Journal of Chromatography B, 1012 (2016) 162–168

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Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Combination of counter current salting-out homogenous

liquid–liquid extraction and dispersive liquid–liquid microextraction
as a novel microextraction of drugs in urine samples
Reza Akramipour a , Nazir Fattahi b,∗ , Meghdad Pirsaheb b,∗∗ , Simin Gheini a
a
School of Medical, Kermanshah University of Medical Sciences, Kermanshah, Iran
b
Research Center for Environmental Determinants of Health (RCEDH), Kermanshah University of Medical Sciences, Kermanshah, Iran

a r t i c l e i n f o a b s t r a c t

Article history: The counter current salting-out homogenous liquid-liquid extraction (CCSHLLE) joined with the disper-
Received 21 October 2015 sive liquid–liquid microextraction based on solidification of floating organic drop (DLLME–SFO) has been
Received in revised form 17 January 2016 developed as a high preconcentration technique for the determination of different drugs in urine samples.
Accepted 19 January 2016
Amphetamines were employed as model compounds to assess the extraction procedure and were deter-
Available online 22 January 2016
mined by high performance liquid chromatography–ultraviolet detection (HPLC–UV). In this method,
initially, NaCl as a separation reagent is filled into a small column and a mixture of urine and acetonitrile
Keywords:
is passed through the column. By passing the mixture, NaCl is dissolved and the fine droplets of acetoni-
Counter current salting-out homogenous
liquid–liquid extraction
trile are formed due to salting-out effect. The produced droplets go up through the remained mixture
Dispersive liquid–liquid microextraction and collect as a separated layer. Then, the collected acetonitrile is removed with a syringe and mixed
Amphetamines with 30.0 ␮L 1-undecanol (extraction solvent). In the second step, the 5.00 mL K2 CO3 solution (2% w/v) is
Urine analysis rapidly injected into the above mixture placed in a test tube for further DLLME–SFO. Under the optimum
conditions, calibration curves are linear in the range of 1–3000 ␮g L−1 and limit of detections (LODs) are
in the range of 0.5–2 ␮g L−1 . The extraction recoveries and enrichment factors ranged from 78 to 84% and
157 to 168, respectively. Repeatability (intra-day) and reproducibility (inter-day) of method based on
seven replicate measurements of 100 ␮g L−1 of amphetamines were in the range of 3.5–4.5% and 4–5%,
respectively. The method was successfully applied for the determination of amphetamines in the actual
urine samples. The relative recoveries of urine samples spiked with amphetamine and methamphetamine
are 90–108%.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction on the toxicological and pharmaceutical properties of drugs [3].

Sample preparation is an important analytical step especially
for the determination of drugs in complex matrices, commonly
encountered in biological analysis [1]. The solution to this problem
emerged early with the use of separation and extraction techniques,
which offered not only the ability to isolate the target drugs from
the sample solution, thus reducing, controlling or even eliminat-
ing the interferences originally present, but also the opportunity
for these drugs to be pre-concentrated and determined at very
low levels [2]. The separation of drugs or medicines from biologi-
cal matrices is one of the most important objects in investigations
∗ Corresponding author. Fax: +98 833 8263048.
∗∗ Corresponding author.
E-mail addresses: nazir fatahi@yahoo.com (N. Fattahi), mpirsaheb@yahoo.com
(M. Pirsaheb).
Among the biological samples, urine is the primarily preferred spec-
imen for drug testing because specimen collection is simple and
non-invasive and drugs and their metabolites tend to be present
in relatively high concentrations [4]. However, urine matrices are
very complex, and therefore, a suitable sample preparation method
aimed at separating the matrix and enriching the target drugs is
necessary to obtain the reliable analytical results.
Several procedures have been developed for the separation and
preconcentration of different drugs of abuse from biological sam-
ple matrices, such as liquid–liquid extraction (LLE) [5], solid-phase
extraction (SPE) [6–8], solid-phase microextraction (SPME) [9–11],
liquid-phase microextraction (LPME) [12–14], supercritical fluid
extraction (SFE) [15], stir-bar sorptive extraction (SBSE) [16] and
dispersive liquid-liquid microextraction (DLLME) [17–20]. LLE and
SPE are time-consuming and expensive, while LLE method requires
high volume of potentially toxic organic solvents, which is haz-
ardous to health. SPME is also expensive, its fiber is fragile and

