Talanta 161 (2016) 398404

Contents lists available at ScienceDirect

Talanta
journal homepage: www.elsevier.com/locate/talanta

Ion-pair vortex assisted liquid-liquid microextraction with back

extraction coupled with high performance liquid chromatography-UV
for the determination of metformin in plasma
Anas Alshishani a,n, Ahmad Makahleh a,b, Hui Fang Yap a, Elbaleeq Adam Gubartallah a,
Salizawati Muhamad Salhimi c, Bahruddin Saad a
a
b
c

School of Chemical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

Department of Chemistry, Faculty of Science, University of Jordan, 11942 Amman, Jordan
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

art ic l e i nf o

a b s t r a c t

Article history:
Received 1 August 2016
Received in revised form
23 August 2016
Accepted 24 August 2016
Available online 26 August 2016

A new sample preparation method, ion-pair vortex assisted liquid-liquid microextraction (VALLME-BE),
for the determination of a highly polar anti-diabetic drug (metformin) in plasma sample was developed.
The VALLME-BE was performed by diluting the plasma in borate buffer and extracted to 150 mL 1-octanol
containing 0.2 M di-(2-ethylhexyl)phosphoric acid as intermediate phase. The drug was next back-extracted into 20 mL of 0.075 M HCl solution. The effects of pH, ion-pair concentration, type of organic
solvent, volume of extraction phases, ionic strength, vortexing and centrifugation times on the extraction
efciency were investigated. The optimum conditions were at pH 9.3, 60 s vortexing and 2 min centrifugation. The microextract, contained metformin and buformin (internal standard), was directly injected into a HPLC unit using C1 column (250 mm
4.6 mm
10 mm) and detected at 235 nm. The
method was validated and calibration curve was linear with r2 40.99 over the range of 202000 mg L  1.
The limits of detection and quantitation were 1.4 and 4.1 mg L  1, respectively. The accuracy was within
94.8108% of the nominal concentration. The relative standard deviation for inter- and intra-day precision was less than 10.8%. The method was conveniently applied for the determination of metformin in
plasma samples.
& 2016 Elsevier B.V. All rights reserved.

Keywords:
Vortex assisted liquid-liquid microextraction back extraction
Ion-pair
HPLC
Metformin
Blood plasma

1. Introduction
Metformin
(N,N-dimethyl-imidodicarbonimidic
diamide)
(Fig. 1), is the most widely used oral antihyperglycemic drug for
the management of type 2 diabetes. It works by helping to restore
the body's response to insulin and lowering the level of blood
sugar effectively [1]. Apart from its hypoglycemic activity, metformin has been taken to prevent diabetes in people who are at
high risk of diabetic. It is also used in women with polycystic
ovarian syndrome [2]. Metformin is the only oral anti-diabetic
drug besides glibenclamide in the WHO List of Essential Medicines, a list of minimum medicine needed for basic health-care [3].
Metformin was rst described in the scientic literature in 1922 by
Emil Werner and James Bell as a product in the synthesis of N,Ndimethylguanidine. In 1957, Jean Sterne for the rst time tried it
for the treatment of diabetes [4]. It was marketed in Europe in
n

Corresponding author.
E-mail address: anasshishani@gmail.com (A. Alshishani).

http://dx.doi.org/10.1016/j.talanta.2016.08.067
0039-9140/& 2016 Elsevier B.V. All rights reserved.

1970s, but only received FDA approval in 1995 [5]. The bioavailability of metformin is 4060% and the maximum concentration
(Cmax) in blood ranges from 1000 to 1600 mg L  1, after 3 h of oral
administration of 0.5 g dose [6]. Metformin is a strong base (pKa
12.4 [7]) than most basic drugs and is present in the blood in the
protonated form (499.9%) [6]. It is not metabolized in the body
and excreted in the urine without degradation with average
elimination half-life of 5 h [6]. The low value of log P (  1.43) [7,8]
indicates that metformin is of low lipophilicity and therefore
plasma protein binding is negligible [9].
The determination of metformin in plasma is important for
studying the pharmacokinetics of this drug for general drug
monitoring. Several separation methods for the determination of
metformin in blood plasma have been reported. High performance
liquid chromatography either with ultraviolet (UV) [1012] or
mass spectroscopy (MS) detection [1316] is considered the most
widely used technique. Although MS detection offers lower detection limit compared to UV, HPLC-UV is less expensive, more
widely available and within the budget of average laboratories.

A. Alshishani et al. / Talanta 161 (2016) 398404

399

extraction of charged analyte [31], but has not been used in three
phase microextraction.
The use of ion-pair reagent with VALLME-BE for the extraction
of charged polar compounds such as metformin is for the rst time
explored. We coined this approach as ion-pair VALLME-BE based
on terminologies as suggested by Jana andrejov et al. [32]. The
high enrichment of the analyte associated with the microextraction facilitate the determination of metformin using the widely
available and cheap UV-detector and also using ordinary glasswares. Also, it is the rst report on the microextraction of metformin without the need of derivatization.

Fig. 1. Chemical structures of metformin, buformin and di-(2-ethylhexyl)phosphoric acid (DEHPA).

