Anal Bioanal Chem (2006) 384: 14471461 DOI 10.

1007/s00216-005-0242-z

REVIEW

Jos Benito Quintana . Isaac Rodrguez

Strategies for the microextraction of polar organic contaminants in water samples

Received: 4 October 2005 / Revised: 14 November 2005 / Accepted: 18 November 2005 / Published online: 22 February 2006 # Springer-Verlag 2006

Abstract In this paper the most recent developments in the microextraction of polar analytes from aqueous environmental samples are critically reviewed. The particularities of different microextraction approaches, mainly solidphase microextraction (SPME), stir-bar-sorptive extraction (SBSE), and liquid-phase microextraction (LPME), and their suitability for use in combination with chromatographic or electrically driven separation techniques for determination of polar species are discussed. The compatibility of microextraction techniques, especially SPME, with different derivatisation strategies enabling GC determination of polar analytes and improving their extractability is revised. In addition to the use of derivatisation reactions, the possibility of enhancing the yield of solidphase microextraction methods for polar analytes by using new coatings and/or larger amounts of sorbent is also considered. Finally, attention is also focussed on describing the versatility of LPME in its different possible formats and its ability to improve selectivity in the extraction of polar analytes with acid-base properties by using separation membranes and buffer solutions, instead of organic solvents, as the acceptor solution. Keywords Solid-phase microextraction . Liquid-phase microextraction . Single-drop microextraction . Membrane extraction . Stir-bar-sorptive extraction . Polar organic contaminants . Water analysis

Introduction
Until the mid-90s, organic trace analysis of water was mainly focussed on persistent organic pollutants (POP) such as PCB, PAH, organochlorine pesticides, etc. Some of these are highly hydrophobic compounds, easily bioaccumulated and biomagnified through the trophic chain in the aquatic system. Fortunately, nowadays, most of these have been banned and their environmental concentrations are strictly controlled. There is now, in contrast, increasing interest on the fate and role of polar organic contaminants in the aqueous environment. Many of these compounds are employed as household chemicals. Several pharmaceutical drugs, disinfection agents, pesticides, and different personal care products can be included in this group. Although, in general, polar species are not bioaccumulative, some are resistant to degradation during conventional wastewater treatment. This, and their excellent water solubility and continuous discharge into the environment, facilitates the ubiquitous distribution of some polar species in aquatic media. As an example, EDTA has been found to be the most abundant anthropogenic organic compound in German surface waters [1]. Surface water is used very often as a source of drinking water. As a consequence, concentrations of EDTA between 1 and 5 g L1 have been reported for tap water in Germany [1]. In parallel with these findings analytical chemistry also is evolving toward more environmentally responsible policies, searching for sample-preparation approaches with lower consumption of toxic organic solvents to minimise the generation of hazardous residues and health risks for operators. This was partially achieved in the first instance by the commercialisation of SPE, which replaced LLE, and was further improved by the popularisation of SPME [2, 3], a completely solvent free technique. The development of SPME has been followed by other microextraction techniques, for example stir-bar-sorptive extraction (SBSE) [4], liquid-phase microextraction (LPME) [57], and membrane extraction techniques [8, 9]. All share a common ideaanalytes are extracted into an acceptor medium (sorbent or solution) by equilibrium

J. B. Quintana (*) Department of Water Quality Control, Technical University of Berlin, Sekr KF 4, Strasse des 17 Juni 135, 10623 Berlin, Germany e-mail: sttito@usc.es Tel.: +49-30-31424281 Fax: +49-30-31423850 J. B. Quintana . I. Rodrguez Departamento de Qumica Analtica, Nutricin e Bromatoloxa, Facultade de Qumica, Instituto de Investigacin e Anlise Alimentario, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain

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processes. Thus, in microextraction techniques only a fraction of the analytes contained in the sample is recovered, in contrast with SPE and LLE which are exhaustive processes. In summary, solvent-free extraction techniques are more sustainable and easily implemented, and also reduce exposure of the analyst to solvents. Apart from these advantages they enable more selectivity in sample preparation than exhaustive extraction approaches such as SPE or LLE. The objective of this paper is to critically review some key features of microextraction strategies suitable for analysis of polar organic compounds in water, including those aspects that require further investigation and/or may be subject to important development in the future. The target analytes considered are medium to high polarity speciespharmaceuticals, personal care products, pesticides, disinfection by-products, amines, and phenolic compounds, etc.

Solid-phase microextraction (SPME)

Although SPME, especially in combination with GC, is a well established sample-preparation technique, its application to the determination of polar analytes in water samples is still an emerging field. Although PA and CW-DVB coatings are commercially available, extraction efficiency for highly polar analytes is still limited and the development of more polar coatings is of interest. Some other difficulties of this type of analysis are the need for derivatisation before GC determination and problems associated with LC determination, for example the stability of some coatings when exposed to organic solvents and peak broadening during on-line desorption of the analytes in the SPME-LC desorption interface. These three aspects of SPME are considered separately in the following paragraphs. Basic principles of the technique, and general guidelines, are not discussed because they have been discussed in detail in several books and reviews (e.g. [2, 3, 10]). Development of new coatings In recent years growing interest in the determination of polar compounds has fostered the development of new sorbents for solid-phase extraction (SPE), as recently reviewed [11]. This has been achieved by different research groups and also by several private companies, leading to much competence. For SPME, however, the situation is quite differentthere is no such competence commercially and, thus, only research groups are currently developing different sorbents for incorporation into microextraction fibres. Some of these are specifically intended for the determination of polar analytes in water samples. The most recent research on different sorbents is considered here, with emphasis on possible future developments toward analysis of polar compounds.

