Microchemical Journal 147 (2019) 635–642

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Microchemical Journal
journal homepage: www.elsevier.com/locate/microc

Green micellar HPLC-fluorescence method for simultaneous determination T

of metoprolol and amlodipine in their combined dosage form: Application
on metoprolol in spiked human plasma
Mokhtar M. Mabrouk, Sherin F. Hammad, Samah F. El-Malla, Eman A. Elshenawy
⁎

Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Tanta University, Tanta, 31527, Egypt

ARTICLE INFO ABSTRACT

Keywords: A fast, sensitive and reliable green micellar liquid chromatographic method has been developed and validated
Metoprolol for simultaneous determination of metoprolol (MET) and amlodipine (AMD). Separation was performed on X-
Amlodipine Bridge ODS column (150 × 4.6 mm, 5 μm particle size) using a micellar mobile phase consisting of 100 mM
Green micellar liquid chromatography sodium dodecyl sulfate, 20 mM sodium dihydrogen phosphate buffer pH 3.0 and 10% v/v n-butanol. The
Fluorescence
fluorescence detector was set at 275/ 303 nm (excitation/ emission) for MET and 364/ 455 nm (excitation/
Human plasma
Green analytical procedure index (GAPI)
emission) for AMD. A linear response was obtained over the ranges of 0.1–10 μg/mL and 0.2–2 μg/mL for MET
and AMD, respectively. The method reached limits of detection of 20 ng/mL for MET and 50 ng/mL for AMD.
The proposed method was successfully employed for the determination of the two drugs in their combined
pharmaceutical dosage form and in addition, it was extended for the determination of MET in spiked human
plasma. Furthermore, the green character of the developed method was evaluated using the new tool “green
analytical procedure index (GAPI)”. The developed method was verified to be an excellent green method.

1. Introduction alternative to conventional reversed phase liquid chromatography.

Cardiovascular diseases including angina, heart attacks, stroke,
hypertension and heart failure remain the leading cause of death
globally [1,2]. The current treatment approach is combining cardio-
vascular drugs from different classes in small doses in order to achieve a
synergistic effect and reduce the side effects [3–5]. Metoprolol (MET)
(Fig. 1a), ( ± )-1-[4-(2-methoxyethyl)phenoxy]-3-[(1-methylethyl)
amino]propan-2-ol, is a cardioselective β1-adrenergic blocker that is
used for the treatment of hypertension, arrhythmias, heart failure and
coronary artery diseases [6,7]. Amlodipine (AMD) (Fig. 1b), 3-Ethyl 5-
methyl 2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6-
methylpyridine-3,5-dicarboxylate, belongs to the 1,4-dihydropyridine
subclass of calcium channel blockers. It is clinically used in the treat-
ment of angina and hypertension [6,8,9]. A combination of MET and
AMD has shown a beneficial effect in the treatment of hypertension and
angina [10–12].
Micellar liquid chromatography (MLC) is a technique adopting the
requirements of “green chemistry” concept and provides an efficient
Mostly the mobile phase in MLC consists of an aqueous solution of a
surfactant above its critical micellar concentration and a small amount
of organic solvent to improve the efficiency and the elution strength.
The approach of MLC offers many advantages including the separation
of drugs with different nature without the need for gradient elution, the
direct injection of biological samples without laborious sample pre-
paration or protein precipitation and the reduction of organic solvents
consumption [13–15].
A literature survey revealed several analytical techniques for the
simultaneous analysis of MET and AMD in their combined pharma-
ceutical dosage form such as spectrophotometry [16–19], spectro-
fluorimetry [20,21] and chromatography [10,22–29]. These reported
methods have high detection limit values. The reported HPLC and UPLC
methods [10,22–27] are based on the addition of a high percentage of
the hazardous organic modifier acetonitrile (sometimes exceeding 50%
of the mobile phase), in addition the UPLC method [22] uses the gra-
dient elution mode. There is also a report for the determination of MET
and AMD in biological fluids using HPLC-MS [10] that uses methanol in

Abbreviations: MET, metoprolol; AMD, amlodipine; MLC, micellar liquid chromatography; GAPI, green analytical procedure index; SDS, sodium dodecyl sulphate;
NTP, number of theoretical plates; Tf, tailing factor; Rs, resolution; LOD, limit of detection; LOQ, limit of quantitation; S.D., standard deviation; %R.S.D., % relative
standard deviation
⁎
Corresponding author.
E-mail address: eman_a.elshenawy@yahoo.com (E.A. Elshenawy).

