Microchemical Journal 145 (2019) 428–434

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

Voltammetric method for simultaneous determination of ascorbic acid, T

paracetamol and guaifenesin using a sequential experimentation strategy
Hassan A.M. Hendawya, Ahmed M. Ibrahima, , Wafaa S. Hassanb, Abdalla Shalabyb,
⁎

Heba M. El-sayedb
a
National Organization for Drug Control and Research, P.O. Box 29, Cairo, Egypt
b
Department of Analytical Chemistry, Faculty of pharmacy, Zagazig University, Zagazig, Egypt

ARTICLE INFO ABSTRACT

Keywords: Cold is a leading cause of school and work absenteeism. Most cold over-the-counter medicines (OTC) contain
Dual response surface method paracetamol (PA), guaifenesin (GU) and other active ingredients. Ascorbic acid (AS) in pharmaceutical pre-
Fractional factorial design parations is designed for preventing and treating respiratory viral infections. Thus, we report a robust voltam-
Graphene oxide metric method for simultaneous determination of AS, PA and GU in their effervescent dosage form. The ad-
Multi-walled carbon nanotubes
vantages of nanotechnology and chemometrics were combined to enhance the performance of electroanalysis.
Square wave voltammetry
Fractional factorial design (FrFD) and dual response surface method (DRSM) were sequentially conducted. The
method was validated in agreement with ICH guidelines; it showed accuracy over the concentration range of
11.3–108.9, 14.6–140.3, 4.6–43.9 μg ml−1 for AS, PA and GU, respectively. Based on findings, a robust and not
time-consuming procedure was applied.

1. Introduction
Voltammetry is a technique utilized for the analysis of compounds
which undergo oxidation or reduction [1,2]. The electroanalysis de-
pends on the type of electrode material. The carbon nanotubes (CNTs)
have good conductivity, and chemical stability [3]. A plethora of ap-
plications are developed based on CNTs such as electron field emission
sources, batteries, nanoelectronic devices and, biosensor [3–5]. Gra-
phene or graphene oxide (GO) has significant importance in recent
years because of its superior mechanical strength, low density and high
heat conductance [6–9]. A plethora of applications are developed based
on graphene-polymer composites such as biosensors, drug delivery, fuel
cell, and batteries [10–12]. Multi-walled carbon nanotubes (MWCNTs)
increase the surface areas of the electrodes, facilitate the electron
transfers and decrease surface fouling [13–15]. Graphene oxide (GO) is
applied due to its low costs, and its amphiphilic nature [16,17]. The
signal amplification properties of GO and the antifouling properties of
MWCNTs were investigated [18].
Chemometrics are utilized in electroanalysis because of the com-
plexity of the optimization process [19,20]. Experimental factors can be
screened by FrFD. Then, response surface methodology (RSM) begins to
optimize experimental conditions [21,22]. Robust design has become a
natural element in process development that emerged in Japan and
reached the United States around 1980 [23]; it reduces the effects of
input variation on outputs to improve quality [24]. DRSM is used to
make robust process [25–27].
Over-the-counter cold products are widely used. AS is used for
common cold. PA is used for its antipyretic actions [28]. GU is used as
expectorant. It reduces the viscosity of bronchial secretions [29,30].
Fig. 1 shows the chemical structure of investigated drugs. A review of
the literature revealed that various instrument techniques could be used
to analyze them [31–35].
Considering the electrochemical properties of the analytes and the
technical simplicity, we intended to perform a robust voltammetric
method for simultaneous determination of AS, PA and GU in their
pharmaceutical dosage form and to combine the advantages of nano-
technology and chemometrics to enhance the performance of electro-
analysis. For best of our knowledge, no voltammetric method was op-
timized using DRSM to achieve robustness. The electrochemical
characterizations of the modified electrode were investigated using
various techniques. The described method was validated by ICH
guidelines, and the assay results were in good accordance with HPLC
method [36].

