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J. Sep. Sci.

2009, 32, 813 824

F. M. Lancas et al.

813

Fernando M. Lancas1 Maria Eugnia C. Queiroz2 Paula Grossi1 Igor R. B. Olivares1


1

Review Recent developments and applications of stir bar sorptive extraction


The theoretical aspects of stir bar sorptive extraction (SBSE), as well as the recent applications of this technique in pharmaceutical, biomedical, environmental, and food analysis, and recently developed new coating procedures are reviewed. A general overview of the important factors to be evaluated in the optimization of extraction efficiency such as extraction time, matrix pH, ionic strength, effect of organic additives, temperature, agitation, pre-extraction derivatization reactions, influence of proteins, and desorption conditions are discussed. An impressive number of applications using SBSE have been published in different areas including biological, environmental, and food, showing the advantages of this technique over the classical extraction techniques (liquid-liquid extraction (LLE), Soxhlet). Although the different SBSE applications use PDMS phase as sorbent, developments of new phases are necessary for specific applications. In this review, recent SBSE developments are shown with a focus on the development of new instrumental approaches and sorbent phases.
Keywords: Miniaturized extraction / PDMS / Sample preparation / SBSE / Received: November 20, 2008; revised: January 14, 2009; accepted: January 14, 2009 DOI 10.1002/jssc.200800669

Institute of Chemistry at So Carlos/USP, University of So Paulo, So Carlos, SP, Brazil 2 Departamento de Qumica, Faculdade de Filosofia Cincias e Letras de Ribeiro Preto, Universidade de So Paulo, SP, Brazil

1 Introduction
Modern trends in analytical chemistry are toward the simplification, miniaturization of sample preparation, and minimization of organic solvent and sample volumes. In particular, the reduction of solvent consumption in analytical laboratories is expected to contribute significantly to the reduction of analytical cost. New solventless sample-enrichment techniques that allow the direct extraction of solutes from sample have been introduced, such as solid-phase microextraction (SPME), in-tube SPME, and stir bar sorptive extraction (SBSE). These techniques combine extraction and concentration of the solute in a single step, thereby reducing the time required to prepare the samples.

Correspondence: Prof. F. M. Lancas, Institute of Chemistry at So Carlos/USP, University of So Paulo, P.O. Box 780, 13560-970 Av. Trabalhador Socarlense, 400 So Carlos, SP, Brazil E-mail: flancas@iqsc.usp.br Fax: +55-16-33739984 Abbreviations: BP, benzophenone; HS, headspace; IVM, ivermectine; LD, liquid desorption; OCP, organochlorine pesticides; PAH, polycyclic aromatic hydrocarbon; PDMS, polydimethylsiloxane; PPY, polypyrrole; PTV, programmable temperature vaporization; PU, polyurethane; RAM, restricted access material; RSE, refrigerated sorptive extraction; SBSE, stir bar sorptive extraction; SPME, solid-phase microextraction; TD, thermal desorption

Microextraction is usually defined as an extraction technique where the volume of the extracting phase is very small in relation to the volume of the sample; in most cases the extraction is not exhaustive with only a small fraction of the initial solute being extracted for further analysis. The extraction efficiency is determined by the solute partitioning between the sample matrix and the extraction phase. The higher the affinity the solute has for the extraction phase relative to the sample matrix, the higher the amount of solute extracted. Partition is controlled by the physicochemical properties of the solute, the sample matrix, and the extraction phase [1]. SBSE was introduced in 1999 by Baltussen et al. [2]. This sorptive extraction technique is based on the same principles than SPME, i.e., partitioning of solute between the sample matrix and the extraction phase. However, instead of a polymer-coated fiber, stir bars of 10 20 mm coated with 25 125 lL (0.3 1.0 mm layer) PDMS, are used for enrichment of organic compounds from aqueous matrices. A magnetic rod is usually encapsulated in a glass jacket on which a PDMS coating is placed (Fig. 1). Owing to the specific PDMS characteristics, superior extraction performance is encountered for the following reasons. First, analytes are partitioned, or sorbed, into the bulk of the PDMS phase. The sorption is a much weaker process than adsorption, so compounds can be desorbed at lower temperatures, thus minimizing the losses of thermolabile solutes. Second, the retaining
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Figure 1. SBSE stir bar. 1, Magnetic rod; 2, glass jacket; 3, PDMS coating.

capacity of PDMS for a certain compound is not influenced by the presence of high amounts of water or other analytes, since all solutes have their own partitioning equilibrium into the PDMS phase, and displacement does not occur. Third, degradation fragments of PDMS sorbent all contain characteristic silicone mass fragments which can easily be discerned with the use of mass selective detector [2]. The extraction of solutes from the aqueous phase into the extraction medium is controlled by the partition coefficient of the solutes between the PDMS and the aqueous phase. This partitioning coefficient has been correlated with the octanol water distribution coefficients (Ko/w). Although not fully correct, the octanol water distribution coefficient gives a good indication if and how well a given solute can be extracted with SBSE [3]. The distribution coefficient between PDMS and aqueous phase (KPDMS/w) is defined as the ratio between the concentration of a solute in PDMS (CPDMS) over the concentration in water (Cw) at equilibrium. This ratio is equal to the ratio of the mass of the solute in the PDMS (mPDMS) over the mass of the solute in the aqueous phase (mw) times the phase ratio (b with b, Vw/VPDMS) [3] (Eq. 1) Ko=w L KPDMS=w CPDMS mPDMS Cw mw 1