https://doi.org/10.1016/j.microc.2019.03.084
Received 16 December 2018; Received in revised form 26 March 2019; Accepted 27 March 2019
Available online 28 March 2019
0026-265X/ © 2019 Elsevier B.V. All rights reserved.
M.M. Mabrouk, et al.
Fig. 1. The chemical structure of (a) metoprolol and (b) amlodipine.
a ratio of 80% of the mobile phase. According to the United States
Environmental Protection Agency, acetonitrile and methanol are clas-
sified as hazardous solvents [30] that require specialized disposal
treatment steps [31]. Based on this review, there is an obvious need to
develop a sensitive, green analytical method for the simultaneous de-
termination of MET and AMD.
This work describes the first green MLC-fluorescence detection
method for the simultaneous determination of MET and AMD in their
co-formulated dosage form. In addition, this method was successfully
applied for the determination of MET in spiked human plasma without
the need for laborious sample preparation. The proposed method shows
priority over the other reported methods since it reduces organic sol-
vents consumption and reaches lower limit of detection compared to
the previously reported methods (see the data in supplementary in-
formation Table S1). The method was validated according to the ICH
guidelines [32]. The greenness of the proposed method was assessed
using the new tool “green analytical procedure index (GAPI)” [33]. The
GAPI tool evaluates the green character of an entire analytical method
and quantify the environmental impact involved in each step. The Eco-
scale value of the method was calculated by assigning penalty points for
each of the analytical procedure parameters (reagents, hazards, energy
and waste) that deviates from ideal green analysis [33–36].
2. Material and methods
2.1. Reagents
MET (99.2%) and AMD (99.4%) were kindly provided by Sigma
Pharmaceutical Industries (Quesna, El Monofeya, Egypt). Chemicals
used, including sodium dodecyl sulphate (SDS), sodium dihydrogen
phosphate and phosphoric acid, were of analytical reagent grade.
Solvents including methanol, ethanol, isopropanol and n-butanol
(HPLC grade) were purchased from Fischer scientific, Germany.
Excipients used in the preparation of tablets include avicel, cellulose,
lactose, talc powder, starch, and magnesium stearate (ADWIC Co,
Egypt). Human blank plasma was obtained from Mansoura University
Hospitals (Mansoura, Egypt).
2.2. Apparatus
Separation was performed on Agilent™ 1260 Infinity HPLC system
equipped with Agilent 1260 Infinity quaternary pump (G1311C),
Agilent 1260 Infinity thermostated column compartment (G13b16A),
Agilent 1260 Infinity autosampler with reliable injections from 0.1 to
100 μL (G1329B) and Agilent 1260 Infinity fluorescence detector
(G1321C). Data was recorded and analyzed with Agilent OpenLAB CDS
Chemstation Edition software. HANNA pH 211 Microprocessor pH-
meter with double junction glass electrode was used to adjust the pH.
2.3. Chromatographic conditions
Chromatographic separations were achieved on X-Bridge ™ C18
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Microchemical Journal 147 (2019) 635–642
reversed phase column (150 × 4.6 mm, 5 μm). Chromatographic se-
parations were performed using a micellar mobile phase consisting of
100 mM SDS, 20 mM sodium dihydrogen phosphate buffer pH 3.0 and
10% v/v n-butanol. The mobile phase was prepared by dissolving the
appropriate amount of SDS and sodium dihydrogen phosphate in water
and the pH was adjusted to pH 3.0 using phosphoric acid, then mixed
with n-butanol in a ratio of 90:10. The mobile phase was filtered
through 0.22 μm nylon membrane filter (Millipore, Ireland) prior to
use. The flow rate was 1.5 mL/min at 40 °C and the injection volume
was 50 μL. The fluorescence detector was set at 275 nm (excitation) and
303 nm (emission) for the first 5 min for MET detection then 364 nm
(excitation) and 455 nm (emission) till the end of the run for AMD
detection. The column was equilibrated for 15 min prior to assay.
2.4. Analytical procedures
Stock solutions (0.5 mg/mL) of MET and AMD were prepared se-
parately by dissolving 25.0 mg of the studied drugs in n-butanol in a
50 mL volumetric flask. A standard solution of 100 μg/mL of each drug
was prepared by diluting appropriate volume from the corresponding
stock solution with distilled water. AMD is a photosensitive drug, so
AMD solutions were kept away from light. The stability of the stock
solutions is ten days when stored in the refrigerator. Different volumes
of MET and AMD standard solutions were quantitatively measured and
transferred into a series of 10 mL volumetric flasks and diluted to the
specified volume with distilled water to obtain a final working con-
centration range of (0.1–10 μg/mL) for MET and (0.2–2 μg/mL) for
AMD. Aliquots of 50 μL were injected triplicate under the previously
prescribed chromatographic conditions (see Section 2.3) and the mean
peak area was plotted against the corresponding concentration of each
drug (μg/mL) to obtain the calibration curves and the corresponding
regression equations were estimated. For analysis of the cited drugs in
binary mixtures, accurately measured volumes of MET and AMD stan-
dard solutions (100 μg/mL) were transferred into a series of 10 mL
volumetric flasks, completed to the volume with distilled water and
mixed well to prepare a set of binary mixtures in the ratio of 10:1
(5:0.5, 8:0.8, 10:1, MET: AMD, respectively). Aliquots of 50 μL were
injected triplicate under the previously prescribed chromatographic
conditions (see Section 2.3) and the regression equations were im-
plemented to calculate the percentage recoveries for both drugs.
2.5. Comparison method
Simultaneous analysis of MET and AMD was performed on a 25 cm
C18 column using a mobile phase consisting of 10 mM phosphate buffer
(adjusted to pH 3.0 using triethylamine) and acetonitrile (50:50) and
flow rate of 1 mL/min at 235 nm [24].
2.6. Analysis of the laboratory prepared tablet
The dosage form is not available in the local market, so a laboratory
prepared mixture simulating this dosage form was prepared by
M.M. Mabrouk, et al.
geometric mixing of 500 mg MET, 50 mg AMD, and the following in-
active ingredients: 890 mg avicel, 220 mg cellulose, 220 mg lactose,
60 mg talc powder, 20 mg starch and 40 mg magnesium stearate for the
preparation of ten tablets. An accurately weighed amount of the pre-
pared tablets containing 50 mg of MET and 5 mg of AMD was trans-
ferred into a 50 mL volumetric flask and 25 mL n-butanol was added,
sonicated for 15 min and completed to the mark using n-butanol. This
solution was filtered, the first portion of the filtrate was discarded then
4 mL of the filtrate was transferred to a 100 mL volumetric flask and
completed with distilled water to the volume. Finally, 2 mL of this so-
