n pfor clinicanalysisHiroyuki Kataoka

ecessse mendshround rdvanarmtracevier

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therapy to be optimized and provides the toxicology.

analysis. Over 80% of analysis time is

Trends Trends in Analytical Chemistry, Vol. 22, No. 4, 2003actions, the pharmacokinetics in specialpopulations and relationships betweenbasis for studies on patient compliance,bioavailability, pharmacokinetics andgenetics, organ function and the inu-ences of co-medication.The quantitative and qualitative analy-

sis of drugs and metabolites is extensivelyapplied in pharmacokinetic studies. Forthe approval of a new drug, variables,such as the time to reach a maximumconcentration in the plasma, clearanceand bioavailability, have to be known.For example, pharmacokinetic inter-

However, despite the advances in thedevelopment of highly ecient analyticalinstrumentation for the endpoint determi-nation of analytes in biological samplesand pharmaceutical products, samplepre-treatment is usually necessary inorder to extract, isolate and concentratethe analytes of interest from complexmatrixes because most analytical instru-ments cannot handle the matrix directly.In general, the analytical process is divi-ded into ve steps: sampling; sample pre-paration; separation; detection; and, data

*Tel.:+81-66-294-2974;Fax:+81-86-294-2974;

E-mail: kataoka@pheasant.

pharm.okayama-u.ac.jp1. Introduction

The requirement to analyze drugs in bio-logical samples and pharmaceutical pro-ducts is becoming more and morefrequent with the development of moreselective and more eective drugs andwith our need to understand more abouttheir therapeutic and toxic eects.Knowledge of drug levels in body uids,suchas serumandurine, allowspharmaco-

Hiroyuki Kataoka*

Hiroyuki Kataoka, Faculty of

Pharmaceutical Sciences,

Okayama University,Tsushima, Okayama

700-8530, Japan

School of Pharmacy,Shujitsu University,

Nishigawara, Okayama

703-8516, Japands in sampleal and pharm

ary to isolate the desired components fromost analytical instruments cannot handle thein sample preparation include miniaturiza-ghput performance, on-line coupling witheduction in solvent volume and time. Thisces in sample preparation techniques foraceutical analysis, with special focus on in-tion and related new techniques.Science B.V.

ystem; Drug analysis; High-throughput technique;ion; Sample preparation; Solid-phase extraction;reparationaceutical

the concentration of drug and pharmaco-logical eect are investigated in post-marketing surveillance. In addition, thera-peutic drug monitoring (TDM) is used as atool for the improvement of drug therapy.Drugs of abuse, illicit drugs and intoxi-

cation by drugs and poisons are analyzedin clinical and forensic toxicology. Thescreening and conrmation of drugs ofabuse in body uids is also important forthe detection and treatment of their usersand the control of drug addicts followingwithdrawal therapy.Biological materials and pharma-

ceutical products are complex and oftencontain proteins, salts, acids, bases andorganic compounds with similar proper-ties to the analytes. In addition, the ana-lytes often exist at low concentration insamples. Depending on the origins ofsamples and analytical objectives, druganalyses have been carried out usingvarious analytical instruments in manycircumstances such as clinical controlfor diagnosis and treatment of diseases,doping control, forensic analysis andhkataoka@shujitsu.ac.jpSample preparation is ncomplex matrices, becaumatrix directly. Recent trtion, automation, high-tanalytical instruments aarticle reviews recent aforensic, clinical and phtube solid-phase microex# 2003 Published by Els

Keywords: Automated on-In-tube solid-phase microeSolid-phase microextractionNew tre232 0165-9936/03/$ - see front matter# 2003 Published by Elsevier Science B.V. doi:10.1016/S0165-9936(03)00402-3

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Trends in Analytical Chemistry, Vol. 22, No. 4, 2003 Trendsspent on the sampling and sample-preparation steps.Furthermore, the quality of these steps is a key factor indetermining the success of analysis from complexmatrices, such as biological samples, so it is no exag-geration to say that choice of an appropriate sample-preparation method greatly inuences the reliabilityand accuracy of the analysis.Sample preparation can include clean-up procedures

for very complex (dirty) samples. This step must alsobring the analytes to a suitable concentration level. Theisolation and measurement of organic compounds pre-sent in a biological matrix, especially at low concen-tration, presents a signicant analytical challenge. Theobjectives of the analytical method will indicate howmuch eort will be necessary for sample preparation.For example, TDM usually requires specicity to distin-guish the drug to be monitored from similar com-pounds, metabolites or co-administered drugs. Bycontrast, a pharmacokinetic study of a potential drugcandidate requires a specic, sensitive analyticalmethod. As a result, the following features are impor-tant in carrying out an ecient sample preparation:

1. sample loss is minimal and a good yield of theanalyte of interest can be recovered;

2. coexistingcomponentscanberemovedeciently;3. problems do not occur in the chromatography

system;4. the procedure can be carried out conveniently

and quickly;5. the cost of analysis is low.

However, previous sample-preparation techniqueshave involved various drawbacks, such as complicated,time-consuming procedures, large amounts of sampleand organic solvent and diculty in automation. Forexample, if a long time is required for sample prepara-tion, this limits the number of samples, and multi-stepprocedures are prone to lose analytes. Furthermore, useof harmful chemicals and large amounts of solventcause environmental pollution, health hazards tolaboratory personnel and extra operational costs forwaste treatment. Ideally, sample-preparation techni-ques should be fast, easy to use, inexpensive and com-patible with a range of analytical instruments.As shown in Fig. 1, solvent extraction [1^4], solid-

