1. health and fitness
2. drugs

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Salt-assisted LLE combined with field-amplified
sample stacking in CE for improved
determination of beta blocker drugs in
human urine
Background: A simple and sensitive CE method was developed and validated for the ana­lysis of some beta blockers
in human urine. Methods: In this study, salting-out assisted LLE combined with field-amplified sample stacking
method was employed for biological sample clean-up and sensitivity enhancement in CE. Results: Under the
optimal conditions good linearity (r2 ≥0.998) was obtained, within 0.025–1 µg/ml for propranolol and metoprolol,
and within 0.05–1 µg/ml for carvedilol in urine samples. LODs and LLOQs ranged from 0.005 to 0.015 µg/ml, and
from 0.025 to 0.05 µg/ml, respectively. The RSDs of intra- and inter-day ana­lysis of examined compounds were
less than 4.0%. The recoveries were in the range of 98–119%. Conclusion: The validated method is successfully
applied to determine propranolol, metoprolol and carvedilol in human urine samples obtained from the patients
who received these drugs.
The beta blockers are an extremely important
class of cardiovascular drugs that are mainly used
to treat various cardiovascular disorders, such as
hypertension and chronic heart failure [1]. They
were also recommended as the first-line therapy
for hypertension by all Joint National Committees [2]. The use of beta blockers was forbidden
by the Medical Commission of the International
Olympic Committee because these drugs reduce
the cardiac rhythm by blocking the beta-receptors
in the heart; this can be useful in sports where
aiming is important [3]. For the detection of beta
blockers in urine samples, the minimum required
performance limit set by the World Anti-doping
Agency is 0.5 µg/ml [101,102]. Therefore, quantifications of these drugs in biological fluids are
required in therapeutic drug monitoring and
doping tests. Different techniques, namely TLC
[4], fluorescence [5,6], GC [7,8], GC–MS [9–11],
HPLC with UV/fluorescence/photodiode array/
amperometric detection and MS [12–16], capillary
zone electrophoresis (CZE) [17] and micellar
electrokinetic chromatography [18,19] have been
used for the determination of beta blockers in biological fluids. A number of GC and LC methods
are compared in Table 1 [8,9,11,20–26]. GC methods usually require a tedious derivatization to
improve chromatographic properties and laborintensive pretreatment procedures for obtaining
high detection sensitivity. LC is the more popular
method, but with some drawbacks such as the
matrix effect. Therefore, it has to be coupled with
a preconcentration method, such as SPE, LLE
and so on. Large amounts of potentially toxic
or hazardous solvents are also required for these
extractions. In addition, in most cases, MS has
been used as a detection system. It is clear that
employing MS detection in an analytical setup restricts its routine ana­lysis applications and
does not attract more attention due to its high
cost. The results of LC–UV methods published
after 2010, are comparable and/or better than
our findings. However, it should be mentioned
that there are some limitations for CE methods
when compared with LC methods. Lower sensitivity is usually expected from CE methods.
CE is extensively used for routine ana­lysis as an
attractive alternative to HPLC because of its small
sample requirements [27], high resolving power
and short ana­lysis time [28–30]. Therefore, using
CE in routine ana­lysis offers a fast method development with low operational costs. The UV/
Vis detector is the most popular detector used
in CZE but possesses a relatively high LOD of
10-5–10-6 mol dm-3 due to the detector’s short path
length and also due to nanoliter injection volumes
[31]. Therefore, for trace ana­lysis applications, the
amount of analyte injected into the capillary or
the detector sensitivity has to be increased [32].
Detection sensitivity can be improved by using
a Z-shaped cell for UV detection to increase the
path length of the detector, or by using sensitive
10.4155/BIO.13.303 © 2014 Future Science Ltd
Bioanalysis (2014) 6(3), 319–334
Rana Fazeli-Bakhtiyari1,2 ,
Mohammad Hossein
Sorouraddin2 , Mir Ali
Farajzadeh2 , Mohammad
Hossein Somi1 &
Abolghasem Jouyban*3
Biomedical Analysis Lab, Liver &
Gastrointestinal Diseases Research
Center, Tabriz University of Medical
Sciences, Tabriz, Iran
2
Department of Analytical Chemistry,
Faculty of Chemistry, University of
Tabriz, Tabriz, Iran
3
Drug Applied Research Center &
Faculty of Pharmacy, Tabriz University
of Medical Sciences, Tabriz, Iran
*Author for correspondence:
Tel.: +98 411 337 9323
Fax: +98 411 336 3231
E-mail: [email protected]
1
ISSN 1757-6180
319
R esearch A rticle |
Key Terms
Beta blockers: Popular class
of cardiovascular drugs that are
mostly used for treatment of
hypertension, angina pectoris,
cardiac arrhythmias, and so on.
Therapeutic drug
monitoring: Subspecialty of
clinical pharmacy used for
measuring drug concentrations
in plasma, serum or blood, in
order to enhance treatment
efficacy, reduce toxicity or assist
with diagnosis.
Doping tests: Technical
analyses to determine the
presence or absence of
prohibited substances in a
biological specimen such as
urine, blood, saliva and so on.
Capillary zone
electrophoresis: Analytical
tool in which species are
separated based on their
charge-to-size ratio in the
interior of an electrolyte-filled
capillary.
Field-amplified sample
stacking: Powerful online
sample preconcentration
method that improves detection
sensitivity by using the
conductivity difference between
sample zone and the capillary
zone electrophoresis buffer.
Method validation: Process
of testing an analytical and
bioanalytical method to assess
its performance and limitations.
In this way, validation is carried
out according to US FDA and
ICH recommendations.
320
Fazeli-Bakhtiyari, Sorouraddin, Farajzadeh, Somi & Jouyban
Table 1. Summary of previous chromatographic methods based on GC and LC for
determination of selected drugs in urine samples.
