Journal of Electroanalytical Chemistry 764 (2016) 93–103

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Journal of Electroanalytical Chemistry

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Voltammetric determination of paracetamol, tramadol and caffeine using

poly(Nile blue) modified glassy carbon electrode
S. Chitravathi ⁎, N. Munichandraiah
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India

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

Article history: A poly(Nile blue) modified glassy carbon electrode (PNBMGCE) was fabricated by electropolymerisation of Nile
Received 25 August 2015 blue (NB) monomer using cyclic voltammetry (CV) and was used for the determination of paracetamol (ACOP),
Received in revised form 18 January 2016 tramadol (TRA) and caffeine (CAF). The electrochemical investigations showed that PNB — film formed on the
Accepted 19 January 2016
surface of glassy carbon electrode (GCE) improved the electroactive surface area and displayed a remarkable in-
Available online 21 January 2016
crease in the peak current and a substantial decrease in over potential of ACOP, TRA and CAF when compared to
Keywords:
bare GCE. The dependence of peak current and potential on pH, sweep rate and concentration were also investi-
Paracetamol gated at the surface of PNBMGCE. It showed good sensitivity and selectivity in a wide linear range from 2.0 × 10–7
Tramadol to 1.62 × 10–5 M, 1.0 × 10–6 to 3.1 × 10–4 M and 8.0 × 10–7 to 2.0 × 10–5 M, with detection limits of 0.08, 0.5 and
Caffeine 0.1 μM, for ACOP, TRA and CAF, respectively. The PNBMGCE was also successfully applied for the determination of
Cyclic voltammetry ACOP, TRA and CAF in pharmaceutical dosage forms.
Differential pulse voltammetry © 2016 Elsevier B.V. All rights reserved.

1. Introduction sample pre-treatment procedure before detection that makes them

Paracetamol (acetaminophen, N-acetyl-p-aminophenol, Scheme 1)
is a long-established and most widely used antipyretic and analgesic
drug. It is commonly used to reduce fever, relieve cough, cold, and
pain including muscular aches, chronic pain, migraine headache, back-
ache, toothache and other minor aches and pains. [1]. Tramadol, (1R,
2R)-2-[(dimethylamino) methyl]-1-(3-methoxyphenyl) cyclohexanol,
Scheme 1], is a centrally acting analgesic, used primarily to treat moder-
ate to severe pain [2]. Caffeine (1, 3, 5-trimethylxanthine, Scheme 1) is a
natural alkaloid belonging to N-methyl derivatives of xanthine. It is the
most widely used psychoactive substance to stimulate central nervous
system (CNS), cardiovascular system and also has many important
physiological effects, such as diuresis and gastric acid secretion [3–5].
In general, limited use of these drugs does not exhibit any harmful
side effects. However, their overdose and the chronic use produce
toxic metabolite accumulation that will cause nervousness, trembling,
nausea, seizures, restlessness, insomnia, headaches, kidney and liver
damage [6–8]. Thus, it is very important to analyse these drugs in phar-
maceutical preparations.
Several techniques have been reported for their assay in biological
and pharmaceutical samples including a number of high-performance
liquid chromatography (HPLC) [9–11], gas chromatography with mass
spectrometry (GC–MS) [12,13] liquid chromatography with mass spec-
trometry (LC–MS) [3,14–16] and spectrophotometry [17–21]. However,
these methods are expensive, time-consuming and involve a tedious
⁎ Corresponding author.
E-mail address: chitrasgowda@gmail.com (S. Chitravathi).
unsuitable for a routine analysis. Hence, it is necessary to develop a
sensitive, simple and efficient method of detection of these drugs.
Electrochemical methods of analysis have shown to be accurate, sen-
sitive and cost-effective. Various electrochemical methods are available
for the analysis of ACOP, TRA and CAF, either individually or in combina-
tion [22–37]. However, according to literature the efficacy and tolerabil-
ity of these drugs were proven to be better in combination of all the
three drugs [38], compared to individual drug action, to relieve pain, re-
duce adverse drug reaction and less medication [39–43]. Considering
the importance of these drugs on human health and their complemen-
tary mechanism of action, it is important to develop a simple, fast, sen-
sitive, and accurate electrochemical method for determination of these
drugs. But, direct oxidation of these compounds at bare electrode occurs
at high overpotentials. So, a decrease in the overpotential along with
increase in current sensitivity can be achieved with chemically mod-
ified electrodes (CMEs). Hence, we planned to develop a CME that
lowers the oxidation potentials of these drugs without affecting their
determination.
CMEs prepared by electropolymerisation of dyes such as phenothia-
zines, phenazines, and phenoxazines have several advantages such
as simple one-step preparation, high stability, and reproducibility [44,
45]. These dyes can be electropolymerised from aqueous solutions
to obtain electrochemically active polymers. Nile blue, one of the
phenoxazine dyes, is a well-known water-soluble electroactive mole-
cule. Electrochemical polymerisation of NB gives a semiconducting
Poly(Nile Blue) (PNB) film. This PNB had been used as a mediator for
the electrocatalytic oxidation of nicotinamide coenzymes, NADH and
NADPH [46–48], ADH [49], electrochemical investigation of Carbidopa