Furthermore, the sensitivity disadvantage of UV detection can be

negated using sample preparation protocols with good enrichment
factor.
Protein precipitation (PP) is the most used sample preparation
technique for the determination of metformin in blood plasma
[1214,17]. Despite being simple, PP lacks sensitivity [18] and it is
not effective in removing endogenous components [11,19]. Furthermore, time-consuming evaporation step is required if preconcentration is needed. On other hand, liquid-liquid extraction
(LLE) is not feasible due to the high polarity of metformin. However, a number of researchers tried to overcome this problem either by using LLE in conjunction with ion-pair [19,20], subjecting
to derivatization to convert metformin into less polar species [21],
or by conducting the extraction at high pH [11,22]. The LLE procedure is simple but it requires the use of large amounts of environmentally harmful solvents.
The use of solid phase extraction (SPE) has also been reported
[16,23,24]. The SPE technique is more selective than LLE as matrix
components were not co-extracted which reected in lower
quantitation limits (5 mg L  1 [24]) compared to the LLE technique
(7.840 mg L  1 [21,22]). The main drawbacks of the SPE method
are the multi-steps involved, require evaporation, time consuming
and relatively expensive cartridges are required.
Microextraction techniques are making in-roads as serious alternative to replace the traditional sample preparation methods in
bio-analysis [25] due to their many desirable features such as the
high enrichments, compatibility with analytical instruments and
consuming signicantly less solvents and reagents. To date, the
only published microextraction technique for the determination of
metformin in blood plasma were based on hollow ber-liquid
phase microextraction (HF-LPME) [26,27]. In both reports, metformin was derivatized to form less polar products, facilitating its
extraction into the aqueous phase in the lumen of the hollow ber
[26]. Despite the great promise of these approaches, the use of
hollow bers is not appealing to the majority of users who are not
specialized in microextraction. This prompted us to consider microextraction that uses ordinary glassware and simple instruments
that are readily available even for an average analytical laboratory.
In this regards, the liquid phase microextraction with vortex assisted and its modications seem to meet this requirement.
The vortex-assisted liquid-liquid microextraction with back
extraction (VALLME-BE) method (a three phase microextraction
technique) has recently been proposed to overcome the problems
that are associated with the vortex-assisted liquid-liquid microextraction (VALLME) (a two phase microextraction technique)
such as selectivity and the compatibility of the nal extract with
reversed phase HPLC or CE analysis [2830]. However, the
VALLME-BE technique is not suitable for polar analyte. Ion-pair
reagent in two phase microextraction was reported for the

2. Experimental
2.1. Chemicals and reagents
Metformin hydrochloride was kindly donated by Dar Al-Dawa
Investment (Naur, Jordan). Buformin hydrochloride (internal
standard (IS), Fig. 1) was purchased from Wako Pure Chemicals
Industries (Osaka, Japan). Chemicals and reagents used were obtained from the following sources: HPLC-grade acetonitrile
(499.99%), triethylamine, phosphoric acid (85%, w/w) and 1-octanol were purchased from Merck (Darmstadt, Germany); di-(2ethylhexyl)phosphoric acid (DEHPA, ion pair reagent, Fig. 1),
1-hexanol, 1-heptanol, sodium phosphate monobasic monohydrate was purchased from Sigma-Aldrich (St. Louis, MO, USA);
hydrochloric acid (37%, w/w) was from Quality Reagent Chemicals
(QReC, Auckland, New Zealand); disodium tetraborate decahydrate
was from BDH Chemicals (London, England). Ultrapure water
(resistivity, 18.2 M cm) was produced by Millipore water purication system (Molsheim, France) and used throughout for the
preparation of solutions. Plasma sample was donated by the
Center for Drug Research, Universiti Sains Malaysia, Penang, Malaysia. Borate buffer was prepared by adjusting the pH of disodium
borate solution (3 mM) to pH 9.3 with NaOH solution (0.2 M). Ionpair solution (0.2 M) was prepared by dissolving appropriate
amount of DEHPA in 1-octanol.
2.2. Instrumentation
A Hitachi LC-6200 intelligent pump (Tokyo, Japan) equipped
with a Hitachi L-4250 UV/Vis detector (Tokyo, Japan) was used.
Sample was injected via a Rheodyne 7125 injection valve (Cotati,
CA, USA), with a 10 mL loop. The detector wavelength was set at
235 nm. The separation was carried out using Spherisorb C1 column (250 mm
4.6 mm, 10 mm) purchased from Phenomenex
(Torrance, CA, USA). Data acquisition was obtained from eDAQ
(Denistone East, Australia) and performed with PowerChrom
software (version 2.6.11) for processing and analysis of the data.
Mobile phase composition was acetonitrile:20 mM monobasic
phosphate buffer:triethylamine (20:80:0.1 v/v/v). The ow rate
was 1.5 mL min  1. The mobile phase was ltered using nylon
membrane lter (0.22 mm) from Agilent Technologies (Waldbronn,
Germany) and was degassed for 10 min before use.
2.3. Preparation of standard and sample solutions
Metformin and buformin stock solutions (200 and 10 mg L  1,
respectively) were prepared by dissolving the desired amounts in
water and stored at 4 C. Working standard solutions were prepared in 10 mL volumetric ask by appropriate dilution of metformin stock solution and addition of IS solution (50 mL) then topup to the mark with borate buffer (pH 9.3). The working solution
was then subjected to the microextraction procedure (Section 2.4).
Plasma was prepared by spiking 0.5 mL with IS solution (50 mL)

