Anal Bioanal Chem (2007) 388:13811391
DOI 10.1007/s00216-007-1294-z
REVIEW
Recent developments in extraction procedures relevant
to analytical toxicology
Sarah M. R. Wille & Willy E. E. Lambert
Received: 9 January 2007 / Revised: 3 April 2007 / Accepted: 4 April 2007 / Published online: 28 April 2007
# Springer-Verlag 2007
Abstract Sample preparation is an important step in the
development of an analytical method but is often regarded as
time-consuming, laborious work. Optimum sample prepara-
tion leads to enhanced selectivity and sensitivity, however, and
reduces amounts of interfering matrix compounds, resulting in
less signal suppression or enhancement. Recent developments
in extraction techniques that could be of interest in clinical and
forensic toxicology, for example liquidliquid, solid-phase,
and headspace extraction, are summarized in this review. The
advantages and disadvantages of several extraction techniques
are discussed, to enable the reader to choose an appropriate
method of extraction for his or her application. Attention is
paid to current trends in analytical toxicology, for example
miniaturization, high throughput, and automation.
Keywords Solid-phase extraction .
Liquidliquid extraction . Automation . Review .
Gas chromatography . Liquid chromatography
Introduction
Optimization of the extraction of the compounds of interest
from, for example, the biological matrix is an important step
in the development of an analytical method, because it will
affect the overall sensitivity and selectivity of the method.
Sample preparation will not only lead to highly concentrated
extracts, but can remove potentially interfering matrix
compounds, resulting in enhanced selectivity and a more
reproducible method independent of variations in the sample
S. M. R. Wille : W. E. E. Lambert (*)
Laboratory of Toxicology, Ghent University,
Harelbekestraat 72,
9000 Gent, Belgium
e-mail: Willy.Lambert@UGent.be
matrix. This is of particular importance in LCMS analysis,
in which suppression or enhancement of the mass spectro-
metric signal can occur when matrix components coelute
with an analyte. Despite all these advantages, sample
preparation is still regarded as time-consuming, laborious
work, and there is much interest in simplifying this step.
Conventionally, liquidliquid and solid-phase extraction
(LLE and SPE) are chosen for non-volatile compounds
whereas headspace extraction is the state of the art for volatile
compounds. Increasing attention has recently been devoted to
reducing sample volume, analysis time, and cost, and to
eliminating the use of (chlorinated) solvents; automation is
also encouraged to reduce the workload. Methods such as
solid-phase micro-extraction (SPME) and liquid-phase micro-
extraction (LPME), and use of restricted access materials
(RAM) are, therefore, also increasing in popularity in
analytical toxicology. This paper reviews sample-preparation
techniques used in forensic and clinical, but not environmen-
tal, toxicology published since 2000, and discusses the trends
in this important analytical step. SPME will not be covered
because a critical review of this technique is presented
elsewhere in this issue. The basic principles of the reviewed
extraction techniques will not be discussed here and we refer
the reader to comprehensive textbooks on solid-phase
extraction and supercritical-fluid extraction techniques [13]
Extraction procedures for non-volatile compounds
Liquidliquid extraction (LLE)
LLE using organic solvents
Liquidliquid extraction is still frequently used in analytical
toxicology, especially for (urgent) screening purposes,
1382
when analysis of a wide range of (unknown) compounds
rather than target analysis is the objective [412]. Devel-
opment of an LLE procedure is, moreover, not time-
consuming, although it is difficult to automate, requires
high-purity solvents, and can result in the formation of
emulsions with incomplete phase separation, the last of
which leads to impure extracts. Safe disposal of toxic
solvents may also be problematic and expensive [13].
Liquid-phase microextraction (also referred to as solvent
microextraction, liquidliquid microextraction and single-
drop microextraction) was therefore introduced in 1996. It
is based on suspension of a single droplet of organic
solvent, from the end of a microsyringe needle, in an
aqueous solution. The droplet, containing analytes extracted
on the basis of passive diffusion, is injected into the GC or
HPLC. He and Kang recently developed a method for
three-phase liquidliquidliquid microextraction of meth-
amphetamine and amphetamine from urine in which
organic solvent containing the acceptor droplet was applied
on top of the sample [14]. These methods are not always
robust, however, because the droplets may be lost during
extraction. To overcome this, membrane extraction tech-
niques have been developed. Microporous-membrane
liquidliquid extraction entails use of a two-phase system
in which one aqueous phase and one organic phase are
separated by a microporous hydrophobic membrane. The
organic solvent fills the pores of the membrane and enables
direct contact of the two phases without their mixing and,
therefore, without emulsion formation. The supported
liquid membrane extraction (SLM) consists of a three-
phase system in which analytes in one aqueous phase can
be extracted into another aqueous phase through an organic
phase held between the aqueous phases by a porous,
hydrophobic membrane [1518]. Pedersen-Bjergaard and
Rasmussen developed a SLM device in which the organic
solvent is immobilised within the pores of a disposable
polypropylene hollow fibre; the analytes are extracted from
biological samples and trapped in a protected micro-extract
(550 L). As a result, consumption of organic solvents is
low and clean and highly concentrated extracts are obtained
[19]. Pedersen-Bjergaard and Rasmussen have reviewed the
use of LPME for extraction of a wide range of drugs from
plasma, whole blood, urine, and human milk [19].