lution was transferred into a 10 mL volumetric flask and diluted to the
specified volume with distilled water to obtain a solution containing
8.0 μg/mL MET and 0.8 μg/mL AMD. The same procedure was repeated
on another five portions of the laboratory prepared tablets. Aliquots of
50 μL were injected under the previously prescribed chromatographic
conditions (see Section 2.3). The percentage concentration found of
MET and AMD in the tablets was derived using the corresponding re-
gression equation.
2.7. Calibration curve for MET in human plasma
Aliquots of 1 mL blank human plasma were transferred into a series
of 10 mL volumetric flasks, spiked with different concentrations of
MET, vortex mixed and completed to volume with the mobile phase to
obtain a concentration range of (100–280 ng/mL). The solutions were
mixed well and filtered through 0.45 μm cellulose acetate syringe filter
then aliquots of 50 μL were injected triplicate under the previously
prescribed chromatographic conditions (see Section 2.3). The mean
peak area was plotted against the corresponding concentration (ng/mL)
to obtain the calibration curve and the regression equation was derived.
A parallel blank experiment was performed.
2.8. Analysis of MET in human plasma
Aliquots of 1 mL human plasma were transferred into a series of
10 mL volumetric flasks, spiked with three concentration levels of MET,
vortex mixed and completed to volume with the mobile phase and
mixed well. The solutions were filtered through 0.45 μm cellulose
acetate syringe filter then aliquots of 50 μL were injected triplicate
under the previously prescribed chromatographic conditions (see
Section 2.3) and the percentage recoveries were calculated using the
corresponding regression equation. This procedure was repeated on
three consecutive days to assess the precision.
3. Results and discussion
3.1. Method development
Different mobile phases were tried in order to achieve the best se-
paration between MET and AMD in a short run time. Mobile phases
consisting of water with different organic modifiers (e.g. methanol,
ethanol or acetonitrile) in different ratios were tried. Since AMD is a
lipophilic drug (Log P = 3.0) [37], a high organic ratio was needed to
elute AMD in a reasonable retention time, however it resulted in an
unretained peak of MET (Capacity factor (k') < 2) as it is a hydrophilic
drug (Log P = 1.9) [37]. Replacing water with different buffers such as
phosphate, acetate or triethylamine buffers at different pH values
(pH 3.0–7.0) was tried. It resulted in a decreased retention time of
AMD, but the unretained peak of MET remained a problem. This may be
explained by that both MET and AMD are basic drugs having pka values
of 9.7 and 8.6, respectively, so they are ionized at the acidic pH range
[37,38]. In order to overcome this problem, a green micellar mobile
phase consisting of 100 mM SDS, 20 mM sodium dihydrogen phosphate
buffer pH 3.0 and 10% v/v n-butanol was used. It achieved a good re-
solution between the two drugs in a reasonable retention time where
MET was eluted first at 3.46 min and AMD was eluted at 6.35 min. This
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Microchemical Journal 147 (2019) 635–642
can be attributed to that partitioning equilibria in MLC are governed by
hydrophobic forces and electrostatic interaction with the surfactant-
modified stationary phase [39]. At mobile phase pH, both MET, and
AMD will be positively charged since they are basic compounds and
will be retained by the electrostatic attraction to the modified sta-
tionary phase. However, the hydrophobic interactions of AMD with the
modified stationary phase are stronger than that of MET (as AMD is
more lipophilic), so MET is eluted first then AMD.
3.2. Method optimization
The major limitation of MLC is the low chromatographic efficiency,
which is caused by poor mass transfer between micelles and a surfac-
tant-modified stationary phase [15,39]. Efficiency in MLC can be en-
hanced by the addition of organic modifiers to the micellar mobile
phase, increasing the column temperature, and decreasing the con-
centration of surfactant [15,39]. Thus, different experimental para-
meters affecting the chromatographic separation, efficiency and reten-
tion time were carefully studied and optimized. These parameters
include: concentration of surfactant, pH, temperature and type and
concentration of organic modifier.
3.2.1. The concentration of surfactant
It is reported that shorter retention times are obtained by increasing
the surfactant concentration, but the chromatographic efficiency dete-
riorates significantly compared to conventional RPLC [39]. Accord-
ingly, different concentrations of SDS ranging from 50 to 150 mM were
studied in terms of number of theoretical plates (NTP) and capacity
factor (K′) (Fig. S1). Decreasing SDS concentration resulted in in-
creasing NTP for both drugs, but below 100 mM SDS the K′ of AMD was
unacceptable (> 10) and a long retention time was obtained
(> 15 min). Thus, SDS concentration of 100 mM was chosen as the
optimum.
3.2.2. pH
For separating ionizable solutes, variations in the mobile phase pH
may easily lead to dramatic variations in selectivity, retention time and
efficiency [40,41]. The ionization degree for MET and AMD is the same,
so selectivity will not change by varying the pH [39]. Thus, the effect of
pH was studied in terms of efficiency over the range 2.5–7.0 (Fig. S2).
The NTP increased for both MET and AMD with lowering the pH,
reaching its highest value at pH 3.0, then decreased at pH 2.5. So, the
optimum pH was chosen to be 3.0.
3.2.3. Temperature
The column temperature was changed from 25 °C to 45 °C.
Increasing the temperature led to an increase in the NTP for both drugs.
At 40 °C, NTP of AMD was acceptable (> 2000). Increasing temperature
to 45 °C increased NTP but also increased the tailing factor (Tf) for both
AMD and MET. Thus, the optimal temperature was chosen to be 40 °C
(Fig. S3).
3.2.4. Effect of organic modifier
Micellar mobile phases containing a weak organic modifier such as
methanol, ethanol or isopropanol led to long retention times of AMD
due to its high lipophilicity (log P = 3) [39,42–44]. The retention time
decreased by using n-butanol which has greater elution power. The
concentration of n-butanol was investigated from 2 to 10% (Fig. S4).
The retention time decreased with increasing the concentration. A
concentration of 10% was chosen as it gave the shortest retention time
and the highest sensitivity (in terms of peak height).
3.3. System suitability test parameters
After optimization of all experimental parameters affecting the
chromatographic separation, the optimum chromatographic conditions
M.M. Mabrouk, et al. Microchemical Journal 147 (2019) 635–642

