but the capital cost of MSPD is much lower than that for PLE and Soxhlet. Different combinations of normal phase sorbents and elution solvents were evaluated by Criado et al. [102], in terms of extraction yield and lipids removal efciency, for the isolation of PCBs from butter, chicken and beef fat. Under optimal conditions, the sample was dispersed on Florisil and transferred to the top of a solid-phase extraction cartridge of Florisil. Non-coplanar PCBs were quantitatively eluted with 15 mL of n-hexane. Coplanar and non-coplanar congeners were eluted with n-hexanedichloromethane (90:10). In this case, 1 g of acidic silica was also placed in the bottom of the SPE cartridge, in addition to the 5 g of Florisil. With this combination of sorbents, the small amount of fat which elutes from Florisil was

oxidized by the silica layer, leading to cleaner extracts, which were directly analysed by GCMS and GCECD. Mart nez et al. [103] reported the extraction of PCBs and PBDEs from different biota material (beef fat, chicken fat, turbot sh muscle and dogsh liver) using a MSPD extraction cartridge of C18 and n-hexane as elution solvent. PCBs and PBDEs were fractionated on a second cartridge containing silica. The same research group [104] developed a multiresidue method for the trace analysis of 15 OPPs from several pesticides, PBDEs, PCBs and PBBs in marine species (turbot, clam, mussel and cockcle). The analytical procedure is based on MSPD of the sample using C18 as sorbent and basic alumina, in the bottom part of the MSPD column to clean-up with subsequent elution with hexane. 3. Extract clean-up Whichever technique is used for extraction, various matrix components such as lipids, carotenoids, pigments and resins are frequently present in the extract and must be eliminated to permit a more denitive identication and quantication of lower levels of analyte and to minimize deterioration of chromatographic performance. Thus, the removal of co-extracted matrix components is critical and so different clean-up procedures have been developed to minimise their negative effects. Moreover, the clean-up step is usually necessary to remove not only the bulk of the co-extracted material, but also those compounds closely related to the analytes that could potentially interfere in the nal determination. In this latter case adequate separation schemes or fractionation processes to allow for isolation of sub-groups of compounds (fractionation of the extract into different classes of compound) has to be carried out. Let us revise the most common strategies for extract clean-up. 3.1. Classical liquid adsorption chromatography Classical liquid adsorption chromatography is still the dominant technique for purication and fractionation of biota extracts. This classic technique is used in an off-line mode and involves passing extracts through several adsorbent columns prepared in the laboratory or through solid-phase extraction cartridges. Liquid adsorption chromatography can discriminate between the target compounds and the matrix components to a degree that depends on the selectivity of the sorbent (sorbents) used. Alumina, silica gel and Florisil columns in different mesh sizes, levels of activity and column sizes (either separately or in combination) are widely used. Sometimes, an alkaline treatment (saponication) or a treatment with sulphuric acid is necessary prior to, or in conjunction with, adsorption columns to remove the bulk of co-extracted lipids (Table 1). Ofcial EPA Methods 3630C, 3610B and 3620B (using silica gel, alumina and Florisil Cleanup, respectively) have been approved for the purication of organic extracts from solid environmental samples [2]. Pena et al. [72] reported the use of silica gel cartridges to clean-up sh tissue extracts containing PAHs after alkaline lipids digestion. Similarly, extracts of different vegetables and fruits (lettuce, tomato, cabbage, apple, grape and pear) were puried on a deactivated silica gel (15% water) column before PAHs