⁎
Corresponding author.
E-mail address: dr.ahmed_analytical@yahoo.com (A.M. Ibrahim).

https://doi.org/10.1016/j.microc.2018.11.010
Received 10 October 2018; Received in revised form 29 October 2018; Accepted 5 November 2018
Available online 06 November 2018
0026-265X/ © 2018 Elsevier B.V. All rights reserved.
H.A.M. Hendawy et al.
Fig. 1. Chemical structure of A) AS, B) PA, and C) GU.
2. Material and methods
2.1. Instrumentation
Metrohm electroanalyzer Model 797VA Computrace was used for
voltammetric experiments. Three electrodes consisted of a GO/
MWCNTs/CPE, an Ag/AgCl, and a platinum wire electrodes. Scanning
electron microscopy (SEM) was performed using a JSM-6700F scanning
electron microscope (Electro Co.). Electrochemical impedance spec-
troscopy was performed using a Gamry-750 system and a lock-in-am-
plifier between 0.1 Hz and 100 kHz with amplitude of 5 mV in 0.1 M
KCL containing 5 mM K4Fe(CN)6. HPLC analysis was performed using A
Dionex UltiMate 3000 HPLC system (ThermoScientific) equipped with a
MWD-30000 (RS) detector, a TCC-3X00(RS) column compartment
oven, a LPG-3400SD Quaternary pump, and remote injector.
Chromatographic experiment was isocratic mode on a C18 column,
ThermoHypersil (250 mm × 4.6 mm I.D., 5 μ m). A JENWAY 3510 pH
meter was used for pH measurements.
2.2. Reagents and solutions
Ultrapure water was obtained from a Milli-Q® system. All other
reagents used were of analytical grade. AS (99.8%), PA (99.7%) and GU
(99.5%) were supplied by NODCAR.A graphite powder (particle di-
mension 20 μm) and MWCNTs (O.D. × I.D. × L 10–20 nm × 5–10 nm
× 0.5–200 μm, ≥95% carbon content) were purchased from
Sigma–Aldrich. Glycine, sodium chloride, potassium chloride, hydro-
chloric acid, anhydrous sodium acetate, glacial acetic acid, citric acid,
sodium hydroxide, sodium dihydrogen orthophosphate, disodium hy-
drogen orthophosphate, methanol (HPLC grade), orthophosphoric acid
85%, boric acid, low density paraffin (99%), aspartame, povidone K25,
sodium bicarbonate, disodium edetate and potassium ferrocyanide tri-
hydrate were purchased from Merck. Citric acid anhydrous and tartaric
acid anhydrous were purchased from ADWIC.
G.C.MOL sachets containing 250 mg of AS, 325 mg of PA and
100 mg GU were purchased from the local market.
A blank solution was prepared by dissolving sodium bicarbonate,
citric acid anhydrous, tartaric acid anhydrous, povidone K25, aspar-
tame, and disodium edetate in methanol in order to obtain 24.2, 9.6,
12.3, 1.2, 0.7 and 0.03 mg ml−1, respectively. This solution contains an
equivalent amount of the major excipients as compared to G.C.MOL
sachets for which the method was developed for. Stock solutions were
prepared in methanol containing 2.5, 3.25, 1.0 mg ml−1 of AS, PA and
GU, respectively. All stock solutions were protected from light.
Calibration standards were prepared by dilution of the stock standard
solutions with phosphate buffer solutions PBS (0.1 M, pH 3.0).
429
Microchemical Journal 145 (2019) 428–434
2.3. Preliminary experiment
2.3.1. Fabrication of GO/MWCNTs/CPE
GO was synthesized by Hummer's method [37], and then MWCNTs
were dispersed with GO in ultrapure water by adding 50 mg MWCNTs
and 50 mg GO in 100 mL volumetric flask (1:1) [38]. The percentage
composition of GO/MWCNTs was studied. The modified GO/MWCNTs/
CPE electrode was prepared by mixing graphite powder (60%), modi-
fier (10%) and paraffin oil (30%). The paste surface was smoothed with
a weighing paper, and then rinsed carefully with ultrapure water.
2.3.2. Effect of solution pH value and supporting electrolytes
The SWV experiments were conducted in 0.04 M Britton-Robinson
(BR) buffer over the pH range 2–8. Five supporting electrolytes: PBS,
acetate, BR, citrate and glycine buffer were studied for selection of the
most appropriate supporting electrolyte.
2.4. The sequential strategy
FrFDs are subset of the experimental runs of a full factorial design
that enable the goals of the experiment to be met with the most ef-
fective use of resources [21,39]. Fractional designs are expressed using
the notation 2k–p k is the number of factors investigated, and p describes
the size of the fraction of the full factorial used [39]. Myers emphasized
the goal of optimizing the mean and the variance simultaneously could
be achieved by DRSM [27,40].The following steps are proposed for
DRSM [40,41]:
1. Developing the experimental design, conducting the experiments,
and collecting data.
2. Fitting response surfaces for the mean (μ) and standard deviation (σ)
responses separately.
The critical method parameters (CMPs) and the critical method at-
tributes (CMAs) were studied. FrFD was initially applied to investigate
