Journal of Chromatographic Science 2015;53:1217– 1221
doi:10.1093/chromsci/bmu178 Advance Access publication February 23, 2015
Article
Electromembrane Extraction of Organic Acid Compounds in Biological Samples Followed
by High-Performance Liquid Chromatography
Mostafa Khajeh1*, Mohammad Shakeri1, Zahra Bameri Natavan1, Zahra Safaei Moghaddam1, Mousa Bohlooli2 and
A. A. Moosavi-Movahedi3
1
Department of Chemistry, University of Zabol, PO Box 98615-538, Zabol 98615-538, Iran, 2Department of Biology, University of Zabol,
PO Box 98615-538, Zabol 98615-538, Iran, and 3Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
*Author to whom correspondence should be addressed. Email: [email protected]
Received 5 July 2014; revised 14 November 2014
Electromembrane extraction (EME) coupled with high-performance
liquid chromatography was developed for determination of organic
compounds including citric, tartaric and oxalic acid in biological samples. Organic compounds moved from aqueous samples, through a
thin layer of 1-octanol immobilized in the pores of a porous handmade polypropylene tube, and into a basic aqueous acceptor solution
present inside the lumen of the tube. This new set-up for EME has a
future potential such as simple, cheap and fast sample preparation
technique for extraction of organic compounds in various complicated matrices. The pH of acceptor phase, extraction time, voltage, ionic
strength, temperature and stirring speed were studied and optimized.
Optimum conditions were: the pH of acceptor phase, 7; extraction
time, 30 min; voltage, 30 V and stirring speed, 500 rpm. At the optimum conditions, the preconcentration factors of 175– 200, the limits
of detection of 1.9 – 3.1 mg L21 were obtained for the analytes. The
developed procedure was then applied to the extraction and determination of organic acid compounds from biological samples.
Introduction
A number of pathological states result increasing the organic acid
levels in blood. The increasing the level of some organic acids
which called acidaemia can lead to a decrease in blood pH
(i.e., acidosis), to acidotic coma and even to death (1). Organic
acidemias, also known as organic acidurias, are a group of disorders characterized by increased excretion of organic acids in
urine. They result primarily from deficiencies of specific
enzymes in the breakdown pathways of amino acids or from
enzyme deficiencies in b oxidation of fatty acids or carbohydrate
metabolism. Abnormal organic acid levels also are found in
the urine of some patients with mitochondrial disease (2).
Therefore, it is essential to be able to precisely determine organic
acids present in biological samples.
In 2006, Pedersen-Bjergaard et al. introduced a novel microextraction method which called electromembrane extraction
(EME) (3). This method has been demonstrated for extraction
of various acidic or basic compounds (4 – 8). In this method, a
hollow fiber made of porous polypropylene where the porous
wall is impregnated with organic solvent, is used as supported
liquid membrane (SLM). An aqueous solution is filled inside the
lumen of the fiber serving as the acceptor solution, and the fiber
is placed into an aqueous solution. The driving force in EME is an
applied electrical potential over the SLM. One of the electrodes is
placed in the acceptor solution inside the lumen of the fiber,
while the other electrode is located in the sample solution (4).
Charged analytes in the solution are drawn across the SLM
towards the electrode of opposite charge in the acceptor solution. In this study, the hand-made polypropylene tube was used
instead hollow fiber.
The aims of this work are: (i) to use of hand-made polypropylene tube for EME of organic acids from biological samples and (ii)
to develop a simple and cheap method for extraction of analytes.
Finally, high-performance liquid chromatography (HPLC) with
diode array detection was applied for analysis of acceptor
solution.
Experimental
Reagents and samples
Citric acid, tartaric acid, oxalic acid, 1-octanol, acetonitrile,
chloroform and methanol were obtained from Merck
(Darmstadt, Germany). A stock standard solution of organic
acids (1000 mg L21) was prepared in ultrapure water and stored
in glass-stoppered bottles at 48C. Dilutions of stock solutions in
water were used to optimize the experimental conditions.
Human plasma was obtained from Iranian Blood Transfusion
Organization (Zabol, Iran). Urine samples were collected from
two young persons. The samples were stored at 248C, thawed
and shaken before extraction.
The extraction process was carried out using a hand-made instrument in our laboratory which has been shown in Figure 1. A
polypropylene tube with internal diameter of 2 mm and wall
thickness of 150 mm was taken. Then, the pores were created
on the tube wall by a needle with tip diameter of 0.2 mm. This
instrument was simple and cheap.
Equipment for EME
The equipment used for the extraction method is shown in
Figure 1. The electrodes used were platinum wires with diameters of 0.2 mm obtained from Pars Pelatine (Tehran, Iran). The
electrodes were coupled to a power supply model PS-305D
with a programmable voltage in the range of 0 – 30 V (China).