phase extraction (SPE) [3^8], solid-phase microextrac-tion (SPME) [8^22], supercritical uid extraction (SFE)[8,23] and membrane-based extraction (MBE)[2,5,7,24^27] are the main sample-preparation techni-ques for gaseous, liquid and solid samples. Traditionalliquid-liquid extraction (LLE) faces several limitations,namely choosing a solvent, which is non-miscible withthe sample, diculty in extracting polar and ioniccompounds from water, the need for large volumes oforganic solvent which results in a diluted extract.To prevent these drawbacks, two major techniqueshave merged ^ SPE and SPME ^ which can produceclean extracts for analysis very eciently. In SPE, thesample is percolated through a solid phase, whichretains the solutes of interest. They are later eluted withlittle volume. This technique oers the unique advan-tages of high concentration of the nal extract, selectiv-ity, and a wide choice of the solid phase, enabling theextraction of virtually all compounds from aqueous ororganic matrices. In addition, several systems are nowavailable (manual, multiple cartridge systems, disks andmulti-well plates) with possible automation and couplingto chromatography. The main drawback is that thenal extract solvent is sometimes incompatible with theanalytical system, so that solvent has to be evaporatedand the residue then dissolved in a suitable solvent.This problem can be overcome by SPME, which is a

really solvent-free technique. A polymer-coated ber,on which the investigated compound adsorbs, is placedin the sample or in its headspace. The compound can bedesorbed, for example in a gas chromatograph (GC),making this technique very useful. As shown in Fig. 1,SPME is also available for the extraction from gaseous,liquid and solid samples and the analytes extracted can

Figure 1. Main extraction techniques for gaseous, liquid and solidsamples.e directly introduced into the GC. The SPME techniqueay be combined with liquid-phase separation techni-ues involving specially designed desorption interfacesr fused-silica GC capillary columns.The liquid-phase microextraction (LPME) technique

2,24,28,29], which uses a porous polypropyleneollow ber as an extraction device, is used in drugnalysis of biological matrices.Immunoanity extraction (IAE) [30^32] and mole-ularly imprinted polymer (MIP)-based extraction [33^6] are applied to SPE and SPME methods as speciccient sample-preparation techniques. These tech-iques, which are based on adsorption or partitioningf analytes, are responsible for removing the majority ohe biological material of interest from the samplehttp://www.elsevier.com/locate/trac 233

robotic liquid-handling workstation. Each well has a

Trends Trends in Analytical Chemistry, Vol. 22, No. 4, 2003lter composed of inert diatomaceous earth particles,allowing continuous and ecient extraction of ana-lytes between the aqueous biological sample and theorganic extraction solvent. The extraction time wasrelatively short.Although the pace of development of SFE for drug

analysis has fallen, Kline et al. [45] recently reported itsuse for extracting selected anti-inammatory drugs inplasma. New variations of membrane techniques aremicrodialysis, which is extensively used in neuro-science research for in-vivo sampling, and electro-dialysis, whereby an electric eld over a dialysismembrane promotes selective transport of chargedcompounds. Recently, Tsai et al. used the dialysis tech-nique in pharmacokinetic studies of unbound cefepimein rat bile [46] and unbound cephaloridine in rat blood[47].LPME is a new solvent-minimized sample-prepara-

tion technique that is quick, inexpensive and minimizesexposure to toxic organic solvents. It is compatible withcapillary GC, CE and HPLC. It can be used for preparingbiological samples for analysis of various drugs, such asThe nal aim of the sample preparation must be toisolate and to purify the analyte and to introduce it intoGC, a high-performance liquid chromatography (HPLC)or capillary electrophoresis (CE) in a manner that iscompatible with each instrument.In this way, preparation of biological samples for

drug analysis is usually carried out in conjunction withthe techniques mentioned above. Furthermore, auto-mated sample-preparation systems are used in conjunc-tion with one or a combination of these techniques.This article reviews the recent advances in the above

sample-preparation techniques for forensic, clinicaland pharmaceutical analysis. Section 2 gives an over-view of current developments in sample-preparationtechniques, considered according to the extractiontype. Section 3 gives an overview of the development ofin-tube SPME techniques, the design of new extractiondevices and their applications to various samples.Details of sample preparation for forensic, clinical andpharmaceutical analysis are also described in books[37^40].

2. Sample-preparation techniques

As mentioned above, many approaches to sample pre-paration, such as LLE, SFE, MBE, LPME, SPE and SPME,have been developed.LLE for drug analysis has recently become semi-auto-

mated and multi-well plates can now be used [2,4,41^44]. Peng et al. [42] have reported a fully automated,high-throughput LLE method for preparing biologicalsamples using a 96-well LLE plate and a 96-channelantidepressants [28] and basic drugs [29,48]. Further-more, it can be used to extract protein-bound drugs[48] and chiral drugs [49].In the following sub-sections, we review in detail SPE

and SPME, which are widely used for forensic, clinicaland pharmaceutical analysis. Various new anitymaterials, such as immunosorbents and MIPs, havebeen developed as SPE and SMPE sorbents and areused for specic preparation of samples. Recently,Kataoka and Lord [50] and Wells and Lloyd [51]reviewed the above techniques for clinical andpharmaceutical analysis.

2.1. Solid-phase extractionSPE has been widely adopted for preparing samples inthe analysis of pharmaceuticals and drugs of abuse inbiological matrices. SPE oers the following advantagesover conventional liquid-liquid procedures:

1. higher recovery;2. more eective concentration;3. less organic solvent usage;4. no foaming or emulsion problems;5. shorter sample-preparation time;6. easier operation;7. easier incorporation into an automated process.

SPE is based on the partitioning of compoundsbetween a liquid (sample) phase and solid (extraction)phase whereby the intermolecular forces between thephases inuences retention and elution. Retention mayinvolve non-polar, polar or ionic interactions. The widerange of SPE sorbents available provides a wide range ofinteractions. Various SPE products are now available,such as column cartridges, disks, well plates and micro-bers (for SPME). The primary decision for analysts isthe selection of the sorbent to optimize extraction.Various SPE sorbents are now widely used, such as

diatomaceous earth Extrelut, Chem Elut, Bond Elut Cer-tify and Chromabond mixed-mode columns. Mixed-mode sorbents and restricted access material (RAM)sorbents [52] have also become commercially avail-able. Among these sorbents, C18 is the most popular fordrug analysis. Mixed-phase extraction columns (Bond-Elut Certify, Chromabond, Isolute HCX and TSC) givegood recoveries and allow retention of all functionalgroups and of molecules with a range of polarity. Forexample, Bond-Elut Certify is a mixed phase of C8 andSCX, hydrophobic and suitable for ion exchange; it issuitable for the extraction of basic drugs in blood andurine samples. Usually a column-switching system isused for these extractions. Hopfgartner et al. [7] havereported a dual column-switching system (Fig. 2) foron-line concentration of a GABA receptor modulatorand its metabolites in plasma samples. Since extractionand elution can be simultaneously carried out using234 http://www.elsevier.com/locate/trac

the ionization techniques used in LC/MS/MS..

