American Journal of Anal yt ical Chemistry, 2011, 2, 429-436
doi:10.4236/ajac.2011.24052 Published Online August 2011 (
Copyright © 2011 SciRes. AJAC
Trace Determination of Tamoxi fen in Biological Fluids
Using Hollow Fiber Liquid-Phase Microextraction
Followed by High-Performance Liquid
Chromatography-Ultraviolet Detection
Amir Kashtiaray1, Hadi Farahani2, Sharareh Farhadi1, Bertrand Rochat3, Hamid Reza Sobhi1,3*
1Department o f Chemistry, Tehran Payamenoor University, Tehran, Iran
2Industrial and Enviro nmental Protection Division, Research Institute of Petroleum Industry (RIPI), Tehran, Iran
3Quantitative Mass Spectrometry Facility, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
Received February 27, 2011; revised May 1, 2011; accepted June 17, 2011
The applicability of hollow fiber liquid-phase microextraction (HF-LPME) combined with high-performance
liquid chromatography-ultraviolet detection (HPLC-UV) was evaluated for the extraction and determination
of tamoxifen (TAM) in biological fluids including human urine and plasma. The drug was extracted from a
15 mL aqueous sample (source phase; SP) into an organic phase impregnated in the pores of the hollow fiber
(membrane phase; MP) followed by the back-extraction into a second aqueous solution (receiving phase; RP)
located in the lumen of the hollow fiber. The effects of several factors such as the nature of organic solvent,
compositions of SP and RP solutions, extraction time, ionic strength and stirring rate on the extraction effi-
ciency were examined and optimized. An enrichment factor of 360 along with substantial sample clean up
was obtained under the optimized conditions. The calibration curve showed linearity in the range of 1 -
500 ng·mL–1 and the limit of detection was found to be 0.5 ng·mL–1 in aqueous medium. A reasonable rela-
tive recovery (89%) and satisfactory intra-assay (3.7% - 4.2%, n = 3) and inter-assay (7.5% - 7.8%, n = 3)
precision illustrated good performance of the analytical procedure in spiked human urine and plasma sam-
Keywords: High-Performance Liquid Chromatography-Ultraviolet Detection, Hollow Fiber Liquid-Phase
Microextraction, Human Urine and Plasma Samples, Tamoxifen
1. Introduction
Sample preparation has a direct impact on accuracy, pre-
cision, limits of detection and is a determining step of the
analytical process, especially when traces have to be de-
termined [1-6].
The invention of solid phase microextraction (SPME)
by Pawliszyn and co-workers [7], basically initiated the
interest for microextraction techniques in analytical che-
mistry. SPME satisfies most of the requirements of a
good sample preparation technique, including simplicity
of use, automation, and low consumption of materials [8].
Thus, it has been applied to determine a broad range of
organic compounds in numerous types of samples [9].
An alternative solvent-minimized sample preparation
approach to complement SPME appeared in the middle-
-to-late 1990s [10-12]; liquid-phase microextraction (LP-
ME) utilizes only a small amount of solvent (low micro-
liter range) for concentrating analytes from aqueous sam-
ples. It is simply a miniaturized format of liquid-liquid
extraction (LLE) and overcomes many of its disadvan-
tages as well as some of those of SPME (e.g. non-de-
pendence on a commercial supplier and sample carry-
over). LPME is simple to implement and use, generally
fast, and is characterized by its affordability and reliance
on widely-available apparatus or materials [13]. The ap-
plications of LPME in environmental and biological
analysis have been described in several papers [14-18].
LPME can be classified as two- [12,19,20] and three-
-phase [21-24] categories. Two-phase microextraction is
usually performed by suspending a drop (a few microli-
ters) of organic solvent on the tip of either a Teflon rod
or the needle tip of a microsyringe immersed in the
stirred aqueous sample solution. Analytes are extracted
into the organic solvent and then directly injected into a
gas chromatograph (GC) or high-performance liquid
chromatography (HPLC) for analysis. Hollow fiber (HF)
two-phase microextraction has also been developed to
enhance the extraction efficiency and to stabilize the
extracted solvent microdrop. In three-phase microextrac-
tion, the ionizable analytes in the aqueous sample are
extracted through a thin phase of organic solvent inside
the pores of a polypropylene HF or an organic solvent
layer held within a Teflon ring and then back extracted
into another aqueous acceptor solution. Following this
procedure the acceptor solution could be analyzed by
HPLC or capillary electrophoresis (CE), without further
treatment. It has been proven that HF-LPME is very
useful for extraction of drugs and metabolites from bio-
logical matrices and pollutants from environmental sam-
ples with simultaneous clean-up of the matrices [25-28].