During the extraction, the EME unit was stirred with a stirring
speed in the range of 300–500 rpm by a heater-magnetic stirrer
model 3001 from Heidolph (Kelheim, Germany).
Apparatus
Chromatographic analysis was performed on HPLC system
(Knaver, Berlin, Germany) consisted of a smart line 1000 pump
and a photodiode array detector. The analytical column was a
C18 (model 100-5) reversed-phase column (250 4.6 mm i.d.;
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into the sample solution. The cathode electrode was put directly
into sample solution. After that the electrodes were subsequently
coupled to the power supply and the extraction unit was placed
on a stirrer with various stirring rate (300–500 rpm). The various
voltage (10–30 V) was turned on and extraction was carried out
for various time (20–40 min). Finally, the acceptor solution was
collected by a microsyring and injected into HPLC instrument for
further analysis.
Results
The preconcentration factor (PF) was defined as follow:
PF ¼
Figure 1. Schematic diagram of EME set-up.
Cf:a
;
Ci:s
ð1Þ
where Cf.a is the final concentration in the acceptor phase, Ci.s is
the initial concentration of target compounds in the sample solution and Cf.a was calculated from a calibration graph obtained
from direct injection of target compounds standard solutions.
The extraction recovery (ER%) was defined as the percentage
of the number of mole of target compounds originally present
in the sample (ni.s), which was extracted to the acceptor phase
(nf.a). ER% is defined as follow:
nf:a
Cf :a Va
100 ¼
100;
ni:s
Ci:s Vf
Va
ER% ¼
; PF 100;
Vf
ER% ¼
Figure 2. SEM image of polypropylene tube.
5 mm particle size). Mobile phase was filtered through a Millipore
0.45 mm membrane filter before use. The mobile phase was water
adjusted to a pH 2.10 with perchloric acid (9). The separation
was performed by isocratic elution with a flow rate of
1.0 mL min 21. Quantification of analytes was accomplished by
measuring peak areas at wavelength of 210 nm. Figure 2 shows
the scanning electron microscopy (SEM) of polypropylene
tube. All experiments were performed in triplicate and the
means of values were used for optimization in this study.
Quantification was performed by the integration of the peak
using standard addition method.
Procedure
First, the end of tube was closed with heat. Sample solution containing the organic acid compounds in deionized water and desired pH (6 – 8) was transferred into the sample vial. To
impregnate the organic solution (1-octanol) in the pores of
tube wall, the tube dipped in the solution for 10 s and then the
excess of organic solution was gently wiped away by blowing
with a syringe. According to previous paper (10), 1-octanol selected as the candidate in EME. The upper end of tube was connected to a medical needle tip as a guiding tube which was
inserted through the rubber cap of the vial. Two hundred microliters of basic water (acceptor solution) was introduced into the
lumen of the tube by a microsyringe. The anode electrode was
introduced into lumen of the tube. The tube containing the
anode, SLM and the acceptor solution was afterward directed
1218 Khajeh et al.
ð2Þ
ð3Þ
where Va and Vf represent the volumes of acceptor phase and
sample solution, respectively.
Relative recovery (RR%) was defined as follow
Cfound Creal
RR% ¼
100;
ð4Þ
Cadded
where Cfound, Creal and Cadded are the concentration of the target
compounds after addition of known amount of standard into the
real samples, the concentration of target compounds in real sample, and the concentration of known amount of standard, which
was spiked into the real sample, respectively.
Optimization of procedure
NaCl (0– 1.0%, w/v) was used for studying the influence of ionic
strength and added to the donor phase. Presence of salt caused a
decrease in the ER% and obtaining of undesirable results.
Thereby, the salt was not added to the sample solution in the
next experiment. According to previous work (10, 11), increasing the ionic strength in sample solution increases the ion balance, which leads to reduction of ER%, due to increasing of the
competition among target analytes and other ions for entering
into the acceptor solution. The ion balance is defined as the
total concentration of ions in sample solution to that in acceptor
phase (11).
To pass the target analytes through electrical field, it is necessary to change them to their ionizable forms. Citric, oxalic and
tartaric acid have pKa values of 5.4, 4.14 and 4.25, respectively.
So they are charged in deionized donor phase sample solution
Figure 3. The effect of pH on the ER%. Extraction conditions: voltage, 20 V; time,
20 min; stirring rate, 300 rpm.
Figure 4. The effect of voltage on the ER%. Extraction conditions: pH, 7; time, 20 min;
stirring rate, 300 rpm.
and easily transferred to anodic acceptor solutions under electrical field. According to the literatures, pH in the donor phase is
not highly critical for the EME process (12, 13).
Also, Figure 3 shows that ER% was increased by increase in the
pH value of anodic acceptor solution. This variation occurs due
to easily releasing of the target analytes into the anodic acceptor
solution. However, there are some limitations for the increase of
hydroxide ions concentrations of the anodic acceptor solutions.