The first and probably most important development has been the use of sol-gel technology for coating the silica fibres. The pioneering work was performed by Chong et al. [12], who used this method to prepare sol-gel polydimethylsiloxane (PDMS) fibres. Unlike commercial PDMS fibres the sol-gel variety have the PDMS chemically bonded to the silica core, which increases their thermal stability and surface area by creation of a highly crosslinked network. Titanium and zirconium-based materials have also been prepared recently; these have increased the pH and mechanical stability of SPME fibres [13, 14]. Similarly, a new generation of PDMS fibres based on a Zr alloy core will soon be available commercially from Supelco; these are expected to enhance both extraction efficiency for very acidic and basic compounds, by enabling work with samples adjusted to extremes of pH, and the mechanical stability of the fibre itself, which is especially important when the SPME holder is incorporated in an autosampler device for GC. This sol-gel technology has been further used to prepare poly(ethylene glycol) (PEG) [15], PDMS/DVB [16], PDMS/poly(vinyl alcohol) (PDMS/PVA) [17], and polytetrahydrofuran (PTHF) [18] coated fibres. All of these, with the exception of PEG, are relatively apolar polymers. More polar sol-gel bonded phases have been prepared from crown ethers [1922] and calyx[4]arenes [23, 24]. Use of aromatic crown ethers in the sol-gel network has enabled more efficient HS-SPME of aromatic amines (compared with use of commercial PA fibres), due to -, dipole, and H-bonding interactions [22]. Similar results were also obtained in the concentration of amines by use of an amide-modified calyx[4]arene sorbent [24]. The polarity of the generated coating can be tuned by selecting appropriate monomers for polymerisation. An example of this is the synthesis of a polyacrylate fibre for extraction of 2-chloroethyl ethyl sulfide from soil [25]. In that work, three different monomers were considered: methyl acrylate, methyl methacrylate and butyl methacrylate. The best results were obtained by use of butyl methacrylate (BMA), because both analyte and extracting sorbent are of medium polarity and similar log Kow values. The same monomer (BMA) can be sol-gel copolymerised with DVB, resulting in a polar coating with enhanced surface area and high extraction efficiency, compared with commercial PA and PDMS-DVB fibres, in the determination of polar alcohols and acids in wine samples [26]. By a method similar to that used for BMA-based coatings, Basheer et al. [27] prepared SPME sol-gel fibres coated with oligomeric bisphenolic groups, the hydrophobicity of which is determined by the ratio of ether to free phenolic groups. Inorganic materials, for example anodised metal wires (e.g. CuCl), which can participate in ionic interactions, have also been tested as SPME fibres for concentration of amines [28, 29]. Thermal stability, ease preparation, and relatively low cost are their main advantages. These sorbents are deactivated by water, however, so can be used only with gaseous samples or in headspace (HS) mode, at room temperature, for water samples. These are

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rather unfavourable conditions for extraction of polar analytes with high water solubility and low volatility. The third group of materials prepared for SPME of polar compounds are electropolymerised polypyrrole (PPy), polyphenylpyrrole (PPPy) [3032], and polyaniline [33].

The last of these has been used for SPME of phenols from water samples and PPy and PPPy have been used for intube SPME of basic compounds from water and biological samples, for which they performed better than commercially available polar PEG capillaries [30, 31]. PPy and

Table 1 Overview of different approaches for combining SPME and derivatisation for analysis of polar organic contaminants in water Analytes Aldehydes and ketones Sampling modea Type of derivatisation DS HS HS Alkylthiols Short-chain fatty acids DS DS HS HS HS HS HS HS HS DS DS DS DS HS DS DS DS DS DS In-sample In-sample On-fibre during extraction In-sample In-sample In-sample On-fibre during extraction In-sample In-sample In-sample In-sample In-sample On-fibre after extraction In-port In-sample In-sample On-fibre during extraction On-fibre after extraction On-fibre after extraction On-fibre after extraction On-fibre after extraction On-fibre after extraction Fibre type PDMS PDMS and PDMS-DVB PDMS-DVB PDMS-DVB PDMS-DVB PA PA PA PDMS CAR-PDMS PDMS CW-DVB PDMS PA PDMS PDMS-DVB CW-DVB PA PA PDMS-DVB PA CAR-PDMS Derivatisation reagentb Refs. PFBHA PFBHA PFBHA DNFB NEM PFBBr/PFPDE PDAM Benzyl bromide H2SO4/EtOH DMS HCl/MeOH DMS Diazomethane TMAOH/TMAHS Butyl chloroformate Benzyl bromide PFBBr Diazomethane MTBSTFA Diazomethane MTBSTFA MTBSTFA [43] [43, 44] [45] [46] [47] [49] [49, 124] [48] [51] [52] [50] [125] [49] [49] [54] [53] [53] [55] [58] [56] [40] [57]

Acetic and haloacetic acids

Nonylphenol ethoxylates and carboxylates Long-chain fatty acids Acidic herbicides

Phthalic acid monoesters Acidic drugs Degradation products of warfare agents Oestrogens and anabolic steroids DS DS Phenolic compounds DS DS HS Linear alkylbenzensulfonates DS Perfluorocarboxylic acids DS Basic drugs DS Aromatic amines DS Aliphatic amines HS HS HS
a b

On-fibre after extraction PA and CW-DVB On-fibre after extraction PA On-fibre after extraction PA and PDMS-DVB In-sample PA In-sample CAR-PDMS and PDMS In-port PDMS In-port PDMS In-sample PDMS-DVB In-sample PDMS-DVB In-sample Crown ether sol-gel fibre In-sample PDMS In-sample PA

BSTFA [64] MSTFA [39] MTBSTFA [61] Acetic anhydride [62] Acetic anhydride [63] TMAHS [59] TMAHS [60] Acetic anhydride [65] NaNO2/hydroiodic acid [68] TFBza-suc [22] SIBA [67] PFBAY [66]

HS: headspace sampling; DS: direct sampling PFBHA: pentafluorobenzylhydroxylamine; DNFB: 2,4-dinitrofluorobenzene; NEM: N-ethylmaleimide; PFBBr: pentafluorobenzyl bromide; PFPDE: pentafluorophenyldiazoethane; PDAM: pyrenyldiazomethane; DMS: dimethylsulfate; TMAOH: tetramethylammonium hydroxide ; TMAHS: tetramethylamonium hydrogen sulfate; MTBSTFA: N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; BSTFA: bis-(trimethylsilyl)trifluoroacetamide; MSTFA: N-methyl-N-(trimethylsilyl)trifluoroacetamide; TFBza-suc: tetrafluorobenzoic acid N-hydroxysuccinimide ester; SIBA: N-succinimidyl benzoate; PFBAY: pentafluorobenzyl aldehyde

