813 ftp 2sbse review

J. Sep. Sci. 2009, 32, 813 – 824 F. M. Lancas et al. 813

Fernando M. Lancas1

Maria EugÞnia C. Queiroz2

Paula Grossi1

Igor R. B. Olivares1

1Institute of Chemistry at S¼oCarlos/USP, University of S¼oPaulo, S¼o Carlos, SP, Brazil

2Departamento de Qu�mica,Faculdade de Filosofia CiÞncias eLetras de Ribeir¼o Preto,Universidade de S¼o Paulo, SP,Brazil

Review

Recent developments and applications of stir barsorptive extraction

The theoretical aspects of stir bar sorptive extraction (SBSE), as well as the recentapplications of this technique in pharmaceutical, biomedical, environmental, andfood analysis, and recently developed new coating procedures are reviewed. A gen-eral overview of the important factors to be evaluated in the optimization of extrac-tion efficiency such as extraction time, matrix pH, ionic strength, effect of organicadditives, temperature, agitation, pre-extraction derivatization reactions, influenceof proteins, and desorption conditions are discussed. An impressive number ofapplications using SBSE have been published in different areas including biological,environmental, and food, showing the advantages of this technique over the classi-cal extraction techniques (liquid-liquid extraction (LLE), Soxhlet). Although thedifferent SBSE applications use PDMS phase as sorbent, developments of new phasesare necessary for specific applications. In this review, recent SBSE developments areshown with a focus on the development of new instrumental approaches and sorb-ent 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

1 Introduction

Modern trends in analytical chemistry are toward thesimplification, miniaturization of sample preparation,and minimization of organic solvent and sample vol-umes. In particular, the reduction of solvent consump-tion in analytical laboratories is expected to contributesignificantly to the reduction of analytical cost.

New solventless sample-enrichment techniques thatallow the direct extraction of solutes from sample havebeen introduced, such as solid-phase microextraction(SPME), in-tube SPME, and stir bar sorptive extraction(SBSE). These techniques combine extraction and concen-tration of the solute in a single step, thereby reducingthe time required to prepare the samples.

Microextraction is usually defined as an extractiontechnique where the volume of the extracting phase isvery small in relation to the volume of the sample; inmost cases the extraction is not exhaustive with only asmall fraction of the initial solute being extracted for fur-ther analysis. The extraction efficiency is determined bythe solute partitioning between the sample matrix andthe extraction phase. The higher the affinity the solutehas for the extraction phase relative to the samplematrix, the higher the amount of solute extracted. Parti-tion is controlled by the physicochemical properties ofthe solute, the sample matrix, and the extraction phase[1].

SBSE was introduced in 1999 by Baltussen et al. [2]. Thissorptive extraction technique is based on the same prin-ciples than SPME, i.e., partitioning of solute between thesample matrix and the extraction phase. However,instead of a polymer-coated fiber, stir bars of 10–20 mmcoated with 25 –125 lL (0.3–1.0 mm layer) PDMS, areused for enrichment of organic compounds from aque-ous matrices. A magnetic rod is usually encapsulated in aglass jacket on which a PDMS coating is placed (Fig. 1).

Owing to the specific PDMS characteristics, superiorextraction performance is encountered for the followingreasons. First, analytes are partitioned, or sorbed, intothe bulk of the PDMS phase. The sorption is a muchweaker process than adsorption, so compounds can bedesorbed at lower temperatures, thus minimizing thelosses of thermolabile solutes. Second, the retaining

Correspondence: Prof. F. M. Lancas, Institute of Chemistry atS¼o Carlos/USP, University of S¼o Paulo, P.O. Box 780, 13560-970Av. Trabalhador S¼ocarlense, 400 S¼o Carlos, SP, BrazilE-mail: [email protected]: +55-16-33739984

Abbreviations: BP, benzophenone; HS, headspace; IVM, ivermec-tine; LD, liquid desorption; OCP, organochlorine pesticides;PAH, polycyclic aromatic hydrocarbon; PDMS, polydimethylsi-loxane; PPY, polypyrrole; PTV, programmable temperature va-porization; PU, polyurethane; RAM, restricted access material;RSE, refrigerated sorptive extraction; SBSE, stir bar sorptive ex-traction; SPME, solid-phase microextraction; TD, thermal de-sorption

i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

814 F. M. Lancas et al. J. Sep. Sci. 2009, 32, 813 – 824

capacity of PDMS for a certain compound is not influ-enced by the presence of high amounts of water or otheranalytes, since all solutes have their own partitioningequilibrium into the PDMS phase, and displacementdoes not occur. Third, degradation fragments of PDMSsorbent all contain characteristic silicone mass frag-ments which can easily be discerned with the use of massselective detector [2].