Iaor .s fatq

sanbmodes of o-line and on-line SPE for pre-concentration

Trends in Analytical Chemistry, Vol. 22, No. 4, 2003 Trendsdual trapping columns, toggling between these twostages of operation provides a run cycle time of 3 min.The particles packed within RAM columns are

designed to prevent, or restrict, large macromoleculesfrom accessing the inner adsorption sites of the bondedphase. In this type of column, the internal surface iscovered with a bonded reversed-phase material and theexternal surface is covered with a non-adsorptive, buthydrophilic, material. This dual-phase column allowsfor eective separation of the analyte of interest frommacromolecules in the sample matrix; drugs and othersmall molecules enter the pores of the hydrophobicreversed phase to become partitioned and retained,while proteins and larger matrix components are exclu-ded by the outer, hydrophilic phase and pass through aswaste.Haque and Stewart reported a direct serum injection

method for HPLC determination of selected non-ster-oidal anti-inammatory drugs (NSAIDs) using RAMcolumns [52]. There are some disadvantages for high-throughput considerations for RAM column usage:retention times can be long (>10 min); the columnneeds to be washed between injections; and, the mobilephases required are not always compatible with some of

Figure 2. Schematic diagrams of dual column-switching system.or pre-treatment of analytes and for conventional SPEwhere the MIP is packed into columns or cartridges.Several applications for the use of MIP for biological

samples have appeared. Pre-concentration of bupiva-caine [56] from plasma samples prior to GC analysis hasbeenperformedwithaMIP, and the specicity of theMIP-SPEwashigh comparedwith aC18SPEmethod. In otherexamples, this techniquehas been extended to the extrac-tion of phenytoin in plasma [57], clenbuterol in urine[58] and propranolol in biological samples [59]. All theseexamples demonstrate the high potential of MIP-SPE tobecome a broadly applicable sample-preparation tool.SPE discs [60] also provide a useful, rapid way of

extracting drugs from liquid specimens. In the conven-tional, packed type of solid phase, it is dicult to elutethe analyte of interest using minimal solvent unless theorganic solvent composition is raised to around 100%.Hence, it is necessary to evaporate the eluate to drynessand to redissolve the residue in the HPLC mobile phase.The Empore disk cartridge has a membrane structure,and the elution can be achieved with the HPLC mobilephase. It is then possible to inject the eluate directly intothe HPLC system. The use of a 96-well format for SPEautomated with a robotic liquid-handling system facil-itates high-throughput analysis of biological samples[3,4,7]. Most of the laborious steps in traditional manualextraction procedures can be automated. Recently,various applications of this technique have been repor-ted for drug analysis, such as methotrexate in urine andplasma [61], glybenclamide in serum [62], indolo-carbazole in plasma [63] and oxazepam in plasma [64].

2.2. Solid-phase microextractionSPME, developed by Pawliszyn and co-workers in1990, is a new sample-preparation technique using afused-silica ber coated on the outside with an appro-priate stationary phase [37]. Analyte in a sample isdirectly extracted into the ber coating. In contrast toconventional SPE with packed-bed columns and microor non-micro columns, this arrangement allows combi-nation of all the steps of sample preparation into onestep. The method saves preparation time, solvent anddisposal cost, and can improve the detection limits. Ithas been used routinely in combination with GC andGC/mass spectrometry (GC/MS), and successfullyapplied to a wide variety of compounds in gaseous,liquid and solid samples, especially for the extraction ofvolatile and semi-volatile organic compounds fromenvironmental, biological and food samples [8^22].SPME was also introduced for direct coupling withHPLC and LC/MS in order to analyze weakly volatile orthermally labile compounds not amenable to GC or GC/MS. An SPME/HPLC interface equipped with a specialdesorption chamber is used for solvent desorption priorto HPLC analysis in place of thermal desorption in theinjection port of a GC.Immunosorbents can also be used as SPE cartridgesAE units (LSD ImmunoElute) for lysergic acid diethyl-mide (LSD) are commercially available for the analysisf biological samples [53]. Recently, Clarke et al. [54]eported ultrafast IAE of warfarin by a 2.1-mm-i.dandwich microcolumn containing a 1.1-mm layer on anti-warfarin antibody support. In addition, the IAEechnique was used for specic clean-up of uoro-uinolines [55] from biological samples.MIPs are capable of molecular recognition and aretable enough for long-term storage, easy to preparend inexpensive. Thus, they may be considered to be aew articial anity media. Dierent modes of MIP-ased SPE have been demonstrated, including varioushttp://www.elsevier.com/locate/trac 235

of drugs from biological uids and matrices. For example,

Trends Trends in Analytical Chemistry, Vol. 22, No. 4, 2003In combination with HPLC and LC-MS, it has beenapplied to the analysis of various polar compounds,such as drugs and pesticides. These SPME methods arebased on the adsorption of compounds into the liquidphase coated on the surface of the ber. Moreover, anew SPME/HPLC system, known as in-tube SPME, hasbeen recently developed with an open tubular fused-silica capillary instead of the SPME ber. Details of in-tube SPME are given in Section 3.Another design and concept of SPME has been

recently proposed in the form of Twister, available fromGerstel [20]. In this technique, known as stir-bar sorp-tive extraction (SBSE), a coated magnetic stirring bar isused, with the same PDMS phase as for SPME but in athicker layer (0.3^1.0 mm). This technique is also com-patible with both GC and HPLC desorption procedures.It can theoretically be more sensitive than the SPMEber for certain applications, but it requires a specialdesorption unit and is dicult to automate.The main advantages of SPME are simplicity, rapid-