http://dx.doi.org/10.1016/j.jelechem.2016.01.021
1572-6657/© 2016 Elsevier B.V. All rights reserved.
94 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103

Scheme 1. Chemical structures of the studied compounds.

and Benserazide [50], Levodopa [51], ascorbic acid [52] and also used in Electrochemical Analyser, CHI608C (CH Instruments, Inc. USA) was
biofuel cells [53]. However, given to its good electrochemical properties, used for all electrochemical measurements. A conventional three-
very little work has been done to use PNB for the determination of other electrode system was utilised throughout the experiments. The working
molecules. In view of this, it is considered interesting to examine the electrode was a bare GCE or PNBMGCE (3.0 mm in dia, CH instruments),
mediating ability of PNB in the electrocatalytic oxidation of ACOP, TRA the auxiliary electrode was a platinum foil and reference electrode was a
and CAF. Hence, in the present study, we report the usage of Poly(Nile saturated calomel electrode (SCE) and all the electrode potentials
Blue) modified glassy carbon electrode (PNBMGCE) towards the deter-
mination of ACOP, TRA and CAF using cyclic voltammetry (CV) and dif-
ferential pulse voltammetry (DPV) techniques. The PNBMGCE electrode
was also employed for the determination of ACOP, TRA and CAF in
pharmaceutical tablets. To the best of our knowledge, PNBMGCE for
the determination of ACOP, TRA and CAF is not reported till now.

2. Experimental

2.1. Chemicals and Instrumentation

All chemicals were of analytical grade and were used as received

without any further purification. Paracetamol (≥ 98%) was purchased
from S.D. Fine Chemicals. Tramadol hydrochloride (≥ 99%), caffeine
(≥ 99%) and Nile blue chloride (85%) were purchased from Sigma
Aldrich. All solutions were prepared with double-distilled (DD) water.
Nile blue (85%) was recrystallized three times from water before use.
0.1 M of phosphate buffer solution (PB), which was prepared from
0.1 M K2HPO4 and 0.1 M KH2PO4, was employed as a supporting electro-
lyte. Stock solutions of ACOP, TRA and CAF were freshly prepared prior Fig. 1. Continuous cyclic voltammograms for the electropolymerisation of 0.5 mM Nile
to measurements. blue in 0.1 M (pH 8.0) for 10 cycles.
 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103 95

Scheme 2. Possible structure a NB dimer formed on the surface of GCE.

have been reported versus the SCE at 22 ± 1°C. The pH measurements
were performed using EUTECH cyber scan pH metre. The formation of
PNB on the surface of GCE was confirmed by the Atomic force microsco-
py (AFM) technique with tapping mode and Micromasch NSC35/Al se-
ries probe.
Prior to modification, GCE was polished with 0.05 μm alumina
powder on a polishing pad to a mirror like finish followed by sonication
for about two minutes in double distilled water and dried at room tem-
perature and immediately used for modification. After the modification,
the electrode was removed and rinsed with double distilled water and
then left to dry in air for about 10 min at room temperature before the
electrochemical studies were carried out.
2.2. Analytical procedures
2.3. Sample preparation
Various commercial pharmaceutical tablets containing ACOP, TRA
and CAF were examined. Ten tablets each of Ultracet (Johnson & John-
son, USA, 325 mg of Acetaminophen IP and 37.5 mg of Tramadol Hydro-
chloride IP) and Sinarest (Centaur Pharmaceuticals Pvt Ltd, 500 mg of
Paracetamol IP and 30 mg of Caffeine IP) were separately weighed
and finely powdered using mortar and pestle. An adequate amount of
the homogenous powder, corresponding to the amount of one tablet
for each brand, was weighed and dissolved in water. The solution was
sonicated for 10 min and filtered. An appropriate volume of stock solu-
tion was transferred into 50 mL volumetric flask and made up with buff-
er (pH: 7.0); so that its concentration falls within the linear working
range. The samples were then spiked with appropriate amount of stan-
dard ACOP, TRA and CAF for experiments.