The extraction of solutes from the aqueous phase intothe extraction medium is controlled by the partitioncoefficient of the solutes between the PDMS and theaqueous phase. This partitioning coefficient has beencorrelated with the octanol–water distribution coeffi-cients (Ko/w). Although not fully correct, the octanol–water distribution coefficient gives a good indication ifand how well a given solute can be extracted with SBSE[3].

The distribution coefficient between PDMS and aque-ous phase (KPDMS/w) is defined as the ratio between the con-centration of a solute in PDMS (CPDMS) over the concentra-tion in water (Cw) at equilibrium. This ratio is equal to theratio of the mass of the solute in the PDMS (mPDMS) overthe mass of the solute in the aqueous phase (mw) timesthe phase ratio (b with b, Vw/VPDMS) [3] (Eq. 1)

Ko=w L KPDMS=w ¼CPDMS

Cw¼ mPDMS

mw

6Vw

VPDMS¼ mPDMS

mwb ð1Þ

The recovery, expressed as the ratio of the extractedamount of solute (mPDMS) over the original amount of sol-ute in aqueous phase (mo = mw + mPDMS), is dependentupon the distribution coefficient KPDMS/w and on b, asdescribed in Eq. (2)

mPDMS

mo¼

KPDMS=w=b

1þ ðKPDMS=w=bÞ ð2Þ

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

In SPME, the maximum volume of PDMS coated on tothe fiber is ca. 0.5 lL (film thickness 100 lm). For a typicalsample volume of 10 mL the phase ratio is 26104, imply-ing that quantitative extraction is obtained only for com-pounds for which Ko/w A 105. In SBSE, on the other hand,25 –125 lL PDMS coating are used; the situation is muchmore favorable. A stir bar coated with 100 lL PDMS caneasily be used to extract 10 mL of aqueous sample, lead-ing to a b value equal to 100, which implies that soluteswith a Ko/w in excess of 500 are quantitatively extracted ina PDMS-coated stir bar. This not only renders quantifica-tion straightforward, but also ensures significantly bet-ter sensitivity (increase by a factor of 50–250) for com-pounds for which Ko/w a 105 [2, 4]. SBSE of a liquid sampleis performed by placing a suitable amount of sample in aheadspace (HS) vial or other container. The stir bar isadded and the sample is stirred until the partition equili-brium time is reached. The extraction time is kineticallycontrolled and determined by the sample volume, stir-ring rate, temperature, and stir bar dimensions andmust be optimized for a given application. After extrac-tion, the stir bar is easily removed with forceps, rinsedwith purified water to remove adsorbed sugars, proteins,or other sample components, and dried with lint-free tis-sue. According to David and Sandra [5] rinsing does notcause solute loss, because the sorbed solutes are presentinside the PDMS phase.

For HS sampling, the stir bar can be placed in a liquidor solid sample; special devices to hold the stir bar areavailable [5]. Desorption of the solute from the bar maybe done by either heating or back extraction with a smallvolume of a liquid solvent. When SBSE is combined withGC, thermal desorption (TDS) is the preferred way oncethe bar is inserted in the heated GC injection and theanalytes desorbed to the column for further analysis.Liquid desorption (LD) can be combined with both GCand LC. The stir bar is placed in a small vial, and the de-sorption can be performed by adding either few microli-ters of a proper solvent (GC) or the mobile phase (LC).

LD methodologies showed high sensitivity and enoughreproducibility to permit the quantification of antide-pressants [6], and anticonvulsants in human plasma sam-ples from patients receiving therapeutic dosages [7].

Special TDS systems consist of two programmable tem-perature vaporization (PTV) injectors in series [5]. The firstPTV injector (TDS-2) is the unit in which the stir bar is ther-mally desorbed. The second PTV injector is a CIS-4 used asa cryotrap for cryogenic refocusing of the thermally de-sorbed solutes. During fast TDS (flows up to 100 mL/minare recommended), the CIS-4 is kept at negative tempera-tures (as low as –1008C) with liquid nitrogen. In order toefficiently cryotrap even the most volatile solutes, it isnecessary to pack the CIS-4 liner. After the TDS program iscomplete, the temperature of the CIS-4 is increased fast tohigh temperature. The solutes transfer from the TDS-2 to


Figure 1. SBSE stir bar. 1, Magnetic rod; 2, glass jacket; 3,PDMS coating.

J. Sep. Sci. 2009, 32, 813 – 824 Sample Preparations 815

the CIS-4 and from the cryotrap onto the column is usu-ally performed in the splitless mode. Typical carry-overvalues below 1% are reported [5, 8].

SBSE in combination with TDS-capillary GC provides avery versatile tool for the analysis of organic solutes indifferent fluids. Biological samples are characterized by adiversity of organic bioactive compounds that often con-tain polar groups. Derivatization is the classical way toconvert the analytes into GC-amenable compounds. Thearea of application of SBSE has been extended by usingboth pre-extraction derivatization reactions and in situderivatization, typically with ethyl chloroformate andacetic acid anhydride as esterification and acetylationreagents, respectively [9].