http://dx.doi.org/10.1016/j.jchromb.2016.01.031
1570-0232/© 2016 Elsevier B.V. All rights reserved.
 R. Akramipour et al. / J. Chromatogr. B 1012 (2016) 162–168
has limited lifetime and sample carry-over can be a problem. The
disadvantages of LPME are as follows: fast stirring would tend to
break up the organic drop; air bubble formation; extraction is time-
consuming and equilibrium could not be attained after a long time
in most cases. SFE and SBSE can also be relatively expensive and
time-consuming [21]. In DLLME, the choice of the extraction sol-
vent is its main drawback and solvents with the densities higher
than water are required and further, they are not often compatible
with reverse phase HPLC. In addition, the high density extraction
solvents, being mostly halogenated, are generally hazardous to lab-
oratory personnel and the environment [22,23].
Recently, a new microextraction method was developed, which
is DLLME integrated with the solidification of a floating organic
drop (DLLME–SFO) [24]. In DLLME–SFO, the extraction solvent after
DLLME, was collected in the top of the test tube and was then cooled
by inserting it into an ice bath for 5 min. The solidified extrac-
tion solvent was transferred into a suitable vial and immediately
melted at room temperature; then it was finally injected into a suit-
able instrument. The performance of DLLME–SFO was illustrated by
extraction of different organic and inorganic compounds [25–30].
In the previous research, we applied DLLME–SFO for extraction
and preconcentration of amphetamines in urine samples [23].
Despite many benefits of the DLLME–SFO, the pretreatment and
dilution of urine samples is its main drawback. Because of decrease
in matrix effect, urine samples should be pretreated and diluted
before DLLME–SFO.
Another extraction procedure, namely homogeneous
liquid–liquid extraction (HLLE), utilizes a phase separation phe-
nomenon in a homogeneous solution and a very small collected
phase is resulted. One version of HLLE is salting-out homogenous
liquid–liquid extraction (SHLLE) which has been used for extrac-
tion and preconcentration of the selected analytes from aqueous
samples [31,32]. It is worthy to note that the enrichment factor
using SHLLE is often low, which still cannot be satisfied for the
requirement of the ultra-trace residue analysis. In principle, SHLLE
combined with DLLME can provide a solution to this problem. Fara-
jzadeh and co-workers introduced a new version of SHLLE, namely
counter current salting–out homogenous liquid–liquid extraction
(CCSHLLE) and its combination with DLLME for the extraction
and preconcentration of some pesticides from fruit juices and
aqueous samples [33,34]. Not only does the combination result
in a high enrichment factor, but it can be also used in complex
matrices.
The aim of this work is the combination of CCSHLLE and
DLLME–SFO, as a sample-preparation method for high performance
liquid chromatography (HPLC). Amphetamine (AP) and metham-
phetamine (MA) were chosen as model analytes to investigate the
feasibility of the improved CCSHLLE–DLLME–SFO technique. To the
best of our knowledge, for the first time, the CCSHLLE–DLLME–SFO
is developed and applied to the analysis of amphetamines
in human urine without pretreatment and dilution of the
samples.
2. Experimental
2.1. Reagents and standards
Standards of amphetamines were obtained from Cerilliant
(Round Rock, TX, USA) as 1 mg mL−1 methanol solutions. The
amphetamines stock standard solution was prepared in methanol
at the concentration levels of 1.00 mg L−1 for AP and MA. After-
wards, they were stored in a freezer at −20 ◦ C. Working standard
solutions were prepared daily by diluting the stock solution with
methanol. The ultra-pure water (six times distilled) was pur-
chased from Shahid Ghazi Company (Tabriz, Iran). Methanol (for
163
spectroscopy), acetone (Suprasolv for gas chromatography), ace-
tonitrile (Hyper grade for liquid chromatography), acetic acid,
sodium dihydrogenphosphate, sodium dodecyl sulfate, sodium
chloride, 1-undecanol, n-hexadecane, 2-dodecanol and 1-decanol
were obtained from Merck (Darmstadt, Germany).