Miyaguchi et al. reported another miniaturised proce-
dure, based on a microchip liquidliquid extraction, for
analysis of amphetamine-type stimulants in urine [20]. This
miniaturisation is based on pressure-driven manipulation of
liquids in fluoroalkylated microchannels in a glass micro-
chip. The organic solvent (1-chlorobutane) and the fortified
urine sample were pumped through the microchip, resulting
in an extraction of the compounds into the organic solvent,
which was collected at the end of the microchannels and
analysed by GCFID. Another development in LLE is the
Anal Bioanal Chem (2007) 388:13811391
extent of automation used to reduce laboratory workloads.
Several papers describe the application of robotics in
combination with 96-well format micro-tubes to (semi-)
automatic LLE of drugs such as paclitaxel, ondansetron,
limepiride, cyclosporin, and dextromethorphan in biologi-
cal samples [2126].
Supercritical-fluid extraction (SFE)
A compound becomes a supercritical fluid when main-
tained at a temperature and pressure above its critical point.
Under such conditions the compound is neither a gas nor a
liquid, and the state is best described as intermediate
between the two extremes. Supercritical fluids have the
solvent properties of liquids yet can be transported like
gases. SFE is, thus, a method of extraction that eliminates
the use of (hazardous) organic solvents while still having
good solvating characteristics. The solvating power of the
supercritical fluid can be adjusted by changing the pressure
or temperature, or by adding a solvent modifier, for
example methanol. Two modes of extraction are used
dynamic and static. In the dynamic mode fresh supercritical
fluid is supplied continuously; in the static mode the sample
is extracted with a fixed amount of supercritical fluid
contained in a closed extraction vessel. The analytes
extracted are isolated by trapping on an adsorbent or in a
solvent after depressurisation, or by thermal trapping
(depressurized in a cooled container) [27]. SFE using CO2
modified with methanol is widely used in analytical
toxicology for analysis of hair; several SFE methods have
been used for analysis of drugs of abuse, for example
cocaine, codeine, and morphine [28] or amphetamines [29],
in hair. Other forensic applications include analysis of
compounds on the surface of the hair, because these are
indicative of subject characteristics such as age, race,
gender, or use of hair products [30].
Solid-phase extraction (SPE)
SPE is used to extract and concentrate analytes from a liquid
matrix by partitioning the compounds between a solid phase
and a liquid phase. The objective of SPE is to remove
interfering compounds and to concentrate the analytes with
good recovery and reproducible results. It should also
facilitate rapid and efficient simultaneous processing of many
samples [31]. SPE has disadvantages which include poor
reproducibility, because of differences between batches of
adsorbents, the difficulty of standardizing the use of a
vacuum, and the variable nature of drying steps. With the
development of new (polymeric) extraction supports, inter-
batch reproducibility has been improved, however. SPE
material is, moreover, expensive and optimisation of an SPE
procedure is not always straightforward. An SPE procedure
Anal Bioanal Chem (2007) 388:13811391
consists of four consecutive stepscolumn conditioning,
sample loading, column washing, and elution. When such a
procedure is being developed, a suitable adsorbent material
and suitable washing and eluting solvents must be selected,
in accordance with the characteristics of the analytes and the
matrix, and the purpose of the analysis (screening or target
analysis). The final extract should also be compatible with
the final analytical procedure (HPLC or GC). Several types
of SPE material are available. These are usually packed into
syringe barrels which used in combination with SPE vacuum
manifolds. The volume of the syringe barrel selected
depends on sample volume, and the amount of adsorbent
(between two frits) determines the sample capacity, which is
approximately 5% of the mass of the adsorbent [13]. SPE
disks or membranes were developed to avoid the drawbacks
of classical barrels, for example reduced retention of analytes
because of channelling through the adsorbent bed, which
disturbs solvent flow, slow sample processing because of the
small cross-sectional area, which can be blocked by matrix
compounds, and variation in adsorbent density. They also
require smaller volumes of conditioning and eluting solvents.
This trend of minimizing the volume of the adsorbent bed,
resulting in a small elution volume and thus highly
concentrated extracts, is also apparent from the micro-scale
formats of the SPE pipette tip and the solid-phase micro-
extraction microfibre [13, 31, 32].
