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Microextraction by packed sorbent of
drugs and peptides in biological fluids
Seyed Mosayeb Daryanavard
Licentiate Thesis
Department of Analytical Chemistry
Stockholm University
2013
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© Seyed Mosayeb Daryanavard 2013
ISBN: 978-91-7447-621-7
Printed by Universitetsservice US-AB, Stockholm, Sweden, 2013
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Abstract
Sample preparation as the first step in an analytical procedure has an important role,
particularly in bioanalysis, because of the complexity of biological samples (blood plasma
and urine). Biological matrix such as plasma and blood contains proteins, organic and
inorganic salts, acids, bases and various organic compounds with similar chemistry to the
analytes of interest. Thus the basic concept of a sample preparation method is to convert a real
matrix into a format that is suitable for analysis by an analytical technique. Therefore the
choice of an appropriate sample preparation method greatly influences the reliability and
accuracy of the analysis results.
The aim of this thesis was to develop and validate of microextraction by packed syringe
(MEPS) as a fast, selective, accurate and fully automated sample preparation technique for
determination of BAM peptides in human plasma and local anaesthtics in human plasma and
urine samples using silica and polymer sorbents.
First work presents use of MEPS technique online with LC-MS/MS as a tool for the
quantification of BAM peptide in plasma samples. MEPS technique provides significant
advantages such as the speed and the simplicity of the sample-preparation process. Compared
with other extraction techniques, such as protein precipitation and ultrafiltration, MEPS gave
cleaner samples and higher recovery.
In the second work, MEPS technique was developed by using synthesized molecularly
imprinted polymer (MIP) as a sorbent for selective quantification of a homologous series of
local anaesthetics, containing lidocaine, ropivacaine, mepivacaine, and bupivacaine in human
plasma and urine samples. Compared with other conventional sorbent, the use of MIP
provides high selectivity of the extraction and decrease the matrix effect.
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List of Papers Included in the Thesis
I Microextraction by packed sorbent and liquid chromatography-tandem mass
spectrometry (MEPS-LC-MS/MS) as a tool for quantification of peptides in
plasma samples: Determination of sensory neuron-specific receptors agonist
BAM8-22 and antagonist BAM22-8 in plasma samples.
Nadia Ashri, Mosayeb Daryanavard and Mohamed Abdel-Rehim, Biomedical
Chromatography, Accepted for publication.
II Molecularly imprinted polymer in microextraction by packed sorbent for the
simultaneous determination of lidocaine, ropivacaine, mepivacaine and
bupivacaine in plasma and urine samples.
Mosayeb Daryanavard, Amin Jeppsson-Dadoun, Lars I Andersson, Mahdi
Hashemi, Anders Colmjsö and Mohamed Abdel-Rehim, J. Chromatogr. A,
Submitted.
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Abbreviations
BAM Bovine Adrenal Medulla
ACN Acetonitrile
C2 Ethyl Silica
C8 Octyl Silica
C18 Octadecyl Silica
DMSO Dimethyl Sulfoxide
ENV+ Polystyrene-DivinylBenzene Copolymer
ESI Electrospray Ionization
GC Gas Chromatography
I.D. Inner Diameter
I.S. Internal Standard
LC Liquid Chromatography
LLE Liquid-Liquid Extraction
LOD Limit of Detection
LLOQ Lower Limit of Quantification
LPME Liquid Phase Microextraction
MEPS Microextraction by Packed Sorbent
MeOH Methanol
MIP Molecularly Imprinted Polymer
MS Mass Spectrometry
MS/MS Tandem Mass Spectrometry
Mw Molecular Weight
PPT Protein Precipitation
QC Quality Control Samples
SBSE Stir-Bar Sorptive Extraction
SPE Solid-Phase Extraction
SPME Solid-Phase Microextraction
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Table of Contents 1. Introduction ..................................................................................................................................... 7
1.1. The role of sample preparation in bioanalysis ........................................................................ 7
1.2. Common sample preparation techniques ............................................................................... 7
1.2.1. Protein precipitation (PPT) .............................................................................................. 7
1.2.2. Liquid–liquid extraction (LLE) .......................................................................................... 8
1.2.3. Solid-phase extraction (SPE) ............................................................................................ 8
1.2.4. Miniaturized solid-phase extraction ................................................................................ 9
1.3. Microextraction in packed syringe (MEPS) ........................................................................... 10
1.4. Molecularly imprinted polymers (MIPs) ............................................................................... 11
1.5. Liquid chromatography (LC) ................................................................................................ 12
1.6. Mass spectrometry (MS) ....................................................................................................... 13
1.7. The role of LC-MS/MS in bioanalysis .................................................................................. 14
1.8. Studied analytes ..................................................................................................................... 15
1.8.1. Local anaesthetics ............................................................................................................. 15
1.8.2. Studied peptide ................................................................................................................. 16
2. Results and discussion ....................................................................................................................... 17
2.1. MEPS conditions ......................................................................................................................... 17
2.2. MEPS method development ....................................................................................................... 17
2.2.1. Type of sorbent ................................................................................................................... 18
2.2.2. The washing solution ........................................................................................................... 19
2.2.3. The elution solvent .............................................................................................................. 21
2.3. Selectivity ................................................................................................................................... 22
2.4. Carry-over ................................................................................................................................... 24
2.5. Sample loading cycles ................................................................................................................. 25
2.6. Matrix effect (ME) ...................................................................................................................... 25
3. Conclusions ........................................................................................................................................ 27
References ............................................................................................................................................. 28
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1. Introduction
1.1. The role of sample preparation in bioanalysis
To achieve accurate and precise measurement of drugs and metabolites in biological samples
such as blood, plasma and urine a suitable sample preparation method is required. Despite the
development of highly efficient analytical instrumentation, most of them cannot handle the
complex matrix directly. Therefore, one of the major trends in bioanalysis is to develop fast
and efficient sample preparation procedure to achieve good extraction. The extent of sample
pre-treatment depends on the complexity of the sample, and is especially when the analytes of
interest have similar chemical and physical properties as endogenous compounds in biological
matrices. Traditional sample preparation methods such as protein precipitation (PPT), liquid–
liquid extraction (LLE) and solid phase extraction (SPE) are time and solvent consuming and
of highly cost.