400

A. Alshishani et al. / Talanta 161 (2016) 398404

and was mixed with acetonitrile (1.5 mL). The mixture was next
vortexed (20 s) and then centrifuged (4000 rpm) for 5 min. The
supernatant was transferred to a volumetric ask (10 mL) and topup to the mark with borate buffer (pH 9.3).
2.4. Ion-pair vortex assisted liquid-liquid microextraction with back
extraction (VALLME-BE)
Working standard or sample solution (10 mL) was mixed with
150 mL ion-pair solution (used as intermediate phase). The mixture
was vortexed vigorously (2500 rpm) for 60 s then centrifuged
(4000 rpm) for 2 min to reform the organic layer on the top of the
ask. 50 mL of the organic layer (intermediate phase) was transferred into a centrifuge tube (500 mL) that contained 100 mL of
1-octanol which was next spiked with 0.075 M HCl (20 mL). The
mixture was vortexed (2500 rpm) for 60 s then centrifuged
(4000 rpm) 1 min. Finally, 10 mL (small drop formed at the bottom
of the tube) was directly introduced into the HPLC unit.
2.5. Method validation
Method validation parameters (i.e., selectivity, linearity, limit of
detection (LOD), limit of quantitation (LOQ), precision and recovery) were tested after subjecting the working standard and
spiked plasma solutions to the proposed microextraction procedure. Ratio of peak areas of metformin to buformin was used in
calculations. Selectivity was evaluated by applying the procedure
on two different lots of plasma. Linearity was studied using seven
concentration levels (20, 50, 100, 250, 500, 1000 & 2000 g L  1) of
metformin. LOD and LOQ were calculated using blank response at
signal to noise ratio (S/N) of 3 and 10, respectively. Precision was
demonstrated by determining the inter- and intra- day relative
standard deviation (RSD) of the analysis. The intra-day precision
was performed by analyzing six spiked plasma samples at three
levels (20, 500 and 2000 g L  1) in the same day. While inter-day
precision was performed for ve days. Recovery test was done by
spiking to the plasma samples at three concentrations (20, 500
and 2000 g L  1) using stock standard solutions.

3. Results and discussion

In the current study, the extraction of metformin from the
plasma matrix was achieved by two successive steps: ion-pair
VALLME followed by back extraction. This two-step microextractions conducted under the adopted conditions enhances the sensitivity to about 220-fold which is expressed as the enrichment
factor (EF). The EF was calculated using the following equation:
EF(area of analyte in 20 mL HCl drop)/(area of analyte in
10 mL sample).
Furthermore, the obtained extraction recovery was higher than
the reported results using bromothymol blue (BTB) which has
been previously used as ion-pair reagent for the LLE of metformin
from plasma samples [19,20]. This is due to the solubility of BTB in
the aqueous phase under the studied concentrations.
3.1. Optimization of chromatographic method
A potential problem that can be anticipated from the separation of highly polar compounds such as metformin and buformin
is their rapid elution when separated on C18 columns, causing
interference with solvent and plasma endogenous components
[22]. Common options to achieve good separations for these
compounds is to use normal phase HPLC (not favored), or using
reversed phase that contains ion-pair as additives in the mobile
phase to modify the analyte polarity [11,22,24] or by derivatizing

them into less polar compounds [21]. A few types of analytical

columns were tested: C18, C8, C1 and pentauoro phenyl (PFP) in
this study. The retention time of buformin was always higher than
metformin due to the fact that N- butyl group of buformin contributed to lipophilicity much more than the N,N-dimethyl group
of metformin [33]. When the C1 column was used, good separation was achieved even without using ion-pair reagent due to its
higher polarity than the other stationary phases. To the best of our
knowledge, the use of C1 column for the separation of metformin
was not previously reported.
Different mobile phases were investigated using the C1 column. A marked reduction in retention time was observed as the
buffer type was changed from acetate to phosphate using acetonitrile and buffer mixture which can be deduced to the formation
of hydrophobic ion-pair complex between the positively charged
analyte and the negatively charged acetate. Different compositions
of acetonitrile and phosphate buffer were investigated. As the
buffer ratio increases the retention time decrease and peak efciency improved. Adding 0.1% triethylamine (TEA) to this composition resulted in improved the theoretical plates, reducing the
peak tailings and shorter retention times. The optimum composition was when using acetonitrile: buffer:TEA (20:80:0.1, v/v/v).
The substitution of acetonitrile with methanol causes a signicant
increase in retention time and poor peak shapes which is due to
the higher elution strength of acetonitrile.
3.2. Optimization of IP-VALLLME procedure
The initial experimental conditions applied during the optimization studies were: sample dilution, 10 mL (pH, 9.25); intermediate phase, 0.15 M DEHPA in 1-octanol (150 mL); nal phase,
0.050 M HCl (20 mL); vortex speed, 2500 rpm; vortex time, 45 s;
centrifugation, 3 min (4000 rpm). EF was used during the optimization experiments to evaluate the effect of the various
parameters.
3.2.1. Type of organic solvent
The organic solvent to be used in this work should fulll the
following conditions: (i) the ion-pair reagent should be soluble in
it, (ii) its density should be lighter than water in order to facilitate
micro layer transfer for the micro back-extraction, (iii) minimum
solubility in water in order to maximize reformed layer in minimum time, (iv) low permittivity to promote formation of ion-pair
complexes. Three solvents were investigated: 1-hexanol, 1-heptanol and 1-octanol (density; 0.816, 0.820, 0.823 g mL  1, respectively) [7]. Of the three solvents tested, 1-octanol gave the highest
EF (Fig. 2A) and therefore was chosen. This is due to the increase of
the organic solvent solubility in the aqueous phase as the number
of the carbon chain decrease, thus improved the solubility of the
ion-pair complex in the aqueous phase. For the same reason, the
biggest volume of the reformed layer was using 1-octanol (125 mL),
followed by 1-heptanol (115 mL) and nally 1-hexanol (40 mL).
3.2.2. Ion-pair reagent concentration
DEHPA is noted for its effectiveness in extracting metal ions
[34] and even very hydrophilic cationic species [35]. Its hydrophobicity (log P 6.1) [7] suggest its good solubility in 1-octanol
and thus prevent its dissolution in water which is necessary to
promote the mass transfer. DEHPA serves as a counter ion and also
as uncharged adduct-forming agent [36,37]. The hydrophobic anion (R2POO  ) will form hydrophobic ion-pair complex with the
protonated metformin (MH ). Based on earlier work [36], the
formation of the ion-pair complex and the adduct is believed to
occur as outlined below:


R2PO4(aq)
2(MH  R2PO4)
MH(aq)

(org)

A. Alshishani et al. / Talanta 161 (2016) 398404

205

3.2.3. pH effect
The pH should be at least two units less that the pKa of metformin and more than the pKa value of DEHPA (pKa 5.2) to ensure that all molecules are in the charged form. The optimum pH
was 9.3 (Fig. 2B) and was therefore used for the remaining studies.
Metformin was back-extracted from the 1-octanol by the addition
of HCl which shifts the equilibrium toward the formation of protonated form of metformin (transferred to the aqueous phase) and
neutralized form of DEHPA (remain in the organic phase). Different concentrations of HCl were investigated (0.050, 0.060,
0.075 M), the highest EF was achieved using 0.075 M.

(A)

200

EF

195
190
185
180
175
1-Hexanol

1-Heptanol

1-Octanol

3.2.4. Ionic strength

Increasing the ionic strength decreases the extraction efciency
as the high concentration of ions will hinder the formation of the
desired ion-pair complex [38]. This was tested by adding NaCl (5%
and 10%) to the sample where the EF was reduced signicantly.

Type of organic solvent

250

(B)

200

EF

150
100
50
0
8.00

8.25

8.50

8.75

9.00

9.25

401

9.50

pH
Fig. 2. Enrichment factor of metformin as a function of (A) type of organic solvent,
(B) donor phase pH. Spiked sample, 1000 mg L  1; donor phase, 10 mL; Intermediate
phase, 150 mL; Acceptor Phase, 20 mL.

(MH  R2PO4)(org) nR2PO4H(org)2(MH  R2PO4  nR2PO4H)(org)

It is interesting to note that DEHPA reagent also functions as an
emulsier (due to its surfactant characteristics), which improve
the emulsication in 1-octanol. This forms the basis of the surfactant-assisted liquid-liquid microextraction technique [25]
which resulted in improvement of the mass transfer of the analyte
in a relatively short time.
Different ion-pair reagent concentrations (0.10.3 mol L  1)
were prepared in 1-octanol and tested. The EF did not change
signicantly over the concentrations studied. This is expected
because excess mole ratio was used at all studied concentrations.

3.2.5. Volume of extraction phases

The effect of 1-octanol (100250 mL) and 0.075 M HCl (15
30 mL) volumes on the extraction efciency were studied. The EF
increased as the volume of the intermediate phase was increased
up to 150 mL, while the maximum EF was achieved using 20 mL of
0.075 M HCl. The recovered volume of the 1-octanol after the
extraction was 125 mL (calculated by applying the extraction procedure ten times under the optimum conditions) indicates that
25 mL of the 1-octanol had dissolved in the aqueous phase (solubility of 2.0 g L  1). The increased solubility of 1-octanol found in
the current studies over the reported earlier results (1.2 g L  1 [7])
is attributed to the surfactant properties of DEHPA. The volume
used for the proposed microextraction is much smaller than any
reported liquid extraction procedures for the determination of
metformin from plasma matrix (Table 1).
3.2.6. Vortex and centrifugation times
Vortexing of the immiscible phase during the extraction and
back-extraction is essential in order to form ne droplets which
increase the contact surface area and hence enhance the mass
transfer of the analyte [39]. The vortex speed was kept at the
highest speed (2500 rpm) while the vortexing time was changed
from 30 to 75 s for both extractions steps. As expected, EF increases as the vortex time was increased and an optimum was
achieved after 60 s for both extractions. Centrifugation time (1
3 min) has been investigated and satisfactory separation was
achieved after 2 min and greater.