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PPPy are cationic sorbents, but their selectivity can be tuned by choosing an appropriate counterion. If this is, for example, ClO ; they have some anion-exchange character, 4 whereas this behaviour is lost when bigger anions (e.g. dodecyl sulfate) are used, resulting in more hydrophobic sorbents [32]. Similarly, the selectivity of aniline polymers is determined by their redox state. The less oxidised polymers produce the best results for polar analytes, by enabling hydrogen-bonding interactions through NH groups [34]. A drawback of these electropolymerised materials is, unfortunately, their thermal instability above 200C; they are, therefore, best suited to in-tube SPME and solvent desorption, rather than thermal, for example GC, applications. This limitation might be overcome by use of room-temperature-ionic liquids as SPME coatings. These solvents are virtually non-volatile and their selectivity toward polar analytes can be modified by choosing the appropriate anion and cation components. Preliminary research on SPME with ionic liquids has been conducted by Liu et al. [35]. In their work 1-octyl-3-methylimidazolium hexafluorophosphate ([C8MIM][PF6]) was physically coated on a silica fibre by dipping it in this solvent. Its high viscosity and thermal stability enabled HS-SPME of BTEX from paints and their determination by GC. It is expected that further applications can be extended to polar analytes by the appropriate selection of both the cation and anion, in a way similar to that used with PPy polymers. Ionic liquids have already been evaluated as stationary phases in GC columns and shown to have excellent thermal stability and the ability to separate polar and non-polar compounds [36].

Derivatisation Despite the advances in the development of new SPME sorbents described above, derivatisation of polar analytes is still important, because it enables their hydrophobicity to be increased, which normally leads to higher extraction yields when using commercially available SPME fibres, and improvement of performance when SPME is combined with GC analysis. These benefits are clear when detection limits of applications using SPME and GC for determination of polar compounds [37, 38] are compared with values achieved after incorporation of an additional derivatisation step in the analytical procedure [39, 40]. This aspect has, however, received little attention and, for example, in a review from 2003 on SPME of herbicides in environmental samples [41], it was not even considered, even though some of the target species were difficult to determine by GC without derivatisation. Only recently the combination of SPME and derivatisation has been reviewed in a more general context [42]. Our review focuses on water analysis and considers the most recent applications and achievements. Table 1 summarizes different applications of SPMEderivatisation in gas chromatographic analysis of polar organic compounds in water. Different strategies can be used for sample, on-fibre, and in-port derivatisation.

The first is also the simplest. Derivatisation occurs in the aqueous sample before, or simultaneously with, the extraction step. It improves both the affinity of the parent analytes for the fibre and the efficiency of subsequent GC separation. For obvious reasons this strategy is not suitable for moisture-sensitive reagents; these are, however, compatible with on-fibre derivatisation reactions. On-fibre derivatisation can be performed either by preloading the fibre with the derivatisation agent, so the reaction occurs as soon as analytes are incorporated in the sorbent material (for water-sensitive reagents only the HS mode can be employed) or, alternatively, by first concentrating the analytes in the fibre and then exposing the fibre to the vapour of the derivatisation reagent. In the last strategy (inport derivatisation) polar analytes, with acid-base properties, are extracted in the SPME fibre as ion pairs which are further decomposed, at the high temperatures of the GC injection port, to produce volatile by-products and the alkyl derivatives of the target compounds. The most suitable approach depends on the properties of the analytes and the derivatisation reaction to be performed. The first example considered (Table 1) is the determination of volatile compounds such as aldehydes, ketones, and thiols. Although some of these are neutral and volatile species, because of their reactivity and polarity they must be derivatised to increase their affinity for the SPME fibre and their thermal stability for GC analysis. Aldehydes and ketones can be converted into oximes by reaction with pentafluorobenzylhydroxylamine (PFBHA), either directly in the sample [43, 44] or in a SPME fibre previously loaded with PFBHA, using the HS mode [45]. Experimental results showed that the second option was faster than the first [45]. For determination of thiols, however, in-sample derivatisation is preferred and two derivatisation agents have been explored2,4-dinitrofluorobenzene (DNFB) [46], which introduces appropriate groups for ECD and NPD determination, and N-ethylmaleimide (NEM), which is regarded as being specific for thiols [47]. Whereas serious problems related to matrix effects have been encountered when using DNFB, it seems that reaction with NEM proceeds faster and without such difficulties. For species containing carboxyl groups different derivatisation approaches have been considered, depending on molecular weight, pKa values and polarity. The use of alkyl halides [48, 49] or strongly acidic conditions [5052] enables esterification of carboxyl compounds in water samples. Further SPME must then be performed in HS mode to avoid damage of the coated phase. This strategy is valid for low-molecular-weight analytes but leads to high detection limits for heavier compounds with low vapour pressures, even after derivatisation [53, 54]. This problem can be overcome by first extracting the compounds by direct exposure of the SPME fibre to the sample and then performing on-fibre derivatisation; this enables the use of moisture-sensitive reagents such as diazomethane [55, 56] and silylation agents [40, 57, 58]. In this option sample pH must be adjusted to acidic values to obtain the compounds in their neutral forms. Under these conditions the highest extraction yields are usually obtained with the PA fibre.

