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Literature Thesis

THE APPLICATION OF MICROEXTRACTION

FOR DETERMINATION OF DRUGS

IN BIOLOGICAL SAMPLES

Febri Annuryanti

Supervisor: Dr. Henk Lingeman

Master in Chemistry – Analytical Sciences

University of Amsterdam

MSc Chemistry

Analytical Sciences

Literature Thesis

The Application of Microextraction for Determination

of Drugs in Biological Samples

by

Febri Annuryanti

April 2013

Supervisor:

Dr. Henk Lingeman

Daily Supervisor:

Dr. Wim Th. Kok

i Febri Annuryanti [10145222]

Table of contents :

Table of contents i

Abbreviations ii I. Introduction 1

II. Liquid-phase Microextraction (LPME) 3

2.1 Principle of LPME 3

2.2 Classification of LPME 3

2.2.1 Single-drop microextraction (SDME) 3

2.2.2 Hollow fiber microextraction (HF-LPME) 6

2.2.3 Carrier-mediated HF-LPME 8

2.2.4 Dispersive liquid-liquid membrane (DLLME) 9

2.3 Recovery and enrichment factor 11

2.3.1 In SDME, HF-LPME, and carrier mediated HF-LPME 11

2.3.2 In DLLME 12

2.4 Influence factors on the LPME efficiency 13

2.4.1 Organic solvent 13

2.4.2 Volume of donor and acceptor solution 13

2.4.3 Extraction time 14

2.4.4 pH adjustment 14

2.4.5 Agitation of the sample 15

2.4.6 The addition of salt 15

III. Solid-phase Microextraction (SPME) 16

3.1 Principle of SPME 16

3.2 Classification of SPME 18

3.2.1 Fiber SPME 18

3.2.2 In-tube SPME 19

3.3 Influence factors on the SPME efficiency 20

3.3.1 Agitation method 20 3.3.2 Sample pH 20 3.3.3 Ionic strength 21 3.3.4 Sample temperature 21 3.3.5 Sample derivatization 22

IV. Discussion 23 4.1 Recent applications of LPME for determination of drugs in biological

samples 4.2 Recent applications of SPME for determination of drugs in biological

samples

23

37

V. Conclusion 43 VI. References 45

ii Febri Annuryanti [10145222]

Abbreviations

LLE = Liquid-liquid extraction SPE = Solid-phase extraction

HPLC = High-performance Liquid Chromatography GC = Gas Chromatography CE = Capillary Electrophoresis

SPME = Solid-phase microextraction LPME = Liquid-liquid microextraction

DI = Direct immersion HS = Head space

HS-SDME = Head space single-drop microextraction SDME = Single-drop microextraction

CF-SDME = Continous flow single-drop microextraction DLLME = Dispersive liquid-liquid microextraction PDMS = Polydimethylsiloxane

PA = Polyacrylate PDMS-DVB = Polydimethylsiloxane-divinylbenzene

CW-TPR = Carbowax-templated resin LC-MS = Liquid chromatography-Mass spectrometry GC-FID = Gas-Chromatography-Flame Ionization Detector

NPD = Nitrogen-phosphorus detector LOD = Limit of detection LOQ = Limit of quantification I.S. = Internal standard TSD = Thermionic specific detector

THE APPLICATION OF MICROEXTRACTION FOR THE DETERMINATION OF DRUGS IN BIOLOGICAL SAMPLES

1 Febri Annuryanti [10145222]

Introduction

Analysis of drugs in biological samples is becoming increasingly important due

to the need to understand more about the therapeutic and the toxic effects of drugs

[1,2]. Many advantages are obtained by knowing the drug levels in body fluids such as in

plasma, serum, and urine [1-3]. The data of drug levels can be used to optimize

pharmacotherapy and give the basis for studies on patient compliance [1-3], to perform

routine drug monitoring [1,2], to compare the pharmacokinetics study for release of

new drugs [6], to reveal the influence of co-medication and to monitor the organ

function [2,3]. Furthermore, the screening of drug abuse in body fluids may be used to

identify potential users of illegal drugs and to control drugs addicts following

withdrawal therapy [1,2].

Although there is an advance development of analytical instrumentation for the

determination of analytes in biological fluids, most of the instruments cannot handle

the sample matrices directly because of sample complexity [1-2,4]. Biological samples

may contain acids, bases, proteins, salts and other organic compounds that may have

chemical properties similar to the analyte of interest [3,5,7]. Therefore, sample

preparation becomes a crucial part of analysis in order to extract, isolate, and

concentrate the analytes [1-3,7-8]. In addition to complex matrices, limited sample

volumes and low analyte concentrations have to be considered during sample

preparation [2,7]. In order to get an efficient sample pre-treatment, it is important to

minimize sample loss so the analytes can be recovered in good yield [1,2], coexisting

components can be removed efficiently [1,2], problems do not occur in chromatography

system, the analysis cost is low and the procedure can be performed quickly [1,2].

Conventionally, sample preparation is carried out by liquid-liquid extraction

(LLE) or by solid-phase extraction (SPE) and the final analysis is accomplished by High-

performance Liquid Chromatography (HPLC), Gas Chromatography (GC) or Capillary

Electrophoresis (CE) [3,5-6,9-10]. However, both of LLE and SPE have various drawbacks



such as requires large amounts of organic solvents that are toxic and expensive [8,11],

time-consuming [8], result in hazardous waste [11], tedious [8], laborious, and difficult to

automate [4].

An ideal sample preparation technique should be easy to use, inexpensive, fast

and compatible with a range of analytical instruments [2,4]. To overcome or reduce the

drawbacks of LLE and SPE, miniaturizations have been reported on alternative sample

preparation methods for drug analysis, namely solid-phase micro extraction (SPME)

and liquid-phase micro extraction (LPME) [2,10,12].

This article presents the main principle of SPME and LPME, factors that

affecting SPME and LPME, their application on determination of drugs in biological

fluids, and further prospect of LPME for drug analysis in biological samples.



Liquid-Phase Microextraction (LPME)

2.1 Principle of LPME

LPME is a new sample-preparation technique for the extraction of analytes.

Basically, LPME is performed between a small amount of water immiscible solvent

(known as acceptor phase) and an aqueous phase containing the analyte of interest

(donor phase) [1,4,13,14]. The volume of acceptor phase is usually in the microliter or

submicroliter region, while the donor phase between 0.5-4.0 mL for biological samples

[8,15,16]. Hence, high analyte enrichments are obtained because of the high sample

volume-to-acceptor phase volume ratio [8]. LPME procedures can be divided into static

and dynamic mode. In static mode, the extractant is suspended in a large volume of

sample phase and the extraction of the analytes is passively carried out. In dynamic

mode, extraction occurs by withdrawing aqueous sample into the extraction unit

(usually a micro syringe) that already containing solvent. The aquase phase is then

pushed out of the syringe and this procedure is repeated several times (typically 20

times) so a higher enrichment factors is obtained [4,17,18]. As a sample preparation,

LPME has many advantages. It is rapid, effective, minimize exposure to toxic organic

solvents and inexpensive [1,3]. The LPME concept is also compatible for analysis of drugs

using HPLC, GC or CE [19].

2.2 Classification of LPME

In general, LPME can be divided into single-drop microextraction (SDME), hollow-

fiber microextraction, and dispersive liquid-liquid microextraction (DLLME) [4].

2.2.1 Single-drop microextraction (SDME)

SDME is the simplest form of LPME. It is based on the extraction of analytes

into a small drop of organic solvent that is held at the tip of a micro syringe needle [20].

In a two-phase system, the organic solvent was placed into the aqueous sample and

the analytes are extracted into the organic solvent based on passive diffusion. In a

three-phase system, analytes are extracted from an aqueous sample into the organic



phase. Then, analytes are “back extracted” into a separate aqueous phase [1]. After

extraction, the organic phase is retracted into the needle and the syringe is transferred

for further analysis [4]. In practice, there are three main approaches to perform SDME,

direct immersion (DI)-SDME, head space (HS)-SDME, and continuous flow (CF)-SDME

[6,20].

DI-SDME is a static mode of LPME. It can be done in a two-phase or a three-

phase system (Fig. 1). It is based on the suspension of a single drop of organic solvent

from the tip of a microsyringe needle immersed in the aqueous sample. In a two-phase

system, the analytes can be directly injected into the GC-system after the extraction.

While in a three-phase system, analytes can be injected into the HPLC system for

analysis. The application of DI-SDME is normally restricted to medium polarity, non-

polar analytes and analytes whose polarities can be reduced before extraction. The

main problem of DI-SDME is the instability of the droplet at high stirring rate and the

option of acceptor phase is limited only for water-immiscible solvent [1,4,6].

Furthermore, fast stirring in DI-SDME may form air bubbles when it is applied to

biological samples like plasma. This condition may emulsify organic solvents and

increase the stability problem [6].