ity, solvent elimination, high sensitivity, small samplevolume, relatively low cost and simple automation.Since 1995, a number of SPME methods have beendeveloped to extract drugs from various biological sam-ples, such as urine, serum, plasma, whole blood, salivaand hair. The ber SPME device consists of a berassembly with a built-in extraction ber inside a needleand an assembly holder. In ber SPME, analytes areextracted directly from the sample onto a polymeric sta-tionary phase coated on the ber. When the ber isinserted into the sample, the target analytes partitionfrom the sample matrix into the stationary phase untilequilibrium is reached.Two types of ber SPME techniques can be used to

extract analytes: headspace (HS)-SPME; and, directimmersion (DI)-SPME. In HS-SPME, the ber is exposedin the headspace of gaseous, liquid or solid samples.In DI-SPME, the ber is directly immersed in liquidsamples. Minor variants in the method depend onwhether or not derivatization is applied and in whichphase, the type of sample agitation and whether or notadditives are required to optimize extraction. The berwith concentrated analytes is transferred to an instru-ment for desorption, followed by separation and quanti-tation. HS-SPME and DI-SPME techniques can be usedin combination with any GC, GC/MS, HPLC and LC/MSsystem. The anity of the ber coating for an analyte isthe most important factor in SPME. A suitable polarityand thickness of the ber coating can be selectedaccording to the drugunder investigation.Most drugs inbiological samples are extracted with 100 mm PDMSfor non-polar drugs and either 85 mm PA or a porouspolymer DVB ber.Most SPME applications have related to forensic and

toxicological analysis; however, they demonstrate thepotential of SPME for other drug analyses, such asamphetamines have been extracted from urine [65,66]and hair [67,68] by HS-SPME with PDMS ber underalkaline conditions. Lidocaine has been extracted byDI-SPME with PDMS ber from plasma and urine, andanalyzed by GC-FID [69] and HPLC-UV [70]. As a newapproach, Yuan et al. [71] recently developed animmunoanity-SPME technique for specic extractionof theophylline in serum sample. The immunoanity-SPME ber covalently immobilized a theophylline anti-serum on its surface and thus was used as a selective,sensitive extraction medium. Mullett and Pawliszyn[72] also developed a biocompatible SPME ber coatedwith an alkyl-diol-silica RAM. This ber was able simul-taneously to fractionate the protein component from abiological sample and to extract directly several benzo-diazepines, overcoming the present disadvantages ofdirect sampling in biological matrixes by SPME.

3. In-tube solid-phase microextraction

The new sample-preparation technique of in-tubeSPME [12,15^17,21,22,50] uses an open tubularcapillary as an SPME device. It can be coupled on-linewith HPLC or LC/MS. Although the technique using aGC capillary tube is also known as open-tubular trap-ping, it is coupled on-line with GC [20].In-tube SPME is suitable for automation, and extrac-

tion, desorption and injection can be done con-tinuously using a standard autosampler. Automatedsample-handling procedures not only shorten the totalanalysis time, but they are also more accurate and pre-cise than manual techniques. With the in-tube SPMEtechnique, organic compounds in aqueous samples aredirectly extracted from the sample into the internallycoated stationary phase of a capillary, and then des-orbed by introducing a stream of mobile phase or staticclinical, metabolic and pharmaceutical. Most of themethods shown to date have involved HS techniquesfor volatile drugs, and some methods have employed DIand derivatization for less volatile analytes. SPME hasprovided low detection limits and excellent quantita-tion. Especially in the HS mode, SPME extractions oerthe potential for very clean analyses, with little to nointerference from non-volatile compounds. Because ofthe relatively low partition coecients between polardrugs and the commercially available HPLC/SPMEbers, the application of SPME for the assay of low-volatility drugs and metabolites in plasma may belimited to drugs with high therapeutic concentrations,in the range 1^100 mg/ml. The situation can beimproved with the use of current tandem quadru-pole LC/MS instrumentation, where concentrationsapproaching 1 ng/mL in plasma can be analyzed.There are numerous references to the SPME analysis236 http://www.elsevier.com/locate/trac

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Trends in Analytical Chemistry, Vol. 22, No. 4, 2003 Trendsthe analytes partition from the sample matrix into thestationary phase until equilibrium is almost reached.Subsequently, the extracted analytes are directly des-orbed from the capillary coating by mobile-phase owor by an aspirated desorption solvent after switchingthe six-port valve (Fig. 3B). The desorbed analytes aretransported to the HPLC column for separation, andthen detected with a UV or mass-selective detector(MSD). An injection loop is installed to prevent con-tamination of the metering pump by the sample. Asshown in Fig. 3, capillary connections are facilitated bythe use of a 2.5-cm-long sleeve of 1/16-inch polyetherether ketone (PEEK) tubing at each end of the capillary,and xed by 1/16-inch SS unions (0.25 mm bore stain-less steel nuts) and ferrules. By building in UV, diodearray or uorescence detectors between the HPLC andthe MSD, multi-dimensional and simultaneous multi-detections are also possible, ensuring identication anda xed quantity. Drawing and ejection of the samplesolution, switching of the valves, control of peripheralequipment, such as HPLC and MSD, and analytical dataprocessing are all computer-controlled. As a result,labor can be saved and high precision realized. Further-more, the in-tube SPME technique combined with anLC-MS can handle a wide variety of compounds from

Figure 3. Schematic diagrams of in-tube SPME/LC/MS system. (A) Load

position (extraction) and (B) injection position (desorption).prevent plugging of the capillary column and ow linesby ltering the sample solution before extraction. In thecase of the ber SPME, it is not necessary to remove par-ticles before extraction because they can be removed bywashing the ber with water before insertion into thedesorption chamber of the SPME/HPLC interface. How-ever, the bers should be carefully handled, becausethey are fragile and can be easily broken, and the bercoating can be damaged during insertion and agitation.Furthermore, high-molecular weight compounds, suchas proteins, may adsorb irreversibly onto the ber,thus changing the properties of the stationary phaseand rendering it unusable. However, open tubular GCcapillary columns are very stable and useful as anSPME device for in-tube SPME coupled with HPLC orLC-MS.Another signicant dierence between in-tube SPME

and manual ber SPME/HPLC is the possible decouplingof desorption and injection with in-tube SPME. Withber SPME, analytes are desorbed during injection as themobile phase passes over the ber. With in-tube SPME,analytes are desorbed either by the mobile-phase owor by aspirating desorption solvent from a second vial,which is then transferred to the HPLC column by themobile-phase ow.