Analyte
Pre-treatment method
Analysis
method
LOQ
(ng/ml)
LOD
(ng/ml)
Metoprolol
Propranolol
Enzymatic hydrolysis
LLE
[20]
SPE-SFE
1.2
3.44
8.4
23.3
ND
ND
Metoprolol
Propranolol
Metoprolol
Propranolol
GC–MS (TMS/TFA
derivatives)
GC–MS( boronate
derivatives)
GC–MS
[21]
Enzymatic hydrolysis
SPE
Derivatization
HF-LPME with in situ derivatization
GC–MS
10.8
17.8
30
40
3.6
6.2
GC–MS
ND
N-ethoxycarbonyl derivatization
LLE
N-Methyl-N-(trimethylsilyl)
trifluoroacetamide derivatization
SPE
GC–MS
ND
LC–MS/MS
ND
Dilution
Filteration
CM-LPME
SPE
MLC/FL
ND
HPLC/DAD
UHPLC/UV
DLLME
HPLC/UV
ND
41.4
86.3
44.4
14
Metoprolol
Propranolol
Metoprolol
Propranolol
Carvedilol
Metoprolol
Propranolol
Metoprolol
Propranolol
Propranolol
Carvedilol
Metoprolol
Propranolol
Carvedilol
Ref.
0.08
0.05
1.8
0.6
20
40
50
19.2
11.8
5
13.8
28.8
14.8
4
[8]
[9]
[11]
[22]
[23]
[24]
[25]
[26]
CM-LPME: Carrier-mediated liquid-phase microextraction; DAD: Diode array detector; DLLME: Dispersive liquid–liquid
microextraction; HF-LPME: Hollow-fiber liquid-phase microextraction; MLC/FL: Micellar LC/fluorimetric detection; ND: No
data; SFE: Supercritical fluid extraction; TMS/TFA: Trimethylsilylated/trifluoroacetate.
detection techniques, for example, laser induced
fluorescence [33], chemiluminescence [34], electrochemical and amperometric detectors [35]. Various
preconcentration techniques were investigated to
address this limitation. Electrophoresis-based preconcentration methods including field-amplified
sample injection [36], large volume sample stacking [37,38], isotachophoretic electrophoresis stacking [36], dynamic pH junction stacking [39–41] and
sweeping [42] can all greatly improve the detection
limit [43–45]. The injection method in CE is more
important and different injection modes can be
used. Therefore, selection of the right injection
approach can result in significant improvements
in performance during any method development
[28]. For example, electrokinetic injection provides
a large preconcentration potential through sample
stacking compared with hydrodynamic injection
[46]. Field-amplified sample stacking (FASS) is
a phenomenon through which sample ions accumulate at the boundary [47], which separates the
low conductivity sample plug and the high conductivity background electrolytes (BGEs) [48,49].
Sample pre-treatment should be considered since
Bioanalysis (2014) 6(3)
many of the techniques involving stacking are
critically dependent on the nature of the solvent
from which the analyte is loaded into the capillary [50]. The major challenge in bioana­lysis is to
separate the analytes from the matrix of the biological samples. Because of this, various types of
clean-up procedures are employed to effectively
separate the analyte from the endogenous biological material [51,52]. In addition, in biomedical
investigations, there is an increasing demand for
efficient analytical methods that permit simultaneous separation and quantification of the complex drug matrix with less laboratory work [53].
Protein precipitation (PPT), immiscible LLE, column SPE and dispersive SPE are the most popular
sample preparation methods in bioana­lysis [54].
PPT is simple but supernatant from PPT contains not only analyte(s) of interest but also a lot
of soluble substances (i.e., all endogenous matrix
components) that can interfere with the analytical methods [55,56]. LLE and SPE require a large
amount of toxic and expensive organic solvents,
and are also time-consuming. Therefore, alternative environmentally friendly sample preparation
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future science group
O
NH
H3C
O
OH
O
H3C
†
HO
NH
0.02–0.3
3.10
9.5
O
Physicochemical properties calculated using ACDLabs software [103].
CH3
0.02–0.5
1.79
9.5
O
OH
NH
CH3
CH3
0.02–0.16
4.11
7.5
Carvedilol
Metoprolol
CH3
Propranolol
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Therapeutic range (µg/ml)
Log P
pKa
CZE-DAD
An Agilent 7100 CE system equipped with
a DAD was used. The system was controlled
by a personal computer installed with Agilent
Chemstation software (Waldbronn, Germany).
Molecular structure
„„Instrumentation:
Properties
Experimental
„„Chemicals & materials
Carvedilol, 1-(carbazol-4-yloxy-3-[[2-(Omethoxyphenoxy) ethyl]amino]-2-propranol,
metoprolol, 1-(isopropylamino)-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol and propranolol (as hydrochloride), 1-(isopropylamino)-3-(1naphthyloxy)propan-2-ol) were kindly supplied
by Salehan Chemi, Alborz Darou and Rouzdarou Pharmaceutical Companies (Tehran, Iran),
respectively, (molecular structures, log P, pK a
values and therapeutic levels of these drugs are
reported in Table 2). Methanol, ethanol, 2-propanol and acetonitrile were purchased from
Scharlau (Barcelona, Spain). Phosphoric acid,
tris (hydroxymethyl) aminomethane (Tris),
sodium chloride, hydrochloric acid, and sodium
hydroxide were purchased from Merck Company
(Darmstadt, Germany). Deionized water (Shahid Ghazi Company, Tabriz, Iran) was used for
preparing the buffer and sample solutions.