Kawaguchi et al. [10] developed a high-sensitivity ana-lytical method that uses SBSE with in situ derivatizationand TD-GC-MS for the simultaneous measurement oftrace amounts of phenolic xenoestrogens, such as 2,4-dichlorophenol, 4-tert-butylphenol, 4-tert-octylphenol, 4-nonylphenol technical isomers, pentachlorophenol, andbisphenol A, in human urine samples. The urine samplewas de-conjugated by adding b-glucuronidase and sulfa-tase. Then, protein precipitation was performed by theaddition of ACN. After centrifugation, the supernatantwas diluted with purified water and subjected to SBSEwith 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 tubefilled with silylation agents are placed inside a glass TD,in which the target compound is derivatized during TDfrom PDMS-coated stir bar [11].

The SBSE variables, such as time, temperature, pHmatrix, ionic strength, and desorption conditions havebeen optimized to reach solute partition equilibrium inshorter analyses time, and to obtain adequate analyticalsensitivity. The sample volume, stirring speed, and stirbar dimensions should be maintained constant duringthe optimization.

Matrix pH can be adjusted to optimize the SBSE ofeither acidic or basic solutes. This is related to the factthat, unless ion exchange coating is used, PDMS-SBSE canextract only neutral (nonionic) species from matrix. Byproperly adjusting the pH, weak acids and bases can beconverted to their neutral forms, in which they can beextracted by the PDMS coating.

Increasing the extraction temperature, the distribu-tion constant of solute between the coating and theextraction mixture decreases; however, it may alsoincrease the diffusion by lowering the viscosity, whichshortens the equilibrium extraction time. Consequently,SBSE methods can be optimized by selecting the properextraction temperature, where satisfactory sensitivity isachieved in an acceptable time period.

Queiroz and coworkers [6] described that the sensitiv-ity of SBSE/LC-UV can be improved by diluting the plasma

samples with the buffer solution, in the pH of which thedrugs were partially or totally in the nonionic form. Thesample dilution also favors the stirring SBSE process. Theauthors also observed that the addition of NaCl, increas-ing the ionic strength, reduced the amount extracted forsome solutes; however for others, it did not alter the effi-ciency of the SBSE process. Probably the salt itself inter-acted with the drugs in solution through electrostatic, orion-pairing interactions, thus reducing the ability of thedrugs to move to the SBSE coating [6].

The life-time of a single stir bar is 20 to more than 50extractions, depending on the matrix, and the analyst'scare.

2 Recent applications

After a historical publication about SBSE in 1999 [2], dis-cussing the theory and principles, different applicationswere developed for environmental, food, and biologicalanalysis. A recent review shows extensive SBSE applica-tions [5] in different areas. Recent applications publishedafter this review were searched and are discussed below.Searching about SBSE applications since its initial devel-opment, it is possible to find around 236 papers; in thelast 5 years around 180 papers; in the last 2 years 49,being 21 in biological, 20 in environmental, and 8 infood analysis applications (Fig. 2).

2.1 Recent applications of SBSE for biologicalsamples

An overview of SBSE applications in biological samples isgiven in Table 1. Most of the described methods (Table 1)showed high chromatographic selectivity, linearity, pre-cision, and high sensitivity, well in line with the interna-tional criteria for validation procedures in order toattend the required purposes, such as therapeutic drugmonitoring, clinical toxicology, forensic toxicology,social toxicology, bioavailability, and pharmacokinetics.

2.2 Recent applications of SBSE forenvironmental analysis

A recent review about SBSE applications [5] shows 39references about the applications of this technique inenvironmental analysis principally on traditional envi-ronmental pollutants such as pesticides, polycyclic aro-matic hydrocarbons (PAHs), volatile organic compounds(VOCs), and others, in water samples. A recent searchabout environmental SBSE application shows thatbeyond traditional environmental pollutants, other spe-cific pollutants have been investigated (Table 2).

An example describing new SBSE environmental appli-cations is the analysis of benzophenone (BP) (also known



as diphenylmethanone) sunscreen compounds. Thesecompounds are widely used as UV absorbers and fra-grance retention agents in the manufacture of cosmeticsand pharmaceuticals. Some studies about these com-pounds have revealed the estrogenic activity of BPs. The

effect of BPs on the ecosystem is also a cause for graveconcern. Considering the low concentration and theimportance to evaluate the presence and concentrationof these compounds in the environment, a method forthe simultaneous measurement of BP sunscreen com-


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 Matrix (volume) Analytical system (LOD) Remarks Ref.

