New Trends in Sample Prep

Trends
Trends in Analytical Chemistry, Vol. 22, No. 4, 2003
New trends in sample preparation

for clinical and pharmaceutical
analysis
Hiroyuki Kataoka
Sample preparation is necessary to isolate the desired components from
complex matrices, because most analytical instruments cannot handle the
matrix directly. Recent trends in sample preparation include miniaturization, automation, high-throughput performance, on-line coupling with
analytical instruments and reduction in solvent volume and time. This
article reviews recent advances in sample preparation techniques for
forensic, clinical and pharmaceutical analysis, with special focus on intube solid-phase microextraction and related new techniques.
# 2003 Published by Elsevier Science B.V.
Keywords: Automated on-line system; Drug analysis; High-throughput technique;
In-tube solid-phase microextraction; Sample preparation; Solid-phase extraction;
Solid-phase microextraction
Hiroyuki Kataoka*
Hiroyuki Kataoka, Faculty of
Pharmaceutical Sciences,
Okayama University,
Tsushima, Okayama
700-8530, Japan
School of Pharmacy,
Shujitsu University,
Nishigawara, Okayama
703-8516, Japan
*Tel.:+81-66-294-2974;
Fax:+81-86-294-2974;
E-mail: kataoka@pheasant.
pharm.okayama-u.ac.jp
hkataoka@shujitsu.ac.jp
232
1. Introduction
The requirement to analyze drugs in biological samples and pharmaceutical products is becoming more and more
frequent with the development of more
selective and more eective drugs and
with our need to understand more about
their therapeutic and toxic eects.
Knowledge of drug levels in body uids,
such as serum and urine, allows pharmacotherapy to be optimized and provides the
basis for studies on patient compliance,
bioavailability, pharmacokinetics and
genetics, organ function and the inuences of co-medication.
The quantitative and qualitative analysis of drugs and metabolites is extensively
applied in pharmacokinetic studies. For
the approval of a new drug, variables,
such as the time to reach a maximum
concentration in the plasma, clearance
and bioavailability, have to be known.
For example, pharmacokinetic interactions, the pharmacokinetics in special
populations and relationships between
the concentration of drug and pharmacological eect are investigated in postmarketing surveillance. In addition, therapeutic drug monitoring (TDM) is used as a
tool for the improvement of drug therapy.
Drugs of abuse, illicit drugs and intoxication by drugs and poisons are analyzed
in clinical and forensic toxicology. The
screening and conrmation of drugs of
abuse in body uids is also important for
the detection and treatment of their users
and the control of drug addicts following
withdrawal therapy.
Biological materials and pharmaceutical products are complex and often
contain proteins, salts, acids, bases and
organic compounds with similar properties to the analytes. In addition, the analytes often exist at low concentration in
samples. Depending on the origins of
samples and analytical objectives, drug
analyses have been carried out using
various analytical instruments in many
circumstances such as clinical control
for diagnosis and treatment of diseases,
doping control, forensic analysis and
toxicology.
However, despite the advances in the
development of highly ecient analytical
instrumentation for the endpoint determination of analytes in biological samples
and pharmaceutical products, sample
pre-treatment is usually necessary in
order to extract, isolate and concentrate
the analytes of interest from complex
matrixes because most analytical instruments cannot handle the matrix directly.
In general, the analytical process is divided into ve steps: sampling; sample preparation; separation; detection; and, data
analysis. Over 80% of analysis time is
0165-9936/03/$ - see front matter # 2003 Published by Elsevier Science B.V. doi:10.1016/S0165-9936(03)00402-3

spent on the sampling and sample-preparation steps.
Furthermore, the quality of these steps is a key factor in
determining the success of analysis from complex
matrices, such as biological samples, so it is no exaggeration to say that choice of an appropriate samplepreparation method greatly inuences the reliability
and accuracy of the analysis.
Sample preparation can include clean-up procedures
for very complex (dirty) samples. This step must also
bring the analytes to a suitable concentration level. The
isolation and measurement of organic compounds present in a biological matrix, especially at low concentration, presents a signicant analytical challenge. The
objectives of the analytical method will indicate how
much eort will be necessary for sample preparation.
For example, TDM usually requires specicity to distinguish the drug to be monitored from similar compounds, metabolites or co-administered drugs. By
contrast, a pharmacokinetic study of a potential drug
candidate requires a specic, sensitive analytical
method. As a result, the following features are important in carrying out an ecient sample preparation:
1. sample loss is minimal and a good yield of the
analyte of interest can be recovered;
2. coexisting components can be removed eciently;
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 techniques
have involved various drawbacks, such as complicated,
time-consuming procedures, large amounts of sample
and organic solvent and diculty in automation. For
example, if a long time is required for sample preparation, this limits the number of samples, and multi-step
procedures are prone to lose analytes. Furthermore, use
of harmful chemicals and large amounts of solvent
cause environmental pollution, health hazards to
laboratory personnel and extra operational costs for
waste treatment. Ideally, sample-preparation techniques should be fast, easy to use, inexpensive and compatible with a range of analytical instruments.
As shown in Fig. 1, solvent extraction [1^4], solidphase extraction (SPE) [3^8], solid-phase microextraction (SPME) [8^22], supercritical uid extraction (SFE)
[8,23] and membrane-based extraction (MBE)
[2,5,7,24^27] are the main sample-preparation techniques for gaseous, liquid and solid samples. Traditional
liquid-liquid extraction (LLE) faces several limitations,
namely choosing a solvent, which is non-miscible with
the sample, diculty in extracting polar and ionic
compounds from water, the need for large volumes of
organic solvent which results in a diluted extract.
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Figure 1. Main extraction techniques for gaseous, liquid and solid