the effects of eight CMPs (Table 1S) on the CMA (No of resolved peaks).
The experimental matrix is composed of 32 runs (Table 2S).
After the selection of CMPs (Tables 3S), DRSM was conducted
through CCD (Table 4S). The CMAs for DRSM are presented in Table 5S.
We collected repeated samples for each run (n = 3), where the mean
and the standard deviation were entered as responses.
2.5. Recommended procedure
The solutions were purged with nitrogen for 5 min. A 10 mL volume
of PBS (pH 3.0) was added to the sample cell. We applied cleaning
potential 0.5 V for 10 s. The GO/MWCNTs/CPE was kept at the
H.A.M. Hendawy et al.
Table 1
CMAs models and statistical parameters after AICc forward model selection.
CMAs Regression equation in terms of coded factorsa
2
M_Ip(AS) M_Ip(AS) = 16.91 + 4.21B – 3.15B
S_Ip(AS) S_Ip(AS) = 1.90 + 0.54B
M_Ip(PA) M_Ip(PA) = 54.59–4.06B + 1.45C
S_Ip(PA) S_Ip(PA) = 6.86 + 0.81B
M_Ip(GU) M_Ip(GU) = 5.4 – 0.15A + 0.65B + 0.34C + 0.31AC – 0.31A2
S_Ip(GU) S_Ip(GU) = 0.81 + 0.68B + 0.54B2
Wp(AS) Wp(AS) = 594.8 + 46.94B – 31.11C
a
Significant factors; M_Ip = mean of the oxidation peak current; S_Ip = standard deviation of the oxidation peak current; Wp = width of the oxidation peak
current; A = deposition time; B = frequency; C = deposition potential.
accumulation potential of −0.12 V for 126 s, while the solution was
stirred at about 2000 rpm. The stirring was then stopped and the so-
lution was allowed to rest for 3 s (equilibrium time), after which a scan
was carried out towards positive potentials over the range –0.2 to 1.4 V,
and the SW voltammograms were recorded. The experimental condi-
tions for SWV were amplitude, 0.02 V; frequency, 8.0 Hz; voltage step,
0.005 V; and sweep rate, 0.04 V s−1. All experiments were performed at
25 ± 1 °C.
2.6. Validation
The method was validated according to ICH guidelines. The oxi-
dation peak current (Ip) was the analytical response. The evaluation of
selectivity was done by checking the lack of interference from effer-
vescent granules matrix. The precision was assessed by ANOVA test (six
independent measurements in three successive days). Different regres-
sion models were assessed (three independent measurements for six
calibration standards). The accuracy was assessed by applying the
standard addition technique. The assay results that obtained by de-
scribed method were compared with HPLC method [36].
2.7. Sample preparation
Six sachets of G.C.MOL effervescent granules were mixed; three
portions were weighed (containing 250 mg of AS, 325 mg of PA, and
100 mg of GU), and they dissolved in methanol. The excipients were
separated by filtration; further appropriate dilutions were done with
PBS.
2.8. Computations
VA Computrace version 1.3.1 was used to control the voltammetric
experiments and to acquire data. Design-Expert® trial version 10.0 was
used to experimental designs and statistical data analysis. Statgraphics
Centurion XVII was used to perform Regression analysis.
3. Result and discussion
The voltammetric results showed well defined oxidation peaks at
potentials of 0.175, 0.59 and 1.17 V, corresponding to the oxidation of
AS, PA and GU, respectively (Fig. 2).
3.1. Preliminary experiments
Preliminary experiments were focused on the percentage composi-
tion of modifier, pH value of buffer solutions and type of supporting
electrolyte. Due to the signal amplification properties of GO and the
antifouling properties of MWCNTs were chosen as modifier for CPE
[18].
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Microchemical Journal 145 (2019) 428–434
Adjusted R2 Predicted R2 Adequate precision
0.92 0.90 18.47
0.87 0.85 23.46
0.74 0.70 12.83
0.65 0.62 12.57
0.82 0.74 11.30
0.64 0.59 10.42
0.46 0.38 6.74
Fig. 2. SW voltammograms of blank (…) and spiked ananalytes (−).
3.1.1. Optimization of the percentage of modifier in CPE
The percentage of modifier up to 10% intensified the oxidation
peaks current (Fig. 1S); therefore, it was selected as the optimum per-
centage for preparation of GO/MWCNTs/CPE.
3.1.2. Characterizations of the GO/MWCNTS/CPE
The SEM image of GO/MWCNTS/CPE exhibits large surface area
(Fig. 2S) when compared with graphite flakes [42].
Cyclic voltammetry measurements of Fe(CN)6–3/−4 redox couple in
0.1 M HCL solution were performed (Fig. 3S). CPE showed a redox
couple at the potential values of +0.393 V and +0.298 V corre-
sponding to oxidation of Fe(CN)64− and reduction of Fe(CN)63−, re-
spectively. These peaks were seen at the potential values of +0.375 V
and +0.309 V at GO/MWCNTS/CPE. Peak separation value of Fe
(CN)63−/4– redox couple decreased to (0.066 V) in the presence of GO/
MWCNTS/CPE compared to the value (0.095 V) obtained with CPE.