Table 1 and σ is the standard deviation of the intercept of regression line of the
Results for system suitability tests for the developed HPLC method. calibration curve. The results of LOQ and LOD for MET and AMD are
Parameters MET AMD Reference value [46] shown in Table 2.

Retention time (min) 3.459 6.353 3.4.4. Accuracy

Capacity factor (K′) 2.469 5.371 >2
Accuracy was tested by analysis of three concentration levels, cov-
Resolution (Rs) 6.997 >2
Number of theoretical plates (NTP) 2216 2278 > 2000 ering the specified range in a ratio of 10:1 (5:0.5, 8:0.8, 10:1, MET:
Tailing factor (Tf) 1.297 1.253 ≤2 AMD, respectively), three replicates of each. The mean peak area was
calculated and the concentrations of the two drugs in the binary mix-
tures were derived using the regression equation. The values of the
were assessed for system suitability parameters including capacity percentage recovery indicate high accuracy of the method as shown in
factor (k'), resolution (Rs), number of theoretical plates (NTP) and Table 2.
tailing factor (Tf). The results were satisfactory compared to the values
required for an acceptable method as shown in Table 1. 3.4.5. Precision
Precision was evaluated regarding repeatability and intermediate
3.4. Validation of the method precision. Three different binary mixtures of MET and AMD were
analyzed in the same day “three replicates of each” to assess repeat-
The method was validated following ICH guidelines [32] regarding ability and on three consecutive days to assess intermediate precision.
selectivity and specificity, linearity and range, limit of detection (LOD), The % relative standard deviation (%R.S.D.) values prove the precision
limit of quantitation (LOQ), accuracy, precision and robustness. of the method, see Table 2.

3.4.1. Selectivity and specificity 3.4.6. Robustness

The selectivity of the method was proved by a good resolution be-
tween the two drugs which indicates the ability of the method to de-
termine each drug in the presence of the other without any interference
(Fig. 2). Injection of a placebo solution containing all the tablet ex-
cipients and a blank human plasma sample under the optimum chro-
matographic conditions showed no interfering peaks at the retention
times of MET and AMD (Figs. 3 and 4). These matrix components did
not interfere with the analysis of both drugs by the developed method
indicating its specificity.
3.4.2. Linearity and range
A linear relationship was obtained by plotting mean peak area
against drug concentration for MET and AMD over the range cited in
Table 2. The validity of the method was proved by evaluating the re-
gression lines statistically. The high value of the coefficient of de-
termination indicates the good linearity of the method over the speci-
fied concentration range.
3.4.3. Limit of quantitation (LOQ) and limit of detection (LOD)
The LOQ and LOD were determined based on the calibration curve
where a specific calibration curve was studied using samples containing
the analyte in the range of LOQ and LOD, respectively using the fol-
lowing equations: LOQ = 10 σ/S; LOD = 3.3 σ/S; where: S is the slope
The evaluation of robustness was considered with respect to delib-
erate variations in the chromatographic conditions such as SDS con-
centration, pH, n-butanol concentration, temperature and flow rate.
The chromatographic responses considered were the resolution and the
percentage recoveries of MET and AMD. Table S2 shows the variations
performed and the results indicating the robustness of the method.
3.5. Applications
3.5.1. Application for the determination of MET and AMD in their
combined dosage form
The proposed method was implemented for the analysis of the
studied drugs in their co-formulated tablet (Fig. 3). The percentage
recovery and %R.S.D. values indicate the high accuracy and precision
of the method. The results of the proposed method were compared with
those of the comparison method [24] (Table 3). There was no sig-
nificant difference between the proposed and comparison methods as
indicated by statistical analysis of the results using student's t-test and
the variance ratio F-test.
3.5.2. Application for the determination of MET in spiked human plasma
The developed MLC method was applied successfully for the de-
termination of MET in spiked human plasma without the need for the