samples.
To prevent these drawbacks, two major techniques

have merged ^ SPE and SPME ^ which can produce
clean extracts for analysis very eciently. In SPE, the
sample is percolated through a solid phase, which
retains the solutes of interest. They are later eluted with
little volume. This technique oers the unique advantages of high concentration of the nal extract, selectivity, and a wide choice of the solid phase, enabling the
extraction of virtually all compounds from aqueous or
organic matrices. In addition, several systems are now
available (manual, multiple cartridge systems, disks and
multi-well plates) with possible automation and coupling
to chromatography. The main drawback is that the
nal extract solvent is sometimes incompatible with the
analytical system, so that solvent has to be evaporated
and 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 placed
in the sample or in its headspace. The compound can be
desorbed, 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
be directly introduced into the GC. The SPME technique
may be combined with liquid-phase separation techniques involving specially designed desorption interfaces
or fused-silica GC capillary columns.
The liquid-phase microextraction (LPME) technique
[2,24,28,29], which uses a porous polypropylene
hollow ber as an extraction device, is used in drug
analysis of biological matrices.
Immunoanity extraction (IAE) [30^32] and molecularly imprinted polymer (MIP)-based extraction [33^
36] are applied to SPE and SPME methods as specic,
ecient sample-preparation techniques. These techniques, which are based on adsorption or partitioning
of analytes, are responsible for removing the majority of
the biological material of interest from the sample
matrix prior to analysis.
http://www.elsevier.com/locate/trac
233
Trends
The nal aim of the sample preparation must be to

isolate and to purify the analyte and to introduce it into
GC, a high-performance liquid chromatography (HPLC)
or capillary electrophoresis (CE) in a manner that is
compatible with each instrument.
In this way, preparation of biological samples for
drug analysis is usually carried out in conjunction with
the techniques mentioned above. Furthermore, automated sample-preparation systems are used in conjunction with one or a combination of these techniques.
This article reviews the recent advances in the above
sample-preparation techniques for forensic, clinical
and pharmaceutical analysis. Section 2 gives an overview of current developments in sample-preparation
techniques, considered according to the extraction
type. Section 3 gives an overview of the development of
in-tube SPME techniques, the design of new extraction
devices and their applications to various samples.
Details of sample preparation for forensic, clinical and
pharmaceutical analysis are also described in books
[37^40].
2. Sample-preparation techniques
As mentioned above, many approaches to sample preparation, such as LLE, SFE, MBE, LPME, SPE and SPME,
have been developed.
LLE for drug analysis has recently become semi-automated 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 biological
samples using a 96-well LLE plate and a 96-channel
robotic liquid-handling workstation. Each well has a
lter composed of inert diatomaceous earth particles,
allowing continuous and ecient extraction of analytes between the aqueous biological sample and the
organic extraction solvent. The extraction time was
relatively short.
Although the pace of development of SFE for drug
analysis has fallen, Kline et al. [45] recently reported its
use for extracting selected anti-inammatory drugs in
plasma. New variations of membrane techniques are
microdialysis, which is extensively used in neuroscience research for in-vivo sampling, and electrodialysis, whereby an electric eld over a dialysis
membrane promotes selective transport of charged
compounds. Recently, Tsai et al. used the dialysis technique in pharmacokinetic studies of unbound cefepime
in rat bile [46] and unbound cephaloridine in rat blood
[47].
LPME is a new solvent-minimized sample-preparation technique that is quick, inexpensive and minimizes
exposure to toxic organic solvents. It is compatible with
capillary GC, CE and HPLC. It can be used for preparing
biological samples for analysis of various drugs, such as
234
antidepressants [28] and basic drugs [29,48]. Furthermore, 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, clinical
and pharmaceutical analysis. Various new anity
materials, such as immunosorbents and MIPs, have
been developed as SPE and SMPE sorbents and are
used for specic preparation of samples. Recently,
Kataoka and Lord [50] and Wells and Lloyd [51]
reviewed the above techniques for clinical and
pharmaceutical analysis.
2.1. Solid-phase extraction
SPE has been widely adopted for preparing samples in
the analysis of pharmaceuticals and drugs of abuse in
biological matrices. SPE oers the following advantages
over conventional liquid-liquid procedures:
1.
2.
3.
4.
5.
6.
7.
higher recovery;
more eective concentration;
less organic solvent usage;
no foaming or emulsion problems;
shorter sample-preparation time;
easier operation;
easier incorporation into an automated process.
SPE is based on the partitioning of compounds

between a liquid (sample) phase and solid (extraction)
phase whereby the intermolecular forces between the
phases inuences retention and elution. Retention may
involve non-polar, polar or ionic interactions. The wide
range of SPE sorbents available provides a wide range of
interactions. Various SPE products are now available,
such as column cartridges, disks, well plates and microbers (for SPME). The primary decision for analysts is
the selection of the sorbent to optimize extraction.
Various SPE sorbents are now widely used, such as
diatomaceous earth Extrelut, Chem Elut, Bond Elut Certify and Chromabond mixed-mode columns. Mixedmode sorbents and restricted access material (RAM)
sorbents [52] have also become commercially available. Among these sorbents, C18 is the most popular for
drug analysis. Mixed-phase extraction columns (BondElut Certify, Chromabond, Isolute HCX and TSC) give
good recoveries and allow retention of all functional
groups and of molecules with a range of polarity. For
example, Bond-Elut Certify is a mixed phase of C8 and
SCX, hydrophobic and suitable for ion exchange; it is
suitable for the extraction of basic drugs in blood and
urine samples. Usually a column-switching system is
used for these extractions. Hopfgartner et al. [7] have
reported a dual column-switching system (Fig. 2) for
on-line concentration of a GABA receptor modulator
and its metabolites in plasma samples. Since extraction
and elution can be simultaneously carried out using
Figure 2. Schematic diagrams of dual column-switching system.
dual trapping columns, toggling between these two