PCBs Sperm (1 mL) GC-MS (SIM) 0.1 pg/mL TD, add 9 mL water –methanol [12]Barbiturates Urine (5 mL) GC-MS (SIM) 12 pg/mL TD [13]Phthalates, metabolics Body fluids, infusates

(5 mL)GC-MS (SIM) TD [14]

Steroids, drugs Biological fluids (5 mL) GC-MS TD, hydrolysis, in situ derivatiza-tion, acetic anhydride/ethylchloroformate

[9]

Pharmaceuticals Urine (5 mL) GC-MS (scan) a 1 ng/mL TD, in situ derivatization, aceticanhydride/ethyl chloroformate

[15]

Drugs of abuse Blood, urine, bile (5 mL) GC-MS a 5 lg/L TD, hydrolysis, in situ derivatiza-tion, acetic anhydride/ethylchloroformate

[16]

Phenols Urine, plasma (1 mL) GC-MS (SIM) 0.4–4 ng/L TD [17]Phenols Urine (1 mL), saliva

(500 lL)GC-MS (SIM) 20 pg/mL TD, in situ derivatization, acetic

anhydride[18]

Phenols Plasma (200 lL) GC-MS (SIM) 100 pg/mL TD, in situ derivatization, aceticanhydride

[18]

Caffeine and metabolites Plasma (0.8 mL) LC-UV 25 ng/mL LD, RAM sorbent [14]Chlorophenols Urine (2 mL) GC-MS (SIM) 10 –20 ng/L TD, in situ derivatization, acetic

anhydride[20]

Tuberculostearic acid Sputum (0.5 mL) GC-MS (SIM) 0.2 ng/mL TD, in situ derivatization, ethylchloroformate

[21]

VOCs (aldehydes, ketones) Urine (animal) (0.5 mL),gland excretion (5 mg)

GC-MS (scan) HSSE [22]

Pesticides Breast milk GC-MS TD [23]Phenols (xenoestrogens) Urine (1 mL) GC-MS (SIM) 10 –50 pg/mL TD, in situ derivatization, acetic

anhydride[24]

4-Hydroxynonenal(oxidative stress marker)

Urine (1 mL) GC-MS (SIM) 22 pg/mL TD, in situ derivatization, PFBHA,acetic anhydride

[25]

Estrone, estradiol Urine (1 mL) GC-MS (SIM) 20 –30 pg/mL TD, hydrolysis, in situ derivatiza-tion, acetic anhydride

[10]

Antidepressants Plasma (1 mL) LC-UV, LOQ: 10–40 ng/mL LD, therapeutic drug monitoring [6]Steroid sex hormones Urine LC-DAD 0.062 –0.38 ng/mL LD, SBSEM sorbent [26]Fluoxetine Plasma (1 mL) GC-MS (SIM) 0.46 pg/mL (TD)

10.0 pg/mL (LOD)TD and LD, in situ derivatization,acetic anhydride/ethyl chlorofor-mate

[27]

Anticonvulsants Plasma (1 mL) LC-UV, LOQ: therapeuticinterval

LD, therapeutic drug monitoring [7]

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).



Table 2. Recent applications of SBSE for environmental analysis

Analyte (matrix) Extraction mode(fiber)

Analytical system (LOQor LOD)

Remarks Ref.

13 OCPs (water samples) HS (PDMS) GC-MS (LOD: 0.01 –1.59 ng/g) LD [28]

BP sunscreen compounds and itsderivatives: 2,4-dihydroxybenzophe-none (BP-1); 2-hydroxy-4-methoxyben-zophenone (BP-3); 2-hydroxy-4-meth-oxy-49-methylbenzophenone (BP-10); 2-hydroxybenzophenone (2OH-BP); 3-hy-droxybenzophenone (3OH-BP); and4-hydroxybenzophenone (4OH-BP)(water samples)

DI (PDMS) GC–MS (LOD: 0.5–2 ng/L) In situ derivatization followedby TD

[29]

Fluoranthene (FLT); benzo[k]fluoran-thene (BkF); benzo[b]fluoranthene(BbF); benzo[a]pyrene (BaP); in-deno[1,2,3-cd]pyrene (IcP); benzo[g,h,i]-perylene (BgP); 2-methylanthracene(IS) (water samples)

DI (PDMS) HPTLC-FLD (LOQ: 0.08 –0.44 ng/band dependingon the PAH)

Liquid microdesorption in a300 lL microinsert filled with150 lL ACN

[30]

80 pesticides (organochlorine, carba-mate, organophosphorus, pyrethroid,and others) (water samples)

DI (PDMS) GC-MS (LOD: 2.1 –74 ng/L) The recovery using sequentialSBSE was compared with thoseof conventional SBSE with orwithout salt addition (30%NaCl) (TD was used)

[31]

06 strongly polar phenols(water samples)

DI (VE, vinylpyri-dine-ethylene di-methacrylate)

HPLC/DAD (LOD: 0.98 –2.20lg/L)