N. Fidalgo-Used et al. / Analytica Chimica Acta 590 (2007) 116 Table 1 Methods for the clean-up of POPs in biota extracts: classical liquid adsorption chromatography Compound PAHs PAHs PCBs PCBs, OCPs, PAHs OCPs PBDEs PCBs, OCPs, OPPs PCBs, OCPs PCBs, DDTs PAHs PAHs PAHs PAHs, OCPs, PCBs PBDEs OCPs DDTs PCBs PCBs, OCPs PAHs OCPs PAHs PAHs, PCBs, DDTs POPs tricholorobenzenes PCBs PCBs PCBs OCPs OCPs OCPs OCPs, PCBs PBDEs PBDEs PBDEs PBDEs PBDEs, PCDDs/Fs, PCBs, PBBs PCBs, PCDD/Fs, PCNs PCBs, PCDD/Fs, PCNs OCPs, PCBs, PBDEs PBDEs, PCBs, PBDEs PCBs, PCDDs/Fs PCBs, OCPs, PCDDs/Fs PCBs, PCDDs/Fs, PBDEs PBDEs, PCBs, PCDDs/Fs Matrix Fish tissues Vegetables and fruits Seaweeds Mussels Frogs Mussel, eel, porpoise and cormorant tissues Whale tissues Fish tissues Longn eel tissues Fish liver Mussels Lichens Fish liver Fish muscle, shellsh and sh liver Vegetables and sunowers seeds Plants Clam, spoonbill eggs, sh, mussel and oyster Fish liver, blubber whale and black scabbardsh Plants, mussel, eel and sh tissues Tree leaves Pine needles Mussel and krill Cod liver Fish tissues Mussels Fish tissues Fish tissues Tobacco Vegetation samples Fish tissues Silversh and krill Mussel, eel, porpoise and cormorant Cormorant liver Whale tissues Whitesh and rainbow trout Horse mackerel tissues Sea food Pine needles Guillemots liver and guillemots eggs Fish and blue mussels tissues Ringed seal blubber Lipid biota matrices Bald eagles tissues Fish tissues Beef fat Adsorption column Silica cartridges after alkaline digestion Silica Silica Silica Silica Silica after sulphuric acid treatment Automatic LC silica Silica impregnated with sulphuric acid Acidic Silica + basic silica Alumina Alumina after alkaline digestion Alumina Alumina Alumina Florisil Florisil after sulphuric acid treatment Florisil after sulphuric acid treatment Florisil Florisil Carbon cartridges Alumina cartridges Silica + alumina (3:1) Florisil + carbon + basic alumina Alumina + acidic silica Silica + alumina Sulphuric acid + silica + silver nitrate coated silica + Florisil Neutral silica + acidic silica + ENVI-Carb cartridge Florisil + silica Alumina + Florisil cartridges Alumina + silica + orisil impregnated with KOH Neutral silica + acidic silica Alumina followed by silica Alumina followed by silica after sulphuric acid treatment Acidic silica + neutral silica + basic silica Acidic silica + neutral silica + basic silica followed by alumina Acidic silica + neutral silica + basic silica Acidic silica + neutral silica + basic silica Acidic silica + neutral silica + basic silica Alumina + silica + acidic silica Alumina + silica + acidic silica Alumina + silica + acidic silica Silica + carbon Silica + alumina + acidic silica + carbon Silica + alumina + acidic silica + carbon Automated acidic silica + neutral silica + basic silica followed by basic alumina and carbon Ref. [72] [105] [57] [106,107] [60] [32] [108] [31] [41] [109] [87] [52] [22] [34] [110,111] [79] [85,112] [61,113,114]

[17,48,115,116] [66] [49] [117] [118] [69] [119] [37] [24] [82] [65,67] [31] [23] [32] [35] [120] [121] [122] [93] [44] [123] [33] [124] [86] [125] [46] [83,126]

determination [105]. Crespo and Yusty [57] used also a silica gel column to clean-up seaweed extracts in the determination of PCBs. A column lled with activated silica gel was used for isolation of fractions containing PCBs and OCPs from mussel extracts [106] and fractions containing PCBs, OCPs and PAHs

from extracts of the same type of samples [107]. Ner n et al. [60] tested several adsorbents (3% deactivated silica, 5% deactivated Florisil) as well as their combinations and different elution solvents (in volume and nature) to clean-up frog extracts for the determination of OCPs. Best results were obtained with 3%