Drug free urine sample (blank) collected from healthy volunteer
in our lab was used for the study. Actual human urine samples taken
from four young people who were suspicious to consumption of
amphetamines were stored at −20 ◦ C and analyzed within 48 h after
of collection without any previous treatment or filtration.
2.2. Instrumentation
Quantitative analysis of the amphetamines was performed
on a Knauer HPLC system (Berlin, Germany) equipped with a
Smartline-1000 binary pumps and Smartline-UV-2500 detector
variable wavelength programmable, an on-line solvent vacuum
degasser and manual sample injector fitted with a 20 ␮L injection
loop (model 7725i, Rheodyne, Cotati, CA, USA). Chromatographic
separation was achieved on an ODS-3 column (25 cm × 4.0 mm,
with 5 ␮m particle size) from Waters (Milford, MA, USA). The
mobile phase consisted of 80% buffer containing 10.0 mmol L−1
sodium phosphate monobasic and 0.50 mmol L−1 sodium dode-
cyl sulfate and 20% acetonitrile. The pH of the aqueous buffer in
the mobile phase was adjusted to pH 5.5. A mobile phase flow-
rate of 1.0 mL min−1 was used in isocratic elution mode and the
detection was performed at the wavelength of 210 nm. The Hettich
Zentrifugen (EBA20, Tuttlingen, Germany) was used for centrifu-
gations. Chromatographic data were recorded and analyzed using
Chromgate software version 3.1.
2.3. Extraction procedure
In the first step, a 10-mL glass syringe barrel was cleaned with
pure water and then a frit was placed in the bottom of the barrel
and installed a stopcock. Afterward 4 g NaCl was poured into the
barrel and slightly compressed with the syringe plunger. A 5.0 mL
of urine sample (spiked or not with amphetamines) was mixed
with one milliliter acetonitrile and passed through the barrel at a
flow rate of 0.6 mL min−1 . By passing the above homogenous solu-
tion through the barrel, fine droplets of acetonitrile were formed
at the interface of solid (NaCl) and solution due to dissolution of
salt into solution (salting-out effect). The produced droplets moved
through the remained solution to top of the barrel and floated on
the surface of solution as a separated layer due to lower density
of acetonitrile with respect to water. During this step, the analytes
were extracted into the fine droplets of acetonitrile. After passing all
aqueous solution, the stopcock was closed. The volume of the ace-
tonitrile (separated phase) on the top of remained NaCl solid was
about 0.50 ± 0.03 mL. Subsequently, the organic phase obtained
from the first step was transferred into a 10-mL glass test tube
and 34.0 ␮L 1-undecanol (extraction solvent) was added to the test
tube. Then, K2 CO3 solution (2% w/v, 5.00 mL) were rapidly injected
into a test tube, using a 5.00-mL syringe (gastight, Hamilton, Reno,
NV, USA). A cloudy solution, resulting from the dispersion of the
fine 1-undecanol droplets in the aqueous solution, was formed in
the test tube and the mixtures were centrifuged for 4 min at 4200 g.
Accordingly, the organic solvent droplet was floated on the surface
of the aqueous solution due to its low density. The sample vial was
there after put into an ice bath for 5 min; at this time, the floated
solvent was solidified because of the low melting point (14 ◦ C). The
solidified solvent was transferred into a conical glass sample cup
where it was melted immediately. Finally, 25 ␮L of the extractant
was collected with a syringe and injected onto the HPLC–UV.
164 R. Akramipour et al. / J. Chromatogr. B 1012 (2016) 162–168
Fig 1. Effect of volume of extraction solvent in CCSHLLE step on the enrichment
factor of amphetamines from urine sample. Extraction conditions: sample, 5 mL
urine spiked with 100 ␮g L−1 of each analyte; type of extraction solvent in CCSHLLE
step, acetonitrile; flow rate, 0.6 mL min−1 ; extraction solvent in DLLME–SFO step and
its volume, 1-undecanol, 34.0 ␮L; aqueous phase in DDLME–SFO step, 5 mL K2 CO3
2% w/v; floated phase volume, 25 ± 2 ␮L; room temperature.
3. Results and discussion
In this research, the CCSHLLE and DLLME–SFO conjunction was
designed and employed for extraction of different drugs from urine
samples. To reach a high extraction recovery and enrichment factor