Stirring extraction and stir-ring LD modes were used

[32]

2,4,6-Trichlorophenol, 2,3,4,6-tetra-chlorophenol, 2,4,6-tribromophenol,2,4,6-trichloroanisole, 2,3,4,6-tetra-chloroanisole, 2,4,6-tribromoanisole(water samples)

DI (PDMS) GC/MS/MS (LOD: 0.01–0.71ng/L)

TD [33]

Organophosphorus compoundsanalysis (review)

– LC/MS Recovery: 3–7% [34]

Mercury and tin organometalliccompounds (surface water, sediment,and biological tissue)

HS (PDMS) GC/MS (LOD: sediment: 10 –42 pg/g; water: 0.4–5 ng/L;biota: 12 –32 pg/g

TD [35]

Triclosan (personal care products;biological, and environmental matri-ces)

DI (PDMS) LC/DAD (LOD: 0.1 g/L) LD [36]

Organic UV filters: ethylhexyl salicy-late, homosalate, isoamyl methoxy-cinnamate, 4-methylbenzylidene cam-phor, BP-3, ethylhexyl methoxycinna-mate, ethylhexyl dimethyl PABA,octocrylene, butyl methoxydibenzoyl-methane (water samples)

DI (PDMS) GC/MS (LOD: 0.2–63 ng/L) TD [37]

Endocrine disruptors (water, biosol-ids, and sludge)

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

TD [38]

Insect repellent (water samples) DI (PDMS) GC/MS (LOD: 0.5–150 ng/L) TD [39]


pounds, its derivatives 2,4-dihydroxybenzophenone (BP-1), 2-hydroxy-4-methoxybenzophenone (BP-3), 2-hydroxy-4-methoxy-49-methylbenzophenone (BP-10), 2-hydroxy-benzophenone (2OH-BP), 3-hydroxybenzophenone (3OH-BP), and 4-hydroxybenzophenone (4OH-BP), in water sam-ples was developed using SBSE with in situ derivatizationfollowed by TD-GC-MS. This methodology reached LODbetween 0.5 and 2 ng/L (ppt) for the seven BPs, linearity,and the correlation coefficients higher than 0.990 for allcompounds and recoveries between 102.0 and 128.1%(RSD a 15.4%, n = 6) [29].

Triclosan is other nonconventional environmentalpollutant compound recently evaluated by SBSE. SBSEand LD followed by HPLC with diode array detection(DAD) was proposed for the determination of triclosan inpersonal care products, biological, and environmental

matrices, which is included in the priority pollutants listset by several international regulatory organizations.The analytical performance proved suitable precision(a3.6%), convenient LODs (0.1 mg/L), and excellent lineardynamic range (r2 A 0.9992) from 0.4 to 108.0 lg/L [36].

SBSE is a good alternative to analyze environmentalpollutants at ultra-trace level. Insect repellents, for exam-ple, can affect adversely the environment but theyappear only in low concentrations. An adequate SBSE incombination with TD-GC-MS was applied for the determi-nation of eight insect repellents and synergists in watersamples [39].

Several other developments using SBSE to analyze con-ventional environmental pollutants like pesticides [31,34, 46], phenols [32, 33, 42], PAHs [41, 45], and others arereported. These applications generally use PDMS phase,


Table 2. Continued

Analyte (matrix) Extraction mode(fiber)

Analytical system (LOQor LOD)

Remarks Ref.

Polybrominated diphenyl ethers(PBDEs) and polybrominated biphen-yls (PBBs) (water samples)

DI (PDMS) GC/MS (LOD: 0.2–3.5 ng/L) TD [40]

16 PAHs; 12 polychlorinated biphen-yls (PCBs); 6 phthalate esters (PEs) and3 nonylphenols (NPs) (water samples)


46 acidic and polar organic pollutants(phenols; acidic herbicides; pharma-ceuticals) (water samples)

DI (PDMS) GC/MS (LOD: 1.0–800 ng/L) LD in combination withlarge volume injection (LVI)

[42]

Semi-volatile organic contaminants(PAHs, polychlorinated biphenyls,OCPs and organophosphorus pesti-cides) (marine samples)


Glyoxal and methylglyoxal (environ-mental and biological matrices)

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

In situ derivatization using2,3-diaminonaphthalene(DAN) followed by LD

[44]

15 PAHs (water samples) DI (PDMS) HPLC-fluorescence detection(FLD) (LOD: 0.2–1.5 ng/L)

LD [45]

Alachlor; p,p9-DDE; p,p9-DDD; p,p9-DDT; decachlorobiphenyl; phenan-threne; anthracene; pyrene; benz[a]an-thracene; benzo[a]pyrene; benzo[ghi]-perylene; n-dodecanoic acid;n-tetradecanoic acid; n-octadecanoicacid; diethyl phthalate; benzyl butylphthalate (atmospheric particulatematter)