SPE adsorbents and their modes of interaction
Different types of interaction can occur between the analytes
of interest and the active sites on the adsorbent [13]. These
include both hydrophobic interactions, for example Van Der
Waals forces, and hydrophilic interactions, for example
dipoledipole, induced dipoledipole, hydrogen bonding,
and interaction. Other mechanisms include electrostat-
ic attraction between charged groups on the compound and
a charged group on the adsorbent surface, and molecular
recognition mechanisms [33]. Reversed-phase, normal-
phase, ion-exchange, and immunoaffinity adsorbents, and
molecularly imprinted polymers (MIPs), exploit one of
these interactions only, whereas mixed-mode adsorbents
combine several interaction mechanisms. Restricted-access-
matrix adsorbents (RAM) combine hydrophobic, ionic, or
affinity interactions, and large matrix components (e.g.
proteins) are excluded by appropriate selection of pore size
or by use of chemical repulsion (by applying an appropriate
hydrophilic coating to the adsorbent surface). The mode of
interaction selected depends on the demands of the method,
for example screening or target analysis, the sensitivity
required, and the final composition (for example organic
extract for GC or aqueous extract or HPLC).
Not only are the chemical characteristics of the func-
tional groups on the adsorbent relevant, but also the
1383
characteristics of the backbones to which these functional
groups are attached. The backbones are either silica or
polymer-based. Silica-based columns tend to contain a
limited number of underivatised or free silanols, resulting
in polar, acidic patches on the adsorbent surface. This
results in secondary hydrophilic or ionic interactions when
reversed-phase and ion-exchange columns are used. These
secondary interactions could be of interest, but are not
reproducible, because the extent of end-capping, and thus
the number of free silanols, can change from batch to batch.
For normal-phase adsorbents, secondary hydrophobic inter-
actions can occur, because of the small alkyl chains that
support the functional groups. Silica-based adsorbents with
a wide range of functional groups are available, are
relatively inexpensive, and are stable within a pH range of
approximately 2 to 7.5. Polymeric adsorbents (e.g. styrene
divinylbenzene) are more hydrophobic, more retentive,
stable within the pH range 0 to 14, and no secondary
interactions are observed. Newer polymers, for example
Oasis HLB, combine hydrophilic and hydrophobic inter-
actions. They do not always require conditioning and can
be used to extract analytes over a large range of polarity.
Because of their hydrophilic character, however, they can
retain water, necessitating a longer drying step, especially if
gas chromatography (with derivatisation) is used as the
final analytical method [31, 34].
Reversed-phase adsorbents Reversed-phase adsorbents are
mainly used for extraction of apolar compounds from polar
matrices, for example plasma, by use of hydrophobic
interaction mechanisms. The compounds are eluted with a less
polar organic solvent that disrupts the Van Der Waals forces.
Apolar adsorbents, such as C18, are widely used in clinical and
forensic toxicology because they are broad-range adsorbents
which can be used to extract a wide range of compounds
from a variety of biological samples (e.g. plasma, urine, brain
tissue) [3539]. Qi and Danielson recently developed mini
reversed-phase SPE tubes and extracted dicyclomine and
cyclopentolate from serum. The mini-tubes (0.5 and 1 cm
long with an internal diameter of 1.55 mm) were used to
furnish mini-extracts for their nano-ESI MS system [40].
Adsorption stationary phases and normal-phase
adsorbents Adsorption stationary phases are unmodified
adsorbents such as pure silica, magnesium silicate, diato-
maceous earth [4144] or alumina [4547]. Because silica
and alumina adsorb water they should be kept dry before
use and aqueous matrices should not be used. Water will
deactivate hydrogen-bonding sites, resulting in reduced
retention of the analytes and variable recovery. Diatoma-
ceous earth is a porous material that can act as a support for
the aqueous phase; this mechanism of extraction, called
supported-liquid extraction, is related to conventional LLE
1384
[48, 49], but problems such as emulsion formation are
avoided. Because the extraction is based on partition and
not adsorption, there is still discussion about whether use of
diatomaceous earth should be classified as LLE or SPE.
Extraction with diatomaceous earth is used in systematic
toxicological analysis (screening) [41]. Ninety-six-well
diatomaceous earth plates have been used for extraction of
indolocarbazole and carboxylic acid-based protease inhib-
itors from biological samples [4244].
Normal-phase adsorbents are created when polar func-
tional groups, for example cyano, primary amine, or diol
functionality, are bonded to the silica surface. These
adsorbents can be used to extract polar compounds from
an apolar matrix, as a result of hydrophilic interactions.
Elution with a polar organic solvent is necessary to disrupt
the hydrophilic interactions. In clinical applications these
adsorbents are mostly used to purify apolar extracts (e.g. in
hexane) of solid matrices such as body tissues [13, 33].