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Problems arise for non-volatile species with pKa values below 1 unit. For such species, addition of an ion-pairing agent to the water sample adjusted to a pH above the pKa of the analytes, then direct SPME, is the most convenient solution. The ion-pairing agent also acts as an alkylating agent in the injection port of the chromatograph [59, 60]. For less acidic analytes, e.g. phenols, both on-fibre after extraction [61] and in-sample [62, 63] derivatisation procedures can be employed. The acylation reaction used for derivatisation of these compounds in aqueous media, normally using acetic anhydride, occurs under milder conditions (compared with esterification of carboxylic acids) and, therefore, both headspace and direct SPME approaches are possible. The derivatives obtained are, moreover, more hydrophobic and volatile, which also improves their extraction yield in headspace sampling. Acylation of aliphatic alcohol groups is not possible, however, and for these analytes on-fibre silylation is preferred [39, 64]. So, for example, the on-fibre derivatisation of oestrogens with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) enables their determination at low ng L1 levels in wastewater samples (Fig. 1) [39]. The last group of compounds presented in Table 1 are basic analytes. In-sample acylation [65] or derivatisation with pentafluorobenzyl aldehyde (PFBAY) [66] are also possible for some amines, although some authors decided to investigate specifically designed reagents [22, 67, 68]. Another possibility, which has been used for the determination of amphetamine drugs, entails on-fibre derivatisation of the analytes while they are being headspace-extracted, with the particularity that an insert is used to support the derivatising agent (HFBCl-HFBA) in the HS over the sample (Fig. 2) [69, 70]. This insert contains several holes enabling the analytes to enter it. The SPME fibre is therefore exposed simultaneously to target species and vapour from the derivatisation reagent, but not to the water sample.

In-tube SPME In contrast with the successful combination of SPME and GC, tandem SPME-HPLC has been less extensively used. This is mainly attributed to two shortcomingsthe lack of a wide range of polar coatings (now partially overcome) and problems related to the commercial desorption chamber [71, 72], which often led to peak broadening. Although the design of the desorption chamber has been improved by reducing its internal volume [71], some authors concluded that the best solution is simply to perform an off-line desorption of the SPME fibre with a smaller volume of solvent, injecting only part of the extract into the HPLC system [73]. Obviously, as a result, sensitivity is reduced and automation capability is lost. Another drawback of SPME which has encouraged the development of in-tube SPME was the limited availability of LC solvent-compatible coatings. Even the robust PA polymer, employed in SPME for concentration of polar species, deteriorates when exposed to organic solvents, compromising the extraction of polar compounds. In-tube SPME overcomes this limitation by using commercially available GC capillary columns which are (thermally and chemically) resistant and available in a wide range of polarities [74]. When combined with HPLC, in-tube SPME also facilitates automation of the sample-preparation step [75]. Most forms of on-line coupling of in-tube SPME and LC are based on simple modification of the HP-1100 series autosampler (Agilent Technologies) by installing a GC capillary between the injection loop and the needle (Fig. 3). The original HPChem software can be used to operate the whole system automatically. Diffusion of the analytes from the sample to the sorbent phase is speeded up by aspirating and ejecting it several times through the capillary. In practice, however, equilibrium is almost never achieved in a reasonable time, then non-equilibrium conditions are employed. As an example, Fig. 4a shows the extraction profile obtained from five oestrogenic compounds ex-

Fig. 1 GC-MS-MS chromatogram obtained from oestrone (8 ng L1) and oestradiol (5 ng L1) in an (unspiked) wastewater effluent after SPME and on-fibre derivatisation with MSTFA. Reproduced from Ref. [39], copyright (2004), with permission from Elsevier

1452 Fig. 2 Schematic diagram of simultaneous SPME and onfibre derivatisation of amphetamine compounds. Reproduced from Ref. [70], copyright (2005), with permission from Elsevier

tracted from water; even after 20 draw/eject cycles equilibrium is not reached. The efficiency of the process cannot, furthermore, be improved by increasing the draw/ eject flow rate, because above 100 L min1 there is a reduction on the extraction yield (Fig. 4b) because of the formation of bubbles in the capillary [76]. Finally, after extraction, the compounds are desorbed by the mobile phase, or by drawing a small amount of a solvent from a different vial [75], and analysed by LC. Of commercially available GC coated capillaries, poly (ethylene glycol) coatings (Omegawax, DB-WAX, etc.) have resulted in the best extraction efficiency in the analysis of polar contaminants in aqueous samples (Table 2). Use of the adsorptive coated capillary SupelQ-PLOT (DVB polymeric material) has recently been proved to be more efficient for analysis of oestrogens, because of its large surface area, which improves massFig. 3 Schematic diagram of an in-tube SPME-LC-MS system [75]

transfer kinetics [76], and because oestrogens are of intermediate polarity. Despite the wide availability of coatings and the possibility of automation of this technique, the number of applications to analysis of polar compounds in water by intube SPME-LC is rather small (Table 2). This is probably because, normally, the sample volume considered is limited to 1 mL and the extraction efficiency to a maximum of ca. 30%. As a result, the concentration factors achieved can be regarded as appropriate for biological samples but may not be sufficient for environmental analysis. Obviously, an advantage of in-tube SPME that can be exploited is the selectivity, if performed under appropriate conditions. This is still a major problem for analysis of complex matrices such as wastewater. Even when LC-MS-MS is used as the determination technique, accuracy and detection limits of the method are seriously impaired, because of the presence

1453 Fig. 4 Effects of (a) draw/eject cycle and (b) flow rate on the in-tube SPME of oestrogens. Reprinted from Ref. [76], copyright (2005), with permission from Elsevier

of large amounts of co-extracted species which disturb the efficiency of the electrospray ionisation step [77]. This aspect has not yet been considered in published articles, which focus mostly on maximising the extraction efficiency. This objective can be achieved either by increasing the amount of sample or by increasing the coating/sample ratio. The first option is not normally adopted, because of incompatibilities with some autosampler configurations and the consequent increase in equilibration time. The second can be accomplished in several ways. The simplest is to increase the coating thickness and/or the length of the GC capillary; this, however, has the disadvantage of slower kinetics during both sorption and desorption [75]. Another possibility is the so called wire-in-tube SPME [71]. This entails introducing a narrow wire into the coated capillary so that the sample coating/sample ratio is doubled; a further development of this is to pack the capillary with filaments of a polymeric material ( fibre-in-tube) [71] or with a monolithic sorbent [78]. The last option is the most promising. It operates as a chromatographic enrichment process and not as an equilibrium technique and can be regarded, therefore, as miniaturised SPE rather than SPME. Most of the sorbents employed are quite hydrophobic and have not been tested for extraction of polar analytes. GC capillaries can also be used to perform miniaturised SPE. The analytes can then be desorbed by use of an appropriate solvent [79], or thermally [80], making coupling to GC easier. Although described as in-tube SPME [79, 80] or capillary microextraction [15, 18] by

most authors, it must not be forgotten that this is miniaturised SPE and, therefore, maximum enrichment is determined by breakthrough of the analytes and not solely by kinetic or equilibrium processes [15, 18].