For voltammetric experiments the working electrode (either bare

GCE or PNBMGCE) was placed in 0.1 M PB, pH 7.0 containing desired
concentrations of ACOP, TRA and CAF vs SCE before commencing
the scan. CV and DPV techniques were used for the determination
of ACOP, TRA and CAF and all electroanalytical measurements were
made at room temperature. The CV was performed in the potential
range between + 0.1 and + 1.7 V at a scan rate of 0.05 V/s and the
DPV experiments was carried out between the same potential range at
a pulse height of 0.05 V, pulse width 0.06 s and pulse period 0.5 s.
3. Results and discussion
3.1. Electropolymerization and characterisation of the PNBMGCE
Fig. 1 shows the continuous cyclic voltammograms for the
electropolymerization of 0.5 mM Nile blue in 0.1 M PB (pH 8.0) in the
potential range between − 0.8 to + 1.2 V (vs SCE) at a sweep rate of
0.05 V/s on GCE. During the electropolymerization process two

Fig. 2. (A) EIS spectra of 1.0 mM Fe(CN)3−/4-

6 obtained at bare GCE (open squares of curve ‘a’) and PNBMGCE (open circles of curve ‘b’). Overlaid smooth lines indicate Equivalent circuit
fitting. (B) equivalent electrical circuit. EIS condition: frequency range: 0.01–105 Hz; potential: 0.234 V; perturbation amplitude: 5 mV.
96 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103

Table 1 and the diameter of the semicircle decreases when the PNBMGCE is
Data obtained from Impedance analysis for both bare GCE and PNBMGCE in 1 mM Fe employed. The spectra of Fig. 2A were analysed separately with proper
(CN) 6 3−/4− solution using equivalent circuit fitting.
fitting (smooth lines of curve ‘a’ and ‘b’). The electrical equivalent circuit
Parameter Bare GCE Error (%) PNBMGCE Error (%) model used for fitting is as shown in Fig. 2B. The model includes a solu-
Rs in Ω 67.6 0.95 72.01 0.83 tion resistance, Rs, constant phase element (Q), roughness exponent for
n 0.89 1.3 0.87 1.65 Q i.e. ‘n’ of the PNB film, charge transfer resistance (Rct) and Warburg
Q in F sn-1 1.352 × 10–5 4.5 1.69 × 10–5 2.23 impedance (W). The theoretical spectrum agrees well with the experi-
Rct in kΩ 3200 6.01 980 5.9
mental spectrum. Furthermore, χ2 parameter (which is a judging pa-
W in Ω 0.00023 1.24 0.0002 1.88
rameter for goodness of a least squares fit) of fitting is 2.1 × 10–4.
These results suggest that the fitting procedure is fairly reliable. The
oxidation peaks Pa1 and Pa2 at 0.34 and 0.024 V in the anodic sweep and fitted results are shown in Table 1. Rct values obtained from Fig. 2 for
two reduction peaks Pc1 and Pc2 at 0.114 and 0.44 V in reverse sweep bare GCE and PNBMGCE was 3200 and 980 kΩ respectively. This obser-
are observed respectively. The first redox couple with E0` of ~ 0.39 V vation implies that the interfacial charge transfer resistance (Rct) of the
((Pa1 + Pc2)/2) was due to the redox reaction of NB monomer and the electrode surface has decreased and the charge transfer rate has in-
second redox couple with E0` of ~0.07 V ((Pa2 + Pc1)/2) was due to NB creased with the employment of PNBMGCE.
polymer and the irreversible oxidation peak at ∼0.9 V was due to forma- The formation of PNB on the surface of GCE from 0.5 mM NB mono-
tion of monomer radical. In the electropolymerisation process of NB mer in PB (pH 8.0), is also confirmed by the AFM images, which show a
(Fig. 1), the redox peak current of the NB monomer (E0` of ~0.39 V) de- rough surface (Fig. 3A (2)) compared to the unmodified surface
creases gradually from the first sweep and the redox peak current of NB (Fig. 3A(1)). Thus there is an increase in the surface area of the modified
polymer (E0` of ~0.07 V) increases in consecutive sweeps demonstrating electrode and the sensitivities in voltammetric determinations. The film
the formation of PNB on the surface of GCE. After that, the redox peak thickness of PNB-film deposited on the GCE surface was found to be 50–
current decreases perhaps due to the electrode surface completely 60 nm (Fig. 3B).
being covered with PNB and the thicker film of it may obstruct
electron-transfer process. The schematic representation of the forma- 3.2. Cyclic voltammetric behaviour of ACOP, TRA and CAF at PNBMGCE
tion of PNB on the surface of GCE is proposed as in Scheme. 2. Similar re-
sults for the electrochemical polymerisation of NB were also reported in Cyclic voltammetric behaviour of 0.1 mM ACOP, TRA and CAF
[44,48,49]. at PNBMGCE was studied in comparison with bare GCE in 0.2 M PB,
Fig. 2A shows the electron impedance spectra (EIS) of bare GCE pH 7.0 at a sweep rate of 0.05 V/s. Fig. 4A depicts the cyclic voltammetric
(open squares of curve ‘a’) and PNBMGCE (open circles of curve ‘b’) in responses for electrochemical oxidation of 0.1 mM ACOP at bare
1 mM Fe (CN) 6 3 −/4- solution in the frequency range of 10–2-106 Hz. GCE (solid line) and at PNBMGCE (dotted line). ACOP exhibit a
As such the semicircle diameter (Rct) at higher frequencies in the quasireversible behaviour at bare GCE with anodic and cathodic
Nyquist plots for bare GCE and PNBMGCE shows a significant difference. peaks appearing at 0.504 V (a2) and 0.074 V (c2) and a peak potential
Its value varies when different substances are adsorbed on the electrode separation (ΔEp) of 0.43 V. Whereas at PNBMGCE, the anodic and ca-
[54,55]. A semicircle with a larger diameter is observed for the bare GCE, thodic peaks appear at 0.435 V and 0.286 V, respectively with Δ Ep of