stages of operation provides a run cycle time of 3 min.
The particles packed within RAM columns are
designed to prevent, or restrict, large macromolecules
from accessing the inner adsorption sites of the bonded
phase. In this type of column, the internal surface is
covered with a bonded reversed-phase material and the
external surface is covered with a non-adsorptive, but
hydrophilic, material. This dual-phase column allows
for eective separation of the analyte of interest from
macromolecules in the sample matrix; drugs and other
small molecules enter the pores of the hydrophobic
reversed phase to become partitioned and retained,
while proteins and larger matrix components are excluded by the outer, hydrophilic phase and pass through as
waste.
Haque and Stewart reported a direct serum injection
method for HPLC determination of selected non-steroidal anti-inammatory drugs (NSAIDs) using RAM
columns [52]. There are some disadvantages for highthroughput considerations for RAM column usage:
retention times can be long ( > 10 min); the column
needs to be washed between injections; and, the mobile
phases required are not always compatible with some of
the ionization techniques used in LC/MS/MS.
Immunosorbents can also be used as SPE cartridges.
IAE units (LSD ImmunoElute) for lysergic acid diethylamide (LSD) are commercially available for the analysis
of biological samples [53]. Recently, Clarke et al. [54]
reported ultrafast IAE of warfarin by a 2.1-mm-i.d.
sandwich microcolumn containing a 1.1-mm layer of
an anti-warfarin antibody support. In addition, the IAE
technique was used for specic clean-up of uoroquinolines [55] from biological samples.
MIPs are capable of molecular recognition and are
stable enough for long-term storage, easy to prepare
and inexpensive. Thus, they may be considered to be a
new articial anity media. Dierent modes of MIPbased SPE have been demonstrated, including various
modes of o-line and on-line SPE for pre-concentration
Trends
or pre-treatment of analytes and for conventional SPE
where the MIP is packed into columns or cartridges.
Several applications for the use of MIP for biological
samples have appeared. Pre-concentration of bupivacaine [56] from plasma samples prior to GC analysis has
been performed with a MIP, and the specicity of the MIPSPE was high compared with a C18 SPE method. In other
examples, this technique has been extended to the extraction of phenytoin in plasma [57], clenbuterol in urine
[58] and propranolol in biological samples [59]. All these
examples demonstrate the high potential of MIP-SPE to
become a broadly applicable sample-preparation tool.
SPE discs [60] also provide a useful, rapid way of
extracting drugs from liquid specimens. In the conventional, packed type of solid phase, it is dicult to elute
the analyte of interest using minimal solvent unless the
organic solvent composition is raised to around 100%.
Hence, it is necessary to evaporate the eluate to dryness
and 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 mobile
phase. It is then possible to inject the eluate directly into
the HPLC system. The use of a 96-well format for SPE
automated with a robotic liquid-handling system facilitates high-throughput analysis of biological samples
[3,4,7]. Most of the laborious steps in traditional manual
extraction procedures can be automated. Recently,
various applications of this technique have been reported for drug analysis, such as methotrexate in urine and
plasma [61], glybenclamide in serum [62], indolocarbazole in plasma [63] and oxazepam in plasma [64].
2.2. Solid-phase microextraction
SPME, developed by Pawliszyn and co-workers in
1990, is a new sample-preparation technique using a
fused-silica ber coated on the outside with an appropriate stationary phase [37]. Analyte in a sample is
directly extracted into the ber coating. In contrast to
conventional SPE with packed-bed columns and micro
or non-micro columns, this arrangement allows combination of all the steps of sample preparation into one
step. The method saves preparation time, solvent and
disposal cost, and can improve the detection limits. It
has been used routinely in combination with GC and
GC/mass spectrometry (GC/MS), and successfully
applied to a wide variety of compounds in gaseous,
liquid and solid samples, especially for the extraction of
volatile and semi-volatile organic compounds from
environmental, biological and food samples [8^22].
SPME was also introduced for direct coupling with
HPLC and LC/MS in order to analyze weakly volatile or
thermally labile compounds not amenable to GC or GC/
MS. An SPME/HPLC interface equipped with a special
desorption chamber is used for solvent desorption prior
to HPLC analysis in place of thermal desorption in the
injection port of a GC.
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Trends
In combination with HPLC and LC-MS, it has been