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deactivated silica and n-hexane as eluent. Extracts of different biota samples (mussel, eel, porpoise and cormorant) containing PBDEs were also clean up on a silica gel column after fat destruction with sulphuric acid [32]. Also, automatic liquid chromatography on a silica gel column, using n-hexane as mobile phase, was reported to clean-up extracts of whale tissues in the determination of several POPs [108]. Activated silica gel impregnated with concentrated sulphuric acid was used for clean-up extracts of sh species containing PCBs and acid-stable OCPs [31], while a column with two layers of silica, treated with sulphuric acid and potassium carbonate, respectively, was also reported to be useful to clean-up longn eels extracts for the determination of PCBs and DDTs [41]. Vives et al. [109] used an alumina column to clean-up sh liver extracts. Elution with n-hexanedichloromethane (1:2, v/v) provided the PAHs fraction. Mart nez et al. [87] used alumina for purication of mussels extracts containing PAHs after alkaline digestion. Alumina columns were also applied for purication of lichens extracts containing PAHs [52] for isolation of fractions containing PAHs, OCPs and PCBs from sh liver extracts [22] and for clean-up sh muscle, shellsh tissue and sh liver extracts when determining PBDEs [34]. For the clean-up of OCPs in fresh vegetables [110] and sunower seeds [111] extracts a Florisil column was reported. Also, plant extracts containing DDTs were puried by using a Florisil column after sulphuric treatment [79]. Florisil was also employed to clean-up mussels extracts after sulphuric treatment [112] and different biota (clam, spoonbill eggs, sh, mussel and oyster) extracts [85], prior to the determination of PCBs. Using again a Florisil column the separation of PCBs and OCPs fractions on extracts of sh liver samples [113], whale blubber [114] and black scabbard sh [61] and clean-up of PAHs in plant [115], mussel [48], eel, goldsh, catsh [17] and in other several sh species extracts [116have been reported. On the other hand, Barriada-Pereira et al. [66] compared cartridges lled with four different sorbents: Florisil, a tandem of Florisil and alumina, silica, and carbon to clean-up tree leaves extracts prior to OCPs determinations. Carbon proved to be the sorbent, providing colourless eluates, cleaner chromatograms and lesser interferences. Similarly Florisil, silica and alumina cartridges as well as glass columns (lled with either Florisil, silica or alumina) were also compared for pine needles extracts purication previous to nal determination of PAHs and alumina disposable cartridges were chosen as the most efcient [49]. Silica gel, alumina, aminopropyl-silica, cyanopropyl-silica, Florisil, graphitized nonporous carbon and silica gelalumina mixture (3:1) were used for column chromatographic clean-up of PAHs, PCBs and DDTs in mussel tissue and krill samples in combination with modied supercritical CO2 as the mobile phase [117]. A silica gelalumina (3:1) column was shown to offer the best performance in terms of clean-up efciency [117]. Sometimes it may be necessary to use more than one column (adsorbent) to obtain adequate clean-up and/or fractionation of sample extracts. In this vein, extracts of cod liver containing several POPs were fractionated with Florisil, activated carbon and basic alumine column chromatography [118]. A combination