Table 2. Molecular structures and physicochemical properties of the analytes†.
methods are greatly needed. Recently, salting-out
extraction, which is a type of homogeneous LLE
has received considerable attention by bioanalysts [57]. Salting-out assisted LLE (SALLE) is
performed by more polar solvents (such as acetonitrile, acetone and so on), which are water
miscible. When an appropriate salting-out agent
(e.g., NaCl) is added to a mixture of water and
a water-miscible organic solvent, it leads to the
separation of the solvent from the bulk aqueous
solution and forms a biphasic system, see [56] and
the references within. Experimental results demonstrated that SALLE is simple, fast and environmentally friendly [55]. In the present work,
SALLE-FASS-CE-diode array detector (DAD)
was employed for biological sample clean-up and
sensitivity enhancement in CE (using beta blocker
drugs as model analytes). The effects of chemical
parameters (e.g., buffer ionic strength, pH and
buffer additives) and instrumental parameters
(e.g., applied voltage and temperature) were investigated. After that, sample electrokinetic injection
parameters in FASS were investigated to achieve
improved sensitivity. Finally, method validation
was performed according to US FDA guideline
and then applied to analyze the selected drugs
concentrations in urine samples.
| R esearch A rticle
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Improved determination of beta blocker drugs in human urine
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Fazeli-Bakhtiyari, Sorouraddin, Farajzadeh, Somi & Jouyban
Electro­phoresis and preconcentration experiments were performed in uncoated fused-silica
capillary 50 µm I.D. and 375 µm O.D. (64.5 cm
total length, 56 cm effective length). When a new
capillary was used, the capillary was washed with
sodium hydroxide solution (1.0 M) for 30 min,
deionized water (30 min) and running buffer (30 min). The experiments were performed
after prewashing with sodium hydroxide solution
(0.1 M) followed by deionized water and then
running buffer for 2 min at each step. The BGE
solution consisted of 30 mM Tris and phosphate
buffer at pH 2.2 containing 15% (v/v) methanol
as organic modifier. It should be noted that pH of
the buffer was adjusted to 2.2 with concentrated
phosphoric acid before addition of the organic
solvent. Injection was performed electrokinetically. The injection time, the injection voltage
and the preinjection water plug were investigated. All BGE and sample solutions were filtered
through a 0.20 µm pore size PTFE filter (Chromafil, Germany). Capillaries were thermostated
at 25˚C and the applied voltage was +25 kV.
Online UV detection was performed at 195 and
214 nm where the selected drugs showed sufficient absorbance responses. A Metrohm model
827 pH meter (Herisau, Switzerland) was used
to measure buffer and urine solution pH.
agent/extraction solvent) was added to the solution and vortexed for 1 min. The salting out effect
was used to induce phase separation by addition
of NaCl (1 g) to increase the ionic strength. The
mixture was again vortexed for 1 min. After
centrifugation at 4000 rpm for 5 min, 1 ml of
organic phase supernatant was removed from
the urine sample. Next, 0.2 ml of the collected
organic phase was removed and evaporated to
dryness under a gentle N2 stream. Subsequently,
the dry residue-containing enriched analytes was
reconstituted in 0.1 ml of deionized water and
then analyzed by CZE-DAD. The total sample
preparation time was 18 min; including extraction time (2 min), centrifugation time (5 min),
solvent evaporation time (~10 min) and residue
reconstitution time (1 min).
„„Standard
urine samples collection
Urine samples were obtained from patients
receiving the drugs who had signed consent
forms approved by the ethics committee, Tabriz
University of Medical Sciences. Samples were
collected in polypropylene tubes and stored at
-20°C until ana­lysis.
where Ccoll and C 0 are the concentration of
analyte in the collected phase and the initial concentration of analyte within the sample, respectively. Ccoll was calculated from calibration curves
plotted by direct injection of standard solutions.
Vcoll and Vaq are also the volumes of the collected
phase and sample phase, respectively. It should
be noted here that EFs and ER% of each analyte
was calculated based on starting with 4 ml urine
and extracting the analytes into 1 ml (Table 3). If
whole volumes of the collected phase (i.e., 1 ml)
are evaporated and reconstituted in 0.1 ml,
higher EFs could be obtained, but the matrixinduced interferences are also enriched. In order
to avoid any matrix effect and reduce evaporation time, a portion of the collected phase was
removed, dried and reconstituted in 0.1 ml. The
proposed method was validated with different
urine samples collected at different times of day
and the same matrix effect was observed.
„„Extraction
„„
Assay
„„Calculation
of enrichment factor
& extraction recovery
The enrichment factor (EF) and extraction
recovery (ER%) were obtained by the following
equations:
EF = Ccoll
C0
Equation 1
ER% = EF # Vcoll # 100
Vaq
Equation 2
solutions & biologic samples
A stock solution of three beta blockers (each
1000 mg/l) was prepared in methanol and kept
in a refrigerator at 4°C. Working standard solutions were prepared daily by appropriate dilutions of the stock solution with deionized water.
Spiked urine samples were also prepared daily by
dilution of the stock solution by drug-free urine
samples. It should be noted that the method was
developed using standard solutions of the three
drugs and spiked urine samples were used for
validation studies.
„„Real
procedure
The pH of urine sample was adjusted to 11.3 by
adding drops of 6 mol/l NaOH solution. 4 ml of
this solution was transferred into a 10 ml test tube.
Then, 2 ml acetonitrile (protein precipitating
322
Bioanalysis (2014) 6(3)
validation
The validation studies were carried out according
to FDA recommendations [58]. The calibration,
linearity, LOD, LLOQ, ULOQ, intra- and interday precisions, accuracy, recovery, selectivity,
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Improved determination of beta blocker drugs in human urine
stability (room temperature and freeze–thaw)
and robustness were investigated for each analyte.
The mean of three calibration curves that were
constructed on three nonconsecutive days were
used for linearity studies. All experiments were
replicated three times. LLOQ and ULOQ terms
are defined as the lowest and highest concentration level of calibration curve, while the CV%
of three replications should be less than 20 and
15%, respectively. Also, LODs and LOQs were
calculated from the S/N ratio as three and ten,
respectively, for each drug. For evaluating the
inter-and intra- day precisions five spiked urine
samples in the low, medium and high concentrations of each drug were analyzed by the developed
method on five different days. The accuracy of
method was also examined by computing relative
errors (%) using the following equation:
RE(%) = 100 # c calulated conc. - nominal conc. m
nominal conc.
Equation 3
The relative recoveries were determined for
five human urine samples spiked with three
different concentrations of the examined compounds. In the present study, selectivity of
the developed method was studied by analyzing urine samples spiked with some other coadministered cardiovascular drugs for potential
interferences. To assess room temperature stability, spiked urine samples were left at ambient
temperature for 12 h. The freeze–thaw stability was also assessed after three 12-h freeze–
thaw cycles. Furthermore, the robustness of the
method was evaluated by partial varying of some
effective parameters in FASS method in three
levels, such as running buffer concentration and
its pH, separation temperature and voltage.