In most cases, DI-SDME involves a two-phase extraction mode. However, a

three-phase extraction mode has also been reported for SDME [1]. In a three-phase



extraction, the pH of the donor phase is adjusted to ensure that the analyte is in its

unionized form so it can be extracted into an organic phase whereas the pH of

acceptor phase is kept below the pH of the donor phase to prevent back-extraction

into the organic phase again. Subsequently, the acceptor phase can be transferred to

an HPLC or CE system for final analysis [12].

In HS-SDME (Fig. 2) the analyte is extracted into a microdrop of appropriate

solvent located in the head space of sample solution or in a flowing air sample stream,

which is thermostated at a given temperature for a preset extraction time [4,20]. This

method is most suitable for determination of volatile or semivolatile analytes [1,6]. In

this mode, the analyte is distributed among three phases, the aqueous sample, head

space and organic solvent, and the rate of this extraction is determined by mass

transfer of aqueous phase [1,20]. The advantage of HS-SDME is it allows the use of both

organic solvent and aqueous solvent as acceptor phase because the droplet does not

directly contact with the sample solution. In addition, HS-SDME provides an excellent

clean up for sample with complicated matrix [4,6,20]. The drawback of this method is the

need of solvent with low vapor pressure and low viscosity [20].

CF-SDME (Fig. 3) is a dynamic mode of SDME and was first introduced by Liu

and Lee in 2000 [20]. In this method, a polyetheretherketone (PEEK) connecting tube

hold an organic drop at the outlet tip and immersed in a continuously flowing sample



solution. This PEEK connecting tube acts as the fluid delivery duct and solvent holder.

This method produces a higher concentration factor than static mode of SDME

because the solvent drop makes continuous and full contact with the sample solution

[4,20]. Because of its high concentration factor that can be achieved, only a small volume

of sample is needed for extraction [4]. The disadvantages of this method are the need

of peristaltic pump and extra filtration since complex matrices affect the stability of

solvent drop during the extraction [20].

2.2.2 Hollow fiber liquid-phase microextraction (HF-LPME)

HF-LPME is an alternative concept for LPME. This concept was introduced in

1999 by Pedersen-Bjergaard and Rasmussen to improve the stability and reliability of

LPME [21]. This technique use single, low-cost, disposable porous hollow fiber made of

propylene [22,23]. The advantages of HF-LPME are that the sample can be vibrated or

stirred vigorously without any loss of the extracting liquid and the extracting liquid is

not partly dissolved in the sample during extraction [24]. The small pore size of hollow

fiber allows microfiltration of the samples to yield very clean extracts [25] and the use of

disposable hollow fiber eliminates the possibility of carry over and ensures

reproducibility [25-27]. Particularly, in the three-phase system when both extraction and

back-extraction are included, excellent clean-up has been observed, even in

complicated biological samples [28].



In this system, the extracting liquid is not directly contact with the acceptor

phase. The acceptor phase is contained within the lumen of porous hollow fiber, either

as loop or a rod sealed at the bottom [21,29]. Prior to extraction, the hollow fiber is

dipped in the immiscible organic solvent (like toluene, dihexyl ether or n-octanol) for

several seconds to immobilize the organic solvent in its pores. Alternatively, a small

volume of organic solvent can be injected into the lumen of hollow fiber and

immobilized from the inside of hollow fiber [21]. The organic solvent forms a thin layer

within the wall of the hollow fiber and the excess solvent outside the hollow fiber is

removed by ultra-sonification [23]. Subsequently, the hollow fiber is then placed into a

sample vial that contains the sample of interest. An extensive agitation or stirring of

the sample can be applied to speed up the extraction process [23]. The organic solvents

are used in HF-LPME should be immiscible with water, strongly immobilized in the

pores of hollow fiber, provide an appropriate extraction selectivity, and has a low

volatility, to ensure that it remains within the pores during extraction with no leakage

to the biological samples [22,23].

Like SDME, HF-LPME may be accomplished both in a two-phase or a three-

phase system (Fig. 4) [21-23,27]. In a two-phase system, the analytes are extracted from

the aqueous sample into an organic solvent immobilized in the pores and the lumen of

hollow fiber [24-25]. This technique may be applied for analytes with high solubility in

non-polar organic solvents. Since the pores and the lumen of hollow fiber are filled

with an organic solvent immiscible with water, the final extract may be directly

analyzed with GC, or may be evaporated and reconstituted in an aqueous solution for

analysis with CE or HPLC [25].

In a three-phase system, the analytes are extracted from an aqueous sample

through the thin film of the organic solvent into an aqueous acceptor solution. The thin

film of organic phase serves as a barrier between the donor phase and the acceptor

phase [25]. This extraction mode is limited to acidic or basic analytes with ionizable

functionalities, where the analyte is in its neutral form in the donor phase [22,25]. For

the extraction of acidic compounds, pH in the sample has to be adjusted in acidic

region to promote their extraction into the organic phase, whereas the pH in the



acceptor solution should be high to promote high extraction efficiency from organic

phase into the acceptor phase [22,25]. In contrast for basic compounds, the pH of sample

solution should be in alkaline region and the acidic solution should be utilized within

the lumen of fiber. Following extraction, the acceptor phase is directly analyzed by

HPLC, CE, or MS without any further treatments [22,25].

2.2.3 Carrier-mediated HF-LPME

In two-phase and three-phase HF-LPME, the extraction is based on passive

diffusion, in which the high partition coefficient plays an important role. However,

some analytes, such as very hydrophilic drugs, have poor partition coefficients that

prevent them from being extracted by passive diffusion. In order to enhance the

extraction of hydrophilic drugs, HF-LPME may be accomplished in a carrier-mediated

mode [22,23].

Carrier-mediated, as illustrated in Fig. 5, is an active transport mode of HF-

LPME. In this method, a carrier is added to the sample solution or is dissolved in the

impregnation solvent in the pores of the hollow fiber [25]. The carrier, which is relatively

hydrophobic ion-pair reagent providing acceptable water solubility, forms ion-pairs

with the analyte of interest followed by the extraction of ion-pair complexes into the

organic phase in the pores of hollow fiber. In the contact region of organic phase and

acceptor phase, the analytes are released from the ion-pair complexes into the

acceptor solution, whereas an excessive counter-ions in the acceptor solution form

ion-pairs with the carrier in the contact area. The new ion-pair complexes are then



back-extracted into the donor phase. In the sample solution, the carrier releases the

transporter counter ion and forms an ion pair with a new analyte molecule, and the

cycle is repeated [22,23].

A carboxylic acid with an appropriate hydrophobic moiety may be used as the

carrier (such as octanoic acid) for basic analytes. In the extraction process, the pH of

the sample is adjusted to ensure that the analytes are present in their ionized state in

order to form the ion pair, and the pH of acceptor is adjusted to low value to ensure

that the carrier is not trapped within the phase. Furthermore, the low pH value

provides sufficient protons to serve as counter ion for the carrier [22,23].

2.2.4 Dispersive liquid-liquid microextraction (DLLME)

DLLME is another recent technique of LPME that was introduced by Assadi and

co-workers in 2006 [4,16]. It is based on ternary solvent component system involving an

aqueous sample, a polar water miscible solvent (disperser solvent) and a non-polar

water immiscible solvent (extracting solvent) [6]. The selection of extracting solvents is

based on their density, extraction capability of interest compounds and good

chromatographic behavior. The density of extracting solvent should be higher than



water. Halogenated hydrocarbons such as chlorobenzene, carbon disulfide, carbon

tetrachloride, tetrachloroethylene and chloroform are usually chosen as extracting

solvents [20,30].

The choice of disperser solvent is determined by its ability to miscible in both

extracting solvent and aqueous sample. Methanol, ethanol, acetonitrile and acetone

are mostly used as disperser solvent [20,30].

Figure 6 shows the different step of DLLME. When the mixture of disperser and

extraction solvent is injected into the sample solution, a cloudy solution is produced.

This cloudy solution gives rise to the formation of fine droplets, which are dispersed

throughout the aqueous sample. After the formation of cloudy solution, the surface

area between extracting solvent and the sample solution becomes very large so the

equilibrium state is achieved quickly and the extraction time is relatively short. The

cloudy solution is then cooled and centrifuged to form a sediment phase in the bottom



of conical tube and used for further analysis. DLLME can be coupled with HPLC, GC and

also with atomic absorption spectrometry [16,20].

2.3 Recovery and enrichment factor

2.3.1 In SDME, HF-LPME and carrier mediated HF-LPME

In two-phase SDME and HF-LPME, the analytes are extracted from donor

solution by passive diffusion from directly into the acceptor solution, described in

equation (1). The extraction process in this system depends on the partition between

the acceptor (organic) solution and the donor solution (Ka/d), defined by equation

(2)[11,21].