Tamoxifen (TAM) is an oral non-steroidal anti-
estrogen drug used in the treatment and prevention of
breast cancer [29]. TAM’s primary mechanism of action
is competitive inhibition of the estrogen α-receptor, the-
reby inhibiting growth of malignant breast cells. TAM
administration for 5 years reduces the risk of recurrence
of breast cancer both locally and systemically and im-
proves overall survival rates. Moreover, with the signifi-
cant grow in the use of TAM, the drug monitoring at
trace level is of great importance.
Analytical techniques applied in the determination of
TAM has been well documented, including TLC [30],
GC [31,32], HPLC [33-35], CE [36-38], GC-MS [39],
LC-MS [40,41] and CE-MS [42]. Additionally, some
electrochemical methods such as voltammetry [43] have
also been reported for TAM analysis. Nonetheless, the
drug was determined in complex matrices by these tech-
niques, usually after laborious manipulation of the sam-
ple before the instrumental analysis.
The aim of the present study is to assess the suitability
of HF-LPME technique for the determination of TAM in
biological fluids. The factors affecting the microextrac-
tion efficiency were studied in details and the optimal
conditions were established. The resulting method was
validated for quantitative purposes and then was applied
to spiked real sample analysis in combination with
high-performance liquid chromatography-ultraviolet de-
tection (HPLC-UV).
2. Experimental
2.1. Chemicals and Reagents
All the reagents were of analytical grade. TAM citrate
was kindly donated by Iran Hormone pharmaceutical
company (Tehran, Iran). HPLC-grade methanol, acetoni-
trile, dihexylether, n-octanol, n-dodecane, HCl, NaCl,
and NaOH were purchased from Merck (Darmstadt,
Germany). Phosphate and ammonium acetate buffers
were prepared from phosphoric and glacial acetic acid
and their corresponding salts, respectively (Merck). The
used reagent water was purified with a Milli-Q system
from Millipore (Bedford, MA, USA).
2.2. Preparation of Standard Solutions and Real
A proper amount of TAM citrate was dissolved in me-
thanol to obtain stock solution with a concentration of
1000 mg·L–1. Working standard solutions were freshly
prepared by diluting the standard solutions of the analyte
with the reagent water to the required concentrations.
Both stock and working standard solutions were stored at
4°C in a refrigerator. The concentration of the drug in the
preliminary experiments was 50 ng·mL–1. Human urine
sample was obtained from a healthy female and Iranian
Blood Transfusion Organization (Tehran, Iran) was the
supplier of the plasma sample as well. These samples
were filtered through a 0.45 µm pore-size cellulose ace-
tate membrane filters prior to the extraction.
2.3. Instrumentation
The HPLC system consisted of a Shimadzu (Tokyo, Ja-
pan) LC-10 AV pump, a Rheodyne 7725 injector equ-
ipped with 20 µL sample loop combined with a SPD-10
AV UV-Vis detector. Chromatographic separation was
made on a Phenomenex CLC-C18 (150 mm × 4.6 mm;
5 µm) column under isocratic elution condition. The
mobile phase was a mixture of acetonitrile and ammo-
nium acetate (pH 6.9; 0.05 M) (70/30, v/v) with a flow
rate of 1.0 mL·min–1. UV detection at 254 nm was used
for quantification.
2.4. Extraction Procedure
All the HF-LPME experiments were performed using
Accurel Q 3/2 polypropylene hollow fiber membrane
(600 µm I.D., 200 µm wall thickness, 0.2 µm pore size)
from Membrana (Wuppertal, Germany). The whole fiber
was cut into small segments with the length of 9.0 cm.