Results & discussion
„„Development of separation conditions
Effects of pH, buffer type and concentration,
buffer additives, separation temperature and
voltage were investigated using a one-factor-ata-time approach for the baseline separation of
beta blockers. In order to find the most suitable
buffer composition, three BGE systems (acetate,
phosphate and tris-phosphate) in low pH was
tried. Best separation for the three beta blockers was achieved when using the tris-phosphate.
The pH of buffer plays an important role in the
separation since it determines the extent of ionization of each analyte [59]. Therefore, the choice
of a proper pH is very important to optimize
the separation in CZE. The effect of buffer pH
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Table 3. Quantitative results of salting-out assisted LLE–fieldamplified sample stacking-CE-diode array detector for the selected
drugs in urine samples.
Parameter
Propranolol
Metoprolol
Carvedilol
Linear range (µg/ml)
Slope
Slope standard errors
Intercept
Intercept standard errors
Correlation coefficient (r2)
Number of data points
LOD (µg/ml)
LOQ (µg/ml)
LLOQ (µg/ml)
EFextraction ± SD
0.025–1
0.023
0.001
1.3
0.2
0.998
5
0.008
0.025
0.025
2.8 ± 0.09
0.025–1
0.024
0.002
0.8
0.4
0.999
5
0.005
0.016
0.025
3.2 ± 0.07
0.050–1
0.010
0.002
0.4
0.3
0.999
5
0.015
0.050
0.050
1.4 ± 0.03
EFsample preparation ± SD
5.5 ± 0.2
6.4 ± 0.1
2.74 ± 0.05
SEF
ER% ± SD
200
69.2 ± 2.3
200
79.5 ± 1.8
100
34.3 ± 0.7
EF: Enrichment factor; ER%: Extraction recovery; SEF: Sensitivity enhancement factor.
was evaluated with 30 mM tris-phosphate buffer at pH values varying from 2.2 to 3.7. In this
investigated pH range basic amino alcohols are
fully protonated and migrate under reduced electroosmotic flow. Best resolution was achieved at
pH 2.2. Furthermore, the buffer concentration
effect was studied in the concentrations of 20,
30, 40, 60 and 80 mM. The observed currents
for these ionic strengths were 17.5, 22.5, 27.5,
37.5 and 47.5 µA, respectively. According to
these results, by increasing buffer ionic strength,
current in the capillary was increased and baseline noise was observed. As a result, 30 mM
Tris-phosphate was finally chosen as CZE buffer.
Furthermore, the separation voltage and temperature effect was investigated by performing
runs at increasing voltages (i.e., 20, 25, 27 and
30 kV) and temperatures (i.e., 20, 25, 27 and
30°C). The viscosity of BGE decreases as temperature increases, so runtime can be reduced
by increasing the voltage and temperature
until a deterioration in resolution is observed.
According to the results obtained, 25 kV and
25°C were the optimal voltage and temperature, respectively. The effect of organic solvent
present in the BGE was also investigated with
some solvents such as methanol, ethanol, 2-propanol and acetonitrile. Addition of the organic
solvent resulted in the reduction of the sphere
of hydration of the analytes. As a consequence,
the difference in charge-to-size ratio increased,
resulting in a better resolution of the analytes
[60]. For all solvents, no significant difference
was observed in lower percentage (5%) but in
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Fazeli-Bakhtiyari, Sorouraddin, Farajzadeh, Somi & Jouyban
higher level baseline noise was increased except
for methanol. As well as in the aqueous-organic
buffer system, the mobility decreases with an
increase in organic solvent content of the buffer [61]. On the one hand, separation selectivity
was significantly increased with increasing the
percentage of methanol (from 0 to 15%) and on
the other hand, migration time became longer.
Therefore, 30 mM Tris-phosphate buffer (pH
2.2) containing 15% methanol was selected as
the optimum buffer solution. Under optimum
conditions migration order was; propranolol,
metoprolol and carvedilol, and total runtime was
less than 16 min. The obtained results indicated
that the detection limits of the selected drugs
are too high (10 mg/l), therefore it is necessary
to employ suitable preconcentration methods
in order to improve the poor sensitivity of the
method.
A
Diluted buffer (tenfold dilution)
Acetonitril:water (30:70)
Methanol:water (30:70)
Water
„„Development
of FASS
The possibility of application of FASS for
detection limit enhancement of beta blockers
in CE was investigated. Therefore, we studied
the influence of FASS parameters (the sample
matrix composition, water plug injection time,
sample injection time and voltage) on the introduced sample amount in this method. Low buffer concentration, pure water, binary solvent
mixtures such as methanol:water (30:70) and
acetonitrile:water (30:70) were used as sample
matrices and a mixture of these drugs (10 mg/l
each) was prepared and injected (5 kV for 5 s).
Figure 1A shows that the highest sensitivity can
be obtained when pure water was employed as
sample matrix. When the sample is prepared in
a lower conductivity matrix (than that of the buffer), the analytes will experience a high field zone
in the sample plug, causing faster migration of
B
Propranolol
Metoprolol
Carvedilol
80
400
Peak area (a.u.)
Peak area (a.u.)
500
300
200
100
0
C
Metroprolol
20
Propranolol
Metoprolol
Carvedilol
Carvedilol
0
D
1
3
5
Water plug injection time (s)
Propranolol
Metoprolol
Carvedilol
1500
Peak area (a.u.)
Peak area (a.u.)
40
0
Propanolol
1200
900
600
300
0
60
1200
900
600
300
0
5
10
15
20
Injection time (s)
25
30
5
10
Injection voltage (kV)
12
Figure 1. Optimization of the injection parameters for field-amplified sample stacking. Separation conditions: uncoated
fused-silica capillary, 64.5 cm (effective length 56 cm) × 50 µm I.D.; BGE: 30 mM Tris-phosphate (pH 2.2) containing 15% of methanol;
UV detection at 195 nm; temperature, 25°C; applied voltage, 25 kV. Optimization of (A) sample matrix composition; sample solution:
10 mg/l of each drug injected at 5 kV for 5 s; (B) water plug injection time; sample solution: 1 mg/l of each drug injected at 5 kV for
5 s after preliminary pressure injection of water; (C) sample injection time; sample solution: 1 mg/l of each drug injected at 5 kV after
preliminary pressure injection of water (50 mbar for 1 s); and (D) injection voltage; sample solution: 1 mg/l of each drug injected for
25 s after preliminary pressure injection of water (50 mbar for 1 s). The error bars indicate the maximum and minimum of three
determinations.