A donor A acceptor (organic) solution (1)

K a/d = Ceq.acceptor / Ceq.donor (2)

where Ceq.acceptor is the concentration of analyte in the acceptor (organic phase)

solution at equilibrium and Ceq.donor is the concentration of analyte in the sample at

equilibrium. Based on Eq. (2) and a mass balance of the two-phase LPME system, the

recovery of analyte (R) at equilibrium may be calculated by the following equation [21]:

R = (Ka/d . Va)/{(Ka/d . Va) + Vd} . 100% (3)

where Va is the volume of acceptor solution in the organic phase system (sum of

organic solvent present in the porous wall of the hollow fiber and in the lumen of

hollow fiber and Vd is the volume of donor solution [21].

In three-phase SDME, HF-LPME and carrier mediated HF-LPME, the analytes are

extracted from the aqueous phase by passive diffusion, through the organic phase, and

further into the acceptor solution presents inside the lumen of hollow fiber. This

process may be illustrated by following equation [11,21]:

Adonor Aorganic acceptor Aacceptor(aqueous) acceptor (4)



The total extraction process is affected by both partition coefficient between

the organic phase and the donor solution (Korg/d) and that between the acceptor

solution and the organic phase (Ka/org), defined by equation (5) and (6) [21]:

Korg/d = Ceq.org/Ceq.d (5)

Ka/org = Ceq.a/Ceq.org (6)

where Ceq.org is the analyte concentration at equilibrium in the organic phase, Ceq.d is

the analyte concentration at equilibrium in the donor solution, and Ceq.a is the analyte

concentration at equilibrium in the acceptor solution. The partition coefficient

between the acceptor solution and donor solution is calculated as the product of Korg/d

and Ka/org [11]:

Ka/d = Ceq.a/Ceq.d = Korg/d . Ka/org (7)

The recovery, R, in the three-phase LPME system may be calculated by equation [18]:

R = (Ka/d . Va) / {(Ka/d . Va) + (Korg/d . Vorg) + Vd} . 100 % (8)

where Va is the volume of acceptor phase, Vorg is the volume of organic phase

immobilized in the pores of the hollow fibre and Vd is the volume of the donor solution.

The analyte enrichment (E) in two-phase and three-phase LPME may be

calculated by equation (9) and (10), respectively [18]:

E = (Vd . R) / (Vorg . 100) (9)

E = (Vd . R) / (Va.100) (10)

2.3.2 In DLLME

The recovery, R, in DLLME is defined as the percentage of total analyte amount

(no) extracted to the sediment phase (nsed) [16]:

R = (nsed / no) x 100 = {(Csed x Vsed)/(Co x Vs)} x 100 (11)

where Csed is the analyte concentration in sediment phase, Co is the initial

concentration of analyte, Vsed and Vs are the volumes of sediment phase and sample

solution, respectively.



2.4 Influence factors on the LPME efficiency

There are some factors which affect the method optimization and extraction

efficiency (recovery and enrichment) in liquid phase [11,12,20]:

2.4.1 Organic solvent

The selection of organic solvent is an essential step for an efficient extraction.

The choice of organic solvents should be based on several considerations. Firstly, it

should have good affinity for analyte of interest. Secondly, it should have a low

solubility in water to prevent dissolution into the aqueous phase. Thirdly, the organic

solvents should have low volatility so it will not evaporate during extraction. Fourthly,

it should be stable during extraction time. Finally, the organic solvent should have

excellent GC or LC behavior. In general, several water-immiscible solvents which have

different solubility and polarity may be used as extraction solvent. 1-octanol, di-n-

ethylether, n-hexane, o-xylene and toluene may be used as organic solvent in HF-LPME

[11]. For DI-SDME and DLLME, the density of organic solvent plays an important role in

the extraction process. In DI-SDME, the density of organic solvent should be lower than

water. On the other hand, DLLME requires organic solvent which has density higher

than water. Decane, 1-butanol, isooctane and n-octanol are usually used in SDME,

whereas chlorobenzene, dichlorocarbene and tetrachloride carbon are used in DLLME

[20].

2.4.2 Volume of donor and acceptor solution

The volume of donor and acceptor solutions directly affects the extraction

efficiency. The biological sample volume usually between 0.1 -4 mL, while the volume

of acceptor solution may vary depends on the method of extraction and on the

analytical technique coupled to LPME. The volume of acceptor solution in SDME is

typically in the range of 1.0-10.0 µL because larger drops lead to instability of the

microdrop. In HF-LPME, the extraction volume depends on the length of hollow fiber.

2-8 cm of hollow fiber are usually used in the range of 2.0-25 µL. As for the DLLME, the

volume of acceptor solution is in the range of 10-300 µL. The extraction efficiency and

enrichment factor can be increased by increasing the ratio of acceptor-to-donor phase.



However, enrichment factor will decrease when it exceeds a certain limit. Therefore,

keeping a low extraction volume is necessary to obtain highest selectivity [11,20].

2.4.3 Extraction time

Most of extraction in LPME is a time-dependent process, in which the

extraction efficiency is attained at the equilibrium condition. Accordingly, it is

important to determine the extraction time profile of analyte in order to configure the

equilibrium time. In SDME and HF-LPME, the equilibrium time usually between 30 and

60 minutes without lose of organic solvent [11]. Even though longer extraction times

generally result in increased extraction efficiency, it is not always practical to apply

extended extraction times. Sampling times shorter than the total chromatographic

time is more likely to ensure high sample throughput [20].

Unlike in SDME and HF-LPME, the extraction time in DLLME is not very

important. As the infinitely large surface area between extraction solvent and aqueous

phase forms after the formation of cloudy solution, so the target analytes differ quickly

into the extraction solvent. Therefore, DLLME is a time-independent, which is the most

important advantage of this technique [11].

2.4.4 pH adjustment

pH adjustment can enhance the extraction efficiency, because the dissociation

equilibria is influenced by the solubility of the acidic/basic target analytes. Many

reports show that the pH changes in the donor solution resulted in higher analyte pre-

concentration of analytes in a two-phase and a three-phase LPME.

Particularly in three-phase LPME, adjusting pH in the donor and acceptor phase

is very critical, since it influences the distribution ratio, enrichment factor and

recoveries of target analytes. To obtain high enrichment factor and high recoveries,

the pH of donor solution should be adjusted so the analytes of interest are in their

unionized form. In this form, the solubility of analyte in the donor solution will

decrease and an efficient transfer into the organic phase will be obtained. On the other

hand, the pH of acceptor solution should be adjusted to make the analytes of interest



in their ionized condition, in order to ensure efficient extraction of analytes into the

acceptor solution and to prevent analytes trapped in the organic phase [11].

2.4.5 Agitation of the sample

The main purpose of agitation is to accelerate the extraction kinetics and

enhance the extraction efficiency, since stirring allows the continuous exposure of the

extraction surface to the aqueous sample. Hence, thermodynamic equilibrium can be

achieved in a short time and induces the convection in membrane phase. Sample

agitation can be done in two ways, by stirring or vibrating the sample. Vibrating the

sample solution has more advantages than stirring the sample using magnetic stirrer,

because it eliminates the possibility of analytes being contaminated by the magnetic

stirrer. Furthermore, the use of magnetic stirrer in high stirring rate promotes bubble

formation, solvent evaporation and instability of micro drops [11,20].

2.4.6 The addition of salt

Salt addition is widely used in microextraction to improve the analyte

partitioning into the organic phase by salting-out effect. However, the effect of salt

addition to extraction efficiency may vary from enhancing, not influence to decreasing,

depending on the nature of target analytes. Caution should be given to the presence of

high concentration of salt in sample solution that may change the physical properties

of the extraction film. This condition will decrease the diffusion rate of analyte into the

organic phase [11,20].



Solid-Phase Microextraction (SPME)

3.1 Principle of SPME

SPME is a sample preparation technique that was developed by Pawliszyn and

coworkers in 1990 [31-33]. This technique is simple, rapid, highly sensitive, solvent free,

inexpensive and easy to automate [1-2,34-35]. The basic principle of SPME is the

partitioning of analyte between the sample phase and the coated fiber when the

coated fiber is exposed to the sample for a well-defined period time [10,31]. The

extraction is completed when the analyte concentration has reached distribution

equilibrium between sample matrix and the fiber coating. Once equilibrium is reached,

the extracted amount is constant and it is independent of further increase of

extraction time. The equilibrium condition can be described as [12]:

n = (Kfs . Vf . Vs . Co) / {(Kfs . Vf )+ Vs} (11)

where n is the mass of analyte absorbed by the coating; kfs is the partition coefficient

of analyte between the coating and sample matrix; Co is the initial concentration of a

given analyte in the sample; Vf and Vs are the volume of the coating and the sample,

respectively. When the sample volume is very large, such as in river, production stream

and ambient air (Vs >> Kfs . Vf), equation (11) can be simplified to [12]:

n = Kfs . Vf . Co (12)

It can be seen from equation (12) that the amount of extracted analyte is

independent from sample volume, so it is no need to collect a defined amount of

sample prior to analysis. The amount of extracted analyte will correspond directly to its

concentration in the matrix. This condition is very useful for on-site applications [12].