with the employment of CCSHLLE–DLLME–SFO, the CCSHLLE and
DLLME–SFO conditions must be examined and optimized. Since
the DLLME–SFO conditions had been optimized in our previous
research [23], those results were used in this research. Only the
CCSHLLE conditions together with some notable parameters in the
CCSHLLE–DLLME–SFO combination were studied. The enrichment
factor (EF), the extraction recovery (%ER) and the relative recovery
(%RR) were calculated according to the equations described in our
previous research [23].
3.1. Selection of extraction solvent in CCSHLLE step
In CCSHLLE–DLLME–SFO procedure, the extraction solvent in
CCSHLLE step should be able to play the role as a disperser sol-
vent in the following DLLME–SFO step. For this purpose, acetone,
acetonitrile, methanol, and tetrahydrofuran, displaying this ability,
were selected. The obtained results showed that only acetonitrile
formed a two-phase system, while other solvents could not be sepa-
rated from the aqueous solution by passing through a syringe barrel
filled with NaCl. Therefore acetonitrile was selected as an extraction
solvent for the further studies.
3.2. Selection of extraction solvent volume in CCSHLLE step
For obtaining optimized volume of extraction solvent in CCSH-
LLE step, various experiments were performed by using different
volumes of acetonitrile (i.e., 0.50, 0.75, 1.00, 1.50, 2.00, and 2.50 mL)
and the results are shown in Fig. 1. According to Fig. 1 and consid-
ering the experimental errors on the data points, the enrichment
factor of analytes was found to increase by increasing volume of
acetonitrile up to 1.00 mL; while, further increase in volume of
acetonitrile caused a small decrease in the enrichment factor. This
observation could be attributed to the fact that at lower acetoni-
trile volumes, the cloudy suspension of the 1-undecanol droplets
was not formed well (in DDLME–SFO step), resulting in a decrease
in the enrichment factor. By using more than 1.00 mL acetonitrile,
the solubility of analytes in aqueous phase increases and it causes
a small decrease in the enrichment factor. Also, no collected phase
was obtained in the case of 0.50 mL acetonitrile. Thus, according
Fig. 2. Effect of sample solution pH on amphetamine enrichment factors from urine
using CCSHLLE-DLLME–SFO. Extraction conditions: as in Fig. 1 except acetonitrile
volume which was 1.0 mL.
Fig. 3. Effect of sample solution flow rate in CCSHLLE step on amphetamine enrich-
ment factors from urine. Extraction conditions as in Fig. 2.
to the results, 1.00 mL of acetonitrile was chosen as the optimum
volume of extraction solvent in CCSHLLE step.
3.3. Effect of pH
Amphetamine and methamphetamine form anions in acidic
solution while they are neutral molecules in alkaline solution. In
CCSHLLE step, for investigating the effect of aqueous solution pH
on the performance of extraction, various experiments were per-
formed by different pH of aqueous solution (from 5 to 12). Other
experimental conditions were kept constant. The results are shown
in Fig. 2. It was found that the enrichment factor of amphetamines
increased with the increasing pH from 5 to 9 and is kept constant
upon further increase in pH of samples. As can be seen, when the pH
is high, the acid–base equilibrium for the alkaline amphetamines
shifts significantly toward the neutral forms. On the other hand,
since an aqueous solution of urine is nearly alkaline, within the
optimized pH range (i.e., pH∼10), in this work, the use of an alkaline
solution for the pH adjustment was not needed.
3.4. Effect of the flow rate of the sample solution
The flow rate of the sample solution through the solid (NaCl)
is an important factor because it controls the time of analysis and
extraction recovery. The flow rate of the sample solution must be
low enough to perform an effective salting-out. On the other hand,
it must be high enough not to waste time. The effect of the flow rate
of sample solution was examined from 0.2 to 2 mL min−1 . As it is
illustrated in Fig. 3, the flow rates up to 0.6 mL min−1 have no effect
 R. Akramipour et al. / J. Chromatogr. B 1012 (2016) 162–168 165

