Applications of Solid-phase Microextraction in Food Analysis
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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
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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
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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
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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
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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.
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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
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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
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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
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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].
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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
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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
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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
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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
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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)
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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.
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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.
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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
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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].
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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].
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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].
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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
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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
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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].
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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,
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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].
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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.
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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
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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
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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.
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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
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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.
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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
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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.
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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.
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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.
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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-
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34 Febri Annuryanti [10145222]
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.
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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.
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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.
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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.
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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.
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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.
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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
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41 Febri Annuryanti [10145222]
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.
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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.
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43 Febri Annuryanti [10145222]
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
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44 Febri Annuryanti [10145222]
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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