Ion-exchange adsorbents Extraction with ion-exchange
adsorbents exploits ionic interactions between the analytes
of interest and the functional groups on the adsorbent. By
use of this mechanism, negatively (anion exchange) and
positively (cation exchange) charged compounds can be
extracted from a biological matrix. When this mode of
extraction is used, pH control during the loading, washing,
and elution steps is important. During loading on an anion
(cation)-exchange adsorbent the pH should be two units
higher (lower) than the pKa of the compound and two units
lower (higher) than the pKa of the adsorbent. Under these
conditions more than 99% of the functional groups are
charged. During elution the pH is chosen so that the
functional groups on the compound and/or adsorbent are
not charged, resulting in disruption of the ionic interactions.
Anion-exchange adsorbents contain quaternary amino
groups of pKa>14 or aliphatic aminopropyl groups of pKa
9.8 bonded to a silica surface. For cation exchange the
choice is between a weak (carboxylic acid, pKa 4.8) or strong
(sulfonic acid, pKa<1) exchangers. The ionic strength of the
buffer and the flow-rate should also be controlled. Ion-
exchange adsorbents are used to isolate groups of either
basic or acidic drugs from biological matrices. Several SPE
procedures using ion-exchange have been developed [50
65]. Mixed-mode adsorbents combining cation or anion
exchange with a hydrophobic or polymeric adsorbent are of
interest in drug screening in forensic toxicological analysis
[51]. Because the substances present are not known in
advance, the extraction procedure must isolate a range of
compounds and remove interfering substances from the
matrix. These mixed-mode adsorbents combine several
interaction mechanisms and, by use of different elution
conditions, extracts containing neutral, basic and acidic
drugs can be obtained separately [12, 41, 6671].
Anal Bioanal Chem (2007) 388:13811391
Molecular recognition mechanisms Use of immunoaffinity
adsorbents and molecularly imprinted polymers (MIPs)
exploits molecular-recognition mechanisms for selective
extraction of trace amounts of the analytes of interest from
matrices containing large amounts of interfering com-
pounds. Because immunoaffinity adsorbents and MIPs are
tailor-made with high selectivity for a target molecule or
structural analogues, these adsorbents are not useful for
toxicological screening; they are, instead, used for highly
sensitive and specific target analysis. Several procedures in
which molecular recognition mechanisms are used are
listed in Table 1 [7285].
Immunoaffinity adsorbents consist of biological antibodies
covalently linked to silica, controlled-size glass particles, or
agarose or other soft gels, and result in high affinity and
selective antigenantibody interactions. The preparation of
these adsorbents and optimisation of immunoextraction
techniques have been reviewed elsewhere [8688]. The
antibodies can be compound-specific but usually have some
cross-reactivity with structural analogues [87]. They can also
be denatured by contact with an organic solvent, or by fungi
if stored incorrectly. The adsorbent should therefore be
preserved in buffer solution containing sodium azide and
stored in a cooled environment (4C). These adsorbents
should also be used under mild SPE conditions with regard
to pH and organic content, are expensive, and only a limited
number is commercially available [13, 34].
As a result of these disadvantages MIPs were produced
by using template molecules to create cavities in a polymer
which will recognise the target molecule. Specific
cavities are created with the same dimensions as the analyte
of interest and with the same mechanisms of interaction.
The imprint is obtained by polymerisation of functional and
cross-linking monomers in the presence of the template
molecule [89]. MIPs are stable in organic solvents, and at
higher temperatures and over a wider pH range than
immunoaffinity adsorbents. They are prepared rapidly and
easily [90]. It may, however, be difficult to remove the
target molecules completely and residual template material
may leak into sample extracts, leading to incorrect
quantification, especially at trace levels [89]. This problem
can be overcome by using a structural analogue as template.
The polymer backbone of the adsorbents can also lead to
non-specific hydrophobic binding of matrix compounds,
especially when aqueous matrices are loaded. The analytes
are therefore retained by a mixed-mode mechanism
involving both selective affinity binding with imprints and
non-specific physicochemical adsorption on the polymer
surface [82]. In such circumstances the washing step will
have a critical effect on the selectivity of the extraction.
Caro et al. have reviewed the application of MIPs to the
extraction of several drugs in a variety of biological
samples, for example plasma, urine, and serum [91].
Table 1 Molecular recognition mechanisms: immunoaffinity and molecularly imprinted polymer adsorbents

Compound Sample Hapten Conditioning step Loading step Washing step Eluent Ref.