Table 1
Figures of merit of CCSHLLE–DLLME–SFO in an amphetamines–free urine sample.

Analyte RSDa (%) RSDa (%) EFb ERc (%) LRd (␮g L−1 ) r2 e LODf (␮g L−1 )
(intra-day, n = 7) (inter-day, n = 7)

AP 4.5 5 157 78 5–3000 0.998 2

MA 3.5 4 168 84 1–2000 0.999 0.5
a
RSD at a concentration of 100 ␮g Lfor AP and MA.
b
EF, enrichment factor.
c
ER, extraction recovery.
d
LR, linear range.
e
r2 , determination coefficient.
f
LOD, limit of detection for a S/N = 3.

on enrichment factor of target analytes while, at higher speeds,

the enrichment factor decreased. This behavior can be explained
because the amount of dissolved salt in aqueous phase is decreased
at high flow rates and leads to a decrease in efficiency. Thus, a flow
rate of 0.6 mL min−1 was selected for further studies.

3.5. Analytical figures of merit

The optimized CCSHLLE–DLLME–SFO and HPLC–UV procedure

was validated with respect to limit of detection (LOD), precision
(intra–day and inter–day), linear range (LR), EF, and ER. Table 1
summarizes the analytical characteristics of the optimized method.
The repeatability (intra–day) and reproducibility (inter–day) were
studied by extracting the spiked urine samples (100 ␮g L−1 for each
analyte). The repeatability and reproducibility were calculated to
be in the range of 3.5–4.5% and 4–5%, respectively. Determination
coefficients (r2 ) ranged from 0.998 to 0.999. Good linearities rang-
ing from 5–3000 and 1–2000 ␮g L−1 were obtained for AP and MA,
respectively. The limits of detection, based on a signal-to-noise
ratio (S/N) of 3, were 2 and 0.5 ␮g L−1 for AP and MA, respectively.
Moreover, the EFs and the ERs of AP and MA were 157 and 168, and
78 and 84%, respectively.

3.6. Real sample analysis

To demonstrate the potentiality of the technique, the procedure

was applied for the analysis of various urine samples. The proposed
method was firstly applied to determination of the concentration of
AP and MA in human urine samples, provided by one female volun-
teer in our lab, who was not exposed to any drugs or amphetamines
for at least 6 months. The results from urine samples showed that
they were free of AP and MA. These samples were spiked with
amphetamines standards at different concentration levels to assess
matrix effects. The results of relative recovery of urine samples are
shown in Table 2. As seen, the relative recoveries for AP and MA in
spiked urine samples are between 97 and 102%.
Four actual urine samples taken from male and female young
persons who were suspicious to consumption of amphetamines
Fig. 4. Chromatograms of amphetamines in (A) standard solution of 1000 ␮g L−1
were also subjected to the proposed procedure and analyzed in
concentration, (B) extract of urine sample from a person (26-year-old male) sus-
triplicate. All of urine samples were analyzed within 48 h of col- picious of amphetamines consumption, and (C) extract of the same urine sample
lection. AP was detected in all of the actual urine samples in the spiked with 50 ␮g L−1 of amphetamines. Extraction conditions as in Fig. 2.
range of 58.4–144.8 ␮g L−1 except for urine sample taken from
a 22-year-old female. Also, MA was detected in all of the actual
urine samples in the range of 117.3–286.1 ␮g L−1 . The concentra- 50.0 ␮g L−1 for AP and MA (C). These results demonstrated that the
tion of amphetamines in different actual urine samples are listed matrices of the analyzed actual urine samples possess negligible
in Table 2. The presences of mentioned amphetamines in these effect on the proposed CCSHLLE–DLLME–SFO procedure followed
samples were confirmed by spiking AP and MA at the different by HPLC–UV determination of the amphetamines.
concentration levels. The results of relative recoveries and concen-
trations obtained by analysis of spiked actual urine samples are 3.7. Comparison of the proposed method with other methods
also included in Table 2. Fig. 4 shows the obtained chromatograms
of direct injection of AP and MA standards at concentration level We compared the resulting data of CCSHLLE–DLLME–SFO
of 1000 ␮g L−1 (A), actual urine sample taken from a 26-year-old enrichment of amphetamines from urine samples with our pre-
male (B) and corresponding spiked ones at concentration level of vious work [23] and, also, with the results of other methods.
166 R. Akramipour et al. / J. Chromatogr. B 1012 (2016) 162–168