Vw mPDMS b VPDMS mw

The recovery, expressed as the ratio of the extracted amount of solute (mPDMS) over the original amount of solute in aqueous phase (mo = mw + mPDMS), is dependent upon the distribution coefficient KPDMS/w and on b, as described in Eq. (2) KPDMS=w =b mPDMS 1 KPDMS=w =b mo 2

From Eq. (2), it is important to note that only two terms affect the recovery of an analyte, KPDMS/w and b. The higher the PDMS amount, the lower b and the higher extraction efficiency [3].

In SPME, the maximum volume of PDMS coated on to the fiber is ca. 0.5 lL (film thickness 100 lm). For a typical sample volume of 10 mL the phase ratio is 26104, implying that quantitative extraction is obtained only for compounds for which Ko/w A 105. In SBSE, on the other hand, 25 125 lL PDMS coating are used; the situation is much more favorable. A stir bar coated with 100 lL PDMS can easily be used to extract 10 mL of aqueous sample, leading to a b value equal to 100, which implies that solutes with a Ko/w in excess of 500 are quantitatively extracted in a PDMS-coated stir bar. This not only renders quantification straightforward, but also ensures significantly better sensitivity (increase by a factor of 50 250) for compounds for which Ko/w a 105 [2, 4]. SBSE of a liquid sample is performed by placing a suitable amount of sample in a headspace (HS) vial or other container. The stir bar is added and the sample is stirred until the partition equilibrium time is reached. The extraction time is kinetically controlled and determined by the sample volume, stirring rate, temperature, and stir bar dimensions and must be optimized for a given application. After extraction, the stir bar is easily removed with forceps, rinsed with purified water to remove adsorbed sugars, proteins, or other sample components, and dried with lint-free tissue. According to David and Sandra [5] rinsing does not cause solute loss, because the sorbed solutes are present inside the PDMS phase. For HS sampling, the stir bar can be placed in a liquid or solid sample; special devices to hold the stir bar are available [5]. Desorption of the solute from the bar may be done by either heating or back extraction with a small volume of a liquid solvent. When SBSE is combined with GC, thermal desorption (TDS) is the preferred way once the bar is inserted in the heated GC injection and the analytes desorbed to the column for further analysis. Liquid desorption (LD) can be combined with both GC and LC. The stir bar is placed in a small vial, and the desorption can be performed by adding either few microliters of a proper solvent (GC) or the mobile phase (LC). LD methodologies showed high sensitivity and enough reproducibility to permit the quantification of antidepressants [6], and anticonvulsants in human plasma samples from patients receiving therapeutic dosages [7]. Special TDS systems consist of two programmable temperature vaporization (PTV) injectors in series [5]. The first PTV injector (TDS-2) is the unit in which the stir bar is thermally desorbed. The second PTV injector is a CIS-4 used as a cryotrap for cryogenic refocusing of the thermally desorbed solutes. During fast TDS (flows up to 100 mL/min are recommended), the CIS-4 is kept at negative temperatures (as low as 1008C) with liquid nitrogen. In order to efficiently cryotrap even the most volatile solutes, it is necessary to pack the CIS-4 liner. After the TDS program is complete, the temperature of the CIS-4 is increased fast to high temperature. The solutes transfer from the TDS-2 to
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the CIS-4 and from the cryotrap onto the column is usually performed in the splitless mode. Typical carry-over values below 1% are reported [5, 8]. SBSE in combination with TDS-capillary GC provides a very versatile tool for the analysis of organic solutes in different fluids. Biological samples are characterized by a diversity of organic bioactive compounds that often contain polar groups. Derivatization is the classical way to convert the analytes into GC-amenable compounds. The area of application of SBSE has been extended by using both pre-extraction derivatization reactions and in situ derivatization, typically with ethyl chloroformate and acetic acid anhydride as esterification and acetylation reagents, respectively [9]. Kawaguchi et al. [10] developed a high-sensitivity analytical method that uses SBSE with in situ derivatization and TD-GC-MS for the simultaneous measurement of trace amounts of phenolic xenoestrogens, such as 2,4dichlorophenol, 4-tert-butylphenol, 4-tert-octylphenol, 4nonylphenol technical isomers, pentachlorophenol, and bisphenol A, in human urine samples. The urine sample was de-conjugated by adding b-glucuronidase and sulfatase. Then, protein precipitation was performed by the addition of ACN. After centrifugation, the supernatant was diluted with purified water and subjected to SBSE with in situ derivatization and TD-GC-MS [10]. TD with in-tube derivatization was also developed. After extraction, the stir bar and a glass capillary tube filled with silylation agents are placed inside a glass TD, in which the target compound is derivatized during TD from PDMS-coated stir bar [11]. The SBSE variables, such as time, temperature, pH matrix, ionic strength, and desorption conditions have been optimized to reach solute partition equilibrium in shorter analyses time, and to obtain adequate analytical sensitivity. The sample volume, stirring speed, and stir bar dimensions should be maintained constant during the optimization. Matrix pH can be adjusted to optimize the SBSE of either acidic or basic solutes. This is related to the fact that, unless ion exchange coating is used, PDMS-SBSE can extract only neutral (nonionic) species from matrix. By properly adjusting the pH, weak acids and bases can be converted to their neutral forms, in which they can be extracted by the PDMS coating. Increasing the extraction temperature, the distribution constant of solute between the coating and the extraction mixture decreases; however, it may also increase the diffusion by lowering the viscosity, which shortens the equilibrium extraction time. Consequently, SBSE methods can be optimized by selecting the proper extraction temperature, where satisfactory sensitivity is achieved in an acceptable time period. Queiroz and coworkers [6] described that the sensitivity of SBSE/LC-UV can be improved by diluting the plasma