of 5% deactivated alumine and silica impregnated with sulphuric acid (acidic silica) was used as a clean-up column in the determination of trichlorobenzenes in sh tissues samples [69], while mussel extracts were cleaned-up using a silicaalumina column prior to PCBs determination [119]. Similarly, a combination of sulphuric acid treatment, silica gel, silver nitrate coated silica gel and Florisil was used for purication extracts of sh (mullet) containing PCBs [37]. A multilayer silica column (neutral/acidic) was recommended to clean-up sh extracts for PCBs determinations. That clean-up was followed by a separation of ortho-PCBs from non-ortho-PCBs on a ENVI-Carb cartridge) [24]. Florisil followed by silica gel clean-up was used for extracts of tobacco) containing low polar OCPs [82]. 5% deactivated alumina was added to a Florisil cartridge for purication of OCPs in different vegetation samples (grass, ve species of plants) [65,67]. A column made with superposed layers of alumina, silica and Florisil impregnated with KOH and elution was done with n-hexanedichloromethane (3:1, v/v) [31] was used for the clean-up of non-acid stable OCPs in sh extracts) while a multilayer silica column (neutral/acidic) was applied for fractionation between PCBs and OCPs in extracts of krill and silversh samples [23]. Two separated columns, one packed with 5% deactivated alumine and the other with 3% deactivated silica were used sequentially to clean biota samples (mussel, eel, porpoise and cormorant) extracts containing PBDEs [32] and a similar procedure, but after an additional treatment with sulphuric acid, was used for PBDEs clean-up in cormorants liver extracts [35]. A multilayer silica column (acidic silica /neutral activated silica /basic silica) was proposed for the clean-up of PBDEs in whale tissue extracts [120], while this silica column treatment followed by a basic alumina column was used for extracts purication of whitesh and rainbow trout containing PBDEs [121]. Multilayer silica columns were also reported to clean-up PBDEs, PCDD/Fs, PCBs and polybrominated biphenyls (PBBs) in horse mackerel extracts [122] and PCBs, PCDD/PCDFs and PCNs in seafood [93], and pine needle extracts [44]. The clean-up of OCPs, PCBs and PBDEs in extracts of guillemots livers and guillemot eggs was carried out on a multilayered column packed with deactivated alumina, activated silica and activated silica impregnated with sulphuric acid [123]. The same multilayer column was employed to clean-up PBDEs in sh and blue mussels extracts [33], and PBDEs and PCBs in ringed seal blubber extracts [124]. Coplanar-PCBs, PCDDs and PCDFs in extracts of lipid-rich biological matrices were fractionated by resorting to tandem multilayer silica gel-activated carbon (MLS-AC) column chromatography [86], while PCBs, OCPs and PCDD/DFs in bald eagle tissue extracts were sequentially subjected to silica gel, alumina, acidic silica and activated carbon column chromatography for adequate clean-up and fractionation [125]. Isosaari et al. [46] used a similar procedure for adequate clean-up and fractionation of PCBs, PCNs, PCDD/DFs and PBDEs in extracts of sh tissue. A commercially available automated clean-up procedure based on multi-column clean-up system (Power-Prep, Fluid Management Systems, Waltham, MA, USA) was evaluated for the determination of PBDEs, PCBs, PCDDs and PCDFs in for-

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11

Fig. 3. Schematic diagram of the automated clean-up system. Reprinted with permission from [126].

tied beef fat as quality control samples [83]. The commercially available clean-up system (Fig. 3) based on the use of disposable multilayer silica (acidic, basic and neutral), basic alumine and carbon columns can clean-up ten extracted samples containing up to 1 g of lipids in less than 2 h. The system is made of threeway and six-way electrostatic valves driven by PC software. Valve modules (V1V6) are responsible for the selection of the solvent and columns. The fractionation procedure can be tuned to allow the operator to collect different fractions at different steps of the purication process [83,126,127]. When the extract contains more than 1 g of fat, a high capacity silica column (28 g
Table 2 Methods for the clean-up of POPs in biota extracts: gel-permeation chromatography Compound PCBs, OCPs PAHs PCBs Pesticides PAHs PCBs, PBDEs, PCNs PCBs, OCPs OCPs PCBs, OCPs PBDEs Polyciclic musks PCBs, OCPs OCPs, PCBs, PBDEs OCPs, PCBs, PCDD/Fs PCBs, OCPs, MeSO2 -PCBs PCBs, OCPs PBDEs POPs PBDEs Matrix

acidic, 16 g basic, 6 g neutral) is located before the multilayer silica column (in order to remove a large amount of fat). 3.2. Gel-permeation chromatography (GPC) This technique also referred to as size exclusion chromatography (SEC) and based on molecular size separation. GPC separation is used primarily to fractionate and remove lipidic which elute rst from the column in biota material (>500 A), matrices. Most workers used Bio-Beads S-X3 (200400 mesh) gels in a range of column size and of mobile phases (Table 2).

GPC column Biobeads S-X3 Biobeads S-X3 Biobeads S-X3 Biobeads S-X3 Biobeads S-X3 after Alumina + silica column Silica + Biobeads S-X3 + alumina column Biobeads S-X3 after Florisil column Biobeads S-X3 after Florisil column Biobeads S-X3 after Florisil column Biobeads S-X3 after Florisil column Biobeads S-X3 after Florisil column Biobeads S-X3 after silica column Biobeads S-X3 after silica column Biobeads S-X3 after silica column Biobeads S-X3 after silica and Florisil columns Biobeads S-X3 after alumina column Biobeads S-X3 after silica column Biobeads S-X3 after silica column Biobeads S-X3 after alumina column

Ref. [78] [88] [38] [128] [51] [45] [129] [130] [131] [132] [30] [133,134] [19] [18] [135] [136] [42] [90] [137]