DI (PDMS) GC/MS (LOD: 3.0–100 ng/L) Microwave-assisted extrac-tion – SBSE-TD

[46]

Pyrethroids (water samples) DI (PDMS) GC/MS (LOD: TD: 0.02–1.4 ng/LLD: 0.9 –32.5 ng/L)

Comparison between: TDand LD

[47]

SBSE for trace analysis (review) – – 39 papers references aboutapplications in environ-mental analysis

[5]


but new phases are important to enhance the extractionprocess, as shown by Huang et al. [32] that developed anew phase (poly(vinylpyridine-ethylene dimethacrylate))to extract polar phenols, concluding that this phase canalso 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] reportedexamples of food analysis with 56 references and Ridg-way et al. [48] showed sample preparation techniques forthe determination of trace residues and contaminants infoods containing six references using SBSE.

After publication of these reviews some papers usingSBSE in food analysis were reported. Salvadeo et al. [49]describes the development and application of a new ana-lytical method for the analysis of the volatile fractions ofPesto Genovese. The HSSE-TD-GC-MS analysis can be veryuseful for a careful control of the composition and qual-ity of this valuable niche product.

The biosynthesis of monoterpenes and norisoprenoidsin raspberry fruits was investigated by Hampel et al. [50].

SBSE coupled to LC was successfully applied to deter-mining the resveratrol isomer content of wine, must,and fruit juices. The same time and temperature wereused for the extraction step in the presence of 2.5% (m/v)sodium chloride. LD was performed with 150 lL of a50:50 v/v ACN/1% v/v acetic acid solution in a desorptiontime of 15 min. Linearity was between 0.5 and 50 ng/mLfor trans-resveratrol with a LOD of 0.1 ng/mL, while cis-resveratrol could not be extracted [51].

Determination of six oxazole fungicide residues(hymexazol, drazoxolon, vinclozolin, chlozolinate, oxa-dixyl, and famoxadone) in wine and juices was reported.The best results were achieved at 608C for 30 min withstirring 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–watersolution 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 thecompound. Recoveries obtained for spiked samples weresatisfactory (83–113%) for all compounds. The proposedmethod was successfully applied to the analysis of differ-ent samples, residues of chlozolinate and drazoxolonbeing found in samples of red wine and grape juice,respectively [52].

Oliva et al. [53] also studied several fungicide residues(famoxadone, fenhexamid, fluquinconazole, kresoxim-methyl, quinoxyfen, and trifloxystrobin) in relation tothe aroma composition of monastrell red wines. Thewines obtained in the 13 trials were analyzed by SBSE-GC-MS. The method proposed showed good linearity overthe concentration range tested, with correlation coeffi-cients higher than 0.9 for all analytes. The reproducibil-ity of the method was estimated between 1.87 and18.52%, and repeatability between 1.00 and 11.29%. TheLODs and LOQs of all analytes were lower than the con-centration found in these Monastrell wines [53] (Table 3).

3 SBSE: New developments

Previous session emphasized the fact that SBSE applica-tions have been developed in different areas showinggood results. Considering that only PDMS is available asan extraction phase on commercial stir bars, the largemajority of applications use this coating. AlthoughPDMS shows good results as an extraction phase, it is nec-essary to consider other phases to extend the SBSE appli-cation in order to extract more polar compounds.

New developments involving the SBSE instrumenta-tion is another research area that needs to be focused inorder to improve the extraction process.

Although there are few results about new SBSE phasesand instrumentation developments, it is important toconsider the relevance of these developments to extendand improve the SBSE applications.


Table 3. SBSE applications in food analysis

Analyte (matrix) Extraction mode Analytical system(LOQ or LOD)

Remarks Ref.

Pesticides (vinegars) DI (PDMS) GC-MS (LOD: 0.13 –0.81 lg/L) TD-PTV [55]Volatile fraction (pesto genovese) HSSE (PDMS) GC-MS TD [49]Monoterpenes and norisoprenoids(raspberry fruits)

DI (PDMS) MDGC-MS TD [50]

Resveratrol (wine, juice, and must) DI (PDMS) LC-UV (LOD: 0.1 ng/mL) LD (in situ derivatizationacetic anhydride)

[51]

Fungicides (wines and juice) DI (PDMS) UPLC (LOD: 0.05–2.5 ng/mL) LD [52]Red wines (fungicides) DI (PDMS) GC-MS (LOD: 0.01 –2.03 mg/L) TD [53]Fatty acids (beer) DI (PDMS) GC-FID LD [54]Review of food analysis – – 6 papers [48]Review of SBSE – – 56 papers [5]


3.1 New approaches

Kawaguchi et al. [56] emphasized on the multishot modeusing five stir bars to develop a method for the trace anal-ysis of natural and synthetics estrogens, such as estrone(E1), 17b-estradiol (E2), and 17b-ethynylestradiol (EE), inriver water samples. This method involved SBSE with insitu derivatization followed by TD-GC-MS. Using the mul-tishot mode it was possible to reach low LODs, around0.5 pg/mL, concluding that this simple, accurate, andhighly sensitive method presents the potential to beapplied to determine other analytes in water samples.