samples with the buffer solution, in the pH of which the drugs were partially or totally in the nonionic form. The sample dilution also favors the stirring SBSE process. The authors also observed that the addition of NaCl, increasing the ionic strength, reduced the amount extracted for some solutes; however for others, it did not alter the efficiency of the SBSE process. Probably the salt itself interacted with the drugs in solution through electrostatic, or ion-pairing interactions, thus reducing the ability of the drugs to move to the SBSE coating [6]. The life-time of a single stir bar is 20 to more than 50 extractions, depending on the matrix, and the analyst's care.

2 Recent applications
After a historical publication about SBSE in 1999 [2], discussing the theory and principles, different applications were developed for environmental, food, and biological analysis. A recent review shows extensive SBSE applications [5] in different areas. Recent applications published after this review were searched and are discussed below. Searching about SBSE applications since its initial development, it is possible to find around 236 papers; in the last 5 years around 180 papers; in the last 2 years 49, being 21 in biological, 20 in environmental, and 8 in food analysis applications (Fig. 2).

2.1 Recent applications of SBSE for biological samples


An overview of SBSE applications in biological samples is given in Table 1. Most of the described methods (Table 1) showed high chromatographic selectivity, linearity, precision, and high sensitivity, well in line with the international criteria for validation procedures in order to attend the required purposes, such as therapeutic drug monitoring, clinical toxicology, forensic toxicology, social toxicology, bioavailability, and pharmacokinetics.

2.2 Recent applications of SBSE for environmental analysis


A recent review about SBSE applications [5] shows 39 references about the applications of this technique in environmental analysis principally on traditional environmental pollutants such as pesticides, polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOCs), and others, in water samples. A recent search about environmental SBSE application shows that beyond traditional environmental pollutants, other specific pollutants have been investigated (Table 2). An example describing new SBSE environmental applications is the analysis of benzophenone (BP) (also known
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Figure 2. SBSE published papers (obtained from a search of the keyword SBSE in http://apps.isiknowledge.com).

Table 1. SBSE applications in biological analysis Analyte PCBs Barbiturates Phthalates, metabolics Steroids, drugs Pharmaceuticals Drugs of abuse Phenols Phenols Phenols Caffeine and metabolites Chlorophenols Tuberculostearic acid VOCs (aldehydes, ketones) Pesticides Phenols (xenoestrogens) 4-Hydroxynonenal (oxidative stress marker) Estrone, estradiol Antidepressants Steroid sex hormones Fluoxetine Anticonvulsants Matrix (volume) Sperm (1 mL) Urine (5 mL) Body fluids, infusates (5 mL) Biological fluids (5 mL) Urine (5 mL) Analytical system (LOD) GC-MS (SIM) 0.1 pg/mL GC-MS (SIM) 12 pg/mL GC-MS (SIM) GC-MS GC-MS (scan) a 1 ng/mL Remarks TD, add 9 mL water methanol TD TD TD, hydrolysis, in situ derivatization, acetic anhydride/ethyl chloroformate TD, in situ derivatization, acetic anhydride/ethyl chloroformate TD, hydrolysis, in situ derivatization, acetic anhydride/ethyl chloroformate TD TD, in situ derivatization, acetic anhydride TD, in situ derivatization, acetic anhydride LD, RAM sorbent TD, in situ derivatization, acetic anhydride TD, in situ derivatization, ethyl chloroformate HSSE TD TD, in situ derivatization, acetic anhydride TD, in situ derivatization, PFBHA, acetic anhydride TD, hydrolysis, in situ derivatization, acetic anhydride LD, therapeutic drug monitoring LD, SBSEM sorbent TD and LD, in situ derivatization, acetic anhydride/ethyl chloroformate LD, therapeutic drug monitoring Ref. [12] [13] [14] [9] [15] [16] [17] [18] [18] [14] [20] [21] [22] [23] [24] [25] [10] [6] [26] [27] [7]