In recent years the trends have been towards:
• Increased potential for automation or for on-line methods.
• A more environmentally friendly approach, more greener (less solvents and waste)
• The ability to handle smaller sample volumes.
• More selectivity using more selective material.
Therefore, an ideal sample preparation method should involve a minimum number of working
steps and it should be fully automated. Off-line sample preparation is limiting step to get fast
bioanalysis. This indicates the clear need of on-line sample pretreatment for speeding up the
sample preparation.
1.2. Common sample preparation techniques
1.2.1. Protein precipitation (PPT)
Protein precipitation is widely used sample preparation method in bioanalysis. Protein
precipitation is suitable for plasma or blood in which the analyte concentration is relatively
high.
The most common type of precipitation for proteins is addition of an organic solvent
(methanol, acetonitrile) or salt induced or by changing the pH by adding acids such as
sulphoric acid or hydrochloric acid [1]. The insoluble form which is obtained as a precipitate
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(typically an easy to sediment solid) is subsequently separated from the dissolved components
by appropriate solid-liquid separation techniques such as centrifugation.
Protein precipitation or deproteinization is a relatively simple and minimally eliminates
interferences from large molecules. The advantages of PPT are: simplicity, universal
procedures, normally no pH modification and speed. The disadvantages of PPT are: sample
dilution, ion suppression in MS since the efficiency of protein removal is not complete, high
back pressure and accumulation in LC system [1-4].
1.2.2. Liquid–liquid extraction (LLE)
LLE is one of the oldest and widely used methods for sample preparation. It is based on the
analyete partitioning between two immiscible solutions (aqueous/organic solvents). It has
been widely used for the preparation of aqueous and biological samples for many years [1, 5-
9]. In LLE the analytes are extracted in the uncharged form or as an ion pair by adjusting the
sample pH. Solvent polarity, modifiers, solvent volumes and sample pH have an important
role in LLE.
LLE is labor intensive, time consuming and not easily amenable to automation. LLE
generally requires large amounts of solvent consumption and solvent evaporation, which have
environmental, safety, and health concerns. Finding a suitable solvent system and conditions
may be difficult for simultaneous extraction of analytes with different polarities. There is also
a tendency for emulsion formation, which introduces further complications.
1.2.3. Solid-phase extraction (SPE)
SPE technique was introduced for sample preparation in the mid-1970s [1, 10-15]. In this
technique, first the SPE sorbent is conditioned with methanol and water and then the sample
flows through the SPE column and the analytes are retained on the sorbent (silica based or
polymer). Washing step is required to remove interferences from the analytes of interest from
which they are subsequently eluted with a small amount of solvent. Isolation and enrichment
are thus achieved in a single step. SPE is analogous to offline gradient liquid chromatography
and has overtaken LLE in popularity as it lacks a number of the drawbacks associated with
LLE. Whereas only water-immiscible solvents can be used to isolate the analytes in LLE,
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there are almost no restrictions in the choice of mobile phase for the SPE procedures. Another
advantage of this methodology is the wide sorbent choice, ranging from nonpolar supports to
metal-loaded sorbents with covalent interactions. Cartridge columns with a wide variety of
bonded phase packing materials are commercially available.
Because SPE requires relative small solvent volumes compared to PPT and LLE, common
experimental equipment, provides simple experimental procedure, and a rapid sample
throughput, it has been widely accepted as an alternative to LLE for the sample preparation,
especially for the clean-up and enrichment of organic compounds in water samples.