After the completion of extraction process, the fiber with concentrated analyte are

thermally desorbed in the case of GC or GC-MS, or injected via a sample loop in the

case of HPLC [36].



As shown in Figure 7, SPME device consists of coated fused silica fiber connected

to stainless steel tubing that is used to increase the mechanical strength of the fiber

assembly for repeated sampling. The stainless steel then contained in a specially

design syringe. During extraction, the fiber is first withdrawn into the syringe needle

then lowered into the vial by pressing down the plunger [10].

As seen in equation (11), the extraction efficiency is dependent on the partition

coefficient of the analyte between the coating and sample matrix (Kfs) [37]. Therefore,

the selection of fiber coating plays an important role in SPME [10]. The coating materials

can be liquid polymer, solid sorbent or combination of both, where the extraction

mechanism is quite different between liquid and solid polymer. In liquid coating, the

extraction mechanism is absorption. In absorption mode, the magnitude of analyte

diffusion coefficient allows the molecule to penetrate to the entire volume of the

coating within a well-defined extraction time [37].

On the other hand, solid polymers as coating agent possess complex crystalline

structure, lead to reduce analyte diffusion coefficient within the structure. In this

polymer, the extraction only occurs on the surface of the coating or through an

adsorption mechanism. Consequently, the extraction time for adsorption in solid

polymer is shorter than the absorption mechanism in liquid polymer [37].



The fiber coating selection of microextraction is based on the principle “like

dissolves like” [10,34]. Most of non-polar analytes can be extracted using

polydimethylsiloxane (PDMS), whereas polyacrylate (PA) is more suitable as extracting

agent for polar compounds, such as phenols. Mixed phases such as Carboxen-PDMS,

polydimethylsiloxane-divinylbenzene (PDMS-DVB) and divinylbenzene-carboxen-PDMS

are suitable for the extraction of volatile low-molecular mass [10,37].

The coating thickness is selected based on the efficiency required, the nature of

the analyte, the extraction time, and the molecular mass of analyte. Faster partition

equilibrium can be obtained by using thinner coating while small-molecular mass

compounds can yield high extraction with relatively thick coatings [10,37].

3.2 Classification of SPME

SPME may be performed in two arrangements, fiber SPME and in-tube SPME.

3.2.1 Fiber SPME

Fiber SPME is based on a modified syringe which contains stainless steel micro

tubing within needle. Inside the syringe, there is a fused silica fiber tip coated with

organic polymer. The techniques that usually used are headspace (HS) SPME and direct

immersion (DI) SPME (Fig. 8) [1,2]. The selection of extraction technique depends on the

nature of sample matrix, analyte volatility and affinity of sample between the matrix

and coating [10].

HS-SPME is used for volatile sample or sample that can be made volatile by

moderate heating [1,10]. In this technique, the fiber is placed above the sample so there

is no direct contact between the fiber and the sample [2]. This design can protect the

fiber coating from damage caused by extreme condition (very low or high pH) or large

molecules that tend to foul the coating [1]. In addition, HS-SPME can minimize

contamination on the surface of the fiber, gives cleaner extracts, greater sensitivity

and longer fiber life time. Extraction process of HS-SPME involves three phases

(coating, headspace and sample matrix), in which the limiting step is the transfer time

of analytes from the sample matrix to the head space [35]. Because of the requirements

of a high vapour pressure analytes, the transfer of the fiber to the GC as well as



desorption should be performed immediately after the extraction to minimize the risk

of analyte loss during storage of the loaded fiber.

DI-SPME is used for the extraction of low-to-medium volatility and high-to-

medium polarity [1,37]. In this technique, the fiber is directly immersed in the liquid

samples [2] and the mass transfer rate is determined by diffusion of the analyte in the

coating provided that the sample is “perfectly” agitated [10]. As the sample directly

contact with the fiber, strong acidic or alkaline condition should be avoided. An HF

membrane can be used to protect the SPME fiber from insoluble component in the

sample [1].

3.2.2 In-tube SPME

In-tube SPME is a new sample preparation technique using an open tubular

capillary as an SPME device (Fig. 9). It can be coupled with HPLC, liquid

chromatography-mass spectrometry (LC-MS) or GC and allows the convenient

automation of the extraction process [1,2,6]. In in-tube SPME, aqueous sample that

contains organic compounds can be directly extracted from the sample into the

internally coated stationary phase of a capillary. Subsequently, analytes are desorbed

using a stream of mobile phase. When the analytes are more strongly adsorbed to the

capillary coating, a static desorption solvent can be used [1,2]. Finally, the desorbed

compounds are injected into the column for further analysis. To prevent plugging of



the capillary column and flow lines, the sample solution need to be filtered before

extraction.

The extraction, desorption, and injection in in-tube SPME can be performed

continuously using standard auto sampler. The automation of sampling handling

process not only reduces analysis time, but also provides better sensitivity and

precision than manual techniques. Despite the low extraction yields of in-tube SPME,

this technique may provide reproducibility of extracted compound using an auto

sampler. Moreover, all of the extracts may be introduced into the LC column after in-

tube SPME [1].

3.3 Influence factors on the SPME efficiency

There are some variables which can influence the extraction efficiency in SPME.

3.3.1 Agitation of the sample

Sample agitation is important in order to ensure rapid and efficient extraction.

Agitation accelerates the transfer of analytes from matrix to the coating. Different

agitation methods can be chosen, namely fast sample flow, rapid fiber movement,

stirring and sonication. A suitable agitation method will result in shorter equilibration

time and higher extraction amount of analyte [10,34,37].



3.3.2 pH of sample

The extraction efficiency in SPME is enhanced by fully converting the analytes

into neutral forms because SPME coatings are more efficient to extract neutral forms

of analytes [10]. The adjustment of pH sample can be done by adding buffers to the

sample to prevent ionization of the sample. A high pH value and a lower pH value are

efficient to improve the extraction of basic and acid compounds, respectively. For

molecules possessing both acidic and basic functionalities, the optimum pH for

extraction must be determined empirically. The determination of optimum pH of the

sample should be between the stability ranges of the coatings and an extreme pH

value should only be used in HS-SPME mode owing to the potential fiber deterioration

when DI-SPME is used [37].

3.3.3 Ionic strength

The addition of salt influences the partition coefficient of analyte (Kfs). By

adding salt into the sample solution, the ionic strength will increase and the aqueous

solubility of sample will decrease (Kfs increase). This salting out effect causes the

analytes more easily to pass from the sample onto the coating. However, in some

cases, the salt addition may improve the extraction efficiency for both the target and

interfering compounds [37]. In this case, the effect of salt addition on the analyte

extraction depends on the nature of target analyte and the salt concentration.

Therefore, for a particular target analyte and sample matrix, experiments are needed

to determine the effect of adding salt on extraction efficiency. Generally, the addition

of salts is preferred for HS-SPME because fiber coating are prone to deteriorate during

agitation in DI-SPME. The salts commonly used to increase extraction efficiency are

(NH4)2SO4, Na2CO3, K2CO3 and NaCl [10,37].

3.3.4 Sample temperature

Extraction temperature should be considered during SPME. Increasing the

sample temperature may help to release the sample into the headspace and increase

analyte diffusion coefficient, which leads to an increase in the extraction rate or the

mass transfer rate onto the fiber coating. Nevertheless, as the temperature increases,



the fiber coating begins to lose its ability to adsorb analytes and the distribution

constant of sample between matrix and coating decreases. As a result, the sensitivity

and the analyte recovery at equilibrium condition are decreased. Furthermore, an

extremely high sample temperature may result in decomposition of some compounds

and creation of other component artifacts. As increased sample temperature affects

both diffusion coefficient and distribution constant, the optimum sample temperature

will depend on the physicochemical properties of target analytes [37].

3.3.5 Sample derivatization

Derivatization is commonly used in SPME-GC applications. Analyte

derivatization is used to transform an original compound into a product that has

different physicochemical properties. This step is important for the analysis of non-

volatile, polar, and ionic species which are difficult to extract and tend to react with

the injection port and analytical column. Some examples of derivatizing agents are

trimethyloxonium tetrafluoroborate, pentafluorobenzaldehyde, and bis(trimethylsylil)

trifluoroacetamide [37,38]. Two derivatization strategies that can enhance the extraction

efficiency are as follows:

a. Pre-extraction derivatization

In this method, the derivatizing agent is added to the sample matrix. Pre-extraction

derivatization is used for underivatized highly polar target analytes which do not

have a high affinity toward the commercially polar fiber coating. Accordingly, the

analytes must be first converted into less polar derivatives before SPME process

[37].

b. Simultaneous extraction and derivatization

This derivatization involves the loading of a derivatizing agent onto the fiber

followed by fiber exposure to the sample matrix, allowing simultaneous

derivatization and extraction processes to occur. Subsequently, the derivatized

analogs desorbed into an analytical instrument for further analysis. The loading

procedure for derivatizing agent needs to be optimized and factors such as

reagent’s vapor pressure, volatility and affinity toward the coating must be

considered [37].