324
Bioanalysis (2014) 6(3)
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Improved determination of beta blocker drugs in human urine
the analytes until they reach the boundary of the
BGE zone. When the analytes pass the boundary
between the sample and the buffer, they experience a loss in velocity and stack a sharp band
because of the significant decrease of the field
strength in the electrophoresis buffer [47]. In general, a pre-injection plug can provide a trap in
which the analytes are collected prior to being
separated in the BGE; consequently, the stacking
efficiency becomes larger [62]. Furthermore, the
influence of the hydrodynamic injection of water
plug prior to electrokinetic sample injection (5 s
at +5 kV) was studied. The obtained results presented in Figure 1B, indicate that the peak areas
increase with increasing the injection time from
0 to 1 s and then decrease thereafter. Hence, a
short plug of water (50 mbar × 1 s) was selected
for subsequent experiments. Here, we employed
electrokinetic injection for sample introduction
in CE because of its ability to concentrate the
sample in CE capillary. Therefore, the effect of
injection time and voltage on peak areas of the
three beta blockers was tested. Injection time
and voltage were varied from 5 to 30 s and 5 to
12 kV, respectively. Longer injection times and
A
higher injection voltages resulted in greater peak
areas [41]. As shown in Figure 1C & D, a significant increase in the peak areas was observed with
increasing injection time in the range of 5–25 s
and injection voltage within 5–10 kV. Therefore,
25 s and 10 kV were chosen as injection time and
voltage, respectively. Under obtained FASS-CE
conditions (i.e., 30 mM Tris-phosphate, 15% v/v
methanol, pH 2.2, pure water as the sample solution and electrokinetic injection with 10 kV for
25 s after preliminary pressure injection of water
for 1 s) linearity was assessed within 25–5000 ng/
ml. The standard electropherogram of the
selected drugs are displayed in Figure 2A & B.
Comparing Figure 2A & D, reveals that FASS is
strongly affected by the matrix effect, since the
concentration of the analytes in Figure 2D is a
quarter of those in Figure 2A , however, the peak
heights are 1/20 of the standard solutions. It
should be added that although a ten-times diluted
(1 mg/l) concentration of the standard solutions
was used in FASS-CE-DAD method and a sensitivity enhancement factor of 200-, 200- and
100-fold was achieved for propranolol, metoprolol and carvedilol, respectively. The sensitivity
C
12
118
98
2
3
78
58
1
38
18
Absorbance (mAU)
Absorbance (mAU)
138
-2
0
2
4
B
6 8 10 12 14 16 18
Migration time (min)
98
78
58
38
18
-2
0
2
4 6 8 10 12 14 16 18
Migration time (min)
8
6
4
2
8
10
12
14
16
Migration time (min)
8
10
12
14
16
Migration time (min)
D
Absorbance (mAU)
118
10
0
138
Absorbance (mAU)
| R esearch A rticle
16
14
12
10
8
6
4
2
0
Figure 2. Typical electrophoregrams. Using (A) water samples spiked with 1 mg/l of selected
drugs obtained under optimal field-amplified sample stacking-CE-diode array detector (DAD)
(sample solution injected at 10 kV for 25 s after preliminary pressure injection of water [50 mbar for
1 s]); (B) water samples spiked with 10 mg/l of selected drugs obtained under optimal capillary
zone electrophoresis-DAD (sample solution injected at 5 kV for 5 s); (C) blank human urine; and
(D) blank human urine spiked with 250 ng/ml of selected drugs obtained under optimal
salting-out assisted LLE–field-amplified sample stacking-CE-DAD. Separation conditions are the
same as in Figure 1. Peaks: (1) propranolol; (2) metoprolol; and (3) carvedilol.
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Fazeli-Bakhtiyari, Sorouraddin, Farajzadeh, Somi & Jouyban
enhancement factor values were calculated for
each drug using the following equation:
SEFheight =
Height with field amplified sample injection
# dilution
Height with conventional injection
Equation 4
Method validation
„„Linearity & calibration curves
Method validation is a process that demonstrates whether the method will successfully
meet or exceed the minimum standards recommended in the FDA guideline for accuracy,
precision, selectivity, specificity, stability and
robustness [63]. Calibration was performed in
blank urine samples spiked with five different
concentrations of each drug. As mentioned
in the previous section, 4 ml of these solutions were subjected to SALLE method before
FASS-CE ana­lysis. Linearity was tested within
25–5000 ng/ml for standard calibration curve
and 25–1000 ng/ml for matrix calibration
curves. Higher concentrations were not tested
as this linear range was wide enough for clinical applications. Mean of regression equations,
correlation coefficients of calibration curves
constructed on three different days and corresponding validation parameters (i.e., linear
range, LOD, LOQ and LLOQ ) are listed
in Table 3.
„„Precision
& accuracy
Intra- and inter-day precision (expressed as
RSD%) along with accuracy (expressed as
RE%) of the method were determined as
described in the experimental section. All
RSD% values were less than 4.0% and acceptable range of accuracy was obtained (Table 4).
These results indicate that the developed
method is both accurate and precise.
„„Recovery
Relative recoveries (expressed as RR%) of the
tested drugs from spiked urine samples were measured at three concentrations – high (500 ng/ml),
medium (250 ng/ml) and low (50 ng/ml). The
obtained results are presented in Table 5. The
calculated recoveries are in range of 98% (for
propranolol) to 119% (for carvedilol).