Figure 4. Extraction of analytes by (A) ber SPME and (B) in-tube SPME.esorption solvent when the analytes are more stronglydsorbed to the capillary coating. Desorbed compoundsre nally injected into the column for analysis.

.1. Automated on-line in-tube SPME systemig. 3 is a schematic diagram of an automated in-tubePME/LC-MS system using an Agilent 1100 series LC/SD. The standard autosampler of this equipment isuitable for construction of an on-line, in-tube SPME/C-MS system.Saito et al. [22] have constructed an in-tube SPMEystem using two Microfeeder MF-2 pumps equippedith MS-GAN microsyringes. As shown in Fig. 3Ahile under computer control, the injection syringeepeatedly draws and ejects sample from the vial, andw to high molecular weight and from high to lowolatility. In addition, automatic processing of a largeumber of samples is possible by the autosampler with-ut carryover, because the injection needle and capil-ry column are washed in methanol and the mobilehase before the sample is extracted.Fig. 4 shows the transfer of the analytes in the extrac-

ion process of SPME. Although the theories behindber and in-tube SPME methods are similar [12,16,37]he signicant dierence between these methods is thatith ber SPME, the analytes are adsorbed on the outerurface of the ber from agitated sample solution, andith in-tube SPME, they are adsorbed on the inner sur-ce of the capillary column from owing sample solu-http://www.elsevier.com/locate/trac 237

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Trends Trends in Analytical Chemistry, Vol. 22, No. 4, 2003Figure 5. Eects of (A) capillary column (B) sample pH and

Capillary column: 60 cm0.25 mm i.d., 0.25 i`m lm thickness. SPME condcycle, 220; draw/eject volume, 30 mL; draw/eject rate, 100 mL/min.draw/eject cycle on the in-tube SPME of several compounds.

compounds, 0.51.0 mg/mL; sample pH, 8.5 (50 mM Tris-HCl); draw/ejectThe ber SPME/HPLC method also has the advantagef eliminating the solvent front peak from the chroma-ogram, but peak broadening is sometimes observedecause analytes can be slowly desorbed from the ber.ith in-tube SPME, peak broadening is comparatively

mall, because analytes are completely desorbed beforejection.

.2. Optimization of in-tube SPMEn-tube SPME is an extraction method by transfer ofnalyte; it depends on the distribution coecient of thenalyte as well as the anity for the ber SPME, and it isportant to raise the distribution factor in the stationary

hase in order to obtain rapid, high extraction eciency.n in-tube SPME, the amount of analyte extracted intohe stationary phase of the capillary column depends onctors such as the polarity of the capillary coating, theumberandvolumeof draw/eject cycles, and sample pH.For in-tube SPME, there are several commerciallyvailable capillary columns. The columns have dierent

properties depending on the selectivity of the stationaryphase, internal diameter, length and lm thickness. Forexample, a low-polarity column with a methyl-siliconliquid phase selectively retains hydrophobic compoundsand a high-polarity column with a polyethylene-glycoliquid phase selectively retains hydrophilic compounds.The extraction eciencies of several commercia

capillary columns have been evaluated. As shown inFig. 5A, Omegawax with a polyethylene-glycol liquidphase was suitable for extracting relatively highly polarcompounds. Although the extraction yields are low, it ispossible to extract the compounds reproducibly by usingan autosampler, and to introduce all of the extractsinto the LC column after in-tube SPME. The internadiameter, length and lm thickness of the column andother dimensions aect the amount of sample that canbe loaded and the amount of compound that can beextracted, and should be chosen carefully.If the dimensions are increased, although the load

and amount extracted may increase, the extension o238 http://www.elsevier.com/locate/trac

Trends in Analytical Chemistry, Vol. 22, No. 4, 2003 Trendssample band-width causes peak broadening and tail-ing. In addition, if the lm thickness of the stationaryphase is large, large amounts of compound can beextracted, but quantitative desorption of the compoundfrom the capillary column may be dicult. From ourexperience, a 50^60 cm length of capillary columnand/or narrow-bore column in which the lm thicknessis comparatively small aords large extracted amountswith minimal peak tailing and without carryover.Although capillary columnswith a chemical bonded or

cross-linked liquid phase are very stable for water andorganic solvent, they readily deteriorate in the presenceof strong inorganic acids or strong alkalis. However,the capillary column is generally stable for the mobilephase usually used in HPLC. For instance, an Omega-wax column was repeatedly used over 500 times with-out a reduction in the extraction eciency [73^75].Generally, it is possible to increase the extraction e-

ciency of an analyte into a stationary phase in SPME bychanging the pH and the salt level of the sample solu-tion. Acidic and basic compounds can be extractedeectively from acidic and alkaline sample solutions,respectively. However, the stability of the compound atthe sample-solution pHmust be checked beforehand.As shown in Fig. 5B, the extraction eciency of some

basic drugs was optimal at pH 8.5 (Tris-HCl buer), andthe optimum concentration of buer solution was50^100mM. Although salting out increases the extrac-tion eciency in ber SPME, the salt deposits can clogup the column in in-tube SPME. Furthermore, the pre-sence of a hydrophilic solvent, such as methanol, in thesample decreases the extraction eciency because itincreases the solubility of the compound in the sample.However, the extraction eciency is little inuenced bya methanol concentration of 5% or less. The amount ofcompound extracted into the stationary phase dependsupon the concentration of the compound in the sample.The draw/eject volume and the number of sample

solutions aect the extracted amount and depend onthe capacity of the column. Complete equilibriumextraction is generally not obtained for any of the ana-lytes, because the analytes are partially desorbed intothe mobile phase during the ejection step. Although asthe draw/eject volume increases, the amount extractedincreases independently, the band-width extends andthe peak becomes broad.In our experiments, the optimum draw/eject volume