Blood, urine, bile (5 mL) GC-MS a 5 lg/L Urine, plasma (1 mL) Urine (1 mL), saliva (500 lL) Plasma (200 lL) Plasma (0.8 mL) Urine (2 mL) Sputum (0.5 mL) GC-MS (SIM) 0.4 4 ng/L GC-MS (SIM) 20 pg/mL GC-MS (SIM) 100 pg/mL LC-UV 25 ng/mL GC-MS (SIM) 10 20 ng/L GC-MS (SIM) 0.2 ng/mL

Urine (animal) (0.5 mL), GC-MS (scan) gland excretion (5 mg) Breast milk GC-MS Urine (1 mL) GC-MS (SIM) 10 50 pg/mL Urine (1 mL) Urine (1 mL) Plasma (1 mL) Urine Plasma (1 mL) Plasma (1 mL) GC-MS (SIM) 22 pg/mL GC-MS (SIM) 20 30 pg/mL LC-UV, LOQ: 10 40 ng/mL LC-DAD 0.062 0.38 ng/mL GC-MS (SIM) 0.46 pg/mL (TD) 10.0 pg/mL (LOD) LC-UV, LOQ: therapeutic interval

PCBs, polychlorinated biphenyls; RAM, restricted access material; PFBHA, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine; TD, thermal desorption; LD, liquid desorption; SBSEM, poly(methacrylic acid stearyl ester-ethylene dimethacrylate).

as diphenylmethanone) sunscreen compounds. These compounds are widely used as UV absorbers and fragrance retention agents in the manufacture of cosmetics and pharmaceuticals. Some studies about these compounds have revealed the estrogenic activity of BPs. The

effect of BPs on the ecosystem is also a cause for grave concern. Considering the low concentration and the importance to evaluate the presence and concentration of these compounds in the environment, a method for the simultaneous measurement of BP sunscreen comwww.jss-journal.com

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Table 2. Recent applications of SBSE for environmental analysis Analyte (matrix) 13 OCPs (water samples) Extraction mode (fiber) HS (PDMS) Analytical system (LOQ or LOD) Remarks Ref. [28]

GC-MS (LOD: 0.01 1.59 ng/g) LD GC MS (LOD: 0.5 2 ng/L)

DI (PDMS) BP sunscreen compounds and its derivatives: 2,4-dihydroxybenzophenone (BP-1); 2-hydroxy-4-methoxybenzophenone (BP-3); 2-hydroxy-4-methoxy-49-methylbenzophenone (BP-10); 2hydroxybenzophenone (2OH-BP); 3-hydroxybenzophenone (3OH-BP); and 4-hydroxybenzophenone (4OH-BP) (water samples) Fluoranthene (FLT); benzo[k]fluoran- DI (PDMS) thene (BkF); benzo[b]fluoranthene (BbF); benzo[a]pyrene (BaP); indeno[1,2,3-cd]pyrene (IcP); benzo[g,h,i]perylene (BgP); 2-methylanthracene (IS) (water samples) 80 pesticides (organochlorine, carba- DI (PDMS) mate, organophosphorus, pyrethroid, and others) (water samples)

In situ derivatization followed [29] by TD

HPTLC-FLD (LOQ: 0.08 0.44 ng/band depending on the PAH)

Liquid microdesorption in a [30] 300 lL microinsert filled with 150 lL ACN

GC-MS (LOD: 2.1 74 ng/L)

The recovery using sequential [31] SBSE was compared with those of conventional SBSE with or without salt addition (30% NaCl) (TD was used) Stirring extraction and stirring LD modes were used TD [32]

06 strongly polar phenols (water samples)

DI (VE, vinylpyridine-ethylene dimethacrylate)

HPLC/DAD (LOD: 0.98 2.20 lg/L) GC/MS/MS (LOD: 0.01 0.71 ng/L)

2,4,6-Trichlorophenol, 2,3,4,6-tetra- DI (PDMS) chlorophenol, 2,4,6-tribromophenol, 2,4,6-trichloroanisole, 2,3,4,6-tetrachloroanisole, 2,4,6-tribromoanisole (water samples) Organophosphorus compounds analysis (review)

[33]

LC/MS GC/MS (LOD: sediment: 10 42 pg/g; water: 0.4 5 ng/L; biota: 12 32 pg/g LC/DAD (LOD: 0.1 g/L)

Recovery: 3 7% TD

[34] [35]

Mercury and tin organometallic HS (PDMS) compounds (surface water, sediment, and biological tissue) Triclosan (personal care products; DI (PDMS) biological, and environmental matrices) Organic UV filters: ethylhexyl salicy- DI (PDMS) late, homosalate, isoamyl methoxycinnamate, 4-methylbenzylidene camphor, BP-3, ethylhexyl methoxycinnamate, ethylhexyl dimethyl PABA, octocrylene, butyl methoxydibenzoylmethane (water samples) Endocrine disruptors (water, biosolids, and sludge) Insect repellent (water samples) DI (PDMS)