One end of each resulting hollow fiber was heat-sealed
using a soldering iron. A 25 µL syringe model 702 NR
from Hamilton (Bonaduz, Switzerland) was employed to
introduce the receiving phase (RP) solution into the lu-
men of the hollow fiber, to suspend the hollow fiber and
also to inject the extracted analyte at the end of the ex-
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
traction into the HPLC loop. Extraction and injection
processes were performed in the following steps: 1) 15 mL
of the aqueous sample solution (source phase; SP) was
transferred into a 16 mL glass vial containing a 10 mm ×
4 mm magnetic stirring bar; 2) the vial was placed on a
magnetic stirrer model ZMS 74 from ZAG Chimi Com-
pany (Tehran, Iran); 3) a carefully measured portion of
25 µL of the receiving phase was injected into the hollow
fiber; 4) the fiber was submerged in the organic solution
(membrane phase; MP) for 5 s and then into the reagent
water for 5 s for washing the extra organic solution from
the surface of the fiber; 5) the fiber was bent into a
U-shape and together with a small part of the supporting
syringe needle was submerged in the sample solution; 6)
the vial was covered with Para Film and stirred for a
prescribed time period; 7) at the end of the extraction
time, the hollow fiber was removed from the sample so-
lution, and its closed end was cut and the receiving phase
was withdrawn into the syringe; 8) finally 24 µL of the
receiving phase was injected into the HPLC. In initial
experiments, the volumes of SP and RP solutions were
15 mL and 25 µL, respectively. Also, to obtain suitable
signals in the optimization experiments, relatively high
concentration of aqueous solution of TAM (50 ng·mL–1)
was used. All the experiments were done at room tem-
perature and the SP was stirred at a rate of 1000 rpm for
60 min.
2.5. Calculations
The enrichment factor (EF) and percent extraction of the
drug were calculated by the following equations:
EF = C RP, final / C SP, initial (1)
Extraction (%) = EF × VRP / VSP × 100 (2)
where C RP, final and C SP, initial are the final and initial con-
centrations of the drug in RP and SP, respectively. C
RP,final of the extracted drug was calculated from the cali-
bration curve. V SP and V RP are the volumes of SP and
RP, respectively.
3. Results and Discussion
3.1. Basic Principle of the Extraction
In three-phase LPME, the analyte is extracted from the
aqueous sample solution (SP) into the organic phase
immobilized within the pores of the hollow fiber known
as membrane phase (MP) and then it is back-extracted
into RP located inside the hollow fiber. For an analyte
such as A, the extraction process can be written as:
RP (3)
The initial amount of analyte, ni, is equal to the sum of
individual amounts of analyte present in all the phases
during the whole extraction process:
n i
= n SP + n MP + n RP (4)
where n SP is the amount of analyte in the SP solution, n
MP the amount of analyte in the MP solution and n RP is
the amount of analyte in the RP solution. At the equilib-
rium condition, Equation (3) can be written as:
C i V SP = C eq.SP V SP + C eq.MP V MP + C eq.RP V RP (5)
where C i is the initial concentration of analyte, C eq.SP, C
eq.MP and C eq.RP are analyte concentrations in the SP, MP
and RP solutions at equilibrium condition, respectively.
V SP, V MP and V RP are the volumes of the source, mem-
brane and receiving phases, respectively. It is worth not-
ing that in all cases, the analytical signal was recorded as
the function of extraction percent (%) versus each pa-
rameter regarding the optimization process.
3.2. Organic Solvent Selection
Selection of the solvent should be based on comparison
of selectivity, extraction efficiency and the level of tox-
icity. In addition, the polarity of the organic phase should
be similar to that of the polypropylene fiber so that it can
be easily immobilized within the pores of the fiber. This
function greatly affects the performance of HF-LPME,
since extraction occurs on the surface of the immobilized
solvent. Three different organic solvents (i.e. dihexy-
lether, n-octanol and n-dodecane) were used in the pre-
sent work as organic membrane solvents. Based on the
results, the best solvent proved to be dihexylether. Thus,
dihexylether was chosen as the membrane solvent in the
subsequent studies.