Immunoaffinity adsorbent
Oestradiol, oestrone, oestriol Urine (4 mL) Oestradiol-BSA Sample centrifuged 1 mL 0.01 mol L1 300 L 80% 72
PBS MeOH
Morphine, morphine-3--D- Blood (150 L) N-Aminopropylnormorphine, Sample diluted with water, 41 mL PBS (pH 8), 61 mL MeOH 73
glucuronide, morphine-6-- (postmortem) N-Aminopropylnormorphine methanol, PBS (pH 8), sonicated, 0.5 mL water, 0.5 mL water (95:5)
D-glucuronide glucuronidated at position 3 centrifuged acetonewater (95:5)
or 6 coupled to BSA
Chlorophenols Urine (10 mL) 5-KLH conjugate 5 mL 70% EtOH or (Hydrolysed) urine sample diluted 5 mL 20% EtOH or 3 mL 70% EtOH 74
0.05 mol L1 glycine-HCl (2 or 3-fold) 5 mL PBS PBS
(pH 3), 5 mL PBS
Anal Bioanal Chem (2007) 388:13811391

Molecularly imprinted polymer

Clenbuterol, bromobuterol, Urine (calf) 1 mL MeOH, 1 mL water, Sample 1 mL water, 1 mL 21 mL MeOH 75
mapenterol, tulobuterol, 1 mL sodium acetate 25 ACN1% acetic acid 10% acetic acid
ractopamine, isoxsuprine, nmol L1, pH 6.7
salmeterol, formoterol
Ropivacaine Plasma (50 L) Bupivacaine 50 L MeOH, 50 L water Sample diluted 1:1 with water 50 L water 20 L MeOH 76
0.4% NH4OH
Dopamine, serotonin, Urine (4.5 mL) Dopamine HCl Sample (4.5 mL) diluted with 5 mL MeOHwater 5 mL MeOH1% 77
salbutamol, epinephrine, water (1 mL) and MeOH (4:1) acetic acid
isoproterenol (4.5 mL)
Enrofloxacin, ciprofloxacin Urine, liver (pig) Enrofloxacin 6 mL MeOH MeOH extract of sample (Oasis) 6 mL ACNacetic acid 3 mL ACN4% 78
(95:5) formic acid
Tetracycline, oxytetracycline Pig kidney (5 g) Tetracycline, oxytetracycline 10 mL waterNaOH Tissue extract adjusted to pH 2 3 mL ACN 20 mL MeOH 79
(pH 11) (buffer, homogenised, 10% 1 mol L1
centrifuged, filtration) aq. KOH
Cefathiamidine Plasma (1 mL) Cefathiamidine 8 mL water Plasma extract dried and 2 mL water30% 80
redissolved in 1 mL water (C18) acetic acid
Naproxen Urine (25 mL) Naproxen 6 mL ACNwateracetic Sample filtered, acidified to pH 3 2 mL ACN 3 mL ACN1% 81
acid (6:3:1) acetic acid
Bupivacaine, ropivacaine Plasma (400 L) Pentycaine 1 mL MeOH, 1 mL water Sample diluted with IS, citrate 2 mL MeOHwater 2 mL ACNwater 82
buffer pH 5, containing 0.1% (1/4), 0.5 mL ACN triethylamine
Tween 20 and 10% ethanol ethanol (9/1) (46:3:1)
Caffeine Urine (1 mL) Caffeine 2 mL water, 1 mL MeOH Sample diluted with alkaline Alkaline buffer (pH 9) 1 mL MeOH 83
buffer (pH 9)
Verapamil, gallopamil Urine, plasma Verapamil Sample extract (100% ACN after ACNammonium 84
RAM) acetate (0.01
mol L1, pH 3)
Alprenolol Rat plasma Propranolol 20 mmol L1 phosphate Sample 20 mmol L1 20 mmol L1 85
(50 L) buffer (pH 5.8)ACN phosphate buffer pH phosphate buffer
(80:20) 5.8ACN (80:20) pH 3.2ACN

Abbreviations: ACN, acetonitrile; BSA, bovine serum albumin; 5-KLH conjugate, 3-(3-hydroxy-2,4,6-trichlorophenyl)propanoic acid coupled to keyhole limpet haemocyanin; PBS, phosphate-buffered saline
1385
1386 Anal Bioanal Chem (2007) 388:13811391

Restricted-access matrix adsorbents Restricted-access ma-
trix (RAM) adsorbents are used in clinical toxicology to
exclude large molecules such as proteins and to extract low-
molecular-mass analytes by use of hydrophobic, ionic, or
affinity interactions. Access to the retentive part of the
adsorbent by high-molecular-weight compounds is restrict-
ed by either a physical or a chemical diffusion barrier [89].
The pore diameter is the physical barrier and the chemical
barrier is a protein or polymer network [92]. Because RAM
adsorbents do not retain or denature proteins they enable
direct injection of plasma or serum samples on to a
chromatographic column (in HPLC) without rapid deterio-
ration of chromatographic performance or clogging of the
column. Use of RAM adsorbents in pre-analytical and/or
analytical columns thus facilitates automation [92, 93].