applied to the analysis of various polar compounds,
such as drugs and pesticides. These SPME methods are
based on the adsorption of compounds into the liquid
phase coated on the surface of the ber. Moreover, a
new SPME/HPLC system, known as in-tube SPME, has
been recently developed with an open tubular fusedsilica capillary instead of the SPME ber. Details of intube SPME are given in Section 3.
Another design and concept of SPME has been
recently proposed in the form of Twister, available from
Gerstel [20]. In this technique, known as stir-bar sorptive extraction (SBSE), a coated magnetic stirring bar is
used, with the same PDMS phase as for SPME but in a
thicker layer (0.3^1.0 mm). This technique is also compatible with both GC and HPLC desorption procedures.
It can theoretically be more sensitive than the SPME
ber for certain applications, but it requires a special
desorption unit and is dicult to automate.
The main advantages of SPME are simplicity, rapidity, solvent elimination, high sensitivity, small sample
volume, relatively low cost and simple automation.
Since 1995, a number of SPME methods have been
developed to extract drugs from various biological samples, such as urine, serum, plasma, whole blood, saliva
and hair. The ber SPME device consists of a ber
assembly with a built-in extraction ber inside a needle
and an assembly holder. In ber SPME, analytes are
extracted directly from the sample onto a polymeric stationary phase coated on the ber. When the ber is
inserted into the sample, the target analytes partition
from the sample matrix into the stationary phase until
equilibrium is reached.
Two types of ber SPME techniques can be used to
extract analytes: headspace (HS)-SPME; and, direct
immersion (DI)-SPME. In HS-SPME, the ber is exposed
in the headspace of gaseous, liquid or solid samples.
In DI-SPME, the ber is directly immersed in liquid
samples. Minor variants in the method depend on
whether or not derivatization is applied and in which
phase, the type of sample agitation and whether or not
additives are required to optimize extraction. The ber
with concentrated analytes is transferred to an instrument for desorption, followed by separation and quantitation. HS-SPME and DI-SPME techniques can be used
in combination with any GC, GC/MS, HPLC and LC/MS
system. The anity of the ber coating for an analyte is
the most important factor in SPME. A suitable polarity
and thickness of the ber coating can be selected
according to the drug under investigation. Most drugs in
biological samples are extracted with 100 mm PDMS
for non-polar drugs and either 85 mm PA or a porous
polymer DVB ber.
Most SPME applications have related to forensic and
toxicological analysis; however, they demonstrate the
potential of SPME for other drug analyses, such as
236
clinical, metabolic and pharmaceutical. Most of the

methods shown to date have involved HS techniques
for volatile drugs, and some methods have employed DI
and derivatization for less volatile analytes. SPME has
provided low detection limits and excellent quantitation. Especially in the HS mode, SPME extractions oer
the potential for very clean analyses, with little to no
interference from non-volatile compounds. Because of
the relatively low partition coecients between polar
drugs and the commercially available HPLC/SPME
bers, the application of SPME for the assay of lowvolatility drugs and metabolites in plasma may be
limited to drugs with high therapeutic concentrations,
in the range 1^100 mg/ml. The situation can be
improved with the use of current tandem quadrupole LC/MS instrumentation, where concentrations
approaching 1 ng/mL in plasma can be analyzed.
There are numerous references to the SPME analysis
of drugs from biological uids and matrices. For example,
amphetamines have been extracted from urine [65,66]
and hair [67,68] by HS-SPME with PDMS ber under
alkaline conditions. Lidocaine has been extracted by
DI-SPME with PDMS ber from plasma and urine, and
analyzed by GC-FID [69] and HPLC-UV [70]. As a new
approach, Yuan et al. [71] recently developed an
immunoanity-SPME technique for specic extraction
of theophylline in serum sample. The immunoanitySPME ber covalently immobilized a theophylline antiserum on its surface and thus was used as a selective,
sensitive extraction medium. Mullett and Pawliszyn
[72] also developed a biocompatible SPME ber coated
with an alkyl-diol-silica RAM. This ber was able simultaneously to fractionate the protein component from a
biological sample and to extract directly several benzodiazepines, overcoming the present disadvantages of
direct sampling in biological matrixes by SPME.
3. In-tube solid-phase microextraction

The new sample-preparation technique of in-tube
SPME [12,15^17,21,22,50] uses an open tubular
capillary as an SPME device. It can be coupled on-line
with HPLC or LC/MS. Although the technique using a
GC capillary tube is also known as open-tubular trapping, it is coupled on-line with GC [20].
In-tube SPME is suitable for automation, and extraction, desorption and injection can be done continuously using a standard autosampler. Automated
sample-handling procedures not only shorten the total
analysis time, but they are also more accurate and precise than manual techniques. With the in-tube SPME
technique, organic compounds in aqueous samples are
directly extracted from the sample into the internally
coated stationary phase of a capillary, and then desorbed by introducing a stream of mobile phase or static
Trends
3.1. Automated on-line in-tube SPME system

Fig. 3 is a schematic diagram of an automated in-tube
SPME/LC-MS system using an Agilent 1100 series LC/
MSD. The standard autosampler of this equipment is
suitable for construction of an on-line, in-tube SPME/
LC-MS system.
Saito et al. [22] have constructed an in-tube SPME
system using two Microfeeder MF-2 pumps equipped
with MS-GAN microsyringes. As shown in Fig. 3A,
while under computer control, the injection syringe
repeatedly draws and ejects sample from the vial, and
the analytes partition from the sample matrix into the
stationary phase until equilibrium is almost reached.
Subsequently, the extracted analytes are directly desorbed from the capillary coating by mobile-phase ow
or by an aspirated desorption solvent after switching
the six-port valve (Fig. 3B). The desorbed analytes are
transported to the HPLC column for separation, and
then detected with a UV or mass-selective detector
(MSD). An injection loop is installed to prevent contamination of the metering pump by the sample. As
shown in Fig. 3, capillary connections are facilitated by
the use of a 2.5-cm-long sleeve of 1/16-inch polyether
ether ketone (PEEK) tubing at each end of the capillary,
and xed by 1/16-inch SS unions (0.25 mm bore stainless steel nuts) and ferrules. By building in UV, diode
array or uorescence detectors between the HPLC and
the MSD, multi-dimensional and simultaneous multidetections are also possible, ensuring identication and
a xed quantity. Drawing and ejection of the sample
solution, switching of the valves, control of peripheral
equipment, such as HPLC and MSD, and analytical data
processing are all computer-controlled. As a result,
labor can be saved and high precision realized. Furthermore, the in-tube SPME technique combined with an
LC-MS can handle a wide variety of compounds from
low to high molecular weight and from high to low