Table 1
Comparison between the proposed IP-VALLLME-HPLC method and some reported methods for the determination of metformin in blood plasma.
Extraction technique

Extraction organic
solvents

Evaporation step
included

Extraction time (min)

Technique Stationary
phase

RT (min) LOQ
(mg L  1)

Ref.

IP-VALLLME

150 mL of IP/1-Octanol

No

10

LC-UV

C1

3.1

4.1

LLE Derivatization
IP-LLE
IP-LLE
LLE High pH

2 mL of DCM
5 mL Diethylether/DCM
2 mL Chloroform
3 mL 1-butanol/ hexane
(50:50)
3 mL DCM:Amyl Alcohol
(9:1)
Dihexyl ether (HF
saturation)
2 mL Acetonitrile
1 mL Methanola
1 mL Methanola

Yes
No
Yes (2 steps)
Yes

68 evaporation time
22
5 evaporation time
7.5 evaporation time

LC-UV
LC-UV
CE-UV
LC-UV

C18
C18 (IP)

C18 (IP)

15
3.3

10

40
10
250
7.8

This
work
[21]
[19]
[20]
[22]

Yes

19 evaporation time

LC-MS

C18

1.8

10

[15]

Yes

45 evaporation time

LC-UV

C18

5.5

1.7

[26]

Yes (2 steps)
Yes
No

10 evaporation time
NA
NA

LC-UV
LC-UV
LC-MS

Phenyl
C18 (IP)
SCX

7.5
4.9
4.5

30
5
10

[12]
[24]
[16]

LLE High pH
HF-LPME
Derivatization
PP
SPE-IP
SPE Cation- Exchange

Abbreviations: LLE: Liquid-liquid extraction, PP: Protein precipitation IP: Ion pair, IP-VALLLME: Ion pair vortex assisted liquid-liquid-liquid microextraction, DCM: Dichloromethane, SCX: Strong cation exchange.
a

Elution volume.

402

A. Alshishani et al. / Talanta 161 (2016) 398404

120

Table 2
Accuracy and precision data for plasma samples (n 6).

20
500
2000

100

Accuracy (%)

Precision (%RSD)

Inter-day

Intra-day

Inter-day

Intra-day

108
94.8
100

98.2
104
103

10.2
3.30
4.50

10.8
8.70
4.40

3.3. Method validation

Recovery (%)

Spiked concentration (g L  1)

80
60
40
20
0

40

50

60

70

80

90

100

110

intermediate phase volume (L)

In order to demonstrate the suitability of the method, the following validation merits were investigated: linearity, LOD, LOQ,
precision, accuracy and selectivity. The selectivity of the method
was veried as no interference from plasma endogenous components was observed for metformin or the IS using two different
lots of plasma. Calibration curve was linear (r2 0.9988) over the
studied range (20 2000 mg L  1). The LOD and LOQ were 1.4 and
4.1 mg L  1, respectively. The low value achieved is largely contributed to the successive extractions into smaller volumes in
addition to the effective removal of endogenous interferences from
the protein precipitation procedure. Inter- and intra-day precision
results (%RSD) for three different concentration levels (20, 500 and
2000 mg L  1) were less than 10.8% (Table 2).
3.4. Analysis of plasma samples
The application of the developed VALLME-BE method on real
samples was demonstrated by analyzing blood plasma that was
spiked with metformin and the IS at physiological levels. Generally, ion-pair extraction is not preferred in bioanalysis due to the
presence of large and variable amount of interfering cations [37].
However in this work, acceptable accuracy for the determination
was obtained (94.8108.5%) (Table 2). In order to achieve that, one
volume of plasma sample was mixed with three volumes of
acetonitrile in order to precipitate proteins prior to performing the
extraction procedure. Acetonitrile was found to be a suitable
plasma protein precipitant, particularly at volume ratios 2:1
(precipitant: plasma) or more [40]. The addition of the ACN from
protein precipitation did not signicantly enhance the extraction.
This was demonstrated in this work by comparing the EF of
standard solution with and without addition of 1.5 mL ACN (as in
protein precipitation). This may be due to the fact that metformin
is a high polar compound and the mass transfer to the 1-octanol is
predominantly by the ion-pairing process.
During the initial stages, poor recoveries (o13%) were encountered when all the intermediate phase was used for the micro
back-extraction step. A systematic investigation using different
volumes of intermediate phase for the micro back extraction reveals that the recovery signicantly decreases with increasing
volume of the intermediate phase (Fig. 3). This phenomenon is
attributed to the co-extraction of metal ions (e.g., Na , K , Ca2 )
from the plasma by the ion-pair reagent (DEHPA). This is not
surprising as DEHPA is widely used for the extraction of metal ions
[34,41]. Furthermore, the presence of these co-extracted metal
ions in the aqueous phase reduces the mass transfer of metformin
due to the salting-out effect (solubility of metal ions in the aqueous phase is greater than that for metformin and buformin). In
order to overcome this problem, 50 mL of the intermediate phase
was used for the micro-back extraction which resulted in good
recoveries (94.8108%). 100 mL of 1-octanol was added to ensure
that the volume used in the optimization experiments were the
same as for plasma analysis.
Buformin was used as IS in order to compensate for sample to
sample variation during extraction and it was selected because it is

