1

Microextraction by packed sorbent of

drugs and peptides in biological fluids

Seyed Mosayeb Daryanavard

Licentiate Thesis

Department of Analytical Chemistry

Stockholm University

2013

2

© Seyed Mosayeb Daryanavard 2013

ISBN: 978-91-7447-621-7

Printed by Universitetsservice US-AB, Stockholm, Sweden, 2013

3

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.

4

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.

5

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

6

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

7

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

8

(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,

9

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].

10

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.

11

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.

12

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

13

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].

14

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

15

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.

16

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

17

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.

18

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

19

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

20

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

21

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

References

[1] Ashri N, Abdel-Rehim M, Sample treatment based on extraction techniques in biological matrices.

Bioanalysis (2011) 3, 2003-2018.

[2] Souverain S, Rudaz S, Veuthey J L, Protein precipitation for the ana lysis of a drug cocktail in

plasma by LC–ESI–MS. J. Pharm. Biomed. Anal. (2004) 35, 913–920.

[3] Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R, Optimization of protein precipitation based

upon effectiveness of protein removal and ionization effect in liquid chromatography–tandem mass

spectrometry. J. Chromatogr. B (2003) 785, 263–275.

[4] Biddlecombe R A, Pleasance S, Automated protein precipitation by filtration in the 96-well format.

J. Chromatogr. B (1999) 785, 263–275.

[5] Schutze H G, Quebedeaux W A, Lochte H L, Liquid-liquid extraction in the separation of

petroleum acids (Review). Industrial and Engineering Chemistry (1938) 10, 675-677.

[6] Remane D, Meyer M R, Peters F T, Wissenbach D K, Maurer H H, Fast and simple procedure for

liquid–liquid extraction of 136 analytes from different drug classes for development of a liquid

chromatographic–tandem mass spectrometric quantification method in human blood plasma. Anal.

Bioanal. Chem. (2010) 397, 2303–2314.

[7] Højskov C S, Heickendorff L, Møller H J. High-throughput liquid–liquid extraction and LC–

MS/MS assay for determination of circulating 25 (OH) vitamin D3 and D2 in the routine clinical

laboratory. Anal. Chim. Acta (2010) 411, 114–116.

[8] Han D, Chen C, Zhang C, Zhang Y, Tang X, Determination of mangiferin in rat plasma by liquid-

liquid extraction with UPLC–MS/MS. J. Pharm. Biomed. Anal. (2010) 51, 260–263.

[9] Whelpton R. Pharmaceutical ana lysis/sample preparation. In: Encyclopaedia of analytical science

(Second Ed.). Elsevier Ltd (2005) 107–116.

[10] RE Majors, Sample preparation for HPLC and gas chromatography using solid-phase extraction.

LC-GC (1986) 4, 972-984.

[11] Hennion M C, Solid-phase extraction: method development, sorbents, and coupling with liquid

chromatography. J. Chromatogr. A (1999) 856, 3–54.

[12] Poole C F, New trends in solid-phase extraction. Trends Anal. Chem. (2003) 22, 362–373.

[13] Wells D A, High Throughput Bioanalytical Sample Preparation. Elsevier, Amsterdam, The

Netherlands (2003).

[14] Mitra S, Sample Preparation Techniques in Analytical Chemistry. (2003) Wiley, NY, USA.

[15] Koohpaei A R, Shahtaheri S J, Ganjalic M R, Rahimi Forushanie A, Golbabaei F. Optimization

of solid-phase extraction using developed modern sorbent for trace determination of ametryn in

environmental matrices. J. Anal. Chem. (2010) 65, 694–698.

[16] Farid E A, Analyses of pesticides and their metabolites in foods and drinks. Trends Anal. Chem.

(2001) 20, 649-661.

29

[17] Arthur C L, Killam L M, Buchholz K D, Pawliszyn J, Berg J R, Automation and optimization of

solid-phase microextraction. Ana.l Chem. (1992) 64, 1960-1966.