Discussion

4.1. Recent applications of LPME for determination of drugs in biological samples

Single-drop microextraction has become a very popular LPME technique because

it is inexpensive, easy to operate and nearly solvent-free. There are few publications

on SDME extraction for drugs analysis in biological samples. Yao et al. [39] developed a

single drop LPME combined with HPLC-UV detector for the simultaneous analysis of

local anesthetics, lidocaine, bupivacaine, and tetracaine. Organic solvent o-dibutyl

phthalate was selected to extract local anesthetics in human urine sample because it is

compatible with the mobile phase of HPLC. The mobile phase consisted of (A) a

mixture of acetonitrile and triathylamine aqueous solution (11 mM)-0.1% phosphoric

acid aqueous solution (10:90,v/v) and (B) a mixture of acetonitrile and triathylamine

aqueous solution (20mM)- 0.1% phosphoric acid aqueous solution (50:50,v/v). 6 mL of

urine is made alkaline to pH 11 with 1.0 M NaOH and then extracted using 1 µL of o-

dibutyl phthalate. Higher enrichment factor (more than 86.0 fold) and significant

sample clean up were achieved within 30 min under the optimized extraction

condition (160 rpm of the stirring rate at 30oC). No matrix effects occur during the

extraction and the method was applied to urine sample from a patient who was

treated with extradural anaesthesia of lidocaine, bupivacaine, and tetracaine. Figure

10 shows the chromatogram of urine sample analysis. The result reveals that the

method is selective and sensitive enough to allow determination of lidocaine,

bupivacaine, and tetracaine in urine. This method may be applicable for drug

monitoring, forensic toxicology, and medico-legal practices.



In 2005, Gioti et al.[40] reported the analysis of hyperforin and hypericins

(hypericin and pseudohypericin) in biological fluids using single-drop LPME in

conjunction with HPLC-fluorescence detector. Those drugs are the extracts of St.

John’s Wort (Hypericum perforatum L.), which has been known for many medicinal

properties such as hepatic disorders, gastric ulcers, anti-inflammatory, anti-microbial,

anti-viral, anti-depressant, and anti-cancer agent. Many methods have been developed

for the measurement of hypericins and hyperforin in a variety of biological media, but

most of the methods employed hitherto that require non polar organic solvents where

hyperforin is unstable. Hence, the author proposed a new option for analysis of

hypericins and hyperforin in biological samples in order to reduce the steps required

prior to analysis and increasing the sensitivity. In this method, urine samples were

filtered before use in extraction to remove the suspended particles, while plasma

samples were mixed with methanol to precipitate protein. The pH of the sample was

adjusted to 6.0 prior to extraction. A mixture of n-octanol:chloroform (7:3 v/v) was

chosen as organic drop to avoid drop dislodgement and improve extraction yield of

hypericins and hyperforin. Extraction was held for 15 min with the stirring rate of 150



rpm at 40oC and no salt addition. After extraction, the organic solvent drop was

transferred to a micro vial and made up to 30 µl with methanol. Using isocratic

reversed-phase HPLC with methanol and phosphate buffer solution (pH 2.2) as mobile

phase (95:5, v/v), a complete analysis of urine and plasma samples can be performed

within 22 and 25 min, respectively. The author claims that the method is selective,

flexible and amenable to improvements towards improving identifications and LOQs.

Ebrahimzadeh et al. has determined fentanyl, a potent synthetic narcotic

analgesic, in plasma, urine and waste water using SDME combined with HPLC-UV [41].

To diminish matrix effect, plasma and urine sample were diluted with water at 1:5 and

1:1 ratios, respectively. The procedure is based on three-phase SDME, where fentanyl

was extracted from 3.6 mL samples solution containing 0.01 M NaOH into 100 µL n-

octane, then back extracted into 5 µL of 1x10-3 M HClO4. Others optimum experimental

conditions were stirring rate of 1000 rpm for 30 min in pre-extraction and 700 rpm for

20 min in back extraction; extraction temperature at 30 0C; and no salt addition. Within

optimum condition, enrichment factor of 355 was obtained. Table 1 compares the

proposed method with alternative methods for the extraction of fentanyl from

biological fluids. Although the proposed method has higher LOD (0.1 ng/mL) than

others, but reliable measurements of fentanyl can be performed with lower cost.

Table 1. Comparison of figures of merit of the proposal method with other methods applied for the analysis of fentanyl (from Ref. 27)

Method Sample preparation

LOD (ng/mL) r LDR (ng/mL)

Proposed LLLME 0.1 0.9998 0.5-1000 GC-NPD LLE 0.1 0.997 0.5-50 GC-MS SPE 0.0025 0.997 0.05-0.15 GC-MS HS-SPME 0.03 0.996 0.1-2000 GC-MS SDME < 0.075 0.9855 0.1-10

LC-MS/MS LLE 0.02 - 0.02-10 HPLC-UV LLE 0.2 0.996 0-2

He and Kang extracted a popular drug of abuse, methamphetamine and

amphetamine from urine samples by coupling three-phase SDME with HPLC-UV [42].

The author used method from other previous studies with some modification in

organic solvent volume and HPLC syringe. Instead of using Teflon ring in organic phase

and a Teflon sleeve on the tip of syringe needle, they used larger volume of organic



solvent and larger HPLC syringe. Urine samples were diluted with pure water (1:1)

before use to overcome unstable acceptor drop caused by interferences of co-

extractives in urine samples. The optimized extraction condition were 6.0 mL sample

solution containing 0.5 M NaOH, 400 µL n-hexane as organic phase, 5 µL 0.02 M H3PO4

as acceptor phase, 40 min pre-extraction followed by 40 min back-extraction with the

simultaneous extraction. The enrichment factor was found 730 and 500 for

methamphetamine and amphetamine, respectively. Figure 11 shows the

chromatogram result. Unknown peaks are found, with one tiny peak overlapped with

methamphetamine in spiked urine sample. The author reveals that the tiny peak could

be neglected since the area only 2% of amphetamine. Moreover, better selectivity of

the method could be improved by using mass spectrometer as detector. This method

exhibited low detection limit (0.5 µg/L), a wide linear range (1.0-1500 µg/L) and a good

repeatability (RSD < 5%). The wide linear range made of this method can be applied in

the initial screening test and confirmatory test of drug abuse.



The use of DLLME for analyte extraction in biological samples is limited because

of some reasons. First, the production of sediment phase for injection in analytical

instrument is not possible due to the interaction of matrix sample with the organic

solvents. Serial dilutions of sample may be used to procedure sediment phase, but this

procedure may alter the inherent property of matrix. Secondly, DLLME is only

applicable for the samples containing high concentration of analytes [16]. However,

some experiments for determining drugs in biological fluids were done using DLLME.

The application of DLLME combined with GC-FID was developed for separation

and determination of tricyclic antidepressants drugs, amitryptiline and nortryptiline, in

water samples by Yahdi and co-workers [43]. The performance of proposed method was

evaluated by determining amitryptiline and nortryptiline in human plasma. Prior to

extraction, amitryptiline and nortryptiline were liberated from protein plasma by

adding 1.0 mL methanol to 0.5 mL plasma sample. The samples then centrifuged for 15

min at 1000 rpm. 0.05 mL of supernatant was transferred to vial tube and diluted with

water to 5.0 mL. Subsequently, samples were basified using 1 M NaOH to the pH 12. A

mixture of 1.0 mL methanol (disperser solvent) and 18.0 µL of carbon tetrachloride

(extraction solvent) were injected rapidly into plasma samples and followed by gently

shaken of the mixture. Afterward, the mixture was centrifuged for 10 min at 1000 rpm

to form sediment phase. Finally, 2.0 µl of sediment phase was injected into GC. The

limit of detections was 0.07 and 0.02 µg/mL for amytriptyline and nortryptiline,

respectively. According to author, this method provides high recovery and enrichment

factor within a very short time.

Xiong et al. [44] proposed a DLLME combined with HPLC-UV for the

determination of three psychotropic drugs (amitryptiline, clomipramine, and

thioridazine) in urine samples. Prior to extraction, urine sample was centrifuged for 15

min at 4000 rpm. The supernatant was filtrated through a 0.45 µm filter and 10 M

NaOH was added to adjust the pH to 10. Subsequently, 0.50 mL of acetonitrile

(disperser solvent) and 20 µL of tetrachloride (extraction solvent) were rapidly injected

into 5.0 mL of urine sample and formed a cloudy solution. The cloudy solution was

gently shaken and followed by centrifugation at 4000 rpm for 3 min. A different



phenomenon was observed between the aqueous standard and urine samples after

the centrifugation process. For aqueous standard, a small droplet of carbon

tetrachloride was sediment in the bottom of the conical test tube. While for urine

sample, white lipidic solid was sediment in the bottom of the conical tube. The white

lipidic solid in urine sample might be due to co-sedimentation of the matrixes (such as

carbamide and uric acid) in urine at high pH values. The white lipidic solid was

dissolved in 200 µL of acetonitrile and then filtrated through a 0.45 µm membrane to

discard the white floccules in the extract urine. Finally, extract was injected into HPLC

for further analysis. The proposed method was applied to two urine samples collected

from two female patients who taken some psychotropic drugs combinations including

amitryptilline and clomipramine, respectively. The chromatogram result is shown in

Figure 12. As can be seen, the presence of major endogenous components, coexisting

drugs and their metabolites in urine sample has no obvious influence on the

determination of target anaytes. This result reveals that the proposed method has a

good selectivity for the analysis of the analytes and the method can be used in clinical

situations.