was 30^40 mL for tested drugs in the case of a capillarycolumn with a length of 60 cm and an internal dia-meter of 0.25 mm. Under these conditions, the extrac-tion eciency did not increase even at high volumes.Although an increase in the number of draw/ejectcycles can enhance the extraction eciency, peakbroadening is often observed in this case. In addition,the draw/eject speed corresponds to the agitation speedof ber SPME, and the extraction eciency increaseswith the speed. However, in our experience, the optimalow rate of draw/eject cycles was 50^-100 mL/min.Below this level, extraction required an inconvenientlylong time, and, above this level, bubbles formed insidethe capillary, reducing the extraction eciency.It is ideal to carry out draw/eject of the sample solu-

tion until the compound reaches distribution equili-brium, in order to obtain maximum extraction amount.However, it is possible to cease extraction before equili-brium to reduce the analysis time, if sucient sensitiv-ity is obtained. The extraction time depends on thevolume, speed and the number of cycles of the draw/eject, and these conditions must be xed in order toobtain a quantitative reproducibility. In Fig. 5C, theequilibrium was nearly reached over 10^15 draw/ejectcycles at 100 mL/min for a 30 mL sample, although thiswill dier, depending on the compound.As for desorption of the compound from the capillary

column, there are twomethods:

the dynamic method desorbs into the owingmobile phase;

the static method desorbs into a solvent aspi-rated from the outside.

Static desorption is preferable when the analytes aremore strongly adsorbed to the capillary coating.In each case, it is necessary to carry out quick, perfect

desorption with a minimum volume of solvent. In thecase of a capillary column with length 60 cm and inter-nal diameter 0.25 mm, the desorption is usually carriedout by aspirating 40 mL, depending on the capacity ofthe column.For the static method, it is also necessary to consider

the solubility of the compound and its miscibility withthe mobile phase.For the dynamic method, it is possible to desorb

directly into the owing mobile phase after switchingthe six-port valve. Still, although carryover may beobserved after the analysis of highly concentratedsamples, it is possible to wash the injection needle andthe capillary column by draw/eject of methanol and themobile phase several times prior to the next analysis.Thereby, carryover in in-tube SPME is lower or elimi-nated compared with that in ber SPME. Furthermore,it is possible to automatically carry out extractionand desorption operations using an overall injectionprogram.

3.3. Forensic, clinical and pharmaceutical analysisKataoka et al. [73] developed an automated in-tubeSPME coupled with LC-ESI-MS (positive ion mode, SIM)for nine b-blockers. The optimum extraction conditionswere 15 draw/eject cycles of 30 mL of sample in Tris-HClbuer (pH 8.5) at a ow-rate of 100 mL/min using anOmegawax 250 capillary.http://www.elsevier.com/locate/trac 239

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Trends Trends in Analytical Chemistry, Vol. 22, No. 4, 2003Figure 6. Total ion and SIM chromatograms obtained from standard

SPME/LC/MS-SIM. (A) Standard solution containing 200 ng/mL propran

5-hydroxypropranolol and N-desisopropylpropranolol. (B) Clinical serum

i.d., 3 mm particle size); mobile phase, acetonitrile/methanol/water/aceticrun. ESI+-MS conditions: nebulizer gas, N2 (40 psi); drying gas N2Peaks: 1 = 5-hydroxypropranolol (m/z 276); 2 = 4-hydroxypropranolol (m

(m/z 218); 5 = propranolol (m/z 260). (Reprinted with permission from [73ranolol and its metabolites, and a clinical serum sample by in-tube

50 ng/mL 4-hydroxypropranolol and 7-hydroxypropranolol, 20 ng/mL

le (100 mL). LC conditions: column, Hypersil BDS C18 (5.0 cm2.1 mm(15:15:70:1); ow-rate, program from 0.25 to 0.45 mL/min for a 20 min/min, 350C); fragmentor voltage, 70 V; capillary voltage, 3500 V;6); 3 = 7-hydroxypropranolol (m/z 276); 4 = N-desisopropylpropranolol

1999 American Chemical Society).The b-blockers extracted in the capillary were easilyesorbed into the mobile phase by the dynamic deso-ption technique. Using this method, the detection limitas 0.1^1.2 ng/mL (S/N=3), and the linearity was inhe 2^100 ng/mL range. This method can be directlypplied to diluted urine and ultraltered serum withoutterferences. Recoveries of b-blockers added to urinend serum samples were higher than 71%, and repro-ucibility was good with a relative standard deviationRSD) of 7.6% or less. Furthermore, this method can beuccessfully applied to the determination of propranololnd its metabolites in serum of patients administeredropranolol (Fig. 6).Kataoka et al. [74] also developed an automated in-

ube SPME coupled with LC-ESI-MS (positive ion mode,IM) for analyzing drugs of abuse, including ampheta-ine, methamphetamine and their methylenedioxynalogues. The optimum extraction conditions were 15raw/eject cycles of 35 mL of sample in Tris-HCl buerpH 8.5) at a ow-rate of 100 mL/min using an Omega-ax 250 capillary.The stimulants extracted in the capillary were easilyesorbed with a mobile phase by the dynamic desorp-

tion technique. Using this method, the detection limitwas 0.2^0.8 ng/mL (S/N=3), and linearity wasobtained in the range of 2^100 ng/mL. This methodcan be directly applied to the diluted urine sampleswithout interferences. Recoveries of stimulants addedto urine samples were over 80%, and it was possible toanalyze them reproducibly with a RSD of 7.9% or lessIn addition, the within-day and between-day variationsfor analysis of spiked urine samples were 0.9^3.0% and2.1^6.0%, respectively.In addition, the in-tube SPME method of analyzing

ranitidine was developed using an Omegawax capillary[76] and directly applied to tablet and urine sampleswithout interferences. On the other hand, Supel-QPLOT coated with the porous divinylbenzene polymerwas also used as a conventional capillary columnYuan et al. [77] reported an automated in-tube SPMEcoupled with LC-ESI-MS of 7 benzodiazepines using thiscapillary. The optimum extraction conditions were 10draw/eject cycles of 30 mL of sample in Tris-HCl buer(pH 8.5) at a ow-rate of 300 mL/min. The benzodiaze-pines extracted in the capillary were easily desorbedwithmobile phase by the dynamic desorption technique240 http://www.elsevier.com/locate/trac

organic polymer or silica with a unique ow-through

Trends in Analytical Chemistry, Vol. 22, No. 4, 2003 TrendsUsing this method, the detection limits of these com-pounds were 0.02^2 ng/mL (S/N=3), and the linearitieswere obtained in the 0.5^500 ng/mL range. Thismethod was directly applied to urine and serumsamples without interferences.