Stir-bar sorptive extraction (SBSE)

SBSE was developed at the Research Institute of Chromatography (Kortrijk, Belgium) [4] and commercialised by Gerstel (Mlheim, Germany) in 1999 under the name Twister. Analytes are concentrated using a magnetic stirbar coated with PDMS which is placed in the liquid sample. This format results in a significant increase in the volume of the extraction phasefrom approximately 0.5 L for an SPME fibre (100 m PDMS) to ca 126 L for SBSE, although the stir-bars most commonly used (10 mm length, 0.5 mm coating thickness) have a PDMS volume of ca 24 L, which is still a 50-fold increase. As a consequence, the yield of the extraction process is much greater when using a stir-bar rather than an SPME fibre, both coated with PDMS. This effect is even more important for species of medium polarity than for very hydrophobic and very hydrophilic species [4]. The greater coating area of magnetic stir bars does, on the other hand, also somewhat limit the applicability of the techniquethe extraction kinetics are slower than for SPME fibres, a thermal desorption unit is necessary for transfer of the analytes from the Twister bars to the head of the GC column, and very few publications describe its coupling to

Table 2 Applications of in-tube SPME combined with HPLC for determination of polar analytes in water samples Analyte Phenylurea herbicides Phenoxy acid herbicides Carbamate pesticides Phenols, -blockers, aromatic compounds Oestrogens Organoarsenic species
a

Capillary coating Omegawax, SBP-5, PPya, PMPya DB-WAX Omegawax 250 PPya Supel-Q-PLOT PPya

Detection LC-UV, LC-MS LC-MS LC-UV, LC-UV LC-UV LC-MS-MS LC-MS

Refs. [126, 127] [128] [74, 129, 130] [31, 131] [76] [132]

Non-commercial coatings: PPy: polypyrrole; PMPy: polymethylpyrrole

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LC, because it requires relatively large solvent volumes for desorption and, obviously, on-line connection of both techniques is not possible. Because of the apolar character of PDMS, SBSE has mainly been successfully applied to the determination of medium to non-polar pesticides [81, 82]; it fails in the extraction of more polar compounds [82, 83] unless they have previously been derivatised. As in SPME, both insample acylation (normally acetylation) and in-port silylation strategies can be considered. In-sample acetylation can be used for extraction of analytes containing phenolic moieties: e.g. hydroxy-PAH [84], chlorophenols and alkylphenols [8589], and oestrogens [90]. In-port silylation has been used for determination of alkylphenols [91] by incorporating a small glass capillary tube containing the derivatising agent (0.5 L BSTFA) with the PDMS-coated stir bar in the desorption chamber. The advantage of this reaction over acylation is that in-port silylation can be used to derivatise a wider range of functional groups and not only phenol moieties; acylation, on the other hand, enables the Kow of the analyte to be increased, improving the extraction yield. Both approaches can be combined to improve sensitivity in the determination of compounds containing aromatic hydroxyl and other polar groups which react with silylation reagents. This has been applied to the determination of oestradiol [92]after acetylation of the phenolic moiety the affinity of the hormone for the PDMS polymer increases; silylation of the aliphatic hydroxyl during desorption of the stir bar further improves the peak shape and thus the minimum amount detectable by GC-MS. In-sample derivatisation of polar analytes to produce more hydrophobic species is not always possible, however, and for the most polar compounds extraction is still difficult with PDMS-coated stir bars [82, 83]. To overcome this limitation a new dual-phase stir bar has been produced and commercialized by Gerstel. It consists of an empty PDMS tube (1 cm long, 0.5-1 mm thick) which can be filled with the desired sorbent (Fig. 5). Only carbonaceous materials have yet been tested as filling sorbents. The results obtained revealed important enhancement of the extraction yield for polar compounds (flavour compounds and pesticides) compared with those achieved using the empty PDMS tube [93]. Although additional studies are still necessary to assess the durability of this device, cost of extraction, and potential carry-over problems for less volatile species, because of the high retention capacity of carbonaceous sorbents, it is expected this dual-phase

configuration will help to increase the range of applications of the SBSE technique. An interesting alternative to commercial stir bars (i.e. Twister), based on the use of disposable PDMS-coated rods, has been proposed by Montero, Popp and coworkers [94, 95]. These rods have the advantage that they can be cut to the desired length in the laboratory. These rods are available in diameters from 1 to 5 mm. This determines the ratio sorbent volume/sorbent amount and thus the kinetics of the extraction process. Because they are very inexpensive, they can be discarded after each use, reducing the possibility of cross-contamination between samples and the need for cleaning. Although such rods have been used for determination of hydrophobic analytes only (PCB, chlorobenzenes, and PAH), it seems reasonable that a similar strategy could be employed for more polar compounds by selection of more polar materials.

Liquid-phase and membrane microextraction

The last technique considered in this review is liquid-phase microextraction (LPME), which is the result of applying the principles of SPME, i.e. reduced organic solvent consumption and equilibrium extraction, to LLE. There are, in practice, several means of achieving these objectives, depending on whether the extracting solution is directly in contact with the sample or separated by a polymeric membrane which can be held in different configurations. In LPME, furthermore, two-phase and three-phase systems are possible; the latter can be regarded as a micro liquid-liquid extraction-back-extraction system which exploits the acid-base character of the analytes to achieve simultaneous enrichment and clean-up. The most representative configurations are presented in Fig. 6. More details of their theoretical aspects and general applications can be found in recent reviews focussing on the different modalities of LPMEsingle drop microextraction (SDME) [6], membrane-based extractions [8, 9], and hollow-fibre liquid-phase microextraction (HFLPME) [5, 7]. Applications of LPME to the extraction of polar analytes from water samples are summarized in Tables 3 and 4; the applications are discussed below, where two-phase and three-phase systems are treated separately. Two-phase systems Applications of two-phase LPME in water analysis are normally restricted to medium polarity and non-polar analytes and those whose polarities can be reduced before extraction. The reason is simplethe extracting solvent or mixture (acceptor solution) cannot be miscible with water. As a consequence, two-phase LPME is best suited to GC analysis (Table 3) in contrast with three-phase systems which are often employed in combination with LC or CE (Table 4). The simplest way to perform two-phase LPME is the single-drop microextraction mode (SDME). In this ap-

Fig. 5 Dual-phase stir bar for SBSE. Reprinted from Ref. [93], copyright (2005), with permission from Elsevier

1455

Fig. 6 Different liquid-phase microextraction systems: (a) twophase single-drop microextraction (2P-SDME), from Ref. [6], copyright (2002), with permission from Elsevier; (b) three-phase single-drop microextraction (3P-SDME), from Ref. [114], copyright (2002), with permission from Elsevier; (c) hollow-fibre liquid-phase microextraction (HF-LPME) in the rod configuration, from Ref.