Fig. 2. HPLC Chromatogram of 0.5 μg/mL of metoprolol (MET) and 2 μg/mL of amlodipine (AMD) in a mixture using a mobile phase composed of 100 mM SDS,
20 mM sodium dihydrogen phosphate buffer pH 3.0 and 10% v/v n-butanol.

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M.M. Mabrouk, et al. Microchemical Journal 147 (2019) 635–642

Fig. 3. HPLC Chromatograms of (a) laboratory prepared mixture of 8 μg/mL of metoprolol (MET) and 0.8 μg/mL of amlodipine (AMD) with tablet excipients (with
adjusted scale illustration for AMD peak); (b) placebo, using a mobile phase composed of 100 mM SDS, 20 mM sodium dihydrogen phosphate buffer pH 3.0 and 10%
v/v n-butanol.

Fig. 4. HPLC Chromatograms of (a) 0.28 μg/mL of metoprolol (MET) in spiked human plasma; (b) blank plasma, using a mobile phase composed of 100 mM SDS,
20 mM sodium dihydrogen phosphate buffer pH 3.0 and 10% v/v n-butanol.

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M.M. Mabrouk, et al. Microchemical Journal 147 (2019) 635–642

Table 2
Validation parameters for the determination of MET and AMD by the proposed micellar HPLC method.
Parameter MET AMD

Concentration range (μg/mL) 0.1–10 0.2–2

Coefficient of determination (R2) 0.9999 0.9997
Slope 1353.570 208.066
Intercept 41.795 1.248
LOD (μg/mL) 0.020 0.048
LOQ (μg/mL) 0.061 0.146
Sa 11.164 0.756
Sb 26.081 0.708
Sy/x 30.648 2.539
Accuracy (mean % 100.156 ± 0.330 100.026 ± 0.863
recoverya ± %RSD)

Precision Conc. taken (μg/mL) Mean conc. foundb (μg/mL) ± %RSD Conc. taken (μg/mL) Mean conc. found b
(μg/mL) ± %RSD

Intra-day precision 5 4.996 ± 0.430 0.5 0.506 ± 0.668

8 7.930 ± 0.358 0.8 0.796 ± 0.488
10 9.932 ± 0.599 1 1.000 ± 0.777
Inter-day precision 5 5.032 ± 0.788 0.5 0.504 ± 0.536
8 8.011 ± 1.056 0.8 0.794 ± 0.451
10 10.047 ± 1.526 1 1.005 ± 0.696
a
Average of nine readings (3 concentrations × 3 times).
b
Average of three replicate determinations for each concentration.

laborious pretreatment steps for elimination of interfering substances. Table 4

The plasma calibration curve was linear over the range (100–280 ng/ Recovery and precision data for determination of MET in spiked human plasma.
mL) with the following regression equation, y = 1.478 x + 32.989 Conc. added Mean conc. founda ± S.D. % Recovery % R.S.D.
(R2 = 0.9993); where y is the mean peak area, x is the concentration of (ng/mL) (ng/mL)
the drug in ng/mL and R2 is the coefficient of determination. The results
in Table 4 indicate good accuracy and precision of the developed Intra-day 150 147.916 ± 0.002 98.610 1.240
200 199.625 ± 0.001 99.812 0.572
method. 250 247.211 ± 0.001 98.884 0.541
Inter-day 150 147.398 ± 0.001 98.265 0.335
3.6. Assessment of greenness of the method 200 199.823 ± 0.002 99.912 0.765
250 248.198 ± 0.001 99.279 0.398
The criteria of ideal green analysis include elimination or minimal a
Average of three replicate determinations for each concentration.
use of reagents, reduced energy consumption and no waste generation.
In many analytical processes, elimination of the use of reagents cannot
to have an analytical Eco-Scale value of 80 indicating an excellent green
be applicable, thus some practices can be adopted in order to make such
HPLC method.
procedures greener such as replacement of toxic reagents by their safer
and easily degradable equivalents, eliminating or reducing the hazard
of wastes and reducing the number of stages in a given analytical 4. Conclusion
procedure [33,34,45]. The green character of the developed method
was assessed by calculating its analytical Eco-Scale using the recently The present study describes a simple, accurate and precise green
reported tool termed GAPI [33] (Table S3). The analytical Eco-Scale MLC method with fluorescence detection that was successfully em-
calculation is based on that the ideal green analysis has a value of 100 ployed for the simultaneous analysis of MET and AMD in their phar-
and penalty points are assigned for each of the analytical procedure maceutical dosage form. The method reached a LOQ of 60 ng/mL for
parameters (reagents, hazards, energy and waste) that deviates from MET and 150 ng/mL for AMD. The developed method, owing to its high
ideal green analysis [33,35,36]. The developed MLC method was found sensitivity, enabled the determination of MET in spiked human plasma