Moreover, oxidation and reduction peak currents of Fe (CN)63−/4–
redox couple increased with GO/MWCNTS/CPE according to CPE (two
fold); the results indicate that the modification increases the electron
transfer rate of redox couple. The difference between the oxidation and
reduction baseline currents was another different behavior of GO/
MWCNTS/CPE from CPE. This indicates that the modification increases
the capacitive character of CPE. This situation may arise from the high
surface area of GO/MWCNTS/CPE compared to CPE.
The active area of the electrode was obtained by cyclic voltammetry
measurements at different scan rates (Fig. 4S). For a reversible process,
the following Randles–Sevcik equation can be used [43,44].
IP = (2.69 × 105) A n3/2 DR1/2 C0 v1/2
The active area was calculated to be 0.013, 0.032 cm2 for CPE, and
GO/MWCNTs/CPE respectively (2.5 fold increment).
SWV measurements were also evaluated in buffer solution (PBS,
H.A.M. Hendawy et al.
pH 3.0) containing the analytes (Fig. 5S).Table 6S shows oxidation
peaks potential and currents of three analytes at GO/MWCNTS/CPE
and CPE (n = 3). The anodic overpotential of the electrode process
decreased due to the improvement of kinetics of electron transfer for
the analytes at the GO/MWCNTs/CPE. Modification also increased the
oxidation peaks current of the analytes compared to CPE because of the
increase of surface area and high electrical conductivity of GO/
MWCNTS/CPE (statistically significant difference at p = 0.05).
Moreover, modification increased reproducibility of the results (low
variance); this is due to antifouling properties of MWCNTs as shown in
Table 6S (statistically significant difference at p = 0.05 for AS and PA).
EIS was applied for further characterization of the different mod-
ified electrodes. The typical Nyquist plots of EIS were shown in Fig. 6S.
The values of charge transfer resistance (Rct) were obtained from the
semicircle diameters according to an equivalent circuit (inset in
Fig. 6S). The Rct were of 207.7 Ω, 98.4 Ω, 30.43 Ω and 38.4 Ω for CPE,
5%, 10% and 15% of added modifier, respectively. The smallest Rct
value showed the excellent electrical properties of the 10% modifica-
tion.
3.1.3. Influence of buffer pH and supporting electrolytes
Buffer pH and type of supporting electrolytes are key parameters in
voltammetry. The number of resolved peaks decreased above pH 8
because the oxidation of PA became less due to the presence of the
phenoxide [45], and electrostatic repulsion between AS anions with the
surface of GO/MWCNTS/CPE could be anticipated [46,47]. The max-
imum peaks current were at pH 3; therefore, it was the optimal buffer
pH (Fig. 7S). The maximum peaks current were in PBS (Fig. 8S).
3.2. Sequential strategy for the optimization
It is convenient to choose screening design to reduce the number of
tests [48]. RSM begins when the important factors are identified. This
sequential strategy obtained the desired conditions.
3.2.1. Screening design
FrFD finds out the significant factors on the studied response [48].
Fig. 3. One factor effects plots for CMPs of FrFD.
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Microchemical Journal 145 (2019) 428–434
The F-value of 13.93 indicates that the model was significant
(Table 7S). The one factor effects plots show the linear effect of chan-
ging the level of a single factor (Fig. 3). It was revealed that the sur-
factant, the cleaning potential, the deposition time, the voltage step,
and the amplitude were statistically significant. Voltage step, amplitude
and surfactant had a significant negative effect and as anticipated de-
creasing its value resulted in the improvement of resolution. Equili-
brium time, deposition potential and frequency did not have a sig-
nificant effect, while deposition time and applying cleaning potential
had significant positive effect. Frequency, deposition time and deposi-
tion potential were further investigated using DRSM.
3.2.2. Dual response approach
DRSM can find settings robust to unidentified sources of noise. The
optimization using DRSM through CCD was performed. CCD is often
formed in this way.
= b0 + b1 x1 + b2 x2 + b11 x12 + b22 x 22 + b12 x1 x2.
The modeling of three CMPs using a CCD was generated. The data
were fitted to a first-order or a quadratic equation to produce predictive
models relating the CMPs and CMAs as shown in Table 1. The insig-
nificant terms were eliminated from the model through a corrected
Akaike Information Criterion (AICc) with forward model selection ap-
proach to obtain realistic model [49]. The models terms of CMAs were
statistically significant (p-value < 0.05) and lack of fit (LoF) values