[139], copyright (2001), with permission from Elsevier; (d) hollowfibre liquid-phase microextraction (HF-LPME) in the U-shape configuration, from Ref. [142], copyright (2000) with permission from Wiley-VCH and authors; and (e) flat-membrane microextraction (MMLLE and SLME), from Ref. [119], copyright (2002), with permission from Elsevier

proach, analytes are extracted from the stirred aqueous sample into a drop of organic solvent (ca 1-3 L) suspended from the needle of a microsyringe (Fig. 6a). After a given time the drop is retracted into the syringe and injected into the chromatographic system for analysis. Toluene is the solvent most often used, because it is has some polar character and very low water solubility [96], Table 3. Mixtures of CH2Cl2 and CCl4 have also been

successfully applied to the extraction of organophosphorus compounds [97]; these solutions are, however, more prone to dissolve or become dislodged when long extraction periods are used. A final group of solvents with much potential in SDME are ionic liquids. These have mainly been used for the extraction of PAH, alkylphenolic compounds, and chloroanilines, before LC determination [98100]. As discussed in the section on SPME, their

1456 Table 3 Applications of two-phase LPME and membrane microextraction to the determination polar species in water samples Analyte Nitroaromatic explosives Techniquea E.F.b Acceptor phase N.R. N.R. N.R. N.R. N.R. 33 213 100 163 190 163 N.R. 146 528 415 N.R. 208 42 250 Toluene Toluene Toluene and n-hexane Toluene CCl2H2:CCl4 (3:1) Toluene Toluene 1-Octanol Toluene 1-Octanol [C6MIM][PF6] Ethyl acetate Hexyl acetate NaOH 1 mol L1 1-Octanol [C4MIM][PF6] Toluene Toluene Chlorobutane (10% heptanoic acid) Detection GC-MS GC-MS LC-UV, GC-MS GC-FTD GC-MS GC-ECD GC-ECD LC-UV GC-MS GC-MS LC-FLD GC-MS GC-MS CE GC-MS LC-UV GC-MS GC-MS LC-UV Refs. [133] [134] [102, 135, 136] [104] [97] [96] [105, 106] [119, 137] [108, 138] [138] [99] [108, 111] [109] [101] [138] [100] [103, 110] [103] [107]

2P-SDME 2P-HF-LPME Organophosphorus and carbamate pesticides 2P-SDME 2P-HF-LPME Organophosphorus warfare agents 2P-SDME Antifouling biocides 2P-SDME Fungicides 2P-HF-LPME MMLLE Alkylphenols 2P-HF-LPME 2P-HF-LPME 2P-SDME Phenols 2P-HF-LPME 2P-SDME HS-SDME Acidic pharmaceuticals 2P-HF-LPME Aromatic amines HS-SDME Triazine herbicides 2P-HF-LPME 2P-SDME Cationic surfactants MMLLE

N.R., not reported; [C6MIM][PF6], 1-hexyl-3-methylimidazolium hexafluorophosphate; [C4MIM][PF6], 1-butyl-3-methylimidazolium hexafluorophosphate a 2P, two-phase; HS, headspace; SDME, single-drop microextraction; HF-LPME, hollow-fibre liquid-phase microextraction; MMLLE, microporous membrane liquid-liquid extraction b Enrichment factor: ratio between analyte concentration in the acceptor solution and its initial concentration in the sample. The maximum value for the group of analytes considered in each application is reported

Table 4 Applications of three-phase LPME and membrane microextraction to the determination polar species in water samples Analyte Fungicides Phenols Techniquea E.F.b 67 400 100 300 234 15,000d 100 490 60 60 3,000 6,000d 500 250 400 390 140 N.R. Organic solventc Detection Refs. [119, 137] [139141] [113, 115] [115] [112, 122, 142] [122] [142] [143] [120] [117] [118] [123, 144] [144] [114] [114, 116] [145, 146] [146] [121]

SLME 3P-HF-LPME 3P-SDME 3P-SDME Acidic pharmaceuticals 3P-HF-LPME 3P-HF-LPME 3P-HF-LPME Phenoxy herbicides 3P-HF-LPME Glyphosate metabolites SLME Haloacetic acids SLME 3P-HF-LPME Aromatic amines 3P-HF-LPME 3P-HF-LPME 3P-SDME 3P-SDME Triazine herbicides SLME SLME Bipyridilium herbicides SLME

DHE with 15% TOPO LC-UV 1-Octanol LC-UV, MEKC Hexane LC-UV Heptane-toluene (1:1) LC-UV 1-Octanol LC-MS-MS 1-Octanol LC-UV DHE CE 1-Octanol LC-UV DHE doped with methyltrioctylammonium chloride CE DHE (5% TOPO) LC-UV DHE doped with DEHP LC-UV Benzyl alcohol-ethyl acetate (8:2) LC-UV DHE LC-UV DHE LC-UV Benzyl alcohol-ethyl acetate (2:1) LC-UV DHE LC-UV DHE doped with TOPO LC-UV DHE doped with DEHP LC-UV

DHE, dihexyl ether; TOPO, trioctyl phosphine oxide; DEHP, di(2-ethylhexyl) phosphoric acid a 3P, three-phase; HF-LPME, hollow-fibre liquid-phase microextraction; SLME, supported-liquid membrane extraction b Enrichment factor: ratio between analyte concentration in the acceptor solution and its initial concentration in the sample c Solvent impregnating the membrane pores d Values achieved using multiple LPME