volatility. In addition, automatic processing of a large
number of samples is possible by the autosampler without carryover, because the injection needle and capillary column are washed in methanol and the mobile
phase before the sample is extracted.
Fig. 4 shows the transfer of the analytes in the extraction process of SPME. Although the theories behind
ber and in-tube SPME methods are similar [12,16,37],
the signicant dierence between these methods is that,
with ber SPME, the analytes are adsorbed on the outer
surface of the ber from agitated sample solution, and,
with in-tube SPME, they are adsorbed on the inner surface of the capillary column from owing sample solution. Therefore, with in-tube SPME, it is necessary to
prevent plugging of the capillary column and ow lines
by ltering the sample solution before extraction. In the
case of the ber SPME, it is not necessary to remove particles before extraction because they can be removed by
washing the ber with water before insertion into the
desorption chamber of the SPME/HPLC interface. However, the bers should be carefully handled, because
they are fragile and can be easily broken, and the ber
coating can be damaged during insertion and agitation.
Furthermore, high-molecular weight compounds, such
as proteins, may adsorb irreversibly onto the ber,
thus changing the properties of the stationary phase
and rendering it unusable. However, open tubular GC
capillary columns are very stable and useful as an
SPME device for in-tube SPME coupled with HPLC or
LC-MS.
Another signicant dierence between in-tube SPME
and manual ber SPME/HPLC is the possible decoupling
of desorption and injection with in-tube SPME. With
ber SPME, analytes are desorbed during injection as the
mobile phase passes over the ber. With in-tube SPME,
analytes are desorbed either by the mobile-phase ow
or by aspirating desorption solvent from a second vial,
which is then transferred to the HPLC column by the
mobile-phase ow.
Figure 3. Schematic diagrams of in-tube SPME/LC/MS system. (A) Load

position (extraction) and (B) injection position (desorption).
Figure 4. Extraction of analytes by (A) ber SPME and (B) in-tube SPME.
desorption solvent when the analytes are more strongly

adsorbed to the capillary coating. Desorbed compounds
are nally injected into the column for analysis.
237
Trends
The ber SPME/HPLC method also has the advantage

of eliminating the solvent front peak from the chromatogram, but peak broadening is sometimes observed
because analytes can be slowly desorbed from the ber.
With in-tube SPME, peak broadening is comparatively
small, because analytes are completely desorbed before
injection.
3.2. Optimization of in-tube SPME
In-tube SPME is an extraction method by transfer of
analyte; it depends on the distribution coecient of the
analyte as well as the anity for the ber SPME, and it is
important to raise the distribution factor in the stationary
phase in order to obtain rapid, high extraction eciency.
In in-tube SPME, the amount of analyte extracted into
the stationary phase of the capillary column depends on
factors such as the polarity of the capillary coating, the
number and volume of draw/eject cycles, and sample pH.
For in-tube SPME, there are several commercially
available capillary columns. The columns have dierent
properties depending on the selectivity of the stationary

phase, internal diameter, length and lm thickness. For
example, a low-polarity column with a methyl-silicon
liquid phase selectively retains hydrophobic compounds,
and a high-polarity column with a polyethylene-glycol
liquid phase selectively retains hydrophilic compounds.
The extraction eciencies of several commercial
capillary columns have been evaluated. As shown in
Fig. 5A, Omegawax with a polyethylene-glycol liquid
phase was suitable for extracting relatively highly polar
compounds. Although the extraction yields are low, it is
possible to extract the compounds reproducibly by using
an autosampler, and to introduce all of the extracts
into the LC column after in-tube SPME. The internal
diameter, length and lm thickness of the column and
other dimensions aect the amount of sample that can
be loaded and the amount of compound that can be
extracted, and should be chosen carefully.
If the dimensions are increased, although the load
and amount extracted may increase, the extension of
Figure 5. Eects of (A) capillary column (B) sample pH and (C) draw/eject cycle on the in-tube SPME of several compounds.
Capillary column: 60 cm0.25 mm i.d., 0.25 i`m lm thickness. SPME conditions: compounds, 0.51.0 mg/mL; sample pH, 8.5 (50 mM Tris-HCl); draw/eject
cycle, 220; draw/eject volume, 30 mL; draw/eject rate, 100 mL/min.
238