Table 3
Application of the proposed method and comparison method for the determination of the studied drugs in their laboratory prepared mixture with excipients.
Conc. taken (μg/mL) Proposed method Comparison method [26]

% Recovery % Recovery

MET AMD MET AMD MET AMD

8 0.8 99.893 100.118 99.020 99.540
99.511 98.979 99.800 98.390
99.673 101.439 99.350 101.180
99.695 100.298 99.810 100.320
99.708 101.199 99.400 101.410
99.515 99.877 99.700 99.210
Mean ± S.D. 99.666 ± 0.142 100.318 ± 0.902 99.513 ± 0.312 100.008 ± 1.176
%R.S.D. 0.143 0.899
t-testa 1.070 (2.228) 0.515 (2.228)
F-testa 4.815 (5.050) 1.703 (5.050)

a
Values in parenthesis are the tabulated t- and F- values at p = 0.05.

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M.M. Mabrouk, et al.
without complicated pretreatment steps reducing the cost and time of
analysis. This method reduced the consumption of organic solvents
meeting the requirements of “green chemistry”.
Conflict of interest
All authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or
animals performed by any of the authors.
Funding sources
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.microc.2019.03.084.
References
[1] O. González, N. Ferreirós, M.E. Blanco, R.M. Alonso, Cardiovascular drug de-
termination in bioanalysis: an update, Bioanalysis 7 (2015) 2399–2417, https://doi.
org/10.4155/bio.15.163.
[2] WHO, Health topics: Cardiovascular diseases. http://www.who.int/topics/
cardiovascular_diseases/en/, 2016 (accessed 20 November 2018).
[3] X. Wan, P. Ma, X. Zhang, A promising choice in hypertension treatment: fixed-dose
combinations, Asian J. Pharm. Sci. 9 (2014) 1–7, https://doi.org/10.1016/j.ajps.
2013.12.005.
[4] S.G. Mallat, H.S. Itani, B.Y. Tanios, Current perspectives on combination therapy in
the management of hypertension, Integr. Blood Press. Control 6 (2013) 69–78,
https://doi.org/10.2147/IBPC.S33985.
[5] B. Canbakan, Rational approaches to the treatment of hypertension: drug therapy-
monotherapy, combination, or fixed-dose combination? Kidney Int. Suppl. 3 (2013)
349–351, https://doi.org/10.1038/kisup.2013.75.
[6] A. Brayfield, Martindale: The Complete Drug Reference, 38th ed., Pharmaceutical
Press, London, 2014.
[7] J. Emami, M.R. Kazemali, Design and in vitro evaluation of a novel controlled onset
extended-release delivery system of metoprolol tartrate, Res. Pharm. Sci. 11 (2016)
81–92.
[8] H. Fares, J.J. DiNicolantonio, J.H. O'Keefe, C.J. Lavie, Amlodipine in hypertension:
a first-line agent with efficacy for improving blood pressure and patient outcomes,
Open Heart. 3 (2016) e000473, , https://doi.org/10.1136/openhrt-2016-000473.
[9] A.L. Wang, C. Iadecola, G. Wang, New generations of dihydropyridines for treat-
ment of hypertension, J. Geriatr. Cardiol. 14 (2017) 67–72, https://doi.org/10.
11909/j.issn.1671-5411.2017.01.006.
[10] A.K. Sarkar, D. Ghosh, A. Das, P.S. Selvan, K.V. Gowda, U. Mandal, A. Bose,
S. Agarwal, U. Bhaumik, T.K. Pal, Simultaneous determination of metoprolol suc-
cinate and amlodipine besylate in human plasma by liquid chromato-
graphy–tandem mass spectrometry method and its application in bioequivalence
study, J. Chromatogr. B 873 (2008) 77–85, https://doi.org/10.1016/j.jchromb.
2008.07.040.
[11] N.S. Rao, A. Oomman, P.L. Bindumathi, V. Sharma, S. Rao, L.S. Moodahadu,
A. Patnaik, B.R.N. Kumar, Efficacy and tolerability of fixed dose combination of
metoprolol and amlodipine in indian patients with essential hypertension, J. Mid-
life Health 4 (2013) 160–166, https://doi.org/10.4103/0976-7800.119000.
[12] A. Pareek, N.B. Chandurkar, R. Sharma, D. Tiwari, B.S. Gupta, Efficacy and toler-
ability of a fixed-dose combination of metoprolol extended release/amlodipine in
patients with mild-to-moderate hypertension: a randomized, parallel-group, mul-
ticentre comparison with losartan plus amlodipine, Clin. Drug Investig. 30 (2010)
123–131, https://doi.org/10.2165/11531770-000000000-00000.
[13] J. Esteve-Romero, J. Albiol-Chiva, J. Peris-Vicente, A review on development of