Fig. 3. : Effect of volume of intermediate phase on recovery for spiked blood

plasma, 1000 mg L  1; donar phase, 10 mL; acceptor phase, 20 mL.

structurally related to metformin in which the N,N-dimethyl group

is replaced by N-butyl. Buformin was also used by other researchers as IS for the determination of metformin and other biguanides in biological samples [42]. The average absolute recovery
for metformin and buformin from plasma matrix was 92.6% and
86.3%, respectively. Fig. 4 shows typical chromatograms of extracts
from a plasma sample.
3.5. Comparison to previously reported analytical methods
Table 1 summarizes the important analytical characteristics of
the proposed microextraction-HPLC method for the determination
of metformin in blood plasma compared to some previously reported methods. It is readily apparent that the proposed method
has many advantageous features, especially in terms of solvent
consumption, sample preparation time, sensitivity and chromatographic retention time. The VALLME-BE technique consumes
only 150 mL of the organic solvent for each plasma sample. The
good enrichment of the IP-VALLLME method, despite not requiring
derivatization, is translated into low LOQ (4.1 mg L  1), superior
even to MS detection (10 mg L  1) [15,16]. The hollow ber LPME
work [26] has low LOQs (1.7  10 mg L  1) due to the combination
of microextraction and derivatization. The proposed microextraction is simple and does not require evaporation, unlike most reported methods [15,21,22,26] that also involve multi-steps washing, loading and elution with single-use SPE cartridges [16,24]. For
some earlier methods, two evaporation steps were necessary
[12,20]. The sample preparation time required for each sample is
also relatively low (10 min) which is considerably less than the
simple PP procedure of about 10 min (not including the time for
the evaporation and reconstitution) [12], the time increases when
derivatization is required to about 45 and 68 min [21,26]. The
retention time of metformin (3.1 min) is less than all the reported
UV detection methods, only those using MS detection reported
shorter retention (1.8 min) due the selective detection based on
mass/charge ratio.

4. Conclusion
The use of ion-pair with vortex assisted liquid-liquid microextraction is for the rst time reported. It was successfully applied
for the determination of metformin (highly polar compound) in
plasma sample. DEHPA was found to be a very suitable ion-pair
reagent. For the rst time, metformin was successfully separated
on C1 analytical column.. All in all, the proposed microextractionHPLC method provides a truly satisfying procedure for the determination of metformin in plasma. The method is selective,
simple, uses readily available glasswares and the microextractions
into successively smaller volumes resulted in enrichment factor of

A. Alshishani et al. / Talanta 161 (2016) 398404

403

Fig. 4. HPLC-UV chromatograms corresponding to (a) 20 mg L  1 spiked plasma, (b) 2000 mg L  1, (c) blank plasma and (d) plasma spiked with IS only after undergoing
microextraction procedure. Column; Phenomenex C1 (250 mm
4.6 mm), 5 mm. mobile phase; 8:2 buffer:ACN, 235 nm, ow rate; 1.5 mL min  1.

220 fold. Similar strategy can be considered for the determination

of other polar compounds in biological and other matrices.

Acknowledgments
Financial support to Universiti Sains Malaysia via Research
University
Grant
(1001/PKIMIA/811201)
is
gratefully
acknowledged.

References
[1] C.J. Dunn, D.H. Peters, Metformin, Drugs 49 (1995) 721749, http://dx.doi.org/
10.2165/00003495-199549050-00007.
[2] H. Nasri, M. Ra, Metformin: current knowledge, J. Res. Med. Sci. 19 (2014)
658664.
[3] The International Pharmacopoeia, 19th WHO Model List of Essential Medicines, http://www.who.int/medicines/publications/essentialmedicines/en
2015, pp. 143, http://dx.doi.org/10.1016/S1473-3099(14)70780-7.
[4] S. J., Du nouveau dans les antidi- abetiques. La NN dimethylamine guanyl
guanide (N.N.D.G.)., Maroc. Med. 36, 1957, pp. 12951296.
[5] Food and Drug Administration Website, No Title, Electron. Orange B., 1995.
http://www.fda.gov/cder/ob/default.htm/ (accessed 01.04.16).
[6] G.G. Graham, J. Punt, M. Arora, R.O. Day, M.P. Doogue, J.K. Duong, et al., Clinical
pharmacokinetics of metformin, Clin. Pharmacokinet. 50 (2011) 8198, http:
//dx.doi.org/10.2165/11534750-000000000-00000.
[7] A.C.S. (ACS) SciFinder, No Title, (n.d.). https://scinder.cas.org/scinder (accessed 01.01.16).
[8] D.B. Jack, Handbook of Clinical Pharmacokinetic Data, MacMillan Publishers,
London, 1992.