sample band-width causes peak broadening and tailing. In addition, if the lm thickness of the stationary
phase is large, large amounts of compound can be
extracted, but quantitative desorption of the compound
from the capillary column may be dicult. From our
experience, a 50^60 cm length of capillary column
and/or narrow-bore column in which the lm thickness
is comparatively small aords large extracted amounts
with minimal peak tailing and without carryover.
Although capillary columns with a chemical bonded or
cross-linked liquid phase are very stable for water and
organic solvent, they readily deteriorate in the presence
of strong inorganic acids or strong alkalis. However,
the capillary column is generally stable for the mobile
phase usually used in HPLC. For instance, an Omegawax column was repeatedly used over 500 times without a reduction in the extraction eciency [73^75].
Generally, it is possible to increase the extraction eciency of an analyte into a stationary phase in SPME by
changing the pH and the salt level of the sample solution. Acidic and basic compounds can be extracted
eectively from acidic and alkaline sample solutions,
respectively. However, the stability of the compound at
the sample-solution pH must be checked beforehand.
As shown in Fig. 5B, the extraction eciency of some
basic drugs was optimal at pH 8.5 (Tris-HCl buer), and
the optimum concentration of buer solution was
50^100 mM. Although salting out increases the extraction eciency in ber SPME, the salt deposits can clog
up the column in in-tube SPME. Furthermore, the presence of a hydrophilic solvent, such as methanol, in the
sample decreases the extraction eciency because it
increases the solubility of the compound in the sample.
However, the extraction eciency is little inuenced by
a methanol concentration of 5% or less. The amount of
compound extracted into the stationary phase depends
upon the concentration of the compound in the sample.
The draw/eject volume and the number of sample
solutions aect the extracted amount and depend on
the capacity of the column. Complete equilibrium
extraction is generally not obtained for any of the analytes, because the analytes are partially desorbed into
the mobile phase during the ejection step. Although as
the draw/eject volume increases, the amount extracted
increases independently, the band-width extends and
the peak becomes broad.
In our experiments, the optimum draw/eject volume
was 30^40 mL for tested drugs in the case of a capillary
column with a length of 60 cm and an internal diameter of 0.25 mm. Under these conditions, the extraction eciency did not increase even at high volumes.
Although an increase in the number of draw/eject
cycles can enhance the extraction eciency, peak
broadening is often observed in this case. In addition,
the draw/eject speed corresponds to the agitation speed
of ber SPME, and the extraction eciency increases
Trends
with the speed. However, in our experience, the optimal
ow rate of draw/eject cycles was 50^-100 mL/min.
Below this level, extraction required an inconveniently
long time, and, above this level, bubbles formed inside
the capillary, reducing the extraction eciency.
It is ideal to carry out draw/eject of the sample solution until the compound reaches distribution equilibrium, in order to obtain maximum extraction amount.
However, it is possible to cease extraction before equilibrium to reduce the analysis time, if sucient sensitivity is obtained. The extraction time depends on the
volume, speed and the number of cycles of the draw/
eject, and these conditions must be xed in order to
obtain a quantitative reproducibility. In Fig. 5C, the
equilibrium was nearly reached over 10^15 draw/eject
cycles at 100 mL/min for a 30 mL sample, although this
will dier, depending on the compound.
As for desorption of the compound from the capillary
column, there are two methods:
the dynamic method desorbs into the owing
mobile phase;
the static method desorbs into a solvent aspirated from the outside.
Static desorption is preferable when the analytes are
more 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 the
case of a capillary column with length 60 cm and internal diameter 0.25 mm, the desorption is usually carried
out by aspirating 40 mL, depending on the capacity of
the column.
For the static method, it is also necessary to consider
the solubility of the compound and its miscibility with
the mobile phase.
For the dynamic method, it is possible to desorb
directly into the owing mobile phase after switching
the six-port valve. Still, although carryover may be
observed after the analysis of highly concentrated
samples, it is possible to wash the injection needle and
the capillary column by draw/eject of methanol and the
mobile phase several times prior to the next analysis.
Thereby, carryover in in-tube SPME is lower or eliminated compared with that in ber SPME. Furthermore,
it is possible to automatically carry out extraction
and desorption operations using an overall injection
program.
3.3. Forensic, clinical and pharmaceutical analysis
Kataoka et al. [73] developed an automated in-tube
SPME coupled with LC-ESI-MS (positive ion mode, SIM)
for nine b-blockers. The optimum extraction conditions
were 15 draw/eject cycles of 30 mL of sample in Tris-HCl
buer (pH 8.5) at a ow-rate of 100 mL/min using an
Omegawax 250 capillary.
239
Trends
The b-blockers extracted in the capillary were easily

desorbed into the mobile phase by the dynamic desorption technique. Using this method, the detection limit
was 0.1^1.2 ng/mL (S/N=3), and the linearity was in
the 2^100 ng/mL range. This method can be directly
applied to diluted urine and ultraltered serum without
interferences. Recoveries of b-blockers added to urine
and serum samples were higher than 71%, and reproducibility was good with a relative standard deviation
(RSD) of 7.6% or less. Furthermore, this method can be
successfully applied to the determination of propranolol
and its metabolites in serum of patients administered
propranolol (Fig. 6).
Kataoka et al. [74] also developed an automated intube SPME coupled with LC-ESI-MS (positive ion mode,
SIM) for analyzing drugs of abuse, including amphetamine, methamphetamine and their methylenedioxy
analogues. The optimum extraction conditions were 15
draw/eject cycles of 35 mL of sample in Tris-HCl buer
(pH 8.5) at a ow-rate of 100 mL/min using an Omegawax 250 capillary.
The stimulants extracted in the capillary were easily
desorbed with a mobile phase by the dynamic desorp-
tion technique. Using this method, the detection limit