As a rule of thumb, solvent volumes between 4 and 8 times the bed volume are needed to
ensure appropriate conditioning, washing, and elution. SPE cartridges require prior
conditioning to remove any impurities from them and make the sorbent compatible with the
sample solvent and allow sample retention.
More recently, miniaturized SPE formats have been developed. These allow use of smaller
sample volumes and elution volumes, provide large flow area, and avoid channeling in
cartridge between conditioning and prior sample application.
1.2.4. Miniaturized solid-phase extraction
The main format in SPE is the syringe-barrel and cartridge type. The SPE cartridge is a small
plastic or glass open-ended container filled with adsorptive particles of various types and
adsorption characteristics. Limitations of packed SPE conventional cartridges include
restricted flow-rates and plugging of the top frit when handling water-containing suspensed
solids such as surface water or wastewater. Cartridge design has certain disadvantages for
water analysis. For example, the cross-sectional area is small, therefore sample processing
rates are slow and the tolerance to blockage by particles and adsorbed matrix components is
low, and channeling reduces the capacity.
In recent years, a green approach for the development of sample preparation techniques has
attracted much attention by analytical chemists. Miniaturized SPE formats such as SPE disks
and pipette tips by minimizing the extracting phases to a micro scale, leads to inexpensive,
fast and environmentally friendly analysis with high enrichment factor. The sorbent particles
embedded in the disks are smaller than those found in the cartridges. One of the disadvantages
of using disks instead of cartridges is the decrease in the breakthrough volume, mainly for
more polar compounds. For this reason, disks are used when there is a strong interaction
between the analyte and the sorbent [16].
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The automation of SPE began to drive new formats to cope with the automated systems. New
formats for SPE provide reduced bed masses, high-throughput capabilities and greater
convenience for method development. A small mass of the bed allows faster method
development, reduced solvent volume and shorter sample preparation times. The transfer of
manual methods to automated methods has been improved by the advent of removable,
flexible cartridges that can be used manually or in an automated environment.
Further, miniaturization resulted in development of new extraction techniques such as solid-
phase microextraction (SPME) [17], stir-bar sorptive extraction (SBSE) [18] and
microextraction by packed sorbent (MEPS).
1.3. Microextraction in packed syringe (MEPS)
MEPS was recently introduced as a novel method for sample preparation, being a
miniaturization of the conventional SPE technique, in which the sample volume, extraction
and washing solvents volumes being greatly reduced compared to SPE. MEPS differs from
commercial SPE in that the packing is integrated directly into the syringe and not into a
separate column. Moreover, the packed syringe can be used several times, more than 100
times using plasma or urine samples, whereas a conventional SPE column is used only once.
MEPS can handle small sample volumes (10 µL plasma, urine, or water) as well as large
volumes (1000 µL).
Fig. 1 Schematic picture of MEPS syringe.
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In MEPS, a small amount of sorbent is packed into a syringe. The sample is slowly withdrawn
through the sorbent and analytes are adsorbed onto the material. A washing step is introduced
to eliminate potential interferences by selective keeping the analytes of interest. Then,
analytes are eluted with an organic solvent, directly into the injector of the instrument. By
contrast, MEPS is carried out in a closed system, so avoiding analyte losses. MEPS opened
the way for on-line coupling to GC or LC, allowing sample preparation and analysis without
any modification of the chromatographic system. Once the sorbent has been pre-conditioned,
the syringe can be connected to the instrument autosampler for further uses. This approach to
sample preparation is very promising for many reasons, as it is an easy-to-use, rapid, fully
automated, on-line procedure that reduces the volumes of solvent and sample. Thus, the cost
of analysis is minimal compared to conventional SPE. MEPS uses the same sorbents as
conventional SPE columns, so any sorbent material such silica based (C2, C8, C18), strong
cation exchanger using sulfonic acid bonded silica, restricted access material (RAM), hilic
carbon, polystyrene-divinylbenzene copolymer (PS-DVB/ENV+) and specially molecular
imprinted polymers (MIPs) can be used. MEPS has been used to extract a wide range of
analytes in different matrices. Several drugs and metabolites have been extracted from
biological samples such as blood, plasma or urine using MEPS technique. [19-40].
1.4. Molecularly imprinted polymers (MIPs)
MIPs are synthetic polymers made with specific recognition sites that are complementary in
shape, size, and functional group, to the analyte of interest. Due to the high specificity to the
target analytes, MIPs can be used to extract and isolate target analytes from complex
mixtures, such as food, environmental and biological samples. The molecular imprinting
technique has been demonstrated to be an effective strategy for preparing robust materials
with specific recognition characteristics [41-44, 57].