„„Selectivity
& specificity
The proposed method showed adequate peak separation for the selected drugs. Representative electropherograms for blank urine sample and spiked
urine with concentration of 250 ng/ml of drugs
are shown in Figure 2C & D. No interference was
observed from urine matrix. But it should be noted
that matrix-induced migration time shifts were
observed when analyzing urine samples. Therefore, shift in migration times is controlled by ana­
lysis of QC samples that were prepared in the same
manner as the test samples. The selectivity of the
method for the selected drugs was also tested by
ana­lysis of some other cardiovascular drugs (e.g.,
amiloride, amiodarone, atenolol, diltiazem, furosemide, hydrochlorothiazide, losartan, verapamil,
sotalol and nifedipine) and most commonly used
drugs such as acetaminophen, diazepam and salicylic acid. Among the tested drugs, acetaminophen, furosemide and hydrochlorthiazide are
acidic drugs and there are no positive charge to be
analyzed using our developed method. Selectivity
tests were performed using urine samples with a
500 ng/ml concentration of each drug. According
to the results obtained (Table 6), only diltiazem
was found to be interfered with carvedilol, which
could be considered as a restriction parameter for
the developed method.
„„Stability
The analyzed drugs showed reliable stability behavior when stored at room temperature
Table 4. Precision and accuracy of the method for determination of the studied drugs in urine samples.
Drug
Nominal concentration
(ng/ml) (n = 5)
Intra-assay precision
(RSD%) (n = 5)
Inter-assay precision Accuracy (RE%)
(RSD%) (n = 5)
Propranolol
50
250
500
50
250
500
50
250
500
2.5
1.2
0.7
2.3
1.3
0.3
3.6
1.6
3.2
4.0
0.4
0.5
0.1
1.3
0.1
2.2
0.7
0.6
Metoprolol
Carvedilol
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Bioanalysis (2014) 6(3)
-1.7
3.0
5.8
15.1
2.0
2.3
19.3
7.2
1.6
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Improved determination of beta blocker drugs in human urine
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Table 5. Relative recoveries obtained by salting-out assisted LLE–field-amplified sample stacking-CE-diode
array detector in urine samples spiked at 50, 250 and 500 ng/ml.
Drug
Nominal concentration (ng/ml)
(n = 5)
Found concentration (ng/ml) ± SD
(n = 5)
Relative recovery (RR%)
± SD
Propranolol
50
250
500
50
250
500
50
250
500
49± 2
257± 1
529 ± 2
58 ± (<0.5)
255± 3
511 ± (<0.5)
60 ± 1
268 ± 2
508 ± 3
98 ± 4
103 ± (<0.5)
106 ± 1
115 ± (<0.5)
102 ± 1
102 ± (<0.5)
119 ± 6
107 ± 1
102 ± 1
Metoprolol
Carvedilol
(25 ± 2.0°C) for 12 h and over three freeze–thaw
cycles. Results are summarized in Table 7. RE%
values for LLOQ and higher concentration were
below 18 and 6%, respectively.
„„Robustness
Robustness testing was carried out by making
small changes in effective chemical (i.e., running
buffer concentration and its pH) and instrumental parameters (i.e., separation temperature and
voltage). The results are shown in Table 8. As can
be seen, there is no significant difference in the
obtained data and indicating that the reported
method is a robust method.
Comparison of the proposed method
with other methods
For a number of GC methods [8,9,11,20,21] cited
in Table 1, more sophisticated instrumentations,
longer pretreatment time and the limited number of quantified analytes could be considered
as restriction factors when compared with the
proposed method especially in routine ana­lysis.
Lower LOD and LOQ of these methods could
be considered as their advantages. It should be
noted that the proposed method provided better
and/or comparable LOD values with the work
of Hartonen and Riekkola [21]. Comparing LC
methods listed in Table 1, LC–MS/MS method
employs highly sophisticated instrumentation
with slightly higher LOD values for some beta
blockers including three investigated drugs
[22]. The micellar LC with fluorescence detection of metoprolol and propranolol is a simple
method, however produced higher LOD values
in comparison with the proposed CE method
[23]. The HPLC/DAD method coupled with
carrier-mediated liquid-phase microextraction
produced similar LOD for propranolol with our
proposed method [24]. The main advantages of
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the proposed method are simple pretreatment
procedure and more analytes detected by CE
method. The reported UHPLC/UV with SPE
was reported for determinations of some analytes
in urine including the beta blockers investigated
in this work. However, the reported LODs and
LOQs were higher or comparable with our findings. The latest work from our group reported the
ana­lysis of HPLC/UV method coupled with dispersive liquid–liquid microextraction for two cardiovascular drugs in plasma and urine samples.
The reported LOD for carvedilol was better than
CE method. As an overall aspect, higher sensitivity and lower LOD and LOQ values are expected
from GC and/or LC methods in comparison
with CE methods. However, as reported above,
in some cases, our obtained results for CE are better than chromatographic findings. Considering
Table 6. Investigation of the interference effect of other drugs with
the studied drugs in urine samples using the proposed method.
Drug
Retention time (min)
Overlap
Acetaminophen
Amiloride
Amiodarone
Atenolol
Carvedilol
Diazepam
Diltiazem
Furosemide
Hydrochlorothiazide
Losartan
Metoprolol
Nifedipine
Propranolol
Salicylic acid
Sotalol
Verapamil
No peak
No peak
No peak
14.13
15.49
17.09
15.65
No peak
No peak
No peak
14.83
No peak
13.8
17.04
14.03
16.69
ND
ND
ND
No
Diltiazem
No
Carvedilol
ND
ND
ND
No
ND
No
No
No
No
ND: No data.
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Fazeli-Bakhtiyari, Sorouraddin, Farajzadeh, Somi & Jouyban
Table 7. Stability data for three beta blockers in urine samples obtained by proposed salting-out assisted
LLE–field-amplified sample stacking-CE-diode array detector method.