was 0.2^0.8 ng/mL (S/N=3), and linearity was
obtained in the range of 2^100 ng/mL. This method
can be directly applied to the diluted urine samples
without interferences. Recoveries of stimulants added
to urine samples were over 80%, and it was possible to
analyze them reproducibly with a RSD of 7.9% or less.
In addition, the within-day and between-day variations
for analysis of spiked urine samples were 0.9^3.0% and
2.1^6.0%, 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 samples
without interferences. On the other hand, Supel-Q
PLOT coated with the porous divinylbenzene polymer
was also used as a conventional capillary column.
Yuan et al. [77] reported an automated in-tube SPME
coupled with LC-ESI-MS of 7 benzodiazepines using this
capillary. The optimum extraction conditions were 10
draw/eject cycles of 30 mL of sample in Tris-HCl buer
(pH 8.5) at a ow-rate of 300 mL/min. The benzodiazepines extracted in the capillary were easily desorbed
with mobile phase by the dynamic desorption technique.
Figure 6. Total ion and SIM chromatograms obtained from standard propranolol and its metabolites, and a clinical serum sample by in-tube
SPME/LC/MS-SIM. (A) Standard solution containing 200 ng/mL propranolol, 50 ng/mL 4-hydroxypropranolol and 7-hydroxypropranolol, 20 ng/mL
5-hydroxypropranolol and N-desisopropylpropranolol. (B) Clinical serum sample (100 mL). LC conditions: column, Hypersil BDS C18 (5.0 cm2.1 mm
i.d., 3 mm particle size); mobile phase, acetonitrile/methanol/water/acetic acid (15:15:70:1); ow-rate, program from 0.25 to 0.45 mL/min for a 20 min
run. ESI+-MS conditions: nebulizer gas, N2 (40 psi); drying gas N2 (10 L/min, 350 C); fragmentor voltage, 70 V; capillary voltage, 3500 V;
Peaks: 1 = 5-hydroxypropranolol (m/z 276); 2 = 4-hydroxypropranolol (m/z 276); 3 = 7-hydroxypropranolol (m/z 276); 4 = N-desisopropylpropranolol
(m/z 218); 5 = propranolol (m/z 260). (Reprinted with permission from [73]. # 1999 American Chemical Society).
240

Using this method, the detection limits of these compounds were 0.02^2 ng/mL (S/N=3), and the linearities
were obtained in the 0.5^500 ng/mL range. This
method was directly applied to urine and serum
samples without interferences.
3.4. Extraction device developed for in-tube SPME
Until now, although the commercial open tubular capillary columns have been used mainly as extraction devices
in in-tube SPME, various SPME devices have been developed to improve extraction eciency and selectivity.
The development of extraction phases better suited to
extraction of relatively polar compounds from aqueous
samples will enhance the sensitivity and the overall utility of the method. Wu et al. took initial steps in this
direction with the development of a new capillary column; the inner wall of commercial fused silica capillary
was coated with polypyrrole (PPY) polymer. The PPYcoated capillary in-tube SPME coupled with LC-ESI-MS
was successfully used in the analysis of b-blockers in
urine and serum [78], stimulants in urine and hair
[79], and verapamil and its metabolites in biological
matrices [80].
Recently, Mullett et al. [81] synthesized a MIP for use
as an in-tube SPME adsorbent. The inherent selectivity
and chemical and physical robustness of the MIP material was demonstrated as an eective stationary phase
material for in-tube SPME. Using a PEEK tube packed
with MIP particles, an automated on-line in-tube SPME/
HPLC system was developed for the selective analysis of
propranolol in serum samples. Pre-concentration of the
sample by the MIP adsorbent increased the sensitivity,
yielding a limit of detection of 0.32 mg/mL by UV detection. Excellent method reproducibility (RSD < 5%) and
column reusability ( > 500 injections) were observed
over a fairly wide linear dynamic range (0.5^-100 mg/
mL) in serum samples.
Mullet et al. [82] also developed an automated biocompatible in-tube SPME method using RAM, alkyldiol-silica (ADS). The use of RAM capillary enabled
direct extraction of several benzodiazepines from serum
sample. The bifunctionality of the ADS extraction phase
prevented fouling of the capillary by protein adsorption
while simultaneously trapping the analytes in the
hydrophobic porous interior. The approach simplied
the apparatus required, compared with existing RAM
column-switching procedures. It also overcame the
existing problem that in-tube SPME requires an ultraltration or other deproteinization step prior to handling biological samples, thus further minimizing
sample-preparation requirements. Mussho et al. [83]
developed an automated solid-phase dynamic extraction (SPDE) as a technique similar to in-tube SPME,
using a hollow needle with an internal coating of PDMS
instead of a capillary column. This technique is suitable
for headspace extraction coupled with GC-MS and can
Trends
be used for the determination of amphetamines and
synthetic 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 polymer
have been developed to increase extraction eciency
[22]. Recently, Saito et al. [84] described an interesting
innovation for in-tube SPME, whereby a ne wire is
incorporated into the lumen of the extraction capillary,
to eectively increase the surface-to-volume ratio in the
analysis. However, it is thought to limit the extraction
eciency. Using this wire-in-tube SPME method
coupled with microcolumn HPLC, tricyclic antidepressants in urine samples were determined in
the 5^500 ng/mL range by UV detection. The preconcentration was between 15-fold and 110-fold that
used in direct injection.
Jinno et al [85] also developed an on-line interface
between the ber-in-tube SPME and CE, and the preconcentration and separation of the above tricyclic
antidepressant drugs were accomplished with the
hyphenated system. As shown in Fig. 7, this method
was successfully applied to a patients urine sample.
More recently, Shintani et al. [86] reported an alternative approach with an in-tube SPME technique using
a monolithic capillary column consisting of one-piece of
organic polymer or silica with a unique ow-through
double-pore structure.
4. Future prospects
As described in this article, sample-preparation techniques for LLE, IAE, LPME, MBE, SPE and SPME have been
developed recently. Of these, SPE is the most popular for
drug analysis and has become an essential tool in
laboratories all over the world. It has also largely
replaced older techniques.
The development of SPE has been fast during the past
decade, with many improvements in formats, automation and the introduction of new phases. Some promising approaches in SPE are based on special packings,
such as RAMs and MIPs.
Multi-well plates are also expected to be widely adopted in the future for automated analytical systems using
a liquid-handling robot. The introduction and implementation of automated 96-well extraction has
brought about high-throughput approaches to the biological sample-preparation techniques of SPE, LLE and
protein precipitation [3,4,7].
SPME is becoming an attractive alternative to SPE and
LLE for some applications. SPME is a solvent-free, concentrating extraction technique, and can be used in the
analysis of drugs and metabolites in biological uids.
As evidenced with SPE, IAE sorbents, RAMs and MIPs
are also expected to be applied as new sorbent materials
241
Trends
Figure 7. Electropherograms of tricyclic antidepressant drugs. (A) Fiber-in-tube SPME/CE analysis of standard mixture (0.5 mg/ml each), (B) direct
CE analysis of standard mixture (50 mg/ml each), (C) ber-in-tube SPME/CE analysis of a patients urine, (D) ber-in-tube SPME/CE analysis of a
controlled-urine spiked by 5 mg/ml amitriptyline. SPME conditions: extraction medium, HM/DB-5; packing density, 52%; extraction ow rate
and time, 80 mL/min12.5 min (1.0 mL); desorption ow rate and time, 4.0 mL/min0.45 min (1.8 mL); desorption solvent, acetonitrile.
Peaks: 1 = desipramine; 2 = nortriptyline; 3 = imipramine; 4 = amitriptyline. (Reprinted with permission from [84]. # 2001 Wiley-VCH Verlag GmbH).
for highly ecient extraction of drugs from various