Fish tissues Spruce needles and sh tissues Coots tissues Animal fat Vegetation Mussels Guillemot, mussels, green sea urchins, sculpin and clams Burbot and trout Salmon Herring gull eggs Marine mammals tissues Perch and seal blubber Biota Fish and whale tissues Polar bear adipose tissue Fish tissues Dolphin and porpoise tissues Biological tissues Trout

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Several key advantages of GPC, over other methods currently available for the clean-up of lipids include unlike typical saponication or concentrated sulphuric acid treatments it is non-destructive, it allows handling larger masses of lipids in each sample (e.g. compared to adsorption columns) and GPC is more applicable than adsorption chromatography to unknown contaminants isolation when little information on the polarity or chemical functionality of the molecule is available. Finally, it can be fully automated. In this vein, an ofcial EPA method (Method 3640A GPC Cleanup) has been approved for the purication of organic extracts from solid environmental samples [2], while Suchan et al. [78] reported the purication of PCBs and OCPs in sh extracts by GPC on a S-X3 Biobeads column with cychlohexaneethyl acetate (1:1, v/v) as mobile phase. The same GPC column but with chloroform as mobile phase was used for the cleanup of PAHs in spruce needles and sh tissue extracts [88]. Also, a S-X3 Biobeads column was used by Dodder et al. [38] with cyclohexanedichloromethane (60:40, v/v) as eluent for the clean-up of PCBs in extracts of coots tissues, and with ethyl acetatecyclopentane (70:30, v/v) for clean-up of animal fat extracts containing pesticides [128]. One main disadvantage of the GPC system is that it is difcult to completely remove all traces of the lipids. Therefore, further clean-up steps are often necessary by resorting to the liquid adsorption chromatography columns mentioned above. Smith et al. [51] used a column of alumina and silica gel before GPC on Bio-Beads S-X3 for the clean-up of PAHs in pasture vegetation extracts while Pan et al. [45] employed a multilayer silica column followed by a Bio-Beads S-X3 column and nally and alumina column for clean-up of PCBs, PCNs and PBDEs in extracts of mussel. PCBs and OCPs in extracts of biota (guillemot, mussels, green sea urchins, sculpin and clams) were puried using a Bio-Beads S-X3 column followed by a Florisil column [129]. A similar procedure was employed for the clean-up of OCPs in extracts of trout and burbot samples [130], OCPs and PCBs in extracts of salmon samples [131], PBDEs in extracts of herring gull eggs [132] and polycyclic musk fragrances in extracts of marine mammals tissue (bear, seal, sea lion, sea otters, dolphins) [30]. A Bio-Beads S-X3 column followed by a column of silica gel was used for clean-up PCBs and OCPs in perch [133] and seal blubber extracts [134], OCPs, PCBs and PBDEs in several biota extracts [19] and OCPs, PCBs and PCDD/Fs in sh and bowhead whale tissue extracts [18]. This combined GPC-silica procedure was combined with a Florisil column for isolation of fractions containing PCBs, OCPs, and MeSO2-PCBs in polar bear adipose tissue extracts [135], while GPC on Bio-Beads S-X3, but followed by alumina column was also used for the clean-up of OCPs and PCBs in sh tissue extracts [136]. PBDEs in extracts of dolphin and porpoise blubber, liver and kidney were cleanedup by GPC (Bio-Beads S-X3) followed by activated silica gel [42] the same procedure and was used for purication of POPs in extracts of biological tissues [90] or followed by an activated alumina column, treatment for purication of PBDEs in trout extracts [137]. Nowadays, high efcient GPC columns are also used for previous clean-up of OPPs in extracts of biota in a classical HPLC system. Moreno et al. [80] evaluated the com-