3.3. Effect of Compositions of SP and RP
The effect of the concentration of NaOH in the SP solu-
tion on EF at the range of 0.0 - 0.1 M was studied. As can
be seen in Figure 1, the EF had its maximum value in
the presence of 0.01 M NaOH (pH 11.8). In subsequent
experiments, the pH of SP solution was adjusted at 11.8
using 4 M of NaOH solution. At this pH, TAM is mostly
in its free form. The dependence of the EF of TAM on
HCl concentration in the RP solution at the concentration
range of 0.0 - 0.1M was also investigated. Based on
Figure 1, it proved that EF had its maximum value in
the presence of 0.01 M HCl (pH 2). It is noteworthy
that degradation of C18 column can be accelerated in
the presence of Cl- ions, thus in further experiments, the
pH of the RP solution was adjusted at 2 using phos-
phate buffer. At this pH, TAM is mostly ionized. Thus,
in the present study, gradient of the pH between the SP
Extraction %
678910 1112 13 14
Extraction %
Receiving phaseSource phase
Figure 1. The effect of pH of source and receiving phase on the extraction efficiency. Extraction conditions: C TAM,
50 ng·mL–1; SP, 15 mL; RP, 25 µL; stirring rate, 1000 rpm; extraction time, 60 min.
and the RP solutions is a driving force for the drug
transport which was in accordance with the already ex-
3.4. Agitation Speed
Like other microextraction techniques, the extraction in
HF-LPME can be enhanced by agitation of the sample
solution. Thereby, reducing the “time” required to attain
thermodynamic equilibrium especially for the higher
molecular mass analytes [20]. In HF-LPME, the organic
solvent is sealed and protected by the hydrophobic hol-
low fiber membrane, so it is easier to handle and it can
tolerate higher stirring speeds. In our experiments, parti-
tioning of the analytes into the organic solvent was en-
hanced by increasing the stirring speed from 250 to 1000
rpm (Figure 2). Thus, the stirring speed with the maxi-
mum value (1000 rpm) was chosen for the rest of the
3.5. Salt Effect
The effect of salt addition on EF was examined by add-
ing sodium chloride to aqueous samples at the concentra-
tion levels of 0% - 3% w/v (Figure 2). The EF of TAM
was decreased by increasing of the salt concentration.
This effect may be due to increased interactions between
the analyte and salt in solution with increasing salt con-
centration. Such interactions would tend to restrict
movement of the analyte from the SP to the membrane
solvent. So, all the subsequent experiments were per-
formed in the absence of salt. It is worth noting that in
the biological samples due to existence of salts, lower
extractions in comparison with the aqueous sample may
be expected.
3.6. Extraction Time
LPME is not an exhaustive extraction technique, thus
maximum sensitivity is attained at equilibrium condition.
On the other hand, complete equilibrium need not be
attained for accurate and precise analysis. However,
choosing an exact extraction time is essential to obtain
good precision [44]. Therefore, extraction time is one of
the most important factors influencing the extraction
efficiency. In this study, EF of the drug was investigated
as a function of time in the range of 15 - 75 min. Then,
EF of the drug was increased by increasing of the extrac-
tion time. As shown in Figure 3, the optimal extraction
time was 60 min. Thus, 60 min was chosen as the extrac-
tion time in the subsequent experiments. It is noteworthy
that the optimum extraction time is dependent on sample
composition and may be re-evaluated for real samples to
obtain suitable EFs.
3.7. Evaluation of the Method Performance
The calibration curves of TAM were plotted in three dif-
ferent sample solutions. For each level, three replicate
extractions were performed under the optimal conditions.
Dynamic linear ranges (DLRs) and limits of detection
(LODs), defined as the analytical signal which is larger
than the blank by multiple three of the variation in the
blank, all were calculated and tabulated in Table 1 . Fur-
thermore, based on Equation (1) the highest attainable
EF was found to be 360 in aqueous medium (at the
Copyright © 2011 SciRes. AJAC
0200 400 60080010001200
Stirring rate (rpm)
Extraction %
Ionic strength (% w/v of NaCl)
Extraction %
rateIonic stren
Figure 2. The effects of salt addition and stirring rate on the extraction efficiency. Extraction conditions: C TAM, 50 ng·mL–1; SP,
15 mL of 0.01 M NaOH (pH 11.8); RP, 25 µL of 0.01 M phosphate buffer (pH 2.0); extraction time, 60 min.