LD

[36]

GC/MS (LOD: 0.2 63 ng/L)

TD

[37]

GC/MS (LOD: solid samples: 0.02 ng/g; water samples: 2 ng/L) GC/MS (LOD: 0.5 150 ng/L)

TD

[38]

DI (PDMS)

TD

[39]

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Table 2. Continued Analyte (matrix) Extraction mode (fiber) Analytical system (LOQ or LOD) GC/MS (LOD: 0.2 3.5 ng/L) Remarks TD Ref. [40]

Polybrominated diphenyl ethers DI (PDMS) (PBDEs) and polybrominated biphenyls (PBBs) (water samples) 16 PAHs; 12 polychlorinated biphen- DI (PDMS) yls (PCBs); 6 phthalate esters (PEs) and 3 nonylphenols (NPs) (water samples) 46 acidic and polar organic pollutants DI (PDMS) (phenols; acidic herbicides; pharmaceuticals) (water samples) Semi-volatile organic contaminants (PAHs, polychlorinated biphenyls, OCPs and organophosphorus pesticides) (marine samples) DI (PDMS)

GC/MS (LOD: 0.05 3.3 ng/L)

TD

[41]

GC/MS (LOD: 1.0 800 ng/L)

LD in combination with large volume injection (LVI) TD

[42]

GC/MS (LOD: 0.1 7.5 ng/L)

[43]

Glyoxal and methylglyoxal (environ- DI (PDMS) mental and biological matrices) 15 PAHs (water samples) DI (PDMS)

HPLC-DAD (LOD: glyoxal: 15 ng/L; methylglyoxal: 25 ng/L)

In situ derivatization using 2,3-diaminonaphthalene (DAN) followed by LD

[44]

HPLC-fluorescence detection LD (FLD) (LOD: 0.2 1.5 ng/L) GC/MS (LOD: 3.0 100 ng/L) Microwave-assisted extraction SBSE-TD

[45] [46]

DI (PDMS) Alachlor; p,p9-DDE; p,p9-DDD; p,p9DDT; decachlorobiphenyl; phenanthrene; anthracene; pyrene; benz[a]anthracene; benzo[a]pyrene; benzo[ghi]perylene; n-dodecanoic acid; n-tetradecanoic acid; n-octadecanoic acid; diethyl phthalate; benzyl butyl phthalate (atmospheric particulate matter) Pyrethroids (water samples) SBSE for trace analysis (review) DI (PDMS)

GC/MS (LOD: TD: 0.02 1.4 ng/LLD: 0.9 32.5 ng/L)

Comparison between: TD and LD 39 papers references about applications in environmental analysis

[47] [5]

pounds, its derivatives 2,4-dihydroxybenzophenone (BP1), 2-hydroxy-4-methoxybenzophenone (BP-3), 2-hydroxy4-methoxy-49-methylbenzophenone (BP-10), 2-hydroxybenzophenone (2OH-BP), 3-hydroxybenzophenone (3OHBP), and 4-hydroxybenzophenone (4OH-BP), in water samples was developed using SBSE with in situ derivatization followed by TD-GC-MS. This methodology reached LOD between 0.5 and 2 ng/L (ppt) for the seven BPs, linearity, and the correlation coefficients higher than 0.990 for all compounds and recoveries between 102.0 and 128.1% (RSD a 15.4%, n = 6) [29]. Triclosan is other nonconventional environmental pollutant compound recently evaluated by SBSE. SBSE and LD followed by HPLC with diode array detection (DAD) was proposed for the determination of triclosan in personal care products, biological, and environmental

matrices, which is included in the priority pollutants list set by several international regulatory organizations. The analytical performance proved suitable precision (a3.6%), convenient LODs (0.1 mg/L), and excellent linear dynamic range (r2 A 0.9992) from 0.4 to 108.0 lg/L [36]. SBSE is a good alternative to analyze environmental pollutants at ultra-trace level. Insect repellents, for example, can affect adversely the environment but they appear only in low concentrations. An adequate SBSE in combination with TD-GC-MS was applied for the determination of eight insect repellents and synergists in water samples [39]. Several other developments using SBSE to analyze conventional environmental pollutants like pesticides [31, 34, 46], phenols [32, 33, 42], PAHs [41, 45], and others are reported. These applications generally use PDMS phase,
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Table 3. SBSE applications in food analysis Analyte (matrix) Pesticides (vinegars) Volatile fraction (pesto genovese) Monoterpenes and norisoprenoids (raspberry fruits) Resveratrol (wine, juice, and must) Fungicides (wines and juice) Red wines (fungicides) Fatty acids (beer) Review of food analysis Review of SBSE Extraction mode DI (PDMS) HSSE (PDMS) DI (PDMS) DI (PDMS) DI (PDMS) DI (PDMS) DI (PDMS) Analytical system (LOQ or LOD) GC-MS (LOD: 0.13 0.81 lg/L) GC-MS MDGC-MS LC-UV (LOD: 0.1 ng/mL) Remarks TD-PTV TD TD Ref. [55] [49] [50] [51] [52] [53] [54] [48] [5]