Drug
Propranolol
Metoprolol
Carvedilol
Nominal
concentration
(ng/ml) (n = 3)
Room temperature stability
Freeze–thaw stability
Found
concentration
(ng/ml) ± SD
Accuracy
(RE%)
Recovery (%)
± SD
Found
concentration
(ng/ml) ± SD
Accuracy
(RE%)
Recovery (%)
± SD
50
250
500
50
250
48 ± 1
257 ± 3
529 ± 4
59 ± 4
255 ± 10
-4
3
6
18
2
96 ± 3
103 ± 1
106 ± 1
118 ± 8
102 ± 4
51 ± 2
256 ± 2
530 ± 4
57 ± 3
248 ± 2
2
2
6
14
-1
102 ± 3
102 ± 1
106 ± 1
114 ± 6
99 ± 1
500
50
250
500
510 ± 4
55 ± 3
262 ± 3
508 ± 5
2
10
5
2
102 ± 1
110 ± 6
105 ± 1
102 ± 1
512 ± 2
55 ± 2
260 ± 5
503 ± 6
2.4
10
4
1
102 ± 1
110 ± 4
104 ± 2
101 ± 1
CE-based methods (Table 9), three FASS-CE
methods [17,64,65] were reported for determination of one beta blocker in urine samples with
higher and/or comparable LOD values. Methods
reported in references [64,65] used more sensitive
detection with simple dilution for metoprolol,
and propranolol [17] was analyzed after head
space-solid-phase microextraction extraction
method, which is comparable with our method,
but it is applied for one drug. CE-based methods
without FASS used more sensitive detectors and
produced higher and/or comparable LODs [53,66].
It should be noted that these LODs were achieved
with sensitive detectors that are not actually commercially available and are difficult to fabricate
in-house. Even MS and LIF coupled to CE are
not as ‘routine’ as the CE-DAD is, which makes
the developed method quite attractive, considering that the SALLE is quite simple yet effective
and FASS requires no ‘add-ons’ to the CE instrument. CE-UV methods with SPE and/or LLE
also produced higher LOD values and analyzed a
single beta blocker [67–69]. Two CE–MS methods
listed in Table 9 produced higher and/or lower
LOD values. The capillary electrochromatography-ESI-MS [70] produced better LOD using a
capillary electrochromatography column, which
are fragile and expensive, and the data was validated in standard solution. The CE–TOF-MS
[71] that employed a microextraction pretreatment
procedure interestingly produced higher LOD
value when compared with our LOD.
„„Application
to real samples
The described method has been applied to the
urine samples of patients under cardiovascular treatment with propranolol, metoprolol or
carvedilol. Those patients also receiving other
Table 8. Evaluation of method robustness for extraction and analysis of three beta
blockers in spiked urine samples with salting-out assisted LLE–field-amplified
sample stacking-CE-diode array detector.
Drug
Level
Nominal
concentration
(ng/ml) (n = 3)
Found concentration Accuracy
(ng/ml) ± SD (n = 3) (RE%)
Recovery (%)
± SD
Propranolol
1
2
3
1
2
3
1
2
3
500
500
500
500
500
500
500
500
500
532 ± 4
532 ± 4
528 ± 5
515 ± 7
511 ± 3
511 ± 4
496 ± 20
502 ± 10
496 ± 4
106 ± 1
106 ± 1
106 ± 1
103 ± 1
102 ± 1
102 ± 1
99 ± 4
100 ± 2
99 ± 1
Metoprolol
Carvedilol
6
6
6
3
2.2
2
-1
0
-1
Level 1: pH = 2.00, separation temperature and voltage: 24°C and 24.5 kV, buffer concentration: 28 mM.
Level 2: pH = 2.20, separation temperature and voltage: 25°C and 25 kV, buffer concentration: 30 mM.
Level 3: pH = 2.40, separation temperature and voltage: 26°C and 25.5 kV, buffer concentration: 32 mM.
328
Bioanalysis (2014) 6(3)
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Improved determination of beta blocker drugs in human urine
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Table 9. Comparison of the proposed salting-out assisted LLE–field-amplified sample stacking-CE-diode array
detector method with other CE methods used in quantification and determination of the studied drugs
in urine.
Drug
CE type
Sample preparation
Sample stacking
method
Validation
LOD
(µmol/l)
Metoprolol
Metoprolol
Propranolol
Metoprolol
CE-ECL
CE-ECL
CE-AD
CE-ECL
CE-EC
CE-DAD
CZE-DAD
Dilution
Dilution
Filtration /dilution
Dilution
FASS
FASS
ND
ND
No method validation
No method validation
No method validation
No method validation
[64]
SPE
HS-SPME-ultrasonic back
extraction
SPE
PPT with methanol
Filtration
Enzymatic hydrolysis
LLE
DLLME
SALLE ND
FASS
No stability, selectivity test
No method validation
0.1†
0.4†
0.05
0.03
0.02
0.1
0.03
ND
ND
Yes
No method validation
[68]
ND
No method validation
0.1
0.0006†
0.001†
0.74
ND
FASS
No method validation
Yes
Propranolol
Propranolol
Carvedilol
Metoprolol
Propranolol
Carvedilol
CZE-UV
pCEC–ESI-MS
Metoprolol
Carvedilol
Metoprolol
Propranolol
CE–TOF-MS
CZE-DAD
CZE-DAD
1.87
0.04
0.02
0.03
Ref.
[65]
[66]
[53]
[67]
[17]
[69]
[70]
[71]
This
work
The validation of these data has been carried out with standard solutions and the urine samples were used to demonstrate the potential application of the method
on urine.
AD: Amperometric detection; CE-EC: CE-electrochemical; CZE: Capillary zone electrophoresis; DAD: Diode array detector; ECL: Electrochemiluminescence detection;
FASS: Field-amplified sample stacking; HS-SPME: Head space-solid phase microextraction; ND: No data; pCEC: Pressure-assisted capillary electrochromatography;
PPT: Protein precipitation; SALLE: Salting-out assisted LLE extraction.
†
drugs (e.g., furosemide, spironolactone, lactulose, pantoprazole, albumin, metronidazole, warfarin, heparin, ceftriaxone, metconazole, losartan
and so on) presented in Table 10. Also electrophoregrams of two patients (numbers 1 and
3) are shown in Figure 3A–D. The results were
confirmed with a simple experiment such as the
standard addition method. For this purpose, two
different real samples were spiked with 100, 250
and 500 ng/ml of selected drugs. Urine samples
were collected from healthy people: before drug
administration and in 3 h (sample 1) and 6 h
(sample 2) periods after an oral dose of 40 mg
of propranolol. The concentration of propranolol was calculated as 67 ± 1 and 150 ±(<0.5) ng/
ml. According to the obtained results, good relative recoveries in the range of 92–114% were
obtained, which indicates that there were low
matrix effects in the analyzed samples (Table 11).