If compounds are highly protein bound, the active sites
of the compounds that would normally interact with the
adsorbent are not available for this interaction. As a result,
the drug is carried through the adsorbent bed by the protein
instead of being retained on the column. The sample should
therefore be passed slowly through the column; this results
in a switching equilibrium between free and protein-bound
compounds. Addition of salt or modification of the pH can
also affect the extent of protein binding. Under these
conditions, however, exact measurement of the initial
amounts of physiologically bound and unbound material
is no longer possible [13, 34].
Souverain et al. have reviewed commercially available
RAM adsorbents and the methods in which they were
applied before 2004 [92], and Cassiano et al. reviewed the
history of the development of RAM adsorbents and their
applications [94]. Methods used after 2004 are listed in
Table 2. The alkyldiol silica (ADS) RAM is the RAM with
a physical barrier most often applied. The macromolecules
that are excluded are not adsorbed on the phase because of
the hydrophilic groups on its surface. Materials with
different internal surfaces are available; the interactions
range from hydrophobic (C4, C8, C18; RP-ADS)[84, 93,
9598] to weak and strong cation-exchange (XDS) [99
101]. Rbeida et al. used RAM adsorbents containing
hydrophilic diol and diethylaminoethyl groups, with weak
anion-exchange capacity, to extract acidic compounds such
as naproxen, ibuprofen, and diclofenac from plasma [100].
Another cation-exchange restricted access material (diol
groups combined with sulfonic acid groups) was developed
by the same researchers for analysis of basic drugs such as

Table 2 Restricted-access material (RAM)

Compound Sample Adsorbent Washing step Eluent Ref.

Cloxacillin Plasma (100 L) LiChrospher 60 XDS Acetic acid (0.005%)MeOH Phosphate buffer (25 mmol L1; 99
(97:3) pH 4)ACN (72:28)
Voriconazole Serum (5 L) LiChrospher RP-8 ADS Formic acid 0.1% in water ACNformic acid 0.1% in water 95
(50:50)
Verapamil, gallopamil Urine, plasma LiChrospher RP-8 ADS Ammonium acetate (0.01 ACN 84
molL1; pH 6)ACN (95:5)
Rofecoxib Plasma (50 L) LiChrospher 60 RP-18 WaterACN (95:5) WaterMeOH (50:50) with 1% 96
ADS acetic acid
Furosemide Diluted serum LiChrospher RP-18 WaterACN (85:15) pH 2.7 WaterACN (75:25) pH 2.3 97
(50 L) ADS (adjusted with H3PO4) (adjusted with H3PO4)
Aspartic, glutamic, Plasma (100 L) LiChrospher 60 XDS Acetic acid (0.005%)MeOH Phosphate buffer (25 mmol L1; 100
ascorbic acids, (97:3) pH 5 pH 2.9)MeOH (98:2)
acetylcysteine
Naproxen, ibuprofen, Plasma (100 L) LiChrospher 60 XDS Sodium acetate (2 mmol L1) Phosphate buffer (25 mmol L1; 100
diclofenac MeOH (97:3) pH 5 pH 2.9)MeOH (98:2)
Fluoxetine Filtered plasma RAM BSA-C18 Water ACNammonium acetate 102
(7 mmol L1) (70:30)TEA (17
mmol L1) pH 5.0
Cyproterone acetate Plasma (30 L) LiChrospher RP-4 ADS WaterACNformic acid Watermethanolformic acid 98
(90:10:0.1) pH 4 (10:90:0.1) pH 4
Atropine Plasma (200 L) LiChrospher 60 XDS 2 mmol L1 lithium ACNpotassium phosphate 101
perchlorateMeOH (97:3) pH 3 buffer (50 mmol L1; pH 3)+
2 mmol L1 1-heptanesulfonic
acid sodium salt (16:84)
Methadone+metabolite Diluted serum LiChrospher RP-4 ADS Ammonium acetateacetic acid Ammonium acetateacetic acid 93
(20 L) (10 mmol L1; pH 6)ACN (10 mmol L1; pH 6)ACN
(95:5) (50:50)

Abbreviations: ACN, acetonitrile; TEA, triethylamine

Anal Bioanal Chem (2007) 388:13811391
atropine [101]. The most important RAMs using a chemical
barrier are the semi-permeable-surface (SPS) adsorbents
which use a polymer as the barrier, protein-coated silica, for
example bovine serum albumin (BSA)-coated adsorbents
[102], and the shielded-hydrophobic-phase (SHP) adsor-
bent, a silica-based material coated with a hydrophobic
network of poly(ethylene oxide) with embedded hydropho-
bic phenyl groups, commercialised as Hisep.