Rodrigues et al. [57] described in a recent paper thedevelopment and evaluation of an improved interface tobe operated under continuous heating, for on-line cou-pling SPME to HPLC. Heating is desirable to increase de-sorption rate and decrease carryover. The resultsobtained have been compared with that obtained by off-line desorption and online desorption without heating.The SPME-HPLC interface described has an inner volumeof just 60 lL, fixation for infinite points, and a novel leaksealing system. When the heating system was used, thearea values of the extracted analytes were almost ten-foldhigher than that obtained using the off-line mode(Fig. 3).

Based on the same interface project an adapted inter-face was used for on-line coupling SBSE to HPLC for theanalysis 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 differencebetween the two interfaces is the volume of the desorp-tion chamber that was increased to 100 lL in the SBSE-LC, in order to better accommodate bars coated withthicker films. The desorption shows to be more efficientwhen compared to the off-line process and the desorp-tion temperature also shows to be important to be opti-mized.

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 aviscous phase and the other a cure agent. For the prepara-tion of the bars different mixtures of the componentswere tested to form the polymer in the desired consis-tence. The extraction optimization for the analysis oforganochlorine pesticides (OCPs) in water samples usingdifferent parameters has been established by a standardequilibrium time of 120 min at 858C. A mixture of ACN–toluene as back extraction solvent promoted a good per-formance to remove the OCPs sorbed in the bar.

A novel technique termed refrigerated sorptive extrac-tion (RSE) describes the development of a device whichallows heating the sample matrix and simultaneouslycooling the bar coating (Grossi, P., Olivares, I. R. B., Lan-cas, F. M., J. Chromatogr. A, submitted November 2008).The RSE system was prepared in a Teflon mold similar tothe one already fully described [28] but with a largerinternal volume to cover ca. 1.5 cm length by 1.0 mm i.d.stainless steel tube with PDMS (Fig. 5). The technique wasused to analyze OCPs in water samples. A comparisonbetween the use or not of the refrigeration was perform-ed. During method development, it has been establishedthat the RSE system employing a film of 81 lL of PDMS,an extraction time of 120 min at 858C, and a mixture ofACN–toluene (80:20) as desorption liquid, shows a goodperformance to analyze OCP in water samples. Withrefrigeration, better chromatogram areas were obtained.For a detailed study, the DDX's compounds were selected.The literature shows that there is an agreement betweenthe theoretical recovery and the experimental data forSBSE and this was also confirmed in RSE by the averagerecoveries obtained [28].


Figure 3. 2-D design of a heated SPME–HPLC interface(adapted from ref. [57]). 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]).


3.2 New phases

A SBSE limitation, in opposition to SPME for which thereare several types of polymeric phases that cover a widerange of polarity available, is that at present this tech-nique is commercialized with only PDMS coating. Thenonpolar polymeric phases cannot recover all types ofanalytes by SBSE, particularly the more polar ones(log KO/W a 3), since they present lower affinities for thePDMS.

More recently, in-house procedures for stir bar coatinghave been developed. Bicchi et al. [58] developed dual-phase twisters using different carbon-based adsorbentsas an additional concentrating phase. The successfulcombination of two concentrating phases enhanced therecovery of volatile and/or polar compounds comparedwith conventional PDMS stir bars. Liu et al. [59] describedthe use of a compact and thermally stable poroushydroxy-terminated phase for the extraction of PAHs, n-alkanes, and phosphorus pesticides from water samples.

Lambert et al. [19] prepared a biocompatible SBSEdevice using an alkyl-diol-silica (ADS) restricted accessmaterial (RAM) as the SBSE coating. The RAM SBSE barwas able to simultaneously fractionate the protein com-ponent from a biological sample, while directly extract-ing caffeine and its metabolites, overcoming the present

disadvantages of direct sampling in biological matrices,such as fouling of the extraction coating by proteins.

Neng et al. [60] proposed polyurethane (PU) foams asnew polymeric phases for SBSE. It was demonstrated thatthese polymers present remarkable stability and excel-lent mechanical resistance for the enrichment of organiccompounds from aqueous samples. The PU foams seemto be a convenient alternative to replace the conven-tional PDMS for the analysis of the more polar metabo-lites by SBSE at the trace level [61] and also enhance theextraction of triazinic herbicides in water samples [62].