parative performance of Envirogel GPC columns (Waters) with C18 , Florisil and alumina cartridges for purication of pesticides in avocado. It was found that GPC offers the best results. Also, two Envirosep-ABC columns (Phenomenex) were connected in series for purication of pesticides in fatty matrices extracts, using acetatecyclohexane (1:1, v/v) as mobile phase [138]. This high performance GPC column was compared with the classical Bio-Beads S-X3 column for the purication of MeSO2 -PCBs and OCPs in extracts of marine mammal tissues [139]. The best results were obtained when the classical GPC column was employed. 3.3. Solid-phase microextraction (SPME) Solid-phase microextraction (SPME) is a relatively recent extraction technique [140,141], mainly suited for aqueous matrices, based on the partition of target analytes between a polymeric stationary phase coating a fused silica ber, and the sample extract. During extraction the coated ber is either directly immersed into the liquid extract or exposed to the headspace above the liquid. After extraction, the analytes, retained in the ber, are thermally desorbed in the injector of a gas chromatograph for GC analysis. SPME represents today a convenient alternative to more conventional extraction methods of sample preparation of liquid samples (i.e. liquid-liquid extraction (LLE) and solid-phase extraction (SPE). SPME eliminates the use of organic solvents, is signicantly quicker and simpler than both LLE and SPE and integrates extraction, preconcentration and sample introduction into a single step [142144]. Since its introduction in 1990 [141] to analyse relatively volatile compounds in the environmental eld, SPME has gained widespread applications for determination of organic pollutants, including POPs, in different types of samples (water, soil, food and biological uids) as reported in several reviews [142,145147]. Water is by far the most widely analysed type of sample by SPMEGC. This is due to the fact that SPME application to more complex matrices, such as biota, is not so forward straight. However, SPME can be used as a simple clean up/enrichment procedure for POPs determinations in vegetal and animal tissues after previous extraction by liquidsolid extraction, as Soxhlet [39], ultrasonic assisted extraction [148,149], MAE [64,150], ASE [96] or SFE [151]. Fidalgo-Used et al. [39] developed a clean-up/enrichment procedure for OCPs in sh tissue samples based on Soxhlet extraction of the OCPs from the sample followed by SPMEGCECD over the corresponding organic extract. A representative chromatogram corresponding to the SPMEGCECD analysis from a sh tissue organic extract spiked at 1 ng mL1 level in the extract (nal concentration in the SPME vial of 0.1 ng mL1 ) and its respective blank (unspiked sh tissue extract) it shown in Fig. 4. A combined analytical method involving sequential ultrasonic extraction, sulfuric acid clean-up and headspace SPMEGCMS was used for the determination of PCBs [148] and OCPs [149] in bird liver. SPMEGC in combination with MAE for the determination of OCPs in

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Fig. 4. Chromatograms obtained by SPMEGCECD of: (a) sh tissue extract spiked with OCPs (nal concentration in the SPME vial of 100 ng L1 ) and (b) unspiked sh tissue extract. Peak assignment: (1) hexachlorobenzene (HCB), (2) -hexachlorocyclohexane (-HCH), (3) HCH, (4) -HCH, (5) -HCH, (6) heptachlor, (7) aldrin, (8) isodrin, (9) dichlorodiphenyldichloroethylene (p,p -DDE), (10) endosulfan , (11) dieldrin, (12) endrin, (13) dichlorodiphenyldichloroethane (p,p -DDD), (14) endosulfan , (15) dichlorodiphenyltrichloroethane (p,p -DDT), (16) methoxychlor. Reprinted with permission from [39].

medicinal plants [150] and a novel ber coating of polyphenylmethylsiloxane (PPMS) was also combined with MAE for the determination of OCPs in Chinese teas [64]. That novel porous solgel PPMS ber was reported to offer higher sensitivity and selectivity for OCPs compounds, higher thermal stability (up to 350 C) and longer life time (adequate use for more than 150 times) than commercial polydimethylsiloxane (PDMS) bers. On the other hand, Wennrich et al. [96] used SPMEGCMS for the determination of OCPs and chlorobenzenes in fruit and vegetables after a pre-extraction of analytes from the sample by ASE. Finally, Rodil et al. [151] have developed a new approach, based on simultaneous SFE-sample clean-up using SPE followed by SPMEGCMS, for the determination of several POPs (OCPs, PCBs, PBBs and PBDs) in cultured marine species. The inuence of several parameters in the efciency of the SPE/SPME combination was investigated by chemometrics approaches and the proposed procedure was validated with IAEA 406 reference material analysis. 4. Integrated extraction and clean-up Several researchers have examined the suitability of integrating the clean-up step into SFE or PLE techniques by resorting to the use of sorbents in the extraction cell which would retain the matrix components (trapping sorbents). In the case of biota