biological samples by SPME. With the development of
more sensitive phases it may be possible to miniaturize
the technique further. As the market for SPME increases in the future, this could lead to the introduction of
disposable, low-cost extraction bers (e.g. in the form of
a carousel) or tubes, as in other areas of sample preparation, e.g. SPE multi-well plates.
The in-tube SPME technique is very eective as a
sample-preparation technique for qualitative and
quantitative analyses. As extraction and concentration
are combined, all of the analyte extracted is introduced
into the analytical system. The main advantages of intube SPME are simplicity, rapidity, solvent elimination,
high sensitivity, small sample volume, relatively low
cost and simple automation. The in-tube SPME technique can be successfully used for polar and non-polar
compounds in liquid samples, and can be coupled easily
with various analytical methods, such as HPLC, LC-MS
and CE. In addition, it is possible to improve the extraction eciency and selectivity by further development of
new capillaries coated or packed with new materials,
and the range of applications will spread on combination with further, dierent analytical instruments.
The trends are clearly towards:
simplifying the work involved in sample preparation;
242
increasing the reliability and precision of sample

preparation; and,
eliminating the clean-up step by using more
selective extraction procedures.
The development of more selective sorbents will
remain an active area of research with respect to IAE
sorbents and MIPs. In particular, MIP antibody mimics
are much easier to synthesize and consequently much
less expensive.
The development of SPE and SPME based on inexpensive MIPs allows anity extraction to be virtually disposable, potentially making widely available anity
extraction with excellent selectivity. Many applications
can be expected in forensic, clinical, pharmaceutical
and biochemical analyses for molecular recognition
techniques, such as:
anity extraction media that may replace biological 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 interest
in the automation of sample preparation for faster
sample-preparation procedures, improved precision

and more cost-eective analyses. The key attractive
features of automated sample preparation techniques
include miniaturization, high throughput, reproducibility and traceability.
In the last decade, new concepts have been developed
to allow the on-line coupling of sample-preparation
devices to separation and detection systems, all of
which are specially designed for automation. In the
future, advances towards better integration of
sampling/sample preparation and instrumental analysis
will allow wider use of automated on-line analysis in
forensic, clinical and pharmaceutical analysis.
Acknowledgements
This work was supported by grants from Shimadzu
Science Foundation, The Yakumo Foundation for
Environmental Sciences, Japan Food Industrial Center,
and Grants-in-Aid for Basic Scientic Research
(B(2), no. 14370729) from the Japanese Society for
Promotion of Science.
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Hiroyuki Kataoka is an associate professor of health chemistry at

the Faculty of Pharmaceutical Sciences in Okayama University,
Okayama, Japan. He will be a full professor of applied analytical chemistry at the School of Pharmacy in Shujitsu University, Okayama,
Japan, from April 2003. He received a MSc degree in 1979 from Osaka
University, Japan, and a PhD (Doctor of Pharmacy) in 1986 from
Tohoku University. From April 1998 to March 1999, he worked with
Professor Janusz Pawliszyn as a postdoctoral research fellow in the
Solid Phase Microextraction Group at University of Waterloo (Waterloo, Ontario, Canada) developing in-tube solid-phase microextraction. His research interests are in developing selective, sensitive
methods for the analysis of biologically active, potentially harmful or
chemically interesting compounds in living systems, foods and the
environment. His present research projects also cover the development of automated sample-preparation methods and applications to
environmental and pharmaceutical elds.

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New Trends in Sample Prep

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Trends

Trends in Analytical Chemistry, Vol. 22, No. 4, 2003