[9] N. Papanas, E. Maltezos, Metformin: a review of its use in the treatment of

type 2 diabetes, Clin. Med. Ther. 1 (2009) 13671381.
[10] S. AbuRuz, J. Millership, J. McElnay, The development and validation of liquid
chromatography method for the simultaneous determination of metformin
and glipizide, gliclazide, glibenclamide or glimperide in plasma, J. Chromatogr.
B Anal. Technol. Biomed. Life Sci. 817 (2005) 277286, http://dx.doi.org/
10.1016/j.jchromb.2004.12.018.
[11] H. Amini, A. Ahmadiani, P. Gazerani, Determination of metformin in human
plasma by high-performance liquid chromatography, J. Chromatogr. B. 824
(2005) 319322, http://dx.doi.org/10.1016/j.jchromb.2005.07.009.
[12] V. Porta, S.G. Schramm, E.K. Kano, E.E. Koono, Y.P. Armando, K. Fukuda, et al.,
HPLC-UV determination of metformin in human plasma for application in
pharmacokinetics and bioequivalence studies, J. Pharm. Biomed. Anal. 46
(2008) 143147, http://dx.doi.org/10.1016/j.jpba.2007.10.007.
[13] Y. Wang, Y. Tang, J. Gu, J.P. Fawcett, X. Bai, Rapid and sensitive liquid chromatography-tandem mass spectrometric method for the quantitation of
metformin in human plasma, J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
808 (2004) 215219, http://dx.doi.org/10.1016/j.jchromb.2004.05.006.
[14] M.A.S. Marques, A. de, S. Soares, O.W. Pinto, P.T.W. Barroso, D.P. Pinto,
M. Ferreira-Filho, et al., Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem
mass spectrometry: application to pharmacokinetic studies, J. Chromatogr. B
852 (2007) 308316, http://dx.doi.org/10.1016/j.jchromb.2007.01.030.
[15] P. Sengupta, U. Bhaumik, A. Ghosh, A.K. Sarkar, B. Chatterjee, A. Bose, et al., LC
MSMS development and validation for simultaneous quantitation of metformin, glimepiride and pioglitazone in human plasma and its application to a
bioequivalence study, Chromatographia 69 (2009) 12431250, http://dx.doi.
org/10.1365/s10337-009-1056-5.
[16] N. Koseki, H. Kawashita, M. Niina, Y. Nagae, N. Masuda, Development and
validation for high selective quantitative determination of metformin in human plasma by cation exchanging with normal-phase LC/MS/MS, J. Pharm.
Biomed. Anal. 36 (2005) 10631072, http://dx.doi.org/10.1016/j.
jpba.2004.09.007.
[17] P. Tuma, Large volume sample stacking for rapid and sensitive determination
of antidiabetic drug metformin in human urine and serum by capillary

404

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

A. Alshishani et al. / Talanta 161 (2016) 398404

electrophoresis with contactless conductivity detection, J. Chromatogr. A 1345

(2014) 207211, http://dx.doi.org/10.1016/j.chroma.2014.04.016.
O. Vesterqvist, F. Nabbie, B. Swanson, Determination of metformin in plasma
by high-performance liquid chromatography after ultraltration, J. Chromatogr. B Biomed. Sci. Appl. 716 (1998) 299304, http://dx.doi.org/10.1016/
S0378-4347(98)00305-3.
J. Keal, A. Somogyi, Rapid and sensitive high-performance liquid chromatographic assay for metformin in plasma and urine using ion-pair extraction
techniques, J. Chromatogr. B Biomed. Sci. Appl. 378 (1986) 503508, http://dx.
doi.org/10.1016/S0378-4347(00)80751-3.
J.-Z. Song, H.-F. Chen, S.-J. Tian, Z.-P. Sun, Determination of metformin in
plasma by capillary electrophoresis using eld-amplied sample stacking
technique, J. Chromatogr. B Biomed. Sci. Appl. 708 (1998) 277283, http://dx.
doi.org/10.1016/S0378-4347(97)00635-X.
F. Tache, V. David, A. Farca, A. Medvedovici, HPLC-DAD determination of
Metformin in human plasma using derivatization with p-nitrobenzoyl chloride in a biphasic system, Microchem. J. 68 (2001) 1319, http://dx.doi.org/
10.1016/S0026-265X(00)00170-3.
R.Q. Gabr, R.S. Padwal, D.R. Brocks, Determination of metformin in human
plasma and urine by high-performance liquid chromatography using small
sample volume and conventional octadecyl silane column, J. Pharm. Pharm.
Sci. 13 (2010) 486494.
E.P.C. Lai, S.Y. Feng, Solid phase extraction-non-aqueous capillary electrophoresis for determination of metformin, phenformin and glyburide in human
plasma, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 843 (2006) 9499,
http://dx.doi.org/10.1016/j.jchromb.2006.05.030.
S. AbuRuz, J. Millership, J. McElnay, Determination of metformin in plasma
using a new ion pair solid phase extraction technique and ion pair liquid
chromatography, J. Chromatogr. B 798 (2003) 203209, http://dx.doi.org/
10.1016/j.jchromb.2003.09.043.
J.A. Ocaa-Gonzlez, R. Fernndez-Torres, M.. Bello-Lpez, M. Ramos-Payn,
New developments in microextraction techniques in bioanalysis. A review,
Anal. Chim. Acta 905 (2016) 823, http://dx.doi.org/10.1016/j.aca.2015.10.041.
G.M. Ben-Hander, A. Makahleh, B. Saad, M.I. Saleh, Hollow ber liquid phase
microextraction with in situ derivatization for the determination of trace
amounts of metformin hydrochloride (anti-diabetic drug) in biological uids,
J. Chromatogr. B 941 (2013) 123130, http://dx.doi.org/10.1016/j.
jchromb.2013.10.007.
G.M. Ben-Hander, A. Makahleh, B. Saad, M.I. Saleh, K.W. Cheng, Sequential
hollow-ber liquid phase microextraction for the determination of rosiglitazone and metformin hydrochloride (anti-diabetic drugs) in biological uids,
Talanta 131 (2015) 590596, http://dx.doi.org/10.1016/j.talanta.2014.08.037.