Table 2
Analysis of blank and actual urine samples.

Human urine samples Analyte Added (␮g L−1 ) Found mean ± SDa (␮g L−1 ) Relative recovery (%)

Taken from healthy volunteer AP 0 n.d.b –

(blank) 100 98.5 ± 4.6 98
200 194.2 ± 11.5 97
MA 0 n.d. –
100 102.3 ± 6.2 102
200 198.0 ± 8.7 99
Taken from a 26-year-old male AP 0 67.4 ± 3.0 –
50 115.0 ± 7.3 95
100 165.5 ± 12.2 98
MA 0 117.3 ± 9.4 –
50 171.2 ± 13.0 108
100 212.6 ± 16.3 95
Taken from a 22-year-old AP 0 n.d. –
female 40 41.2 ± 2.3 103
80 79.1 ± 4.0 99
MA 0 213.4 ± 15.7 –
40 255.0 ± 21.8 104
80 288.5 ± 17.6 94
Taken from a 19-year-old male AP 0 58.4 ± 4.2 –
30 85.8 ± 6.1 91
60 120.5 ± 8.6 103
MA 0 188.0 ± 12.5 –
30 220.3 ± 13.7 108
60 245.9 ± 16.8 96
Taken from a 20-year-old AP 0 144.8 ± 11.4 –
female 20 166.3 ± 12.0 107
40 182.8 ± 11.9 95
MA 0 286.1 ± 17.7 –
20 304.2 ± 18.4 90
40 325.6 ± 21.6 99
a
SD, standard deviation (n = 3).
b
n.d., not detected.

Table 3
Comparison of the proposed method with other analytical techniques for determination of amphetamines in urine samples.

Extraction techniquea Analyteb Linear range LODs RSD (%) EF Extraction Reference
(␮g L−1 ) (␮g L−1 ) time (min)

OCD-GC-MS AP, MA 500–50000 250 1.4–7.7 – 30 [35]

