Microextraction by packed sorbent of drugs and peptides in biological fluids

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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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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 (t

R

). 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 “t

R

” 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

H

2N-Val – Gly – Arg –Pro – Glu – Trp – Trp – Met – Asp – Tyr – Gln – Lys –Arg – Tyr –Gly-OH

BAM 22-8 H

2

N-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

Test solution

C8 C2 ENV+

Peak areas ^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

Recovery % ^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

Peak area 0% Methanol

5% Methanol 10% Methanol 20% Methanol

0 10000 20000 30000 40000 50000 60000

Peak area

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.

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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

Peak area

100 µL 200 µL 300 µL

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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

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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

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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

Peak area

MEPS number of loading cycles

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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

Recovery %

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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.

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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)

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)

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)

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)

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.

Microextraction by packed sorbent of drugs and peptides in biological fluids