The imprinting process normally involves co-polymerization of functional monomers and a
cross-linker in the presence of a template. After polymerization, the functional groups of
monomers are ‘‘frozen’’ in the position where they initially form the complex with the
template by the highly cross-linked polymeric structure. Subsequent removal of the template
leaves a binding site that is complementary in size, shape and functionality to the analyte. In
this way, the imprinted material is endowed with a ‘‘molecular memory’’ and exhibits
specific binding capability for the template and/or structurally- related compounds.
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Molecularly imprinted materials prepared by various methods possess high chemical and
thermal stability, and can be generated rapidly and inexpensively, to make miscellaneous
devices (e.g., catalysts, sensors, solid-phase adsorbents and stationary phases) [10].
Fig. 2 Schematic representation of the molecular imprinting principle [63].
In addition to the SPE materials mentioned above, molecularly imprinted polymers (MIPs)
have been utilized [41-44]. As the MIPs can selectively rebind with the template (analyte) and
its analogous structures, they present specific molecular recognition ability and high binding
affinity to the template molecules. Therefore, the MIPs can be used as adsorbents for the
selective recognition and enrichment of the target analytes in complex matrices.
The selectivity of the MIPs led to improved sample clean-up, which may facilitate down-
stream analytical separation and quantitation. MIPs rely on affinity interactions and
potentially offer a higher degree of sample clean-up efficiency than that achieved using
conventional type SP sorbents. Characteristic for MIPs is its high ligand selectivity and
affinity, where selectivity can be pre-determined for a particular analyte by the choice of
template used for MIP preparation. MIPs may be a viable alternative to the more traditional
sorbents for extraction pre-concentration.
1.5. Liquid chromatography (LC)
Liquid chromatography was described in the early 1900s by the Russian botanist, Mikhail S.
Tswett when he tried to separate compounds from leaf pigments using a column packed with
adsorbent bed and a solvent. The high speed liquid chromatography was recognized in the late
1960’s, when Kirkland, Huber, and Horvath published their work on the development of high
performance liquid chromatography as it is known today, the use of high pressure liquid
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chromatography to generate the flow required for liquid chromatography in packed columns
[45].
Liquid chromatography (LC) defined in its simplest terms can be regarded as the separation of
components of a mixture based upon the rates at which they elute from a stationary phase
typically over a mobile phase gradient. Differing affinities of the mixtures components for the
stationary phase and mobile phase leads to their separation, since certain components will be
more attracted to the mobile phase and will elute quickly whilst others will be retained by the
stationary phase for longer and therefore will elute more slowly, i.e. have a later retention
time (tR). Column chromatography in its simplest form may employ a mobile phase of
constant single composition in a procedure termed isocratic elution. A disadvantage of
isocratic elution is that peak width increases when analyte “tR” increases, thus leading to peak
broadening and flattening for late eluting peaks. In a later development known as gradient
elution, the mobile phase composition is altered during the chromatographic separation
process. In reverse-phase LC employing typical silica based columns, the two mobile phase
components are referred to as solvent ‘A’ and ‘B’, ‘A’ typically being a highly aqueous
solvent which elutes sample components from the stationary phase slowly, while ‘B’ is
typically a highly organic solvent which rapidly elutes sample components off the column’s
stationary phase. The elution gradient steps and time periods between them can be optimized
for specific matrices so that the maximum number of components can be resolved within a
reasonable analysis time that lends itself to high sample throughput.
1.6. Mass spectrometry (MS)
MS in simple terms functions to detect the mass-to-charge ratio (m/z) and abundance of the
various analytes generated during ionisation of a sample extract or chromatographic fraction.
Ionisation is a key step since ions are far more easily manipulated than neutral molecules. The
three principal components found in all varieties of MS are an ionisation source, a mass
analyser and a detector; all three components are maintained under vacuum to optimise the
transmission of ions to the analyser and detector [46]. In spite of the first mass spectrometer
(MS) was constructed by Sir J. J. Thomson for more than hundred ten years ago (1897) for
the determination of mass-to-charge ratios of ions, today it is almost ubiquitous research
instrument [47].
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1.7. The role of LC-MS/MS in bioanalysis
Today, the mass spectrometry (MS) is one of the most powerful analytical techniques,
particularly in pharmaceutical analysis where good selectivity and high sensitivity are often
needed. Liquid chromatography mass spectrometry (LC/MS) has become highly developed
tool for the determination of drugs in biological samples.
Fig. 3 LC-MS/MS system equipped with autosampler.
The more recent developments in ionisation technologies make mass spectrometry an
important tool for biological research. In the pharmaceutical industry the measurement of
drug level in plasma answer key questions that are asked during drug discovery and
development.