Fuh et al. [45] developed DLLME combined with liquid chromatography-

electrospray-tandem mass spectrometry (LC-ES-MS/MS) for the extraction and

determination of 7-aminoflunitrazepam (7-aminoFM2), a biomarker of the hypnotic

flunitrazepam (FM2), in urine sample. The procedure of extraction as follows: Urine

sample spiked with 7-aminoFM2 was basified using 0.2 M ammonia and 5% of NaCl

was added to increase the extraction efficiency. The precipitate formed was then

separated by centrifuging at 3500 rpm for 10 min. 5 ml of clear supernatant was taken

and the mixture of 500 µL of isopropyl alcohol (dispersive solvent) and 250 µL of

dichloromethane (extraction solvent) was rapidly injected into it. The mixture was

gently shaken and a cloudy solution was formed. The phases were separated by

centrifuging at 4000 rpm for 10 min. Later, the sediment phase was evaporated to

dryness in the concentrator for 20 min. The residue was reconstituted in 30 µl of

mobile phase (acetonitrile:water, 20:80) and 20 µl aliquot was injected into LC-ES-

MS/MS for further analysis. The proposed method was applied for the analysis of

various urine samples to demonstrate the potentiality of the technique. Within the

optimum condition, a good linearity (0.05 – 2.5 ng/ml) and detection limit of 0.025

ng/ml were obtained. A comparison of the proposed method and other methods

shows that the proposed method is specific, simple, and has LOD 40 times higher than

other methods.

In 2007, He and co-workers [46] developed a headspace aqueous drop LPME

combined with HPLC-UV for the determination of methampethamine (MAP) and

amphetamine (AP) in urine samples. Needle tip of an HPLC syringe was modified and

coated with a thin layer of parafilm to enable the formation of a stable aqueous drop.

Prior to extraction, the urine samples were preheated at 80oC for 15 min. To have a

high peak response, urine samples were modified to have 4 M NaOH by adding 8 M

NaOH and added 10% NaCl. 0.05 M H3PO4 solution was used as acceptor phase to

protonate the basic analytes. During 20 min of extraction time, the enrichment factors

were about 400 and 220 for MAP and AP, respectively. This method showed a good

linearity in the concentration range of 1.0-1500 µg/L and repeatability value (RSD) <

5%. The detection limits for both analytes were 0.3 µg/L. Compared with the previous



result using three-phase SDME method [42], this method totally eliminates the use of

organic solvent and significantly shorten the extraction time.

Huang et al. [47] studied the simultaneous extraction and derivatization of

amphetamine (AM) and methylendioxyamphetamine (MDA) in urine samples using

headspace hollow fiber combined with gas chromatography-mass spectrometry (GC-

MS) in the selected ion monitoring (SIM) mode. Prior to extraction and derivatization,

KOH and NaCl were added to urine samples. Since AM and MDA are polar compounds,

derivatizing reagent (pentafluorobenzaldehyde (PFBAY)) was added to the extraction

solvent to increase chromatographic efficiency. The optimal conditions for this method

was as follows: 3.0 µL of 1-nonanol as the extraction solvent; 4M of KOH; sample

agitation, 750 rpm; temperature, 950C; extraction time, 30 min; and NaCl added, 36%.

A good linearity was obtained in the concentration range of 50-350 ng/mL for AM and

50-700 ng/mL for MDA. Low limits of quantitation (0.25 ng/mL and 1.00 ng/mL for AM

and MDA, respectively) and an excellent repeatability (RSD ≤ 4%, n=5) were achieved.

The chromatogram of spiked urine and sample from drug abuser is shown in Figure 13.



Jonsson et al. [48] explored the extraction and preconcentration of salbutamol

and terbutaline in aqueous samples, including urine using HF-LPME containing anionic

carrier (Aliquat 336). The author used HPLC coupled with electrospray ionization

quadrapole ion trap tandem mass spectrometry for quantifying the drugs in urine

samples (ng/L). For analysis of spiked urine samples, 110 µL of urine sample was

diluted to 11 mL and adjusted the pH to 11.70 with 4 mol/L of NaOH solution. 20% of

Aliquat 336 was dissolved in dihexyl ether (impregnation solvent) to obtain higher

enrichment factor. 24 µL of 1 M NaBr was chosen as acceptor phase and the extraction

time was 60 min and 75 min for salbutamol and terbutaline, respectively. Although the

extraction time was relatively long, parallel extraction still can be done. The limit of

detection was 0.5 µg/L for terbutaline and 2.5 µg/L for salbutamol. This low LOD is

useful for tracking the drugs in complex matrices, such as body fluids and

environmental waters.

Ma and co-workers [49] introduced a carrier-mediated three-phase SDME

coupled with HPLC for simultaneous determination of illicit drugs (morphine,

ephedrine, and pethidine) in human urine. These drugs may be used for doping in

sport. Morphine and ephedrine are hydrophilic, while pethidine is hydrophobic. Before

extraction, the pH of the sample was adjusted to 11.4 with 2 M NaOH. 300 µL of

toluene containing 0.10 M Aliquat 336 and 0.2 M HCl (pH 0.7) was used as organic

phase and acceptor solution, respectively. A higher enrichment factor was obtained

with the volume ratio 4.9 mL:1.5 µL of sample and acceptor solution. The sample was

extracted with the stirring rate of 400 rpm at 30oC for 15 min. Under the optimal

conditions, a good linearity (0.1-10 mg/L) and enrichment factors of 202-515 were

obtained for the studied drugs. The limits of detection (LOD) were 0.5 mg/mL for both

morphine and ephedrine, and 0.02 mg/L for pethidine. The LODs were superior to

those obtained with other methods. The author claims that the proposed method is a

feasible, cost-effective, and convenient for quantitative analysis of morphine,

ephedrine, and pethidine in urine samples. Figure 14 gives the chromatograms of

standard solution, urine samples (before and after LPME pretreatment) and LPME

pretreated urine sample spiked with the three drugs.



In 2009, Yamini et al. [50], developed a carrier mediated three-phase HF-LPME

combined with HPLC-UV for simultaneous extraction and determination of some

tetracycline antibiotics, including tetracycline (TCN), oxytetracycline (OTCN) and

doxycycline (DCN), in bovine milk, human plasma, and water sample. For the

extraction of tetracyclines, human plasma was diluted with the deionized water at

ratio 1:3, followed by the addition of 0.05 M Na2HPO4 and adjustment of the pH of

samples (9.1 ≤ pH ≤ 9.5). Subsequently, samples were extracted under the optimized

conditions {10% (w/v) of Aliquat 336 in octanol as organic solvent; 25 µL solution

containing 0.1 M H3PO4 and 1.0 M NaCl (pH=1.6) as receptor phase; stirring rate of 900

rpm and 35 min for the extraction time}. This method exhibits a very low of detection

limit with high pre-concentration factor. The author reveals that the use of mass

detection may improve the limit of detection of tetracyclins from the larger volume of

samples. The extraction setup is simple and has high cleanup effect due to active

transport of analytes. Table 2 shows the high sensitivity of proposed method,

comparing to the traditional methods.