SADLLME-HPLC-UV AP, MA 10–2000 2–3 4.5–5.6 48–56 8 [36]
In-tube SPME-HPLC-UV AP, MA, MDA, MDMA 50–5000 1.4–4 1.9–2.9 – 25 [11]
HS-HF-LPME–GC–MS AP, MDA 50–700 0.25–1c 0.4-4 – 30 [14]
EME-HPLC-DAD AP, MA, MDMA, MDEA, MBDB 50–7000 5–10 5.6–10.2 108–140 7 [37]
HF-LPME-GC-FID AP, MA, MDA, MDMA 30–45000 8–82 6.9–14.1 42–227 20 [38]
LPME-FIA-APCI-MS-MS AP, MA, MDA, MDMA, MDEA – 2–100 – 4–18 15 [39]
SPME-HPLC-FLD AP, MDA 375–10000 250 0.5-20 – >40 [40]
HS-SPME-GC-FID AP, MA 100–10000 3–9 3-9 – 22 [41]
SPE-HPLC-DAD AP, MA, MDA, MDMA 200–20000 100 2.8–10.4 – 15 [42]
MISPE-DLLME-GC-FID MA, MDMA 10–1500 2–18 5.1–6.8 285–427 – [43]
USAEME-GC-FID MDMA, MDA, MDEA, MDPA 0.5–500 0.2–0.4 5.7–9.3 51–102 10 [44]
DLLME-FASI-CZE MDMA, LSD, PCP 10–100 1–4.5 3.5-12 – – [45]
DLLME-SFO-HPLC-UV AP, MA 10–3000 2–8 6.2–7.8 117–125 <10 [23]
CCSHLLE- DLLME-SFO-HPLC-UV AP, MA 1–3000 0.5– 4–5 157–168 <20 Proposed method
a
On-column derivatization (OCD), gas chromatography (GC), mass spectrometry (MS), surfactant-assisted dispersive liquid–liquid microextraction (SADLLME), high-
performance liquid chromatography (HPLC), ultraviolet (UV), solid phase microextraction (SPME), headspace (HS), hollow fiber (HF), liquid phase microextraction (LPME),
electromembrane extraction (EME), diode array detector (DAD), flame ionization detector (FID), flow injection analysis (FIA), atmospheric pressure chemical ionization
(APCI), fluorescence detector (FLD), solid phase extraction (SPE), solid phase extraction (SPE), ultrasound-assisted emulsification microextraction (USAEME), molecularly
imprinted-solid phase extraction (MISPE), Field amplified sample injection (FASI), capillary zone electrophoresis (CZE), dispersive liquid–liquid microextraction (DLLME),
solidification of floating organic drop (SFO), counter current salting-out homogenous liquid-liquid extraction (CCSHLLE).
b
Amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylendioxymethamphetamine (MDMA), 3,4-
methylendioxyethamphetamine (MDEA), methylbenzodioxolylbutanamine (MBDB), 3,4-methylenedioxypropylamphetamine (MDPA), lysergic acid diethylamide
(LSD), phencyclidine (PCP).
c
Limit of quantification.

The linear range, LOD, RSD, EF and extraction time obtained by
CCSHLLE–DLLME–SFO were compared with other reported meth-
ods in order to know the potentiality of the present method for
the determination of amphetamines. The results are summarized
in Table 3. As can be seen RSDs of the proposed method are about
the same with those reported for the other methods and some-
times are better. The LODs of the proposed method are better than
other methods, except for HS–HF–LPME–GC–MS and USAEME-GC-
FID. The linear ranges and analysis time of the proposed method are
relatively superior to those reported before. All these results indi-
cate that the proposed CCSHLLE–DLLME–SFO method is a sensitive,
repeatable and simple technique that can successfully be used for
the preconcentration and determination of the selected drugs in
urine samples.
 R. Akramipour et al. / J. Chromatogr. B 1012 (2016) 162–168
4. Conclusion
In this study, the CCSHLLE was combined with the DLLME–SFO
technique for the determination of drugs in urine samples
prior to analysis by HPLC–UV. Amphetamines were chosen as
model analytes to investigate the feasibility of the improved
CCSHLLE–DLLME–SFO method. This combination not only resulted
in a high enrichment factor, but also it could be used in com-
plex matrices (such as urine, fruit juice and highly saline solution)
without any pretreatment or dilution. As compared with the other
sample preparation methods, the analytical procedure offered
numerous advantages such as simplicity, ease of operation, high
preconcentration factor, low detection limit and relatively short
analysis time. Although the obtained results in this work are related
to determination of amphetamines, the system could be read-
ily applied for the determination of other drugs from complex
biological and pharmaceutical matrices, using different analytical
instruments.
Acknowledgement
The authors thank the Deputy of Research and Technology,
Kermanshah University of Medical Sciences, Kermanshah, Iran for
financial support (Project No. 94119).
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