3.4. Extraction device developed for in-tube SPMEUntil now, although the commercial open tubular capil-lary columnshavebeenusedmainly as extractiondevicesin in-tube SPME, various SPME devices have been devel-oped to improve extractioneciencyand selectivity.The development of extraction phases better suited to

extraction of relatively polar compounds from aqueoussamples will enhance the sensitivity and the overall uti-lity of the method. Wu et al. took initial steps in thisdirection with the development of a new capillary col-umn; the inner wall of commercial fused silica capillarywas coated with polypyrrole (PPY) polymer. The PPY-coated capillary in-tube SPME coupled with LC-ESI-MSwas successfully used in the analysis of b-blockers inurine and serum [78], stimulants in urine and hair[79], and verapamil and its metabolites in biologicalmatrices [80].Recently, Mullett et al. [81] synthesized a MIP for use

as an in-tube SPME adsorbent. The inherent selectivityand chemical and physical robustness of the MIP mate-rial was demonstrated as an eective stationary phasematerial for in-tube SPME. Using a PEEK tube packedwith MIP particles, an automated on-line in-tube SPME/HPLC system was developed for the selective analysis ofpropranolol in serum samples. Pre-concentration of thesample by the MIP adsorbent increased the sensitivity,yielding a limit of detection of 0.32 mg/mL by UV detec-tion. Excellent method reproducibility (RSD500 injections) were observedover a fairly wide linear dynamic range (0.5^-100 mg/mL) in serum samples.Mullet et al. [82] also developed an automated bio-

compatible in-tube SPME method using RAM, alkyl-diol-silica (ADS). The use of RAM capillary enableddirect extraction of several benzodiazepines from serumsample. The bifunctionality of the ADS extraction phaseprevented fouling of the capillary by protein adsorptionwhile simultaneously trapping the analytes in thehydrophobic porous interior. The approach simpliedthe apparatus required, compared with existing RAMcolumn-switching procedures. It also overcame theexisting problem that in-tube SPME requires an ultra-ltration or other deproteinization step prior to hand-ling biological samples, thus further minimizingsample-preparation requirements. Mussho et al. [83]developed an automated solid-phase dynamic extrac-tion (SPDE) as a technique similar to in-tube SPME,using a hollow needle with an internal coating of PDMSinstead of a capillary column. This technique is suitablefor headspace extraction coupled with GC-MS and candouble-pore structure.

4. Future prospects

As described in this article, sample-preparation techni-ques for LLE, IAE, LPME, MBE, SPE and SPME have beendeveloped recently. Of these, SPE is the most popular fordrug analysis and has become an essential tool inlaboratories all over the world. It has also largelyreplaced older techniques.The development of SPE has been fast during the past

decade, with many improvements in formats, automa-tion and the introduction of new phases. Some promis-ing approaches in SPE are based on special packings,such as RAMs andMIPs.Multi-well plates are also expected to be widely adop-

ted in the future for automated analytical systems usinga liquid-handling robot. The introduction and imple-mentation of automated 96-well extraction hasbrought about high-throughput approaches to the bio-logical sample-preparation techniques of SPE, LLE andprotein precipitation [3,4,7].SPME is becoming an attractive alternative to SPE and

LLE for some applications. SPME is a solvent-free, con-centrating extraction technique, and can be used in theanalysis of drugsandmetabolites inbiological uids.As evidenced with SPE, IAE sorbents, RAMs and MIPs

are also expected to be applied as new sorbent materialsbe used for the determination of amphetamines andsynthetic designer drugs in hair samples.However, modied capillary columns inserted with

stainless steel wire and a polyether ether ketone (PEEK)tube packed with brous rigid-rod heterocyclic polymerhave been developed to increase extraction eciency[22]. Recently, Saito et al. [84] described an interestinginnovation for in-tube SPME, whereby a ne wire isincorporated into the lumen of the extraction capillary,to eectively increase the surface-to-volume ratio in theanalysis. However, it is thought to limit the extractioneciency. Using this wire-in-tube SPME methodcoupled with microcolumn HPLC, tricyclic anti-depressants in urine samples were determined inthe 5^500 ng/mL range by UV detection. The pre-concentration was between 15-fold and 110-fold thatused in direct injection.Jinno et al [85] also developed an on-line interface

between the ber-in-tube SPME and CE, and the pre-concentration and separation of the above tricyclicantidepressant drugs were accomplished with thehyphenated system. As shown in Fig. 7, this methodwas successfully applied to a patients urine sample.More recently, Shintani et al. [86] reported an alter-

native approach with an in-tube SPME technique usinga monolithic capillary column consisting of one-piece ofhttp://www.elsevier.com/locate/trac 241

for highly ecient extraction of drugs from variousbmtsdap

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increasing the reliability and precision of sample

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Trends Trends in Analytical Chemistry, Vol. 22, No. 4, 2003Figure 7. Electropherograms of tricyclic antidepressant drugs. (A) Fiber

CE analysis of standard mixture (50 mg/ml each), (C) ber-in-tube SPMcontrolled-urine spiked by 5 mg/ml amitriptyline. SPME conditions: eand time, 80 mL/min12.5 min (1.0 mL); desorption ow rate anPeaks: 1 = desipramine; 2 = nortriptyline; 3 = imipramine; 4 = amitriptbe SPME/CE analysis of standard mixture (0.5 mg/ml each), (B) directanalysis of a patients urine, (D) ber-in-tube SPME/CE analysis of a

tion medium, HM/DB-5; packing density, 52%; extraction ow rate

e, 4.0 mL/min0.45 min (1.8 mL); desorption solvent, acetonitrile.(Reprinted with permission from [84].# 2001 Wiley-VCH Verlag GmbH).iological samples by SPME. With the development ofore sensitive phases it may be possible to miniaturizehe technique further. As the market for SPME increa-es in the future, this could lead to the introduction ofisposable, low-cost extraction bers (e.g. in the form ofcarousel) or tubes, as in other areas of sample pre-aration, e.g. SPEmulti-well plates.The in-tube SPME technique is very eective as aample-preparation technique for qualitative anduantitative analyses. As extraction and concentrationre combined, all of the analyte extracted is introducedto the analytical system. The main advantages of in-

ube SPME are simplicity, rapidity, solvent elimination,igh sensitivity, small sample volume, relatively lowost and simple automation. The in-tube SPME tech-ique can be successfully used for polar and non-polarompounds in liquid samples, and can be coupled easilyith various analytical methods, such as HPLC, LC-MSnd CE. In addition, it is possible to improve the extrac-ion eciency and selectivity by further development ofew capillaries coated or packed with new materials,nd the range of applications will spread on combi-ation with further, dierent analytical instruments.The trends are clearly towards:

simplifying the work involved in sample pre-

preparation; and, eliminating the clean-up step by using more

selective extraction procedures.