[18] Baltussen E, Sandra P, David F, Cramers C, Stir bar sorptive extraction (SBSE), a novel

extraction technique for aqueous samples: Theory and principles. J. Microcolumn Sep. (1999) 11, 737-

747.

[19] Abdel-Rehim M, New trend in sample preparation: On-line microextraction in packed syringe for

liquid and gas chromatography applications I. Determination of local anaesthetics in human plasma

samples using gas chromatography-mass spectrometry, J. Chromatogr B (2004) 801, 317-321.

[20] Abdel-Rehim M, Altun Z, Blomberg L, Microextraction in packed syringe (MEPS) for liquid and

gas chromatographic applications. Part II - Determination of ropivacaine and its metabolites in human

plasma samples using MEPS with liquid chromatography/tandem mass spectrometry. J. Mass Spec.,

(2004) 39, 1488-1493.

[21] Altun Z, Abdel-Rehim M, Blomberg L G, New trends in sample preparation: On-line

microextraction in packed syringe (MEPS) for LC and GC applications: Part III: Determination and

validation of local anaesthetics in human plasma samples using a cation-exchange sorbent, and MEPS-

LC-MS-MS. J. Chromatogr. B (2004) 813, 129-135.

[22] Vita M, Skansen, P, Hassan M, Abdel-Rehim M, Development and validation of a liquid

chromatography and tandem mass spectrometry method for determination of roscovitine in plasma and

urine samples utilizing on-line sample preparation. J. Chromatogr. B (2005) 817, 303-307.

[23] Abdel-Rehim M, Skansen P, Vita M, Hassan Z, Blomberg L, Hassan, M, Microextraction in

packed syringe/liquid chromatography/electrospray tandem mass spectrometry for quantification of

olomoucine in human plasma samples, Anal. Chim. Acta (2005) 539, 35-39.

[24] Altun Z, Blomberg L, Jagerdeo E, Abdel-Rehim M, Drug screening using microextraction in a

packed syringe (MEPS)/mass spectrometry utilizing monolithic, polymer, and silica-based sorbents.

J. Liq. Chromatogr. R. T. (2006) 29, 829-839.

[25] El-Beqqali A, Kussak A, Abdel-Rehim M, Fast and sensitive environmental analysis utilizing

microextraction in packed syringe online with gas chromatography-mass spectrometry: Determination

of polycyclic aromatic hydrocarbons in water. J. Chromatogr. A (2006) 1114, 234-238.

[26] Abdel-rehim M, Andersson L, Altun Z, Blomberg L, Microextraction in packed syringe online

with liquid chromatography-tandem mass spectrometry: Molecularly imprinted polymer as packing

material for MEPS in selective extraction of ropivacaine from plasma, J. Liq. Chromatogr. R. T.

(2006) 29, 1725-1736.

[27] Abdel-Rehim M, Dahlgren M, Blomberg L, Quantification of ropivacaine and its major

metabolites in human urine samples utilizing microextraction in a packed syringe automated with

liquid chromatography-tandem mass spectrometry (MEPS-LC-MS/MS). J. Sep. Sci., (2006) 29, 1658-

1661.

[28] Abdel-Rehim M, Askemark Y, Norsten-Höög C, Pettersson K J, Halldin M, Quantification of 4-

OH-2,6-xylidine and its conjugates in human urine samples utilising microextraction in packed

syringe on-line with liquid chromatography and electrospray tandem mass spectrometry (MEPS-LC-

MS/MS). J. Liq. Chromatogr. R. T. (2006) 29, 2413-2424.

30

[29] El-Beqqali A, Kussak A, Abdel-Rehim M, Determination of dopamine and serotonin in human

urine samples utilizing microextraction online with liquid chromatography/electrospray tandem mass

spectrometry. J. Sep. Sci. (2007) 30, 421-424.