Conclusion
SALLE-FASS-CE method is suitable for screening and quantifying the prescribed beta blockers
in human urine. SALLE was used to reduce the
Table 10. Determination of the target drugs in patients urine samples by the proposed method (results given
as mean results).
N
Gender
Administered drugs
Intake
time
Sampling
time
Concentration
(µg/l)
1
F
9 am
11 pm
22
2
F
9 pm
6 am
66
3
M
9 am
12 am
111
4
M
9 am
11 am
272
5
F
Metoprolol 25 mg
Furosemide, spironolactone, lactulose, pantoprazole, albumin,
metronidazole
Metoprolol 50 mg
Warfarin
Propranolol 20 mg
Furosemide, spironolactone, lactulose, heparin, albumin, pantoprazole
Propranolol 20 mg
Furosemide, ceftriaxone, metconazole, spironolactone, lactulose
Carvedilol 6.25 mg
Losartan
9 am
10 am
LLOQ>
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Fazeli-Bakhtiyari, Sorouraddin, Farajzadeh, Somi & Jouyban
Absorbance (mAU)
R esearch A rticle |
2.5
1.5
A
0.5
-0.5
Absorbance (mAU)
Metoprolol
8
B
9
10
11
12
13
14
15
16
15
16
Migration time (min)
2.5
1.5
Propranolol
0.5
-0.5
C
8 D
9
10
11
12
13
14
Migration time (min)
Figure 3. Electropherograms of propranolol and metoprolol in two real samples analyzed
by salting-out assisted LLE-field-amplified sample stacking-CE-diode array detector.
(A) Blank human urine spiked with 50 ng/ml metoprolol; (B) patient number 1; (C) patient
number 3; (D) blank human urine spiked with 100 ng/ml propranolol obtained under optimal
salting-out assisted LLE–field-amplified sample stacking-CE-diode array detector. Separation
conditions are as in Figure 1.
matrix effect in FASS method. Sample matrix
was the most important parameter in sample
stacking and highest sensitivity was observed
when pure water was used as sample matrix. In
addition, injecting a short plug of water before
sample introduction resulted in greater peak
areas. Therefore, sensitivity enhanced by 200-fold
for propranolol and metoprolol, and 100-fold for
carvedilol. Validation results were satisfactory in
terms of accuracy, precision, selectivity, specificity,
Table 11. Relative recoveries obtained by salting-out assisted LLE-field-amplified sample stacking-CE-diode
array detector method in real samples (sample 1) spiked at 100, 250, and 500 ng/ml.
Real sample
Drug
Concentration added
(ng/ml)
Concentration found ± SD
(ng/ml)
Relative recovery
(%) ± SD (n = 3)
Sample 1
Propranolol
Sample 1
Metoprolol
Sample 1
Carvedilol
Sample 2
Propranolol
NA
100
250
500
NA
100
250
500
NA
100
250
500
NA
100
250
500
67 ± 1
162 ± 2
297 ± 4
529 ± 1
N.D.
95 ± 4
252 ± 8
512 ± 9
N.D.
105 ± 2
240 ± 14
515 ± 12
150 ± (<0.5)
264 ± 1
389 ± (<0.5)
641 ± 2
ND
95 ± 2
92 ± 1
92 ± (<0.5)
ND
95 ± 4
101 ± 3
102 ± 2
ND
105 ± 2
96 ± 6
103 ± 2
ND
114 ± 1
95 ± (<0.5)
98 ± (<0.5)
Analytes contents of samples were subtracted.
NA: Not added; ND: Not detected.
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Bioanalysis (2014) 6(3)
future science group
Improved determination of beta blocker drugs in human urine
stability and robustness. The presented method is
a promising method for routine laboratory ana­
lysis when compared with the other methods.
Finally, this method was successfully applied to
determine these drugs in patient samples.
Future perspective
The validated method is applicable in bioanalytical laboratories for routine low level drug ana­
lysis without the need for a laborious and timeconsuming sample preparation method. In addition, this method could be used for drug abuse
detection in competition antidoping testing.
Acknowledgements
The authors would like to thank the Liver and Gastrointestinal
Diseases Research Center, Tabriz University of Medical
Sciences (Tabriz, Iran) for providing analytical facilities.
| R esearch A rticle
Financial & competing interests disclosure
The authors have no relevant affiliations or financial
involvement with any organization or entity with a
financial interest in or financial conflict with the subject
matter or materials discussed in the manuscript. This
includes employment, consultancies, honoraria, stock
ownership or options, expert t­estimony, grants or patents
received or pending, or royalties.
No writing assistance was utilized in the production of
this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate
institutional review board approval or have followed the
principles outlined in the Declaration of Helsinki for all
human or animal experimental investigations. In addition,
for investigations involving human subjects, informed
consent has been obtained from the participants involved.
Executive summary
Aim
„„
In this study, salting-out assisted LLE combined with field-amplified sample stacking in CE method has been developed for the sensitivity
enhancement of three beta blocker drugs in human urine.
Procedure
„„
Spike urine sample with selected drugs.
„„
Keep at room temperature for 20 min and adjust pH to 11.3.
„„
Add 2 ml acetonitrile to 4 ml spiked urine sample and vortex-mix for 1 min.
„„
Add 1 g NaCl and vortex-mix for 1 min and then centrifugation for 5 min at 4000 rpm.
„„
Remove 0.2 ml of the supernatant and solvent evaporation under a gentle N2 stream.
„„
Reconstitution the residue with 0.1 ml deionized water and transfer to CE instrument.
„„
Inject sample for 25 s at 10 kV after preliminary pressure injection of water (50 mbar for 1 s).
„„
Run electrophoresis for 16 min.
Results & discussion
„„
Poor sensitivity of CE improved using online concentration method named field-amplified sample stacking. In addition, our approach
met US FDA’s requirements and was successfully applied to the ana­lysis of patient’s urine samples.
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