Table 2. Comparison of the proposed method with other published methods for the extraction and determination of TCNs (from ref. 50)

Tetracyclines Sample Method Detection Detection limit

OTCN, TCN, DCN Milk, plasma, water HF-LPME HPLC-UV 0.5-1.0 µg/L

OTCN, TCN, CTCN, DCN

Milk, serum, urine Metal chelate affinity column

CE-UV 1.4-5.3 µg/L

OTCN, TCN, CTCN Urine, plasma LLE as calcium complex

HPLC-UV 1-1.5 mg/L

OTCN, TCN, CTCN Urine, plasma LLE as calcium complex

HPLC-UV 0.25-0.5 mg/L

TCN Plasma Ion-pair extraction with TBA(1)

HPLC-UV 0.2 mg/L

OTCN, TCN Milk Ion-pair extraction with TBA into

CH2Cl2

HPLC-UV 10 µg/L

OTCN, TCN, CTCN Milk Matrix solid phase dispersion

HPLC-UV 0.1 mg/L

OTCN, TCN, CTCN Milk C18 cartridge HPLC-particle

beam MS

0.1 mg/L

OTCN, TCN, CTCN Milk Extraction with 1 M HCl and CH3CN

HPLC-UV 2-4 µg/L

OTCN, TCN, CTCN Urine Addition of 0.2 M KH2PO4

ESI-MS-MS 10 µg/L

OTCN, TCN, CTCN, DCN

Water SPE HPLC-ESI-MS

4-6 ng/L (for 1 L sample)

OTCN, TCN, CTCN, DCN,…

Water On-line SPE LC-MS 0.09 ng/L

(1) Tetrabutyl-ammonium bromide

Ugland et al. [51] used two-phase HF-LPME combined with capillary GC-NPD for

the determination of diazepam and its main metabolite N-desmethyldiazepam in

human urine and plasma. Prior to extraction, 300 µL of 0.1 M phosphate buffer pH 7.5

was added to 3.5 mL urine samples, whereas 200 µL methanol was added to 3 mL

plasma samples to reduce the protein binding of the benzodiazepines. Accordingly,

both of urine and plasma samples were agitated for 30 s. A mixture of butyl acetate: 1-

octanol (1:1 v/v) and a mixture of hexyl ether:1-octanol (1:3 v/v) was used as acceptor

solution for urine and plasma, respectively. The LPME extraction was done by vibrating

the samples at 600 rpm for 50 min. The proposed method provided excellent clean up

of endogenous compounds and a good linearity in the range of 0.5-8.0 nmol/mL for

both drugs. The limit of detection was 0.020 nmol/mL and 0.115 nmol/mL for N-



desmethyldiazepam in urine and plasma, respectively, and 0.025 nmol/mL for

diazepam in plasma.

In 2001, Halverson et al. [52] reported a three-phase HF-LPME combined with CE

UV-detector for the determination of highly hydrophobic drugs. They focused on an

antidepressant drug citalopram (CIT) and its main metabolite N-desmethylcitalopram

(NCIT) as model compounds. The extraction was performed as follows: 1 mL of human

plasma was transferred into vial and diluted with 2.73 mL of pure water. 20 µL of 10

µg/mL I.S. and 250 µL of 2 M NaOH were added to the sample. Polypropylene hollow

fiber was dipped into hexyl ether for impregnation and the excess of solvent was

removed by doing ultra sonification in water bath for 5 s. After impregnation, 25 µL of

20 mM phosphate buffer pH 2.75 was injected into the hollow fiber and the fiber was

subsequently placed in sample solution. Samples were vibrated at 1200 rpm for 60

min. Under the optimized conditions, the extraction recoveries were 76, 62, and 61 %

for CIT, DCIT, and I.S., respectively. Despite the relatively long extraction time, a high

extraction throughput could be achieved due to parallel extraction of 20-30 samples. In

addition, low limit of detections were obtained for both of drugs (5.5 ng/mL for DCIT

and 5 ng/mL for CIT). In Figure 15, the chromatogram of plasma sample from a patient

treated with 40 mg of citalopram is shown. Although the patient was treated also with

trimeprazine and chlorpromazine, only peaks for CIT, DCIT and I.S are detected in the

electropherogram.



Andersen and co-workers[53] published enantiometric determination of

citalopram (CIT) and desmethylcitalopram (DCIT) in plasma using LPME combined with

CE. For extraction process, they used published method from Halverson et al. [52] with

some modifications. Dodecyl acetate was used as organic phase instead of hexyl ether,

because it has better extraction recovery and precision. Shorter fiber (1.8 cm) with a

rodlike configuration was used instead of 8 cm fiber with a hairpin bend. This fiber

modification made ease of liquid handling and also more compatible with smaller

sample. The running buffer for chiral separation was 25 mM sodium phosphate pH 2.5

containing 1%-S-β-CD, 12% ACN and 0.1% PVA. The result shows that the proposed

method (LPME-CE) is a promising tool to determine enantiometric of chiral drugs.

Halvorsen and co-workers[54] extracted amphetamine and its derivatives from

biological matrices by coupling LPME with flow injection tandem mass spectrometry

(FIA-MS-MS). Atmospheric pressure ionization operated in positive mode was used as

ion spray. MS-MS was utilized to identify MDEA and MBDB, since both of analytes have

the same m/z values. In this method, 0.5 mL of urine or whole blood were made

alkaline with 0.5 ml of 1 M NaOH, and all the urine samples were also diluted with

water to 4 mL to reduce the salt concentration. The hollow fiber was dipped in dihexyl

ether to immobilize the solvent in the pores, and excess organic solvent was removed

by ultra sonification. Subsequently, 25 µL of 0.01 M HCl (acceptor solution) was filled

into the hollow fiber. During extraction, the sample was vibrated for 15 min at 1500

rpm. CE system was used to determine the enrichment factors and extraction

recoveries, while FIA-MS-MS was used for the determination of amphetamine sulfate

(A), methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDA), 3,4-

methylendioxyethylamphetamine (MDEA), and N-methyl-1-(3,4-methylene-

dioxyphenil)-2-butanamine (MBDB). The CE results shows that enrichment factors in

whole blood is in the ranges of 6-18 and in the ranges of 4-14 for urine sample,

corresponding to extraction recoveries of 29-89% in whole blood and 20-68% in urine.

The LODs were determined at a signal-to-noise (S/N) level of 5 (Table 3). The author

claims that this method is a promising alternative for rapid screening of drugs in

biological matrices.



Table 3. Detection limit (ng/mL) (S/N = 5)

Whole blood Urine

Amphetamine 8 100

Methamphetamine 5 30

MDA 14 100

MDMA 2 8

MDEA 0.4(a) 4(b) 2(a) 6(b)

MBDB 0.4(a) 4(b) 2(a) 6(b) (a)

After MS-MS, SIM mode; (b) After MS-MS, SRM mode

Another chiral drug determination was developed by Bonato et al. [55]. In this

method, they used antidepressant drug, mirtazapine, in human plasma as a sample

and analyzed using chiral liquid chromatography. Prior to extraction, 0.7 mL of plasma

was made alkaline by adding 0.15 ml NaOH 10 M and diluted with 3.1 mL deionized

water to 4 mL. Hollow fiber was dipped in toluene for 30 s and the excess solvent was

removed by stirring the hollow fiber for 30 s in water. Afterwards, 22 µL of toluene was

used to extract mirtazapine from plasma. The extraction was carried out during 30 min

at 22oC. After extraction, toluene was evaporated to dryness and the residues were

dissolved in 80 µL of mobile phase (hexane:ethanol,98:2 v/v, plus 0.1% diethylamine as

mobile phase). Finally, 50 µL aliquot was injected into LC system. The chromatogram

result shows that there are no interfering peaks and no significant co-elution with

endogenous compounds.



4.2 Recent applications of SPME for determination of drugs in biological samples

The fiber SPME has been successfully applied to drug analysis in biological samples.

In 1998, Reubsaet and co-workers[56] developed a method to determine the

benzodiazepines in human urine and plasma using SPME combined with GC. Several

factors likely to affect the analyte recovery were screened in a fractional factorial

design. The final condition for extraction of oxazepam, diazepam, nordiazepam,

flunitrazepam, and alprazolam were as follows: Octanol was immobilized on a

polyacrylate fiber for 4 min, the extraction took place at pH 6.0 for 15 min. For urine

samples, 0.3 g/mL sodium chloride was added to the solution to increase the

extraction recovery. For plasma samples, 1 M HCl in glycerol was added firstly to

plasma in order to release benzodiazepines from the proteins. Subsequently, TCA was

added to precipitate the protein and then followed by centrifugation. The extraction

recovery in plasma was less than in urine due to high ionic strength in the supernatant

that form an ionic layer around the fiber and causing repulsing of the charged analytes.

According to author, this method offers sufficient enrichment for bioanalysis after a

single dose of high dose of benzodiazepines, but for low dose benzodiazepines as

flunitrazepam, further sensitivity is needed.

Myung and co-workers developed DI-SPME combined with GC-NPD for

determination of pethidine and methadone in human urine [57]. The procedure was

based on the partition of drugs between the coated fiber (100 µm PDMS) and the

aqueous solution during the equilibration time (30 min). To enhance the affinity of the

coated fiber, the pH and the ionic strength of sample solution were set to 11 and 15%

of NaCl, respectively. After extraction, the needle of the coating fiber assembly was

injected directly into the GC injector and desorbed for 1 min. The detection limits of

pethidine and methadone were below 1 ng/mL and the within-day relative standard

deviation (RSD) for both drugs was below 9 %. A typical GC trace from an addict’s urine

sample analyzed using the developed method is presented in Figure 17.