Extraction procedures for volatile compounds
The headspace technique is the best method for extraction for
volatile compounds because it minimizes use of organic
solvents, eliminates non-volatile interferences from the sample
matrix, and is not labour-intensive. The technique can be used
in two modesstatic and dynamic [103]. In the static mode,
partition and eventual equilibrium of the analyte between the
sample and the gas phase occurs in a closed system. After
equilibrium is achieved the vial is pressurised and the
headspace is sampled and injected into the gas chromato-
graph. Static headspace extraction is still popular for volatile
compounds such as fluorinated inhalation anaesthetics,
-hydroxybutyric acid, ethanol, methanol, acetone, and
isopropanol in analytical toxicology [104108]. This tradi-
tional form of headspace analysis has been modified to
enhance the sensitivity, by using concentration effects (HS-
SPME, purge and trap, SPDE, HS-SME). Dynamic headspace
extraction (purge and trap) is accomplished by passing carrier
gas continuously above or through the sample and trapping
evaporated volatile compounds in a cryogenic and/or adsorbent
trap. The trap is then heated to release the analytes, which are
transported to the chromatographic column. Examples of this
procedure have been described for trichloroethane, trichloro-
acetic acid, benzene, toluene, ethylbenzene, xylene, and styrene
[108111]. In solid-phase dynamic extraction (SPDE) the
headspace is passed through a needle coated with polydime-
thylsiloxane, used as an extraction and preconcentration
medium. Musshoff et al. and Lachenmeier et al. published
methods using SPDE to extract drugs of abuse from hair
samples [112, 113]. Headspace solvent micro extraction (HS-
SME), in which a single drop of a high-boiling solvent is
suspended from an injection needle in the headspace above the
sample in a closed vial, has recently been developed [114].
Another means of enhancing sensitivity is to increase the
injection volume by combining a headspace technique with a
cooled inlet or with a cooled GC column, also called cryogenic
focusing or oven trapping, respectively [108, 110, 115117].
Of all the HS techniques, however, headspace solid-phase
micro extraction (HS-SPME) is the method attracting most
interest in forensic and clinical toxicology [108, 118122]; this
technique is discussed elsewhere in this issue.
1387
Automation
Increasing attention is being devoted to the possibility of
automating analytical methods. Automation results in great-
er sample throughput, improved precision and accuracy, and
a minimum of operator intervention, leading to safer sample
handling and time-saving procedures. Carry-over and less
control over the system can result in systematic errors,
however, and analyte stability must be investigated when
samples are analysed sequentially [123].
Several papers describe the application of robotics in
combination with 96-well format micro-tubes to (semi)
automatic LLE of drugs in biological samples [2126, 124,
125]. For SPE analysis robotics are incorporated to reduce
the sample workload [126], and several new SPE formats
and adsorbents have been developed for greater compati-
bility with automation or for greater sample throughput.
Solid-phase-extraction adsorbents that do not need a
conditioning step and new SPE formats such as the 96 or
384-well plates [51, 127138] have been developed for
rapid sample preparation, resulting in high-throughput
analysis [13]. Fully automated liquid-chromatographic
methods using column-switching, are also possible, espe-
cially with developments such as RAM (see section
Restricted-access matrix adsorbents) and large particles
[92, 93, 139] on which biological samples can be injected
without pre-treatment. Gas chromatography or on-line
supercritical-fluid extraction [140] in combination with
headspace extraction and SPME are more robust than when
used in combination with, for example, SPE.
Relevant advantages of the on-line technique include
reduced risk of sample contamination, suitability for full
automation, and analysis of the complete sample (the whole
extract is analysed), leading to improved sensitivity. When
automating a SPE method one must keep in mind that a
manual procedure cannot be transferred without minor
modifications. Pressures, flow-rates, and solvent composi-
tions (stability!) must be optimized again.
On-line systems for liquid chromatography
Although Mauri-Aucejo et al. have described on-line liquid
liquid extraction of amphetamine from urine [141], on-line
solid-phase extraction seems to be gaining popularity. The
most common on-line SPE system for liquid chromatogra-
phy (LC) involves use of a small precolumn located in a six-
port, high-pressure switching valve. This method is referred
to as column-switching. When the sample is injected, it is
preconcentrated on a precolumn that is pressure resistant
and of small dimensions, to avoid band broadening. The
interferences are then washed from the precolumn and
flushed to waste. The valve then switches to elute the
analytes on to the analytical column. To avoid band-
1388
broadening, a back flush desorption mode can be used,
although this may result in blockage of the analytical
column when biological samples are analysed. The col-
umn-switching method has limitations, for example the need
to use small pre-columns containing a limited amount of
adsorbent, resulting in a small breakthrough volume [98,
142149]. An alternative is use of a six-port valve as an
interface for switching arrangements. In this system the
precolumn is not placed in the valve and preconcentration
can be achieved in low or high-pressure mode. The
advantage of this method is that larger pre-columns can be
used [90, 150]. Unlike in off-line SPE, in which different
types of adsorbent are preferred during extraction and
separation, the pre-column adsorbent and the analytical
column should be complementary in on-line mode, because
they must endure the same mobile phases. The choice of
adsorbents is, therefore, restricted.