such sorbents are used mainly to retain the sample fat. However, so far relatively few publications have been published dealing with on-line combined extraction and clean-up procedures based on SFE [53,59,152] or PLE [84,85,89,153,154] treatments. For instance, Ling et al. [53] investigated different sorbents (e.g. Florisil, C18 , silica gel and neutral alumina) as trapping sorbents in SFE for the determination of OCPs in chinese herbal medicine. Florisil was found to provide the most facile and effective integrated clean-up. Two fat retainers, basic alumina and Florisil, were assayed for the lipid-free extraction of PCBs from a model fatty sample using SFE [152] where basic alumina was nally utilized to selectively clean-up PCBs in the fat sample extract. Basic alumina was also used as fat retainer in SFE for the determination of OCPs, PCBs and PBDEs in seal tissue [59]. Bj orklund et al. [84] evaluated the ratios between fat and fat retainer to obtain fat-free extracts. They assayed for ve different fat retainers, including Florisil, basic alumina, neutral alumina, acidic alumina and sulphuric acid-impregnated silica in order to clean-up PCBs in sh using PLE and the use of sulphuric acid-impregnated silica provided the cleanest PCBs extracts. Sulphuric acid-impregnated silica as fat retainer in PLE was also successfully used for the determination of PCBs in several fat containing matrices including lard fat, pork fat, cod liver oil, sh meal, feed poultry and vegetable feedstuff. Four different fat/fat retainer ratios (FFRs) were tested (0.100, 0.075, 0.050 and 0.025) at 50 and 100 C using n-pentane, n-hexane or n-heptane as extraction solvent. No fat was co-extracted when using a FFR ratio of 0.025 and the chromatograms showed very nice base-lines as seen in Fig. 5 [153]. In another approach, the PLE cell was lled up with aluminium oxide, silica gel and magnesium sulphate for the extraction and purication of PAHs in blue mussel and salmon [89], but a further purication by GPC was required. PLE extraction of PCBs from biota tissues (clams, oysters, eggs of spoonbill, mussels and sh) by on-line clean-up with sorbents such as Florisil, silica gel, alumina, 2,3-dihydroxypropoxypropyl (Diol) and cyanopropyl (CN) in the extraction cell has been reported by Gomez-Ariza et al. [85]. The best results were obtained using Florisil to retain

Fig. 5. Inuence of the amount co-extracted fat on the chromatographic behaviour of PCBs. Reprinted with permission from [153].

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coextracted lipids from the matrix. The resulting extract, after pre-concentration, turned out to be clean enough for its direct injection into GCMS and GCECD [85]. Eljarrat et al. [154] used alumina as fat retainer in PLE for the determination of PBDEs and HBCD in sh tissue. 5. Conclusions Sample preparation is still the most critical and timeconsuming step within the overall analytical procedure needed to obtain accurate determination of POPs in environmental biota samples. Due to the peculiarities of these samples (especially the presence of fat and the presence of analytes at trace/ultratrace concentrations) selection of the appropriate techniques to purify and preconcentrate the analytes, along with a careful optimization of the corresponding operational parameters, are the most important aspect to care of. As shown in this review, several alternative extraction techniques have been developed to replace tedious conventional methodology (e.g. Soxhlet) often used in ofcial analytical procedures. Among the new extraction techniques, SFE and PLE offer the advantages of signicantly reducing the amount of organic solvent consumed and easy automation. Moreover, they also provide the possibility for efcient and fast on-line clean-up, by using selective trapping. However, the comparatively high investment cost of these new techniques explains that conventional Soxhlet extraction, in combination with adsorption columns and/or GPC for purication and fractionation of extracts, is still widely used being the sample preparation reference method in numerous applications. Finally, emerging techniques such as MSPD or SPME have only been used in a limited number of applications so far. In any case, the results obtained are promising and show that their application should more likely expand in the near future. Acknowledgments The autors are grateful to the Fundaci on para el fomento en Asturias de la Investigaci on Cient ca Aplicada y la Tecnolog a (FICYT) for the grant to Natalia Fidalgo-Used and nancial support from the Project BQU-2003-04671 from the Ministerio de Ciencia y Tecnolog a, Madrid (Spain). References
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