were statistically insignificant (p-value > 0.05) indicating good
models.
Statistical parameters such as adjusted R2, predicted R2, adequate
precision are presented in Table 1. Predicted R2 measures the variation
in new data explained by the model [49], and their values were in
agreement with the adjusted R2 values. Adequate precision measures
the experimental signal to noise ratio [49]. In this study, the ratio was
found to be > 6.74 in all cases indicating the significance of the models.
Diagnostic plots such as the normal probability plot, the plot of re-
siduals against to the predicted values, residuals against run, and the
predicted values against the actual values are shown in Figs. 9S to 15S.
H.A.M. Hendawy et al.
Fig. 4. Contour plots of CMAs of DRSM.
The diagnostic plots indicate that the models were appropriately fitting
the data.
In the presence of multiple response processes, Derringer's desir-
ability function (D) was used; it was based on a scale of desirability
function ranging from 0 to 1 [50]. Fig. 4 shows the contour plots of
CMAs of DRSM. ANOVA results for reduced models are presented in
Table 8S. The optimization of D led to the following optimum vol-
tammetric conditions: frequency 8 Hz, deposition time 126 s, and de-
position potential −0.12 V (see Fig. 4).
Finally, five voltammetric measurements were analyzed to confirm
the optimum conditions; the differences between the experimental and
the predicted values were < 3.8%.
3.3. Validation
The developed method was selective for simultaneous determina-
tion of AS, PA, and GU since no interfering oxidation peak was observed
at the potential of the well separated AS, PA, and GU as shown in Fig. 2.
Fig. 5 depicts the dependence of oxidation peak current on con-
centration of each analyte (SW Voltammograms are shown in Fig. 16S).
Different regression models were assessed for each analyte in our study
because the Analytical Methods Committee dissuaded from using the
correlation coefficient for linearity test [51,52]. The most appropriate
models were linear, squared Y and square root X transformation and
linear with double reciprocal transformation for AS, PA and GU re-
spectively (Table 2) based on the lack of fit test (LoF) and residual plots.
LoF test determines whether a model is adequate to describe the ob-
served data or not [53]. The models were adequate because the F values
obtained from the LoF test were less than the Ftab at p = 0.05 (3.26) as
presented in Table 2. The distribution of the residuals was random and
homogeneous, which proves that the selected models were appropriate
(Fig. 17S).
The limit of detection (LOD) was estimated to be 3.08, 4.04, and
1.29 μg ml−1 for AS, PA and GU, respectively at a signal to noise ratio of
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Microchemical Journal 145 (2019) 428–434
3σ (σ is the standard deviation of the blank, n = 6).
The limit of quantification (LOQ) was estimated to be 10.16, 13.3,
and 4.26 μg ml−1 for AS, PA and GU, respectively at a signal to noise
ratio of 10σ (n = 6).
The precision is evaluated intra-day (repeatability) and interday
(intermediate precision) through relative standard deviations (RSD).
ANOVA test was used to assess precision [54]. The repeatability RSD
values were 1.6, 1.62, and 2.2 for AS, PA and GU, respectively; the
intermediate precision RSD values were 1.61, 167, and 2.2 for AS, PA
and GU, respectively (Table 9S). The accuracy of the proposed vol-
tammetric method was determined by its recovery during spiked ex-
periments. The results demonstrate the validity of the proposed
methods for the simultaneous determination of AS, PA and GU in ef-
fervescent granules (Table 3).
3.4. Reproducibility and stability of GO/MWCNTs/CPE
SWV measurements were performed with nine different GO/
MWCNTs/CPEs to determine their reproducibility. The relative stan-
dard deviation values were 2.94%, 2.78%, and 2.98% for AS, PA, and
GU, respectively.
Stability is a key factor in the development of electrochemical
sensor; it was examined by keeping the electrode in refrigerator at 4 °C
for a definite period. SWV measurements were performed periodically
for 3 weeks. The voltammetric response decreased only 4.8%, 5.4% and
4.3% for AS, PA, and GU, respectively. The results indicate that GO/
MWCNts/CPE had long term stability.
3.5. Application
The described method was applied for simultaneous determination
of AS, PA and GU in effervescent granules (G.C.MOL). AS, PA and GU
contents were close to claimed contents (Table 4).
The concentrations of three analytes were analyzed by HPLC [36]
H.A.M. Hendawy et al. Microchemical Journal 145 (2019) 428–434