1457

ability to extract more polar analytes may be tuned by selecting the appropriate anions and cations. Ionic liquids have high viscosity and are virtually non-volatile; stable and larger drops can therefore be used, increasing the yield of the extraction. These liquids can, moreover, be used in the headspace mode, with heating of the sample above ambient temperature [100]. Aqueous solutions of sodium hydroxide have also be used for the headspace liquid microextraction (HS-SDME) of semi-volatile phenols from acidified water samples; enrichment factors in excess of 500 have been achieved for some analytes [101]. Although applications of HS-SDME have been included in Table 3, this mode could, theoretically, also be regarded as a threephase system in which volatility differences between the analyte and the other matrix constituents are exploited to achieve a more selective enrichment. Obviously, some drawbacks of LLE have been inherited by SDME. One is the possibility of emulsion formation when dealing with dirty samples, which in SDME would result in drop dissolution. Drop stability is limited and the process requires a dedicated operator, although dynamic automated systems have been proposed [102]. One way of minimizing some of these problems is to use a polymeric membrane which serves as a support for the extracting solvent, enabling the use of larger volumes than in SDME, and a physical barrier between the phases. The membrane is usually made of a porous hydrophobic material (normally polypropylene). In practice, two approaches can be used to perform membrane-based microextractions. One is the use of hollow-fibre (HF) membranes, either in a rod like (Fig. 6c) or U-shape (Fig. 6d) configuration; the other is based on use of a flat membrane (Fig. 6e) to separate the sample (donor) and extracting (acceptor) solutions. The latter is normally referred to as microporous membrane liquid-liquid extraction (MMLLE). Both formats have advantages and limitations. HF-LPME employs inexpensive membranes which are normally discarded after use; automation of the extraction step is difficult, however [5, 7]. In MMLLE applications the membrane is normally reused, with a consequent risk of cross-contamination. This format of LPME is best suited to on-line hyphenation with chromatographic techniques [8, 9]. Direct comparison of HF-LPME and SDME for determination of triazine herbicides [103] showed that HFLPME resulted in higher enrichment factors (Table 3) and cleaner extracts and is also less prone to matrix effects [104] than SDME. This is attributed to the membrane pore structure, which reduces the concentration of high-molecular-weight compounds in the sample extract. As with SDME, applications of two-phase HF-LPME and MMLLE are still limited to medium-polarity compounds and those containing ionisable groups, for example phenols, triazine herbicides, etc. For such compounds, adjustment of sample pH and ionic strength to reduce the solubility of the analytes are expected to improve the yield of the extraction. These effects should be carefully investigated for each group of analytes, because reduction

of the kinetics of the extraction at high ionic strengths and hydrolysis of some compounds at extreme pH might reduce the efficiency of the process [104106]. An alternative, for extraction of ionic species is to form an ion-pair. This strategy, with heptanoic acid as ion-pairing reagent, has been used to extract cationic surfactants by MMLLE [107]. Similar to SPME and SBSE, in-port derivatisation and insyringe derivatisation can also be combined with LPME [108] and SDME [109], respectively, to improve GC determination of some phenolic compounds. Most applications of membrane LPME have been developed using polymers designed for industrial applications; these have to be cut and, sometimes, sealed in the laboratory, often resulting in poor reproducibility. As an alternative, the company Gerstel (Mlheim, Germany) in collaboration with the Environmental Research Centre (UFZ, Leipzig, Germany) has recently commercialised a set of membranes designed for analytical purposes, which can be used in automated operation (MASE). The main difference between this membrane and the hollow fibres employed in HF-LPME is that the MASE membranes are non-porous whereas those normally used in HF-LPME (Accurel Q3/2 from Membrana, Wuppertal, Germany) have a pore size of 0.2 m. MASE is, therefore, a threephase system in which the membrane acts as the real interface. To speed up the mass-transfer kinetics the thickness of the MASE membrane is reduced to 30 m (compared with 200 m for that from Accurel) and the extraction temperature is increased to about 40C [110, 111]. Another difference is that MASE membranes are designed to accommodate up to 1 mL solvent (compared with ca. 10-20 L in HF-LPME using porous membranes); large-volume injection is, therefore, often needed to reduce detection limits. Three-phase systems Three-phase LPME entails extraction of analytes from an aqueous sample to an organic solvent and simultaneous back-extraction from this to the acceptor solution, usually a few microlitres of water at the appropriate pH. The organic solvent is therefore an interface between both aqueous solutions. Simultaneous enrichment and clean-up can be achieved by exploiting the acid-base properties of the analytes, i.e. for acidic analytes the sample is adjusted to a pH below the pKa of the compounds to obtain the neutral species, which are extracted to the organic solvent, and the acceptor solution to a pH above their pKa, so the analytes can then be converted into the more hydrophilic ionic species and trapped in the aqueous extracting solution. In contrast, if the target species is basic in character, the sample is made basic and the acceptor solution is acidic. The result is enhancement of extraction selectivity, an extremely useful means of eliminating interfering compounds when non-selective detectors (for example UV) are employed and of reducing matrix effects during LC-MS analysis of complicated environmental samples, for

1458

example wastewater [112]. As the acceptor solution is aqueous, three-phase LPME is preferably combined with LC and electrodriven separations than to GC. Most significant applications of three-phase LPME for the determination of polar species are summarised in Table 4. As with two-phase LPME, different working modes can be considered. In three-phase SDME, a layer of organic solvent is placed between the sample and the acceptor solution, in direct contact with both. Its volume should be minimised, because it competes with the acceptor solution for the analytes. This can be achieved by using a Teflon ring (Fig. 6b) [113, 114] or a small volumetric flask [115, 116]. This format has been applied to the determination of phenols [113, 115] and aromatic amines [114, 116]. For the first of these non-polar solvents (e.g. hexane, toluene) can be used whereas amines require more polar solvents (an ether, ester, or alcohol). Use of these solvents is, however, more problematic, because they are partially soluble in water and quite volatile and, as a consequence, if long extraction times are used the enrichment factors obtained decrease and precision worsens [114].