LD (in situ derivatization acetic anhydride) UPLC (LOD: 0.05 2.5 ng/mL) LD GC-MS (LOD: 0.01 2.03 mg/L) TD GC-FID LD 6 papers 56 papers

but new phases are important to enhance the extraction process, as shown by Huang et al. [32] that developed a new phase (poly(vinylpyridine-ethylene dimethacrylate)) to extract polar phenols, concluding that this phase can also be applied to analyze other groups of polar analytes.

2.3 Recent applications of SBSE in food analysis


In a recent review on SBSE, David and Sandra [5] reported examples of food analysis with 56 references and Ridgway et al. [48] showed sample preparation techniques for the determination of trace residues and contaminants in foods containing six references using SBSE. After publication of these reviews some papers using SBSE in food analysis were reported. Salvadeo et al. [49] describes the development and application of a new analytical method for the analysis of the volatile fractions of Pesto Genovese. The HSSE-TD-GC-MS analysis can be very useful for a careful control of the composition and quality of this valuable niche product. The biosynthesis of monoterpenes and norisoprenoids in raspberry fruits was investigated by Hampel et al. [50]. SBSE coupled to LC was successfully applied to determining the resveratrol isomer content of wine, must, and fruit juices. The same time and temperature were used for the extraction step in the presence of 2.5% (m/v) sodium chloride. LD was performed with 150 lL of a 50:50 v/v ACN/1% v/v acetic acid solution in a desorption time of 15 min. Linearity was between 0.5 and 50 ng/mL for trans-resveratrol with a LOD of 0.1 ng/mL, while cisresveratrol could not be extracted [51]. Determination of six oxazole fungicide residues (hymexazol, drazoxolon, vinclozolin, chlozolinate, oxadixyl, and famoxadone) in wine and juices was reported. The best results were achieved at 608C for 30 min with stirring at 1700 rpm in the presence of a 0.1 M acetate acetic acid buffer (pH 5) and 20% (m/v) sodium chloride. LD was performed with 100 lL of a 80:20 v/v ACN water solution in a desorption time of 15 min. LODs ranged

from 0.05 to 2.5 lg/L at an S/N of 3, depending on the compound. Recoveries obtained for spiked samples were satisfactory (83 113%) for all compounds. The proposed method was successfully applied to the analysis of different samples, residues of chlozolinate and drazoxolon being found in samples of red wine and grape juice, respectively [52]. Oliva et al. [53] also studied several fungicide residues (famoxadone, fenhexamid, fluquinconazole, kresoximmethyl, quinoxyfen, and trifloxystrobin) in relation to the aroma composition of monastrell red wines. The wines obtained in the 13 trials were analyzed by SBSEGC-MS. The method proposed showed good linearity over the concentration range tested, with correlation coefficients higher than 0.9 for all analytes. The reproducibility of the method was estimated between 1.87 and 18.52%, and repeatability between 1.00 and 11.29%. The LODs and LOQs of all analytes were lower than the concentration found in these Monastrell wines [53] (Table 3).

3 SBSE: New developments


Previous session emphasized the fact that SBSE applications have been developed in different areas showing good results. Considering that only PDMS is available as an extraction phase on commercial stir bars, the large majority of applications use this coating. Although PDMS shows good results as an extraction phase, it is necessary to consider other phases to extend the SBSE application in order to extract more polar compounds. New developments involving the SBSE instrumentation is another research area that needs to be focused in order to improve the extraction process. Although there are few results about new SBSE phases and instrumentation developments, it is important to consider the relevance of these developments to extend and improve the SBSE applications.
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Figure 3. 2-D design of a heated SPME HPLC interface (adapted from ref. [57]).

3.1 New approaches


Kawaguchi et al. [56] emphasized on the multishot mode using five stir bars to develop a method for the trace analysis of natural and synthetics estrogens, such as estrone (E1), 17b-estradiol (E2), and 17b-ethynylestradiol (EE), in river water samples. This method involved SBSE with in situ derivatization followed by TD-GC-MS. Using the multishot mode it was possible to reach low LODs, around 0.5 pg/mL, concluding that this simple, accurate, and highly sensitive method presents the potential to be applied to determine other analytes in water samples. Rodrigues et al. [57] described in a recent paper the development and evaluation of an improved interface to be operated under continuous heating, for on-line coupling SPME to HPLC. Heating is desirable to increase desorption rate and decrease carryover. The results obtained have been compared with that obtained by offline desorption and online desorption without heating. The SPME-HPLC interface described has an inner volume of just 60 lL, fixation for infinite points, and a novel leak sealing system. When the heating system was used, the area values of the extracted analytes were almost ten-fold higher than that obtained using the off-line mode (Fig. 3). Based on the same interface project an adapted interface was used for on-line coupling SBSE to HPLC for the analysis of antidepressants (Nogueira, A. M., Grossi, P., Olivares, I. R. B., Queiroz, M. E., Lancas, F. M., J. Sep. Sci., submitted November 2008). The main difference between the two interfaces is the volume of the desorption chamber that was increased to 100 lL in the SBSELC, in order to better accommodate bars coated with thicker films. The desorption shows to be more efficient when compared to the off-line process and the desorption temperature also shows to be important to be optimized.