Table 2
Calibration function of three analytes.
Parameters AS PA GU

Regression equation Ip = a + bC Ip = a+b C Ip =

1
b
a+
C
a 2.49 −108.63 0.08
b 0.014 104.46 5.25
r 0.992 0.997 0.998
LoF test⁎⁎ 1.44 0.46 0.26

Where LoF = lack of fit; a = intercept; b = slope; r = correlation coefficient;

Ip = oxidation peak current; C = concentration of analytes.
⁎⁎
Non significant values (p > 0.05).

Table 3
Application of the standard addition technique in the determination of AS, PA
and GU by the proposed voltammetric method in the pharmaceutical dosage
form.
Parameter Claimed (μg ml−1) Added (μg ml−1) Recoverya

AS 15 15 98.33
50 100.45
90 101.84
Recovery 100.21
SD 1.77
CV 1.76
PA 20 20 101.08
60 99.03
110 98.67
Recovery 99.59
SD 1.3
CV 1.31
GU 5 5 102.12
20 100.06
35 99.02
Recovery 100.4
SD 1.58
CV 1.57

Where CV = coefficient of variation.

a
For five determinations;

Table 4
Statistical analysis of the results obtained by SWV method for AS, PA, GU
compared with HPLC method.
Parameter AS PA GU

SWV HPLC SWV HPLC SWV HPLC

Mean 101.3 100.5 99.1 100.2 100.9 99.8

SD 1.8 1.2 2.3 1.1 1.85 0.95
n 3
tcal −0.64 0.75 −0.92
Fcal 2.25 4.37 3.79

ttab at p = 0.05 (2.78).

Ftab at p = 0.05 (19).

of the study proved the benefit of applying nanotechnology and che-

mometrics in electroanalysis. A validation approach using ICH guide-
lines proved the reliability of the results. Finally, we applied the de-
Fig. 5. Dependence of oxidation peak current Ip on concentration of A) AS, B)
PA and C) GU.
scribed method to analyze the pharmaceutical dosage form.

for comparison; the results obtained by both methods showed a good
agreement (statistically insignificant difference at p = 0.05).
4. Conclusion
Acknowledgement
The authors thank the Micro-analysis Lab in NODCAR. This research
did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.

A robust voltammetric method for simultaneous determination of

AS, PA and GU in their effervescent dosage form is reported. The results

433
H.A.M. Hendawy et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.microc.2018.11.010.
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