For the first time, delorazepam was determined in urine using SPME

coupled with HPLC-UV by Zambonin et al. [58]. In this experiment, they compared the

performances of Carbowax/templated resin (Carbowax/TPR-100) and a

polydimethylsiloxane/divinylbenzene (PDMS/DVB), indicating the latter as the most

suitable for urine sample analysis. The SPME method involved 13.5 ml of urine, with

1.5 ml phosphate buffer (0.5 M, pH 9.7) to obtain a pH value of 6.5, and the extraction

was carried out at room temperature for 30 min. After sample extraction, the fiber was

statically desorbed for 3 min in acetonitrile/water mixture (40:60 v/v). HPLC analysis

required less than 10 min, and the repeatability was 6.9 ± 0.5 % in concentration range

0.05-0.5 µg/mL. Figure 18 shows the SPME-LC-UV chromatograms obtained from blank

urine sample and a urine sample spiked with 5 ng/ml of delorazepam. As can be seen,

delorazepam is clearly detected and is well resolved from matrix components. The

proposed method is simple and also has a comparable sensitivity with an existing

SPME-LC-MS method for the determination of benzodiazepines.



Lancas and co-workers presented a simultaneous determination of lamotrigine

(LTG) with primidone (PRM), carbamazepine (CBZ), carbamazepine epoxide (CBZE),

Phenobarbital (PB) and phenytoin (PHT) from human plasma using SPME and GC-TSD

[59]. LTG is an anticonvulsant drug and the presence of hepatic-enzyme-inducing

agents, such as CBZ, PB, PHT and PRM can reduce the half life of LTG and a higher dose

may be required. The best procedure for SPME was 1.0 mL of a sample plasma diluted

with buffer (1:3 v/v) followed by modifying with 3.0 mL of a potassium phosphate

buffer (pH 7.0) containing 15% NaCl, and then extracted using 65 µm Carbowax-

divinylbenzene fiber. The extraction was done with the stirring rate of 2500 rpm at

30oC for 15 min. The capillary GC-TSD chromatogram of the SPME extract of human

plasma from a patient who orally administered LTG and CBZ per day is presented in

Figure 19. As apparent, an excellent separation was achieved with a very clean

chromatographic profile. The developed method can be used in pharmacokinetic

studies and routine therapeutic drug monitoring.



The analysis of naproxen in human urine using Carbowax/template resin

(CW/TPR) fiber combined with HPLC—UV was described by Zambonin et al. [60]. The

determination of naproxen glucuronide was also indirectly performed after chemical

and enzymatic hydrolysis of the conjugate. The procedure required a very simple

sample pre-treatment, an isocratic elution and provided a highly selective extraction.

0.15 ml of urine samples were diluted with 15 ml of 10 mM acetic acid pH 3 and 0.05

M NaCl solution. Further, the extractions were performed at 20 0C for 30 min followed

by static desorption in mobile phase for 10 min. Within the optimum condition,

naproxen was clearly detected and could be resolved from matrix components. A good

linearity in the concentration range of 0.2-20 µg/ml and quantitative recoveries of 94.5

± 4.5%, were obtained.

Duan and co-workers developed a HS-SPME coupled with GC-MS for the

determination of tramadol in human plasma [61]. They examined 3 fiber coatings

(PDMS, PA, and PDMS/DVB) and found that PDMS/DVB showed the highest SPME

efficiency for tramadol. 0.5 mL of plasma samples were modified with 0.5 mL NaOH

(0.1 M) and then extracted at temperature 100 0C and stirring rate of 2000 rpm for 30

min. The LOD of tramadol in plasma sample was 0.2 ng/mL and the calibration curve

was linear in the range of 1-400 ng/mL. The method was successfully applied to



determine tramadol in human plasma. Samples were taken from 10 healthy volunteers

after a single oral administration of tramadol 100 mg.

Determination of drugs for depression treatment and other psychiatric

disorders in human urine has been done by Barrio et al. They used citalopram (CIT),

fluoxetine (FLX), and their main metabolites as model compounds and direct

immersion SPME as extraction mode [62]. Prior to extraction, urine samples were

centrifuged at 2500 rpm for 10 min at room temperature and the supernatant was

diluted with water in ratio 1:5 minimize the matrix effect and prevent the

contamination of sample. 3 ml of the diluted samples were put into vials and 150 µL of

5 % acetonitrile and 40 µL of NH3/NH4Cl buffer (pH 9.5, 2M) were added. The

extraction was carried out using CW/TPR fiber. Before extraction, the fiber was

conditioned in the interface with mobile phase for 20 min, followed by immersing the

fiber in water for 5 min and drying for 5 min. The extractions were done by dipping the

fiber to the urine sample for 15 min at room temperature. The results showed the

absence of the interference from endogenous compounds and the present method

was adequate to quantify CIT, FLX and their main metabolites in urine sample of

patients receiving therapeutics dose of citalopram or fluoxetine.

Brown and co-workers provide a validated SPME-GC-MS method for

simultaneous quantification of four club drugs in human urine [63]. These drugs include

gamma-hydroxybuyrate (GHB), ketamine (KET), metamphetamine (MAMP) and

methylendioxymethamphetamine (MDMA). Derivatization prior to GC-MS was done

because all drugs are semi-volatile. Pre-extraction derivatization was chosen to reduce

sample preparation steps and to minimize damage to the column head. The drugs

were spiked in human urine and derivatized using combination of pyridine and

hexylchloroformate. Subsequently, the drugs were extracted using PDMS fiber at 900C

for 20 min. Desorption in the GC injector was done for 1 min at temperature 225oC

using a splitless injection. The LODs and LOQs for GHB, KET, MDMA were 0.1 µg/mL

and 0.5 µg/mL, respectively. While the LOD and LOQ of MAMP was 0.05 µg/mL and 0.1

µg/mL, respectively. Owing to low detection limits, the method is capable of detecting

low amounts of each of these club drugs.



Zambonin et al. developed a simple method for the analysis of beta-adrenergic

drug, clenbuterol, in human urine and serum using SPME and LC [64]. In this

experiment, they used PDMS/DVB fiber for extraction process. pH of urine samples

were modified to 12, and then centrifuged for 10 min at 5000 rpm. 15 ml of

supernatant was subjected to SPME. 0.15 mL of serum samples were diluted with

phosphate buffer (50mM, pH 11.7) in ratio 1:10, stirred and ready for extraction. The

extraction was at 50 0C for 60 min and desorption was performed in static mode for 10

min. A slight modification of mobile phase composition was necessary in urine sample

analysis due to the presence of the unresolved matrix interferences. Figure 20 shows

the SPME-LC-UV chromatograms obtained from urine and serum. The results show

that the analyte is clearly detected and well resolved from matrix components.



Conclusion

The application of LPME and SPME for the determination of drugs in biological

samples has been summarized. As described in the introduction, biological samples

have complex matrices that could disrupt the analysis of drugs. By applying LPME and

SPME as sample preparation method, an excellent clean-up of biological samples can

be obtained. Furthermore, the use of LPME and SPME as extraction technique has a

number of advantages, such as effective, inexpensive and may be utilized as green

chemistry approach because it could reduce the use of organic solvent. Some key

points like the physical properties of analytes, the sample matrix and subsequent

analytical techniques must be considered for the selection of sample preparation

method.

From the means point of view, the use of SPME as microextraction technique

has some drawbacks. SPME fibers are expensive and have limited lifetime, since they

tend to degrade with increased usage. The differences in length and thickness of

commercial fiber coating may result in variation of analyte enrichment from fiber to

fiber, and a partial loss of the coating may occur when a thermal conditioning step

before using SPME is applied which can affect the chromatographic result. A fiber

conditioning before each run is needed to diminish the carry-over effect. Other

drawbacks of SPME are it is identically coupled with GC since only volatile anaytes can

be extracted using SPME and a sample derivatization is needed for less volatile drugs.

Coupling SPME with HPLC or CE is less attractive because it is involving specially

designed desorption interfaces. However, SPME has superiority in extraction time. The

extraction time is usually between 15 and 30 minutes.

The use of LPME may eliminate the drawbacks of SPME. In LPME, especially HF-

LPME, there is no carry-over effect as the hollow fiber is disposable. LPME also can be

used for extraction of most drugs. Depends on the characteristics of the drugs, we can

choose to use a two-phase or a three-phase system to extract the drugs. LPME is also



easily coupled with GC, HPLC or CE, without needed special designed interfaces. For GC

analysis, a two-phase LPME can be chosen as extraction mode, while for HPLC or CE

analysis, a three-phase system is preferred. The main drawback of LPME is a longer

extraction time, which is usually performed in 30-50 min. Nevertheless, a high

throughput is still enabled for LPME by parallel extraction of 20-30 samples. The ease

of LPME as sample preparation method and coupled with other instruments make this

technique is preferred than SPME. This can be seen from increased publications about

LPME year by year.

In my point of view, LPME is a promising tool to analyze drugs in biological

samples. In the future, there will be more research in clinical, pharmaceutical and

toxicological fields utilizing LPME as sample preparation method. However, the

implementation of LPME as sample preparation method is limited by the availability of

commercial equipment. Commercial equipment of LPME that can be fully automated

and compatible with common laboratory robotics and auto-samplers should be

produced for further improvement.



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