LC coupled to single quadrupole gained popularity and became one of the preferred
techniques for analysing polar compounds in biological, environmental and food samples [48,
49]. Single quadrupole offered good sensitivity, but when very complex matrices were
investigated, insufficient selectivity often compromised the unequivocal identification of the
target analytes. Therefore liquid chromatographic techniques coupled to tandem mass
spectrometry (MS/MS) emerged as a new solution for the selectivity and sensitivity required
for some compounds, and it is, nowadays, the most broadly used instrumental techniques in
the quantitative analysis of pharmaceuticals and organic pollutants [49]. Tandem MS/MS is a
term, which comprises a number of mass spectrometers where one stage of the mass
spectrometer is used to isolate an ion and the second stage is used to probe the relationship
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between the first isolated ion and the generated ones. There are different types of instruments
with MS/MS capability like triple quadrupole (QqQ), quadrupole time-of-flight (QqTOF),
quadrupole ion trap (QIT), and quadrupole-linear ion trap (QqLIT). As the quantitation of
organic pollutants at low concentrations involves both high sensitivity and selectivity against
the complex matrix background the best-suited instruments are low resolution machines using
QqQ and QLIT design. LC–MS systems almost exclusively rely on “soft ionization” API
interfaces, namely atmospheric pressure chemical ionization (APCI) and electrospray
ionization (ESI). For compounds of moderate to high polarity, ESI constitutes the most
important ionization technique in MS coupled to LC for the analysis of organic contaminants
(<1000 Da) [49]. MS is just one of many varieties of detector that can be linked to LC.
However, to acquire the volume of information from an analyte required for full structural
elucidation, it is a necessity to employ MS. That is why LC–MS has become a technique
which can be used routinely in a wide variety of analytical laboratories and application areas.
1.8. Studied analytes
1.8.1. Local anaesthetics
Local anaesthetics are weak bases (pKa values: 7.0-8.2) and have a common chemical
structure, consisting of a lipophilic aromatic ring, a link and a hydrophilic amine group; (Fig.
4) and most of these drugs are tertiary amines. They can be classified into two groups based
on the nature of the link: amides [-NH-CO-] and esters [-O-CO-]. The amide-type group is the
most commonly used clinically and includes drugs such as lidocaine, mepivacaine,
ropivacaine and bupivacaine. Local anaesthetic agents cause temporary blockade of nerve
impulses producing insensitivity to painful stimuli in the area supplied by that nerve. Local
anaesthetics work by blocking the inward Na+ current at the sodium ionophore during
depolarization, which prevents propagation of the axonal action potential [50, 51].
Mixtures of local anesthetics used to improve and modify block duration, reduce toxicity and
consequently to attain good synergetic anesthetics effect in clinic [52]. These anesthetic drugs
are extensively metabolized by the liver and excreted in the in human urine [53]. Toxic,
potentially lethal, effects may occur if local anaesthetic agents are administered in excessive
doses or if appropriate doses are accidentally administered intra-vascularly.
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Bupivacaine Lidocaine
Mepivacaine Ropivacaine
Fig. The chemical structure of the local anaesthateics (pKa values: 7.0˗8.2)
1.8.2. Studied peptide
Bovine adrenal medulla (BAM 8-22) is endogenous peptide fragment that is a selective
agonist for the sensory neuron specific receptor; first isolated from bovine adrenal medulla.
BAM 8-22 is a potent ligand for the human SNSR receptors (agonist), while BAM 22-8 is an
antagonist [54].
BAM8-22 is a derivative of BAM22 and a synthesized peptide with 15 amino acids. This
peptide differs from BAM22 in that it does not contain the classical opioid YGGF motif of
BAM22. The BAM8-22 peptide displays longest duration of action compared with other
SNSR agonists that have been synthesized to date [55, 56].
Fig. 5 Amino acid sequence for BAM 8-22 and BAM 22-8
BAM 8-22
H2N-Val – Gly – Arg –Pro – Glu – Trp – Trp – Met – Asp – Tyr – Gln – Lys –Arg – Tyr –Gly-OH
BAM 22-8 H
2N-Gly – Tyr – Arg – Lys – Gln – Tyr – Asp – Met – Trp – Trp – Glu – Pro – Arg – Gly – Val-OH
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2. Results and discussion
2.1. MEPS conditions
In Papers I and II, 250µL gas-tight syringe was used to perform MEPS technique. The solid
sorbent material (2 mg) was manually inserted inside the syringe as a plug. The sorbent
material was tightened by normal polyethylene frits to avoid moving inside the syringe. In
Paper I Silica-C8 was used as sorbent while molecularly imprinted polymer was used in Paper
II.
In general MEPS extraction procedures are similar as in SPE: sample loading, washing, and
elution. In MEPS extraction procedures, additional steps for post-cleaning and re-conditioning
have to be included to enable multiple uses of MEPS sorbent. Before using for the first time,
the sorbent was manually conditioned with methanol followed by water. After activation,
sample loading was performed by taking replicate aliquots of the diluted plasma or urine
sample. This was done by multiple aspirates-dispense cycles through the MEPS syringe. Then
the sorbent was washed once with proper washing solution to remove salts, proteins and other
interfering materials. Afterward, analytes were eluted from the sorbent and injected into LC-
MS system. The sorbent was washed after each extraction using elution solution (4 times)
followed by washing solution (4 times) to avoid carry-over. In addition the last washing
functioned as conditioning step before the next extraction. The same packing bed was used
for about 100 extractions before it was discarded.