The development of more selective sorbents wilremain an active area of research with respect to IAEsorbents and MIPs. In particular, MIP antibody mimicsare much easier to synthesize and consequently muchless expensive.The development of SPE and SPME based on inexpen-

sive MIPs allows anity extraction to be virtually dis-posable, potentially making widely available anityextraction with excellent selectivity. Many applicationscan be expected in forensic, clinical, pharmaceuticaand biochemical analyses for molecular recognitiontechniques, such as:

anity extraction media that may replace bio-logical antibodies;

sensor material that may replace the biochip; novel carriers for capillary electrophoresis; selective membranes; and, other new intelligent materials.

Furthermore, users have increasingly shown interestin the automation of sample preparation for faster242 http://www.elsevier.com/locate/trac

[5] J.R. Veraart, H. Lingeman, U.A.Th. Brinkman, J. Chromatogr.A 856 (1999) 483.

[6] M. Gilar, E.S.P. Bouvier, B.J. Compton, J. Chromatogr. A 909

[14] G.A. Mills, V. Walker, J. Chromatogr. A 902 (2000) 267.[15] H. Kataoka, H. Lord, J. Pawliszyn, J. Chromatogr. A 880

Trends in Analytical Chemistry, Vol. 22, No. 4, 2003 Trends(2000) 35.[16] H. Lord, J. Pawliszyn, J. Chromatogr. A 885 (2000) 153.[17] H. Kataoka, H.L. Lord, J. Pawliszyn, in: I.D. Wilson, T.D.

Adlard, C.F. Poole, M. Cook (Editors), Encyclopedia of Separa-tion Science, Academic Press, London, 2000, p. 4153.

[18] J. Pawliszyn, Adv. Exp. Med. Biol. 488 (2001) 73.[19] F. Augusto, A. Luiz, P. Valente, Trends Anal. Chem. 21 (2002)

428.[20] E. Baltussen, C.A. Cramers, P.J.F. Sandra, Anal. Bioanal.

Chem. 373 (2002) 3.[21] H. Kataoka, Anal. Bioanal. Chem. 373 (2002) 31.[22] Y. Saito, M. Kawazoe, M. Imaizumi, Y. Morishima, Y. Nakao,

K. Hatano, M. Hayashida, K. Jinno, Anal. Sci. 18 (2002) 7.[23] C. Radclie, K. Maguire, B. Lockwood, Biochem, J. Biophys.

Methods 43 (2000) 261.(2001) 111.[7] G. Hopfgartner, C. Husser, M. Zell, Ther. Drug Monit. 24

(2002) 134.[8] C.W. Huie, Anal. Bioanal. Chem. 373 (2002) 23.[9] F. Sporkert, F. Pragst, Forensic Sci. Int. 107 (2000) 129.

[10] G. Theodoridis, E.H.M. Koster, G.J. de Jong, J. Chromatogr. B745 (2000) 49.

[11] N.H. Snow, J. Chromatogr. A 885 (2000) 445.[12] H.L. Lord, J. Pawliszyn, J. Chromatogr. A 902 (2000) 17.[13] S. Ulrich, J. Chromatogr. A 902 (2000) 167.and more cost-eective analyses. The key attractivefeatures of automated sample preparation techniquesinclude miniaturization, high throughput, reproduci-bility and traceability.In the last decade, new concepts have been developed

to allow the on-line coupling of sample-preparationdevices to separation and detection systems, all ofwhich are specially designed for automation. In thefuture, advances towards better integration ofsampling/sample preparation and instrumental analysiswill allow wider use of automated on-line analysis inforensic, clinical and pharmaceutical analysis.

Acknowledgements

This work was supported by grants from ShimadzuScience Foundation, The Yakumo Foundation forEnvironmental Sciences, Japan Food Industrial Center,and Grants-in-Aid for Basic Scientic Research(B(2), no. 14370729) from the Japanese Society forPromotion of Science.

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Hiroyuki Kataoka is an associate professor of health chemistry atthe Faculty of Pharmaceutical Sciences in Okayama University,Okayama, Japan. He will be a full professor of applied analytical chem-istry at the School of Pharmacy in Shujitsu University, Okayama,Japan, from April 2003. He received a MSc degree in 1979 from OsakaUniversity, Japan, and a PhD (Doctor of Pharmacy) in 1986 fromTohoku University. From April 1998 to March 1999, he worked withProfessor Janusz Pawliszyn as a postdoctoral research fellow in theSolid Phase Microextraction Group at University of Waterloo (Water-loo, Ontario, Canada) developing in-tube solid-phase microextrac-tion. His research interests are in developing selective, sensitivemethods for the analysis of biologically active, potentially harmful orchemically interesting compounds in living systems, foods and theenvironment. His present research projects also cover the develop-ment of automated sample-preparation methods and applications toenvironmental and pharmaceutical elds.

Trends Trends in Analytical Chemistry, Vol. 22, No. 4, 2003244 http://www.elsevier.com/locate/trac

New trends in sample preparation for clinical and pharmaceutical analysisIntroductionSample-preparation techniquesSolid-phase extractionSolid-phase microextraction

In-tube solid-phase microextractionAutomated on-line in-tube SPME systemOptimization of in-tube SPMEForensic, clinical and pharmaceutical analysisExtraction device developed for in-tube SPME

Future prospectsAcknowledgementsReferences