Extraction time (min)
Extraction %
Figure 3. The effect of time on the extraction efficiency.
Extraction conditions: C TAM, 50 ng·mL–1; SP, 15 mL of
0.01 M NaOH (pH 11.8); RP, 25 µL of 0.01 M phosphate
buffer (pH 2.0); stirring rate, 1000 rpm.
Table 1. The quantitative data obtained after HF-LPME
and HPLC-UV determination of TAM.
Sample LOD (ng·mL–1) DLR (ng·mL–1)
Aqueous 0.5 1 - 500
Human urine 2.5 10 - 250
Human plasma 5.0 15 - 200
concentration level of 50 ng·mL–1).
3.8. Analysis of Spiked Real Samples
It is apparent that porous hollow fiber functions as a fil-
ter in dirty samples, since particles and also large mole-
cules, which can also be soluble in the organic solvent,
will not be extracted. In this way, the present developed
microextraction technique can be potentially used to ex-
tract drugs from complex matrices, while preventing
co-extraction of other extractable components. In order
to assess the applicability of the extraction method to the
analysis of the drug in spiked real samples with complex
matrices, the spiked urine and plasma samples were ex-
tracted and analyzed using the proposed method under
the optimum conditions which as follows:
3.8.1. Hum an U ri ne
The human urine sample was diluted two times by dou-
ble distilled water. Under the optimum conditions, the
percent relative intra-day and inter-day standard devia-
tions (RSD%) based on three replicate determinations
were 3.7 and 7.5, respectively. The percent relative re-
covery of the drug in spiked human urine sample at
spiking level of 20.0 ng·mL–1 was 89 (Table 2).
3.8.2. Human Plasma
TAM is extensively bounded to plasma proteins (99%)
Table 2. Results obtained for the analysis of TAM in two
spiked biological samples.
Concentration (ng·mL–1)
Found (ng·mL–1)
Relative recovery (%)
Intra-day RSD% (n = 3)
Inter-day RSD% b
ND a
(20.0 ng·mL–1
(25.0 µg·L–1 added)
Concentration (ng·mL–1)
Found (ng·mL–1)
Relative recovery (%)
Intra-day RSD% (n = 3)
Inter-day RSD%b
aNot Detected. bFor three consecutive days.
Copyright © 2011 SciRes. AJAC
[45], and should be librated prior to the extraction. Plas-
ma sample (5 mL) was spiked with particular level of the
drug and vortexed for 3 min. The mixture was added
with 5 mL of acetonitril to disturb the drug protein bind-
ing. The process eventually led to the precipitation of
proteins. Subsequently, the sample was centrifuged at
4000 rpm for 5 min. The whole resulting supernatant
phase was then transferred into a sample vial, followed
by simultaneous dilution (up to 15 mL) and adjustment
of pH at the optimal value (pH = 11.8). Under the opti-
mum conditions, the percent relative intra-day and in-
ter-day standard deviations (RSD %) based on three rep-
licate determinations were 4.2 and 7.8, respectively. The
percent relative recovery of the drug in human plasma
sample at spiking level of 25.0 ng·mL–1 was 90, (Table
2). Figure 4 depicts the chromatograms of the spiked (at
the concentration level of 25.0 ng·mL–1) and non-spiked
plasma samples with TAM under the optimum condi-
tions. The obtained chromatograms revealed that in spite
of complexity of the sample matrix, due to the high sam-
ple clean-up performance, almost no other components
than the target analyte were recovered in the RP solution.
4. Conclusions
The results from this work showed that the HF-LPME
technique in combination with HPLC-UV is a valid
means of enrichment and quantification of TAM at trace
level in spiked human urine and plasma samples. The
established procedure demonstrated good sample clean-
Figure 4. The chromatograms of (a) non-spiked plasma samples with TAM under the optimum conditions, and (b) the spiked
(at the concentration level of 25.0 ng·mL–1).
Copyright © 2011 SciRes. AJAC
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complexity of the sample matrix, due to the high sample
clean-up performance, almost no other components ex-
cept for the target was recovered in the receiving phase
solution. Moreover, excellent extraction recoveries were
achieved demonstrating the fact that the whole determi-
nation is almost independent of the matrix.
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