analytical methods to determine monitorable drugs in serum and urine by micellar
liquid chromatography using direct injection, Anal. Chim. Acta 926 (2016) 1–16,
https://doi.org/10.1016/j.aca.2016.04.026.
[14] K.E. Stępnik, A concise review of applications of micellar liquid chromatography to
study biologically active compounds, Biomed. Chromatogr. 31 (2017) e3741, ,
https://doi.org/10.1002/bmc.3741.
[15] R.N. El-Shaheny, M.H. El-Maghrabey, F.F. Belal, Micellar liquid chromatography
from green analysis perspective, Open Chem. 13 (2015) 877–892, https://doi.org/
10.1515/chem-2015-0101.
[16] S. Hapse, B. Bhagat, S. Mogal, A. Kamod, Spectrophotometric estimation and va-
lidation of metoprolol succinate and amlodipine besylate by different method from
pure and tablet dosage form, Int. J. Pharm. Res. Sch. 5 (2013) 126–131.
641
Microchemical Journal 147 (2019) 635–642
[17] S.M. Dhole, D.R. Chaple, M.T. Harde, Validated UV spectrophotometric method for
simultaneous estimation of metoprolol succinate and amlodipine besylate in their
combined tablet dosage form, Int. J. Anal. Bioanal. Chem. 3 (2013) 82–85, https://
doi.org/10.1016/j.jpba.2007.11.006.
[18] M. Shahlaei, F. Hassanzadeh, J. Emami, E. Sohrabi, L. Saghaie, Simultaneous
spectrophotometric determination of amlodipine and metoprolol by principal
component regression multivariate calibration and comparison with HPLC, J. Rep.
Pharm. Sci. 2 (2013) 179–189.
[19] A. Chabukswar, M. Mohokar, V. Choudhari, S. Sharma, S. Tambe, P. B., Absorption
corrected method and isoabsorptive point method for simultaneous estimation of
metoprolol and amlodipine in their combined dosage form, Int. J. Pharm. Sci. Drug
Res. 4 (2012) 240–244.
[20] M.M. Mabrouk, S.F. Hammad, S.F. El-Malla, E.A. Elshenawy, Simultaneous de-
termination of amlodipine and metoprolol in their combined dosage form using a
synchronous fluorescence spectrofluorimetric method, Luminescence 33 (2017)
364–369, https://doi.org/10.1002/bio.3422.
[21] J. Chen, B.Q. Li, M.L. Xu, X. Wang, Y.H. Jing, H.L. Zhai, Krawtchouk image moment
method for the simultaneous determination of three drugs in human plasma based
on fluorescence three-dimensional spectra, Talanta 161 (2016) 99–104, https://doi.
org/10.1016/j.talanta.2016.08.019.
[22] S. Shitole, M. Gurjar, M. Shah, S. Pimple, G. Bal, R. Patel, Development and vali-
dation of stability-indicating RP-UPLC method for simultaneous determination of
related substances of S (−) amlodipine and S (−) metoprolol succinate in fixed
dose combination tablet dosage form, Chromatogr. Res. Int. 2014 (2014) 874587, ,
https://doi.org/10.1155/2014/874587.
[23] M. Pokala, P.J. Rani, G. Pranathi, M.A. Haque, D. Sireesha, S. Harshini, B. Vasudha,
Development and validation of RP-HPLC method for the simultaneous estimation of
amlodipine and metoprolol in bulk and pharmaceutical dosage form, Indo Am. J.
Pharm. Res. 5 (2015) 3254–3260.
[24] J. Vasavi, M.V. Lakshmi, N.S. Sundari, Rapid RP HPLC method for simultaneous
separation and estimation of metoprolol and amlodipine in combination tablet
formulation, Int. Res. J. Pharm. 4 (2013) 160–165, https://doi.org/10.7897/2230-
8407.04934.
[25] P. Ravisankar, M. Aswini, B.P. Srinivasa, C. Devadasu, R. Sai Ram Aditya,
B. Prathima, G. Bala Guravaiah, Development and validation of RP-HPLC method
for simultaneous determination of metoprolol and amlodipine in tablet dosage
form, J. Chem. Pharm. Sci. 7 (2014) 113–121.
[26] S. Hussain, R.R. Munjewar, M. Farooqui, Development and validation of a si-
multaneous HPLC method for quantification of amlodipine besylate and metoprolol
tartrate in tablets, J. Pharm. Sci. Technol. 1 (2012) 1–5.
[27] D.P. Varma, A.L. Rao, S.C. Dinda, Validated stability indicating HPLC method for
simultaneous determination of amlodipine and metoprolol in bulk drug and phar-
maceutical formulations, Int. J. Res. Pharm. Chem. 2 (2012) 876–884.
[28] P.S. Jain, M.K. Patel, S.B. Bari, S.J. Surana, Development and validation of HPTLC