[30] El-Beqqali A, Kussak A, Blomberg L, Abdel-Rehim M, Microextraction in packed syringe/liquid

chromatography/electrospray tandem mass spectrometry for quantification of acebutolol and

metoprolol in human plasma and urine samples. J. Liq. Chromatogr. R. T., (2007) 30, 575-586.

[31] Said R, Hassan Z, Hassan M, Abdel-Rehim M, Rapid and sensitive method for determination of

cyclophosphamide in patients plasma samples utilizing microextraction by packed sorbent online with

liquid chromatography-tandem mass spectrometry (MEPS-LC-MS/MS). J. Liq. Chromatogr. R. T.

(2008) 31, 683-694.

[32] Abdel-Rehim M, Andersson A, Breitholtz-Emanuelsson A, Sandberg-Ställ M, Brunfelter K,

Pettersson K J, Norsten-Höög C, MEPS as a rapid sample preparation method to handle unstable

compounds in a complex matrix: Determination of AZD3409 in plasma samples utilizing MEPS-LC-

MS-MS. J. Chromatogr. Sci., (2008) 46, 518-523.

[33] Altun Z, Abdel-Rehim M, Study of the factors affecting the performance of microextraction by

packed sorbent (MEPS) using liquid scintillation counter and liquid chromatography-tandem mass

spectrometry. Anal. Chim. Acta (2008) 630, 116-123.

[34] Morales-Cid G, Cárdenas S, Simonet B M, Valcárcel M, Fully automatic sample treatment by

integration of microextraction by packed sorbents into commercial capillary electrophoresis-mass

spectrometry equipment: Application to the determination of fluoroquinolones in urine. Anal. Chem.

(2009) 81, 3188-3193.

[35] Abdel-Rehim M, Recent advaces in microextraction by packed sorbent (MEPS) for bioanalysis,

Invited Paper, J. Chromatogr. A, (2010) 1217, 2569-2580.

[36] Miyaguchi H, Iwata Y T, Kanamori T, Tsujikawa K, Kuwayama K, Inoue H, Rapid identification

and quantification of methamphetamine and amphetamine in hair by gas chromatography/mass

spectrometry coupled with micropulverized extraction, aqueous acetylation and microextraction by

packed sorbent. J. Chromatogr. A (2009) 1216, 4063-4070.

[37] Matysik S, Matysik F M, Microextraction by packed sorbent coupled with gas chromatography-

mass spectrometry: Application to the determination of metabolites of monoterpenes in small volumes

of human urine. Microchimica Acta (2009) 166, 109-114.

[38] Lafay F, Vulliet E, Flament-Waton M M, Contribution of microextraction in packed sorbent for

the analysis of cotinine in human urine by GC-MS. Anal. Bioanal. Chem., (2010) 396, 937-941.

[39] Saracino M A, Tallarico K, Raggi M A, Liquid chromatographic analysis of oxcarbazepine and

its metabolites in plasma and saliva after a novel microextraction by packed sorbent procedure. Anal.

Chim. Acta (2010) 661, 222-228.

[40] De Andrés F, Zougagh M, Castañeda G, Sánchez-Rojas J L, Ríos A, Screening of non-polar

heterocyclic amines in urine by microextraction in packed sorbent-fluorimetric detection and

confirmation by capillary liquid chromatography, Talanta (2011) 83, 1562-1567.

[41] D. Stevenson, Molecular imprinted polymers for solid-phase extraction. Trend. Anal. Chem.,

(1999) 18 (3), 154-158.

31

[42] Chapuis F, Pichon V, Hennion M C, Molecularly imprinted polymers: Developments and

applications of new selective solid-phase extraction materials. LC-GC Europe (2004) 17, 408-417.

[43] Andersson L I, Molecular imprinting: developments and applications in the analytical chemistry

field. J. Chromatogr. B, (2000) 745 3–13.

[44] Andersson L I, Hardenborg E, Sandberg-Ställ M, Möller K, Henriksson J, Bramsby-Sjöström I,

Olsson L I, Abdel-Rehim M, Development of a molecularly imprinted polymer based solid-phase

extraction of local anaesthetics from human plasma. Anal. Chim. Acta (2004) 526, 147–154.