Figure 4. (a) Stainless steel tube and the Teflon mold used for the coating procedure, (b) stir bar already coated, and (c) two parts of the mold and a ruler for dimensions comparison (adapted from ref. [28]).

In-house SBSE was developed using a Teflonm mold (Fig. 4) to cover a magnetic bar with different phases [28]. The PDMS utilized consist of two materials, one being a viscous phase and the other a cure agent. For the preparation of the bars different mixtures of the components were tested to form the polymer in the desired consistence. The extraction optimization for the analysis of organochlorine pesticides (OCPs) in water samples using different parameters has been established by a standard equilibrium time of 120 min at 858C. A mixture of ACN toluene as back extraction solvent promoted a good performance to remove the OCPs sorbed in the bar. A novel technique termed refrigerated sorptive extraction (RSE) describes the development of a device which allows heating the sample matrix and simultaneously cooling the bar coating (Grossi, P., Olivares, I. R. B., Lancas, F. M., J. Chromatogr. A, submitted November 2008). The RSE system was prepared in a Teflon mold similar to the one already fully described [28] but with a larger internal volume to cover ca. 1.5 cm length by 1.0 mm i.d. stainless steel tube with PDMS (Fig. 5). The technique was used to analyze OCPs in water samples. A comparison between the use or not of the refrigeration was performed. During method development, it has been established that the RSE system employing a film of 81 lL of PDMS, an extraction time of 120 min at 858C, and a mixture of ACN toluene (80:20) as desorption liquid, shows a good performance to analyze OCP in water samples. With refrigeration, better chromatogram areas were obtained. For a detailed study, the DDX's compounds were selected. The literature shows that there is an agreement between the theoretical recovery and the experimental data for SBSE and this was also confirmed in RSE by the average recoveries obtained [28].
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Figure 5. RSE system (a) mold; (b) modified HS vial; (c) sealed system; (d) connection with a Teflon ferrule open; (e) connection with a Teflon ferrule closed; (f) complete RSE system (adapted from Grossi, P., Olivares, I. R. B., Lancas, F. M., J. Chromatogr. A, submitted November 2008).

3.2 New phases


A SBSE limitation, in opposition to SPME for which there are several types of polymeric phases that cover a wide range of polarity available, is that at present this technique is commercialized with only PDMS coating. The nonpolar polymeric phases cannot recover all types of analytes by SBSE, particularly the more polar ones (log KO/W a 3), since they present lower affinities for the PDMS. More recently, in-house procedures for stir bar coating have been developed. Bicchi et al. [58] developed dualphase twisters using different carbon-based adsorbents as an additional concentrating phase. The successful combination of two concentrating phases enhanced the recovery of volatile and/or polar compounds compared with conventional PDMS stir bars. Liu et al. [59] described the use of a compact and thermally stable porous hydroxy-terminated phase for the extraction of PAHs, nalkanes, and phosphorus pesticides from water samples. Lambert et al. [19] prepared a biocompatible SBSE device using an alkyl-diol-silica (ADS) restricted access material (RAM) as the SBSE coating. The RAM SBSE bar was able to simultaneously fractionate the protein component from a biological sample, while directly extracting caffeine and its metabolites, overcoming the present

Figure 6. Comparison between data obtained by SBSE/LC assays with PU doped with 5% of the activated carbon ( ), and conventional PDMS ( ). Analytes: (a) anticonvulsants: 1, phenobarbital; 2, epoxide; 3, carbamazepine; 4, methylphenyl-ethyl hydantoin. (b) IVM (adapted from Nogueira, A. M., Grossi, P., Olivares, I. R. B., Queiroz, M. E., Lancas, F. M., J. Sep. Sci., submitted November 2008).

disadvantages of direct sampling in biological matrices, such as fouling of the extraction coating by proteins. Neng et al. [60] proposed polyurethane (PU) foams as new polymeric phases for SBSE. It was demonstrated that these polymers present remarkable stability and excellent mechanical resistance for the enrichment of organic compounds from aqueous samples. The PU foams seem to be a convenient alternative to replace the conventional PDMS for the analysis of the more polar metabolites by SBSE at the trace level [61] and also enhance the extraction of triazinic herbicides in water samples [62]. Nogueira et al. (J. Sep. Sci., submitted November 2008) developed and applied PU doped with activated carbon as polymeric phase for SBSE followed by LC analysis. In this paper antidepressants, anticonvulsants, ivermectine (IVM), and benzoimidazols at therapeutic levels were tested as model pharmaceutical compounds in plasma samples. The comparison of the data obtained by SBSE with the proposed PU polymers and the PDMS is also addressed (Fig. 6). The addition of adsorbent material enhances the number of active sites that participate on the extraction process, increasing the diffusion of the molecules into the pores. The mechanisms of the extracwww.jss-journal.com