On-line SPE-LC is very well suited for analysis of
biological fluids such as urine and plasma, especially in
combination with RAM [98, 143, 144, 151] and large-
particle-size (LPS) [142, 147149] adsorbents. These
adsorbents can be used in a pre-column or can function as
pre-column and analytical column. RAM supports can
exclude macromolecules such as proteins by use of physical
and chemical barriers whereas LPS adsorbents consist of
large particles (30 or 50 m) of silica or polymeric material,
resulting in interaction with the analytes and passage of
Fig. 1 System for automated on-line SPEGCMS. 1, solvent
channels; 2, purge leak restriction; 3, waste; 4, single-piston LC
pump; 5, SPE precolumn; V1V3, Prospect valves; SDU, solvent
delivery unit; SVE, solvent vapour exit; OCI, on-column injector.
(Reprinted from: Louter AJH et al. (1996) J Chromatogr A 725:6783;
Copyright (1996), with permission from Elsevier.) Samples are
concentrated on the SPE precolumn between valves V1 and V2. The
Anal Bioanal Chem (2007) 388:13811391
protein at high mobile-phase flow rates without generating
a high back pressure in the system.
On-line systems for gas chromatography
Whereas on-line coupling of SPE with LC is robust, on-line
coupling of SPE with GC is more complicated, because of
the incompatibility of the aqueous solvents used for SPE
with the stationary phases used for GC. The need for
derivatisation and small injection volumes also makes on-
line SPEGC more complex and can limit the development
of on-line GC procedures in clinical and forensic toxicology.
Several systems have been developed, these use either six-
port valves in combination with a drying gas or a solvent-
vapour exit. In large-volume transfer the solvent evaporation
technique is critical to obtaining efficient chromatography.
On-column and loop-type interfaces are usually used with a
retention gap and fully concurrent solvent evaporation
techniques, respectively (Fig. 1). The on-line configuration
is of interest for biological samples, because of the high
sensitivity and small amount of sample required. Compared
with applications in environmental analysis, however, use in
the biomedical sciences is much less widespread, mainly
because of the need for derivatisation [152]. On-line
derivatisation in the GC column inlet has, however, already
been reported [152]. Hytylinen and Riekkola have
reviewed on-line SPEGC methods for plasma, urine, and
elution solvent is delivered by means of the syringe pump, and the
elution phase is injected on-column, on to the retention gap connected
with a retaining precolumn. This column is connected to the analytical
column and a solvent-vapour exit, which is opened just before
introduction of the eluent into the retention gap and closed just before
full evaporation of the eluent, leaving the volatile analytes in a small
zone before entering the analytical column
Anal Bioanal Chem (2007) 388:13811391
tissue samples. These methods have better detection limits
and repeatability, and shorter analysis time and lower
solvent consumption, than off-line procedures [152].
For practical reasons, on-line SPE combined with GC is
not always robust. Other techniques, for example SFE,
headspace extraction, SPME [153], and microextraction in
a packed syringe (MEPS) [154] thus seem more suited to
on-line GC methods.
Conclusion
Trends in sample preparation in analytical toxicology are to
reduce the use of solvents, to simplify manipulations, and
to reduce the time necessary for sample preparation.
Miniaturisation, high-throughput systems, and automation
are of interest.
In liquidliquid extraction the volume of hazardous
solvents is reduced by such techniques as liquid-phase
microextraction and membrane techniques. Use of super-
critical fluids can even lead to extractions without organic
solvents.
This miniaturisation is also seen in SPE, with the
development of the SPE pipette tip or the SPME technique.
Increasing attention is also being devoted to automation,
because it leads to safer sample handling, higher through-
put, and time-saving. These new automated methods are not
frequently used in routine applications, however, because of
the need for further optimisation, higher instrumental costs,
and increased system complexity. Off-line automatic work-
stations are often used because they reduce workload and
offer flexibility. This flexibility can be relevant in avoiding
problems during derivatisation or because of matrix
precipitation during analysis. The high protein content of
plasma and blood can lead to problems such as precipita-
tion after contact with buffers or solvents, resulting in
blocked SPE tubes and analytical columns. The develop-
ment of RAM and LPS adsorbents can therefore lead to
increased use of on-line methods for analytical toxicology.
Thus, although off-line automation is frequently used in
analytical toxicology, on-line procedures should become
more robust and less complex, and thus become more
valuable in everyday routine analysis. Recent develop-
ments, however, seem very encouraging for the future.
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