method for simultaneous determination of amlodipine besylate and metoprolol
succinate in bulk and tablets, Indian J. Pharm. Sci. 74 (2012) 152–156, https://doi.
org/10.4103/0250-474X.103849.
[29] R. Kakde, N. Bawane, High-performance thin-layer chromatographic method for
simultaneous analysis of metoprolol succinate and amlodipine besylate in phar-
maceutical preparations, J. Planar Chromatogr.–Mod. TLC 22 (2009) 115–119,
https://doi.org/10.1556/jpc.22.2009.2.7.
[30] EPA hazardous waste listings. https://www.epa.gov/hw/user-friendly-reference-
document-hazardous-waste-listings, 2012 (accessed 28 January 2019).
[31] K. Gilomen, H.P. Stauffer, V.R. Meyer, Detoxification of acetonitrile-water wastes
from liquid chromatography, Chromatographia 41 (1995) 488–491, https://doi.
org/10.1007/bf02318626.
[32] ICH harmonised tripartite guidelines, validation of analytical procedures: Text and
methodology Q2(R1). http://www.ich.org/products/guidelines/quality/article/
quality-guidelines.html, 2005 (accessed 15 August 2018).
[33] J. Płotka-Wasylka, A new tool for the evaluation of the analytical procedure: green
analytical procedure index, Talanta 181 (2018) 204–209, https://doi.org/10.1016/
j.talanta.2018.01.013.
[34] M. Tobiszewski, Metrics for green analytical chemistry, Anal. Methods 8 (2016)
2993–2999, https://doi.org/10.1039/C6AY00478D.
[35] A. Gałuszka, Z.M. Migaszewski, P. Konieczka, J. Namieśnik, Analytical eco-scale for
assessing the greenness of analytical procedures, Trends Anal. Chem. 37 (2012)
61–72, https://doi.org/10.1016/j.trac.2012.03.013.
[36] H. Elmansi, F. Belal, Development of an eco-friendly HPLC method for the si-
multaneous determination of three benzodiazepines using green mobile phase,
Microchem. J. 145 (2019) 330–336, https://doi.org/10.1016/j.microc.2018.10.
059.
[37] A.C. Moffat, M.D. Osselton, B. Widdop, J. Watts, Clarke's Analysis of Drugs and
Poisons, 3rd ed., Pharmaceutical Press, London, 2011.
[38] J.B. Lecaillon, N. Febvre, C. Souppart, Influence of solute polarity in column-
switching chromatography for the assay of drugs in plasma and urine, J.
Chromatogr. 317 (1984) 493–506, https://doi.org/10.1016/S0021-9673(01)
91689-9.
[39] M.C. García-Alvarez-Coque, M.J. Ruiz-Angel, S. Carda-Broch, Micellar liquid
chromatography: fundamentals, Anal. Sep. Sci. 2 (2015) 371–406, https://doi.org/
10.1002/9783527678129.assep017.
[40] P.J. Schoenmakers, S.V. Molle, C.M.G. Hayes, L.G.M. Uunk, Effects of pH in re-
versed-phase liquid chromatography, Anal. Chim. Acta 250 (1991) 1–19, https://
doi.org/10.1016/0003-2670(91)85058-Z.
[41] S. Goga, S. Heinisch, J.L. Rocca, Retention and column efficiency in reversed phase
liquid chromatography as a function of pH for optimization purposes,
Chromatographia 48 (1998) 237–244, https://doi.org/10.1007/BF02467677.
M.M. Mabrouk, et al.
[42] T.M. Kalyankar, P.D. Kulkarni, S.J. Wadher, S.S. Pekamwar, Applications of mi-
cellar liquid chromatography in bioanalysis: a review, J. Appl. Pharm. Sci. 4 (2014)
128–134, https://doi.org/10.7324/JAPS.2014.40122.
[43] M.C. García-Alvarez-Coque, M.J. Ruiz-Angel, S. Carda-Broch, Micellar liquid
chromatography: method development and applications, Anal. Sep. Sci. 2 (2015)
407–460, https://doi.org/10.1002/9783527678129.assep018.
[44] M. Walash, F. Belal, N. El-Enany, S. Zayed, Micellar liquid chromatographic de-
termination of felodipine in tablets and human plasma with fluorescence detection:
642
Microchemical Journal 147 (2019) 635–642
application to stability studies and content uniformity testing, Anal. Methods 6
(2014) 3401–3409, https://doi.org/10.1039/C3AY41570H.
[45] M. Tobiszewski, M. Marc, A. Galuszka, J. Namiesnik, Green chemistry metrics with
special reference to green analytical chemistry, Molecules 20 (2015) 10928–10946,
https://doi.org/10.3390/molecules200610928.
[46] Center for drug evaluation and research, FDA reviewer guidance: Validation of
chromatographic methods. http://www.gmp-compliance.org/guidemgr/files/1-6-
13.PDF, 1994 (accessed 28 January 2019).