[45] Touchstone J C, History of chromatography. J. Liq. Chromatogr. & Relat. Technol. (1993) 16,

1647-1665.

[46] Niessen W M, Advances in instrumentation in liquid chromatography– mass spectrometry and

related liquid-introduction techniques. J. Chromatogr. A, (1998) 794, 407–435.

[47] McLafferty F W, A century of progress in molecular mass spectrometry. Annual review of

analytical chemistry, (2011) 4, 1-25.

[48] Petrovic M, Farre M, De Alda M L, Perez S, Postigo C, Kock M, Radjenovic J, Gros M, Barcelo

D, Recent trends in the liquid chromatography–mass spectrometry analysis of organic contaminants in

environmental samples. J. Chromatogr. A, (2010) 1217, 4004-4017.

[49] Smyth W F, Electrospray ionisation mass spectrometric behaviour of selected drugs and their

metabolites. Anal. Chim. Acta, (2003) 492, 1-16.

[50] Columb M O, MacLennan K, Local anaesthetic agents, Anaesthesia and Intensive Care Medicine,

(2007) 8, 159-162.

[51] Lagan G, McLure H A, Review of local anaesthetic agents, Current Anaesthesia and Critical

Care, (2004) 15, 247-254.

[52] Sheng Z R, Wang J K, Practical Clinical Anesthesiology, Liaoning Science and Technology

Press, Shenyang, (1996) 129−158.

[53] Strichartz G R, Berde C B, Local anesthetics, in: R.D. Miller (Ed.), Anesthesia, vol. 1, 4th ed.,

Churchill Livingstone Inc., New York, (1994) 489–521.

[54] Cai Q, Jiang J, Chen T, Hong Y, Sensory neuron-specific receptor agonist BAM8 22 inhibits the

development and expression of toleranc to morphine in rats. Behavioural Brain Research (2007) 178,

154–159.

[55] Lembo P M, Grazzini E, Groblewski T, O’Donnell D, Roy M O, Zhang J, Proenkephalin A gene

products activate a new family of sensory neuron–specific GPCRs. Nat Neurosci (2002) 5, 201–209.

[56] Grazzini E, Puma C, Roy M O, Yu X H, O’Donnell D, Schmidt R, Sensory neuron-specific

receptor activation elicits central and peripheral nociceptive effects in rats. Proc Natl Acad Sci USA

(2004) 101, 7175–7180.

32

[57] Andersson L I, Efficient sample pre-concentration of bupivacaine from human plasma by solid-

phase extraction on molecularly imprinted polymers. Analyst (2000) 125, 1515–1517.

[58] Rossi D T, Zhang N, Automating solid-phase extraction: current aspects and future prospects. J.

Chromatogr. A, (2000) 885, 97-113.

[59] Annesley T M, Ion Suppression in Mass Spectrometry, Clin. Chem. (2003) 49, 1041–1044.

[60] Matuszewski B K, Constanzer M L, Chavez-eng C M, Strategies for the assessment of matrix

effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem. (2003) 75, 3019-

3030.

[61] Van Hout M W J, Hoffland C M, Niederlander H A G, De Jong G J, On-line coupling of solid-

phase extraction with mass spectrometry for the analysis of biological samples. II. Determination of

clenbuterol in urine using multiple-stage mass spectrometry in an ion-trap mass spectrometer. Rapid

Commun. Mass Spectrom. (2000) 14, 2103–2111.

[62] Van Eeckhaut A, Lanckmans K, Sarre S, Smolders I, Michotte Y, Validation of bioanalytical LC–

MS/MS assays: evaluation of matrix effects. J. Chromatogr. B (2009) 877, 2198–2207.

[63] Karsten H, Molecularly imprinted polymers in analytical chemistry. Analyst (2001) 126, 747-756.