M.F. Us, U. Alshana, I. Lubbad, N.G. Gger,
N. Erta, Dispersive liquid-liquid
microextraction based on solidication of oating organic drop combined
with eld-amplied sample injection in capillary electrophoresis for the determination of beta(2)-agonists in bovine urine, Electrophoresis 34 (2013)
854861, http://dx.doi.org/10.1002/elps.201200348.
J.M. Kokosa, Advances in solvent-microextraction techniques, TrAC - Trends
Anal. Chem. 43 (2013) 213, http://dx.doi.org/10.1016/j.trac.2012.09.020.

[30] A.S. Yazdi, Z. EsHaghi, Liquid-liquid-liquid phase microextraction of aromatic

amines in water using crown ethers by high-performance liquid chromatography with monolithic column, Talanta 66 (2005) 664669, http://dx.doi.org/
10.1016/j.talanta.2004.12.026.
[31] A. Hol, A. Arslan Kartal, A. Akdogan, A. Eli, T. Arslan, L. Eli, Ion pair-dispersive
liquid-liquid microextraction coupled to microsample injection system-ame
atomic absorption spectrometry for determination of gold at trace level in real
samples, Acta Chim. Slov. 62 (2015) 196203, http://dx.doi.org/10.17344/
acsi.2014.897.
[32] J. andrejov, N. Campillo, P. Vias, V. Andruch, Classication and terminology
in dispersive liquidliquid microextraction, Microchem. J. 127 (2016) 184186,
http://dx.doi.org/10.1016/j.microc.2016.03.007.
[33] K. Tahara, A. Yonemoto, Y. Yoshiyama, M.A. Toshiya Nakamura, Y. Fujita,
T. Nishikawa, Determination of antihyperglycemic biguanides in serum and
urine using an ion-pair solid-phase extraction technique followed by HPLC-UV
on a pentauorophenylpropyl column and on an octadecyl column, Biomed.
Chromatogr. 20 (2006) 12001205, http://dx.doi.org/10.1002/bmc.
[34] M.B. Shabani, T. Akagi, A. Masuda, Preconcentration of trace rare-earth elements in seawater by complexation with bis(2-ethylhexyl) hydrogen phosphate and 2-ethylhexyl dihydrogen phosphate adsorbed on a C18 cartridge
and determination by inductively coupled plasma mass spectrometry, Anal.
Chem. 64 (1992) 737743, http://dx.doi.org/10.1021/ac00031a008.
[35] G. Hoogewijs, D.L. Massart, Ion-pair extraction of basic drugs with di(2ethylhexyl) phosphoric acid, Anal. Chim. Acta 106 (1979) 271277.
[36] A.B, W.P. van Bennekom, Determinations based on two-phase ion-pair and
metal-complex formation, TrAC Trends Anal. Chem. 4 (1985) 252265, http:
//dx.doi.org/10.1145/2505515.2507827.
[37] E.R. Garrett, J. Tsau, Application of ion-pair methods to drug extraction from
biological uids I: quantitative determination of biguanides in urine, J. Pharm.
Sci. 61 (1972) 14041410, http://dx.doi.org/10.1002/jps.2600610913.
[38] Y.C. Fiamegos, A.P. Kefala, C.D. Stalikas, Ion-pair single-drop microextraction
versus phase-transfer catalytic extraction for the gas chromatographic determination of phenols as tosylated derivatives, J. Chromatogr. A 1190 (2008)
4451, http://dx.doi.org/10.1016/j.chroma.2008.03.010.
[39] E. Yiantzi, E. Psillakis, K. Tyrovola, N. Kalogerakis, Vortex-assisted liquid-liquid
microextraction of octylphenol, nonylphenol and bisphenol-A, Talanta 80
(2010) 20572062, http://dx.doi.org/10.1016/j.talanta.2009.11.005.
[40] C. Polson, P. Sarkar, B. Incledon, V. Raguvaran, R. Grant, Optimization of protein precipitation based upon effectiveness of protein removal and ionization
effect in liquid chromatography-tandem mass spectrometry, J. Chromatogr. B
Anal. Technol. Biomed. Life Sci. 785 (2003) 263275, http://dx.doi.org/10.1016/
S1570-0232(02)00914-5.
[41] K. Larsson, K. Binnemans, Separation of rare earths by split-anion extraction,
Hydrometallurgy 156 (2015) 206214, http://dx.doi.org/10.1016/j.
hydromet.2015.04.020.
[42] M. Zhang, G.A. Moore, M. Lever, S.J. Gardiner, C.M.J. Kirkpatrick, E.J. Begg,
Rapid and simple high-performance liquid chromatographic assay for the
determination of metformin in human plasma and breast milk, J. Chromatogr.
B 766 (2001) 175179 www.elsevier.com.