Nogueira et al. (J. Sep. Sci., submitted November 2008)developed and applied PU doped with activated carbonas polymeric phase for SBSE followed by LC analysis. Inthis paper antidepressants, anticonvulsants, ivermectine(IVM), and benzoimidazols at therapeutic levels weretested as model pharmaceutical compounds in plasmasamples. The comparison of the data obtained by SBSEwith the proposed PU polymers and the PDMS is alsoaddressed (Fig. 6). The addition of adsorbent materialenhances the number of active sites that participate onthe extraction process, increasing the diffusion of themolecules into the pores. The mechanisms of the extrac-


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) completeRSE system (adapted from Grossi, P., Olivares, I. R. B.,Lancas, F. M., J. Chromatogr. A, submitted November2008).

Figure 6. Comparison between data obtained by SBSE/LCassays with PU doped with 5% of the activated carbon (– ),and conventional PDMS (–). Analytes: (a) anticonvulsants:1, phenobarbital; 2, epoxide; 3, carbamazepine; 4, methyl-phenyl-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).


tion into the PU doped with activated carbon phase werebased on both adsorption (activated carbon) and absorp-tion (PU) processes.

To enhance the efficiency yields of the proposed PUbars, the polymer was doped with different percentagesof the activated carbon, which are known to presentstrong adsorptive properties (Fig. 7).

Huang et al. [26] developed a new phase containingpoly(methacrylic acid stearyl ester-ethylene dimethacry-late) for simultaneous determination of six steroid sexhormones in urine.

Solvent bar microextraction (SBME), a novel techniquedeveloped by Jiang and Lee for sample preconcentration,that involves the use of a selected length of a polypropy-lene hollow-fiber membrane was applied to the analysisof trace OCPs in wine samples at fg/mL levels [63].

Modified PDMS as novel stationary SBSE phases wasalso developed by Lan�as and coworkers (Nogueira, A. M.,Grossi, P., Olivares, I. R. B., Queiroz, M. E., Lan�as, F. M., J.Sep. Sci., submitted November 2008). The new phaseswere evaluated with different analytes. For IVM extrac-tion a comparison using a commercial (PDMS) bar andin-house developed bars containing modified PDMS wasdone to test the bar efficiency in bovine plasma. ThePDMS bar with 5% OV-17-OH extracted the compoundwith 4.5 times more efficiency than commercial bar. APDMS bar containing 10% of the DEGS showed almostthe double of the extraction when compared with a com-mercial bar (pure PDMS). The extraction with the OV-17-OH bar presented good efficiency and overcame theextraction performance of the PDMS bar (Fig. 8).

Another material, poly(vinylpyridine-ethylene dimeth-acrylate), was synthesized and selected as SBSE by Huanget al. [32]. The influences of polymerization conditions onthe extraction efficiency were investigated using phenoland p-nitrophenol as target analytes. Based on this, sixstrongly polar phenols present in water samples weredirectly concentrated by the new SBSE phase and deter-mined with HPLC equipped with diode array detector. Incomparison with other extraction methods for phenoliccompound determination, the proposed method is sim-ple, rapid, inexpensive, and stable, and can also be used


Figure 7. Comparison between LC peak areas of analytesextracted by pure PU phase, and PU phase doped with anadsorbent material. Analytes: (a) antidepressants, (b) anti-convulsants, and (c) benzoimidazols (adapted fromNogueira, A. M., Grossi, P., Olivares, I. R. B., Queiroz, M.E., Lancas, F. M., J. Sep. Sci., submitted November 2008).

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


for other groups of polar analytes with little modifica-tion.

A new polymeric coating consisting of a dual-phase,polydimethylsiloxane (PDMS) and polypyrrole (PPY) wasdeveloped for the SBSE of antidepressants (mirtazapine,citalopram, paroxetine, duloxetine, fluoxetine, and ser-traline) from plasma samples, followed by LC analysis(SBSE/LC-UV). The extractions were based on both adsorp-tion (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 to50 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 pre-sented a variation coefficient lower than 15%. The effi-ciency of the SBSE/LC-UV method was proved by analysisof 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 opti-mization, applications, and limitations. A well-knownlimitation of this technique is the fact that only one sorb-ent (PDMS) is commercially available until this manu-script was written. This limits the application of this tech-nique to the analysis of nonpolar and some intermediatepolarity compounds, requiring other steps such as deriva-tization for the analysis of the more polar ones.

On the other hand, in-house polar phases have beensuccessfully used with SBSE, being presented anddescribed in this review. New approaches such as RSE,based upon SBSE concepts were introduced. A novelinterface for on-line SBSE-LC is presented and applica-tions discussed.

Considering its applications in areas such as biologi-cal, environmental, and food safety analysis, described inthis review, joined with developments of new sorbents,interfaces, and analytical approaches, it can be con-cluded that SBSE certainly will occupy an important roleas a major sample preparation microtechnique in thenear future.

The authors would like to acknowledge FAPESP CNPQ and CAPESfor financial support to this research.

The authors declared no conflict of interest.

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