2.2. MEPS method development
Many factors, such as sorbent type, volumes and composition of washing and elution
solutions can affect the performance of MEPS [33, 35]. The recovery of extraction by using
MEPS was measured as the response of processed spiked plasma or urine samples expressed
as peak area and compared as percentage to that of pure standard solution has the same
concentration.
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2.2.1. Type of sorbent
The cartridge bed in MEPS can be packed or coated to provide selective and suitable
sampling conditions. Any sorbent material such silica based (C2, C8, C18), strong cation
exchanger (SCX) using sulfonic acid bonded silica, restricted access material (RAM), hilic,
carbon, polystyrene–divinylbenzene copolymer (ENV+) or molecular imprinted polymers
(MIPs) can be used.
In first Paper, different sorbents (C2, C8 and ENV+) were investigated for the extraction of
BAM8-22 and BAM22-8 from plasma samples by MEPS. As can be seen from Figure 6
MEPS-C8 provided the highest extraction recovery with 90%.
Fig.6 Absolute peak area for BAM8-22 and BAM22-8 extracted from plasma samples by MEPS with different
sorbents.
In the second Paper, molecularly imprinted polymer (MIP) was used as extracting sorbent in
MEPS for the simultaneous determination of four local anaesthetics in plasma and urine
samples. Selectivity can be pre-determined for a certain series of similar analytes sharing a
chemical core structure, or a particular analyte, by the choice of template used for MIP
preparation. In this study a close structural analogue of the target analytes for the MIP
template was used, eliminating any problems with template bleed by the subsequent
chromatographic separation and mass analysis settings of the mass spectrometer [57]. The
studied analytes in Paper II were local anaesthetics (weakly basic) and have pKa values
between 7.0 and 8.2. These drugs in plasma sample may be protein-bonded which reduces the
recovery of extraction. To disrupt the protein binding, the pH of the samples was shifted to
0
500000
1000000
1500000
2000000
2500000
3000000
Testsolution
C8 C2 ENV+
Pea
k ar
eas
BAM8-22
BAM22-8
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about 3 by using 0.1% formic acid. In such pH the studied analyets are positively charged.
Thus, when the type of sorbent was investigated both ion-exchange and hydrophobic
interaction were considered. Sorbent containing strong cation-exchanger and non-polar
functional group was targeted for the extraction of these analytes.
In addition C8, C18, ENV+ and MIP sorbents have been compared. The highest recovery and
cleaner extract were obtained using MIP sorbent compared to the other sorbents. Figure 7
shows the recovery of the studied substances on different investigated solid sorbent.
Fig.7 Extraction recovery of lidocaine, ropivacaine, mepivacaine and bupivacaine from human plasma using
MIP, C8, C8 and ENV+ as sorbents.
2.2.2. The washing solution
The purpose of the washing in MEPS process is to selectively remove unwanted compounds
from the sorbent without losing the analytes. As mentioned before, isolation of weakly basic
analytes from the plasma samples by MEPS is based on ionic and non-polar interactions. In
such case, both the pH and the concentration of organic solvent will have effect on desired
washing performance. In the first Paper, the use of methanol in the washing mixture was
investigated. Despite methanol increased the leakage and decreased the recovery; however,
clean extract was obtained. The lowest amount of leakage, with no interferences and highest
recovery was obtained with the use of 5–10% methanol in water (100 µL of 5% methanol was
used for washing in this study).
In the second Paper, the effect of different washing solutions on recovery was investigated.
Water, 0.1% formic acid in water and methanol in water were investigated using different
volumes (100, 200 and 300 µL). As can be seen from Figure 8, increasing concentration of
0
10
20
30
40
50
60
70
80
90
100
Lidocaine Ropivacaine Mepivacaine Bupivacaine
Rec
ove
ry %
ENV+
MIP
C18
C8
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methanol in the washing solution decreased the recovery. 100µL of aqueous 0.1% formic acid
gave both good recovery and clean extract compared to the other solutions and therefore it
was used as washing solution (see Figure 9).
Fig. 8 Effect of the methanol amount in washing solution on the MS response of analytes.
Fig. 9 Effect of washing solution (0.1% Formic acid in water) volume on the MS response of analytes.