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Figure 8. Comparison of obtained chromatograms of IVM in animal plasma using (a) commercial bar, (b) PDMS modified bar with 10% DEGS, (c) PDMS modified bar with 5% OV-17OH (adapted from Nogueira, A. M., Grossi, P., Olivares, I. R. B., Queiroz, M. E., Lanas, F. M., J. Sep. Sci., submitted November 2008).

Figure 7. Comparison between LC peak areas of analytes extracted by pure PU phase, and PU phase doped with an adsorbent material. Analytes: (a) antidepressants, (b) anticonvulsants, and (c) benzoimidazols (adapted from Nogueira, A. M., Grossi, P., Olivares, I. R. B., Queiroz, M. E., Lancas, F. M., J. Sep. Sci., submitted November 2008).

tion into the PU doped with activated carbon phase were based on both adsorption (activated carbon) and absorption (PU) processes. To enhance the efficiency yields of the proposed PU bars, the polymer was doped with different percentages of the activated carbon, which are known to present strong adsorptive properties (Fig. 7). Huang et al. [26] developed a new phase containing poly(methacrylic acid stearyl ester-ethylene dimethacrylate) for simultaneous determination of six steroid sex hormones in urine.

Solvent bar microextraction (SBME), a novel technique developed by Jiang and Lee for sample preconcentration, that involves the use of a selected length of a polypropylene hollow-fiber membrane was applied to the analysis of trace OCPs in wine samples at fg/mL levels [63]. Modified PDMS as novel stationary SBSE phases was also developed by Lanas and coworkers (Nogueira, A. M., Grossi, P., Olivares, I. R. B., Queiroz, M. E., Lanas, F. M., J. Sep. Sci., submitted November 2008). The new phases were evaluated with different analytes. For IVM extraction a comparison using a commercial (PDMS) bar and in-house developed bars containing modified PDMS was done to test the bar efficiency in bovine plasma. The PDMS bar with 5% OV-17-OH extracted the compound with 4.5 times more efficiency than commercial bar. A PDMS bar containing 10% of the DEGS showed almost the double of the extraction when compared with a commercial bar (pure PDMS). The extraction with the OV-17OH bar presented good efficiency and overcame the extraction performance of the PDMS bar (Fig. 8). Another material, poly(vinylpyridine-ethylene dimethacrylate), was synthesized and selected as SBSE by Huang et al. [32]. The influences of polymerization conditions on the extraction efficiency were investigated using phenol and p-nitrophenol as target analytes. Based on this, six strongly polar phenols present in water samples were directly concentrated by the new SBSE phase and determined with HPLC equipped with diode array detector. In comparison with other extraction methods for phenolic compound determination, the proposed method is simple, rapid, inexpensive, and stable, and can also be used
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for other groups of polar analytes with little modification. A new polymeric coating consisting of a dual-phase, polydimethylsiloxane (PDMS) and polypyrrole (PPY) was developed for the SBSE of antidepressants (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline) from plasma samples, followed by LC analysis (SBSE/LC-UV). The extractions were based on both adsorption (PPY) and sorption (PDMS) mechanisms. The PDMS/ PPY coated stir bar showed high extraction efficiency (sensitivity and selectivity) toward the target analytes. The LOQs of the SBSE/LC-UV method ranged from 20 to 50 ng/mL, and the linear range was from LOQ to 500 ng/ mL, with a determination coefficient higher than 0.99. The interday precision of the SBSE/LC-UV method presented a variation coefficient lower than 15%. The efficiency of the SBSE/LC-UV method was proved by analysis of plasma samples from elderly depressed patients [64].

4 Concluding remarks
In the present review, several aspects of SBSE are reviewed, including the basic theory, experimental parameters optimization, applications, and limitations. A well-known limitation of this technique is the fact that only one sorbent (PDMS) is commercially available until this manuscript was written. This limits the application of this technique to the analysis of nonpolar and some intermediate polarity compounds, requiring other steps such as derivatization for the analysis of the more polar ones. On the other hand, in-house polar phases have been successfully used with SBSE, being presented and described in this review. New approaches such as RSE, based upon SBSE concepts were introduced. A novel interface for on-line SBSE-LC is presented and applications discussed. Considering its applications in areas such as biological, environmental, and food safety analysis, described in this review, joined with developments of new sorbents, interfaces, and analytical approaches, it can be concluded that SBSE certainly will occupy an important role as a major sample preparation microtechnique in the near future. The authors would like to acknowledge FAPESP CNPQ and CAPES for financial support to this research. The authors declared no conflict of interest.

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