Use of porous membranes which contain a trapped organic solvent to separate the sample and acceptor solutions seems a more robust configuration for LPME in three-phase systems. Hollow fibres (three-phase HFLPME) or flat membranes in flow systems, the latter known as supported-liquid membrane extraction (SLME), can be used. The set up of both formats is the same as in a two-phase system (Fig. 6ce) with the difference that the membrane pores have been previously impregnated with the organic solvent. The most commonly used solvents are 1-octanol and dihexyl ether (DHE), because these are relatively polar with low water solubility and volatility and form an immobilised organic phase stable for a relatively long time. For very polar analytes, diffusion through the organic membrane can be enhanced by doping the organic solvent with several modifiers. Thus, trioctylphosphine oxide (TOPO), a strong H-bonding agent, has been used to promote the extractability of haloacetic acids [117, 118] and some fungicide metabolites [119]. For extraction of very basic/cationic and very acidic/anionic analytes an ionpair reagent (also named a carrier) can also be employed, for example methyltrioctylammonium chloride for acidic/

Table 5 Comparison of SPE and several microextraction strategies for determination of acidic pharmaceuticals, oestrogens, and phenols in water samples Extractiona Determination Sample volume (mL) Processing time (min)b Derivatisationc Acidic pharmaceuticals SPE GC-MS SPME GC-MS 2P-LPME GC-MS SPE LC-MS-MS 3P-LPME LC-MS-MS Oestrogens SPE GC-MS-MS SPME GC-MS-MS SBSE GC-MS-MS Matrix effectsd LOD/LOQ (ng L1) Ref.

500 22 5 50 22 2,000 100 50 1,000 1 500 12 10 5 3

90120 60 60 ca 60 45 ca 180 90 240 ca 120 min ca 5 60 30 120 30 20

MTBSTFA On-fibre MTBSTFA No No No MSTFA On-fibre MSTFA In-sample acetylation No No In-sample acetylation In-sample acetylation In-sample acetylation In-port BSTFA In-syringe BSA

No Moderate N.R. No No No Moderate to strong No No Moderate N.R. No No No N.R.

1050 1240 2040 0.86.5 0.542 13 0.23 15 12 312 110 161 110 516 461

[147] [40] [138] [148] [112] [149] [39] [90] [150] [76] [151] [63] [88] [108] [109]

SPE LC-MS-MS IT-SPME LC-MS-MS Phenols SPE GC-MS SPME SBSE GC-MS GC-MS

2P-LPME GC-MS 2P-SDME GC-MS

a

SPE, solid-phase extraction; SPME, solid-phase microextraction; 2P-LPME, two-phase liquid-phase microextraction; 3P-LPME, threephase liquid-phase microextraction; SBSE, stir-bar sorptive extraction; IT-SPME, in-tube solid-phase microextraction; 2P-SDME, two-phase single-drop microextraction b Approximate processing time per sample, including evaporation, derivatisation, clean-up, etc., when needed c MTBSTFA, N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; BSTFA, bis-(trimethylsilyl)trifluoroacetamide; BSA, bis-(trimethylsilyl)acetamide d Matrix effects affecting sample preparation (not determination). N.R., not reported

1459

anionic analytes [120] or diethylhexyl phosphate for cationic/basic ones [121]. The ion pairs formed are extracted into the organic interface and broken by selecting the appropriate pH in the acceptor solution, which releases the free analytes. As shown in Table 4, enrichment factors (defined as the ratio of the analyte concentrations in the acceptor solution and in the sample) up to several hundred can be achieved by use of three-phase LPME. For some compounds these values can be improved substantially by performing a twostep LPME [122, 123]. This is achieved by using the aqueous acceptor solution from the first extraction as the donor phase in the second one, after appropriate pH readjustment (acidification for acidic species).

the matrix effects that complicate quantification by LCMS. Currently, however, its main shortcoming is the use of laboratory-designed extraction devices, which require skilled operators, increase the variability of the results, and slow the spread of the technique outside research laboratories. It is believed that commercialisation of the technique, in a limited number of formats, will overcome such problems.
Acknowledgements Financial support by the Spanish DGICT Ministerio de Educacin y Ciencia (project BQU 2003-02090) and Xunta de Galicia (project PGIDIT03TAM02E) is acknowledged. J.B. Quintana is grateful to Ministerio de Educacin y Ciencia for his postdoctoral grant.

Microextraction strategies comparison and conclusions

As it has been shown throughout this review, the possibility of using microextraction techniques for analysis of polar organic contaminants in aqueous samples has been increased substantially in recent years. As an example, Table 5 compares some characteristics of different microextraction and SPE methods, using GC or LC detection, for determination of three groups of polar compounds (acidic pharmaceuticals, oestrogens, and phenols) in water samples. The first point to notice is that microextraction techniques normally achieve an important decrease not only in organic waste, but also in sample and time consumption. This is very important in the determination of oestrogens, which normally require extraction of very large samples and several steps, including clean-up and solvent evaporation at several stages when SPE is selected as the concentration technique. All the different microextraction techniques available seem to result in similar detection limits. SPME and SDME are the most sensitive techniques toward matrix effects. Although derivatisation of polar analytes can be performed in combination with most microextraction strategies, possibilities are greater with SPME and, therefore, this is often the technique of choice if the analytes are to be detected by GC. Although use of derivatisation reactions improves the extractability of some analytes, the development of new coatings is expected to enhance the applicability of SPME to the determination of polar species. Sol-gel polymerisation seems to be the most promising method for immobilisation of polar sorbents on microextraction fibres. The coated phases obtained can be rinsed with polar solvents and are more stable than those on commercial fibres. They are, therefore, suitable for use in combination with both GC and LC. Lack of alternative coatings to PDMS is clearly limiting the applicability of SBSE to polar compounds. If analytes are to be detected by LC or CE, the most promising technique seems to be three-phase LPME using porous membranes impregnated with the appropriate solvent. This enables simultaneous enrichment and cleanup, important not only for non-selective detection (e.g. UV) but also for MS detection, because it may help to minimise

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