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
Lidocaine Ropivacaine Mepivacaine Bupivacaine
Pe
ak a
rea 0% Methanol
5% Methanol
10% Methanol
20% Methanol
0
10000
20000
30000
40000
50000
60000
Lidocaine Ropivacaine Mepivacaine Bupivacaine
Pe
ak a
rea
100 µL
200 µL
300 µL
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2.2.3. The elution solvent
The proper elution solvent should be able to displace targeted analytes from the sorbent with a
minimum volume. In such case that retention of analaytes is based not only on hydrophobic
intraction but also ion-exchange interaction, a non-polar solvent would not be enough to
distrupt the forces that bind analytes to the sorbent. So, to elute the targeted analytes a solvent
capable of breaking both hydrophobic and ionic forces was needed. Further, in cases when
there are ion-exchange interactions, the pH of elution solvent should be optimized to get
charged-to-uncharged form of the targeted analytes for their elution.
In both Papers I and II, solutions containing methanol, water and ammonium hydroxide were
investigated as elution solutions. The elution efficiency was measured and compared with that
of pure standard solution. The eluting efficiency increased as the methanol content in the
eluent increased.
In Paper I, Highest recovery (>90%) was obtained when using a solution of methanol–water
95:5 (v/v) containing 0.25% ammonium hydroxide, and this was used as elution solution for
validation of the method.
In Paper II, as was observed in Paper I, the eluting efficiency increased as the methanol
content in the eluent increased. A good recovery was obtained when solution of
methanol/water 60:40 (v/v) containing 0.25% ammonium hydroxide was used. In addition the
effect of elution volume on peak response was investigated. As can be seen from Figure 10,
increasing in volume of elution solution increased the recovery, so that 200 µL of elution gave
highest extraction recovery for studied analytes. Dilution effect with using more elution
volume than 200 µL caused to decreasing in analytes response.
22
Fig. 10 Effect of elution solution (0.25% ammonium hydroxide in 60:40 methanol in water) volume on the
response of studied analytes.
2.3. Selectivity
The method selectivity is defined as non-interference from the endogenous substances in the
regions of interest. In Paper I, the selectivity of the assay was assessed, with and without
addition of standard, using six different sources of human plasma blank. In all six individual
sources of blank matrix, the interferences from endogenous plasma compounds were <20% of
the BAM peptides response at the lower limit of quantification (LLOQ). In Paper II, five
different blank plasma and urine specimens were analysed. No interferences were detected at
the same retention time as studied analytes. Representative chromatograms of blank plasma
and spiked plasma at LLOQ (5 nM) for lidocaine, ropivacaine, mepivacaine and bupivacaine
are presented in Figure 11.
0
20000
40000
60000
80000
100000
120000
140000
160000
Lidocaine Robivacaie Mepivacaine Bupivacaine
Pe
ak a
rea
100 µL
200 µL
300 µL
23
Fig. 11 MS chromatograms obtained from blank human plasma and spiked plasma with local anaesthetics at
LLOQ (5 nmol/L).
According to Papers I and II, analysis of BMA peptides in human plasma and local
anaesthetics drugs in both human plasma and urine samples using MEPS technique, no
interfering compounds were detected at the same retention time as the studied compounds.
This indicates a good selectivity for the application of MEPS as sample preparation method in
the analysis of BAM peptides and local anaesthetics drugs using plasma and urine samples
(Figures 11 and 12).
Spiked Plasma ( LLOQ )
Time 2.00 4.00
%
0
100
2.00 4.00
%
0
100
2.00 4.00
%
0
100
2.00 4.00
%
0
100
2.00 4.00
%
0
100
3.93 477
2.37 180
4.88 1388
3.48 350
3.41 190
Retention time Peak area
Lidocaine
Ropivacaine
IS
Mepivacaine
Bupivacaine
Blank Plasma
Time 2.00 4.0
%
0
100
2.00 4.0
%
0
100
2.00 4.0
%
0
100
2.00 4.0
%
0
100
2.00 4.0
%
0
10
Lidocaine
Ropivacaine
IS
Mepivacaine
Bupivacaine
24
Fig. 12 MS chromatograms obtained from blank human plasma and spiked plasma with BAM8-22 at LLOQ (5
nmol/L).
2.4. Carry-over
The carry-over is one of the common problems in bioanalysis. The carry-over is limiting step
for trace analysis giving bad accuracy and precision under method validation. First of all, the
carry-over is a compound depending phenomenon and can be caused not only from MEPS but
also from analyte adsorption to autosampler or LC system or MS interface. This effect
depends on many factors, including adsorption properties of the analytes, apparatus being
employed and sensitivity of the method [58]. When using MS as detector, carry-over can
deteriorate the dynamic range of MS instrument due to the limitation to decrease the level of
limit of detection (LOD). Therefore, it was important to study the carry-over for MEPS.
In Paper I, the carry-over was assessed by making a single injection of a high concentration
sample (highest standard, 3045 nmol/L) followed by three injections of mobile phase. The test
was repeated three times. For BAM8-22, the carry-over to the first sample injected after the
high concentration samples was 2.8%, while the carry-over to the third blank was 0.01%. For
BAM22-8, carry-over to the first sample injected after the high concentration samples was
6.1%, while the carry-over was 0.1% to the fourth blank. At least three blank sample wash
injections were needed to reduce the carry-over from a high concentration sample.