This type of pH adjustment improves the probability of electrolysis reaction and bubble formation on the surface of electrode
(10). In this study, the pH of acceptor phase was selected 7.
According to the results which are shown in Figure 4, applied
voltage is one of the important variables. Increasing the voltage
causes an increase in the number of ions crossing through the
membrane (14 – 17). The ER was increased as the driving force
increased from 10 to 30 V. In the next experiments, the voltage
was selected 30 V.
Time is another factor that can influence the flux of target analytes in EME. Time directly increase (Figure 5) the flux ions and
therefore, increase the ER%. In this study, the extraction time
was selected 30 min.
Stirring speed (Figure 6) plays an essential role in increasing
the kinetic and ER% by increasing the mass transfer and reducing
the thickness of double layer around SLM (16). In this study, the
stirring speed was selected 500 rpm.
Figure 5. The effect of extraction time on the ER%. Extraction conditions: pH, 7;
voltage, 30 V; stirring rate, 300 rpm.
Figure 6. The effect of stirring rate on the ER%. Extraction conditions: pH, 7; voltage,
30 V; extraction time, 30 min.
Temperature is final parameter that can affect the flux of ions
through SLM. Theoretically, increase of temperature decrease
the electrical driving force in EME, while increases diffusion coefficient of ions into SLM (10). However, the results showed that
an increase in temperature led to puncture of SLM, and increase
in bubble formation and arc phenomenon probability because of
increasing the solubility of membrane organic solvent. These results are similar to previous work (10). Therefore, working at
258C can be a good benefit for this method.
Discussion
The analytical figures of merit were obtained under optimal conditions. The results are summarized in Table I. Good linearity was
obtained for all the compounds at concentration within the interval tested (12 – 500 mg L21), with the determination coefficient (R 2) was in the range of 0.9994 – 0.9997. Precision
(RSD%) was studied by performing repeatability and reproducibility studies. The repeatability was studied by intra-day analysis
(n ¼ 10) of an extract from an aqueous sample spiked at a concentration of 50 mg L21, the results obtained are shown in
Table I. In order to evaluate the reproducibility of the technique
10 various aliquots of the aqueous sample were spiked at a
EME of Organic Acid Compounds 1219
Table I
Figures of Merit of the Proposed Method
Compound
Oxalic acid
Tartaric acid
Citric acid
r2
0.9994
0.9997
0.9996
LODa
1.9
3.1
2.8
Table II
Determination of the Organic Compound (mg L21) in Different Biological Samples
Linear
range
(mg L21)
Repeatability
(%)
Reproducibility
(%)
Preconcentration
factor
12–500
12–500
12–500
0.31
1.4
1.1
2.5
1.9
3.2
200
175
192
a
LOD, limit of detection in mg L21.
Sample
Plasma 1
Non-spiked
RR%b
RSD% (n ¼ 3)
Plasma 2
Non-spiked
RR%
RSD% (n ¼ 3)
Urine 1
Non-spiked
RR%
RSD% (n ¼ 3)
Urine 2
Non-spiked
RR%
RSD% (n ¼ 3)
Oxalic acid
Tartaric acid
Citric acid
0.20
98.0
7.2
–a
96.0
5.6
2.3
96.0
6.1
1.44
97.0
6.5
–
96.0
8.9
1.7
98.0
6.8
2.2
97.0
5.3
–
94.0
6.9
8.0
95.0
4.4
0.49
97.0
5.1
–
96.0
6.9
3.1
98.0
8.4
a
Less than LOD.
50 mg L21 of each organic compounds was added to calculate relative recovery (RR%).
b
determination of the target compounds in various biological
samples were carried out. To assess the effects of matrix, the biological samples were spiked with organic compounds at a concentration of 50 mg L21. Figure 7 shows that the chromatograms
of urine sample without and with spiking of organic compounds
(1 mL of real sample (Urine 1) were diluted with 30 mL of distilled water). Table II shows that the results of three replicate
analysis of each real samples obtained by proposed procedure.
Conclusion
In this study, the hand-made polypropylene tube was used instead hollow fiber in EME procedure for determination of organic
compounds in biological samples. Therefore, this procedure can
affect repeatability and reproducibility of extraction due to differences among produced fibers. The optimum conditions
were found to be: extraction time, 30 min; applied voltage,
30 V; pH in acceptor solution, 7; stirring speed, 500 rpm; and
temperature, 258C. Satisfactory RSDs (0.31 – 1.4%) and LODs
(1.9 – 3.1 mg L21), high PF (175 – 200) and good linearity range
(0.9994 – 0.9997) were obtained in this study. The procedure
was successfully applied to analysis of these compounds in real
samples. In addition, no extra cleanup steps were needed in complicated biological samples, which can reduce risk of contaminations because of work with these types of samples.
Figure 7. Chromatogram obtained for a urine samples without (A) and with spiking
(50 mg L21) (B) organic compounds.
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