In Paper II, The system carry-over was investigated by analysing a blank sample after
injection of highest standard (2000 nmol/L). The carry-over was 0.05% or less after first
blank for lidocaine, ropivacaine, mepivacaine and bupivacaine and was close to zero after
25
third blank. Regarding MEPS four washing with elution solution was enough to avoid carry-
over.
2.5. Sample loading cycles
In MEPS the pre-concentration of the analytes on the sorbent phase is affected by the number
of extraction cycles performed and the speed applied. Practically, an aliquot of the volume of
the sample can be drawn up and down through the syringe, once or several times (sample
loading cycles) without discarding it. Sample loading can be done once or more depending on
sample concentration and if pre-concentration of the targeted analytes is required.
In Paper II, the extraction efficiency when using MEPS was examined by testing multiple
loading cycles (n = 1–7) of plasma spiked with local anaesthetics. The relationship between
the MS response as peak area and number of extraction cycles was linear up to 4-5 cycles
(Fig. 13), therefore five sample loadings were found to be satisfactory.
Fig. 13 The MS/MS response (as peak area) as a function of the number of MEPS extraction cycles for
Lidocaine in plasma samples.
2.6. Matrix effect (ME)
Matrix effect is a well-known phenomenon in LC/MS bioanalysis and sometimes is called ion
suppression. A well-defined example of matrix effect or signal suppression was described by
Annesley [59] and Matuszewski et al. [60]. Ion suppresion can give rise to incorrect data
interpretation, i.e. poor accuracy and precision. In addition van Hout et al. [61] showed that
0
1000
2000
3000
4000
5000
0 1 2 3 4 5 6 7 8
Pea
k a
rea
MEPS number of loading cycles
26
the percentage of ion suppression is relates to the matrix/analyte ratio, in another word is
depending on the analyte concentration (ion supression degree α 1/analyte conc.).
Co-eluting endogenous materials in the sample matrix which cause variable responses for the
analytes of interest, generally accepted as matrix effect. These effects are directly related to an
insufficient sample clean-up [62].
In this study in both Papers I and II, ME was calculated by comparing the peak areas of the
analytes which were post-spiked to dry extracted blank plasma and urine with the
concentration of the standard solutions of QC samples. Because the matrix effect is
concentration dependent, the matrix effect was investigated at low and high concentration
levels. Six different sources of pooled blank human plasma and urine were used. The ME% is
calculated as follow:
ME (%) = [(Response of post extracted spiked sample / Response of standard solution) - 1] × 100
Four replicate of plasma and urine samples were analysed and the comparison of matrix to
reference were investigated. In Paper I, the %ME values ranged from 5 to 10% for BAM 8-
22, 22-8 and internal standard. Comparison of MEPS with ultrafiltration (UF) and protein
precipitation (PPT) was carried out for the extraction of BAMs from human plasma samples.
UF and PPT showed low extraction recoveries (50–60%) compared with MEPS (Fig. 14).
MEPS gave clean extract and low background in MS. Complex matrices such as blood;
plasma and urine are potential for ion suppression particularly with electrospray ionization
mass spectrometry [26]. MEPS provides flexibility in different parameters such as washing
solution, elution solution and type of sorbent materials. MEPS technique eliminated salts and
reduced the phospholipids concentration significantly compared to protein precipitation [27].
Fig. 14 Absolute extraction recovery of BAM8-22 using plasma protein precipitation (PPT), ultrafiltration (UF)
and microextraction by packed sorbent (MEPS).
0
20
40
60
80
100
PPT UF MEPS
Rec
ove
ry %
27
The %ME values in Paper II were < 20% utilizing MEPS and this is in accepted range (<
20%). In addition we compared the ME values were obtained from MEPS with plasma
protein precipitation method. The plasma protein precipitation method showed higher matrix
effect (ranged from -22% to 147%).
3. Conclusions
Microextraction by packed sorbent as sample preparation technique was applied for the
determination of BAM peptides in human plasma and local anaesthetics drugs in human
plasma and urine. The results showed that MEPS technique provides significant advantages
such as the speed and the simplicity of the sample-preparation process. Compared with other
extraction techniques (SPE, LLE), MEPS significantly reduces the volume of solvents and
sample needed. The applied sorbent could be used about 100 times before it was discarded.
The carry-over may be almost eliminated by washing the sorbent 3–4 times first with elution
solution and then with washing solution, and furthermore, the use of MEPS can be useful to
eliminate matrix effects.
Also, the results showed that MIP-MEPS and LC-MS/MS can be a good tool for
quantification of drugs in plasma and urine samples. Because MEPS is less time-consuming,
simple and more useful than SPE methods, this technique is an alternative to SPE for the
routine analyses of biological samples in clinical laboratories.
28
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