Fig. 3. A(1) and A(2) 3-dimentional AFM images of the unmodified surface and Poly Nile blue modified surface respectively. (B) 2-dimentional AFM image of poly Nile blue modified
surface.
 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103 97

and anodic peak current is enhanced from 5.1 to 17.4 μA. A significant
negative shift in the peak potential together with an increased current
sensitivity of the anodic peak confirms the electrocatalytic behaviour
of the PNBMGCE.
Fig. 4C shows the cyclic voltammograms of CAF at bare GCE and
PNBMGCE. It is observed that caffeine shows an irreversible oxidation
peak at around 1.53 V with an anodic peak current of 15 μA, whereas
at PNBMGCE, the anodic peak appears at 1.39 V with a peak current of
70 μA. This negative potential shift of 0.14 V and also enhancement in
current can be due to the fast electron transfer kinetics at PNBMGCE
compared to bare GCE.

3.3. Effect of pH

The effect of pH on the oxidation peak currents of 0.1 mM ACOP, TRA

and CAF was studied in PB in the range of pH 2.0 to 9.0, by CV technique
to find the optimum pH of supporting electrolyte which is best suited
for determination of ACOP, TRA and CAF and also the ratio of electrons
and protons involved by employing PNBMGCE. Fig. 5A shows the plot
of pH vs. anodic peak current (ipa) of ACOP, TRA and CAF. It was
observed from figure, that the anodic peak current of ACOP decreases
from pH 2.0 to 5.0 and then increases till pH 7.0 and again decreases
afterwards. No evident peak was observed for TRA in the pH range of
2.0–5.0, however at pH 6.0 the TRA peak appears and the peak current
was maximum at pH 9.0 and then it decreases. CAF gives a well-

Fig. 4. CVs of 0.1 mM each of (A) ACOP (B) TRA and (C) CAF at bare GCE (solid line) and at
PNBMGCE (dash + dotted line) along with CVs of PNBMGCE (dashed line) without the
analyte in 0.1 M PB (pH 7.0) at a sweep rate of 0.05 V/s.

0.15 V. This decrease in the overpotential of about 0.28 V along with en-
hanced peak current response are a clear evidence of the catalytic effect
of the PNBMGCE towards the oxidation of ACOP due to the fast electron
transfer kinetics at PNBMGCE. A small relatively less intensive redox
peak at 0.147 V (a1) and 0.12 V (c1) was also observed which may be
due to the oxidation of small amount of ACOP to N-hydroxy acetamin-
ophen and follow up electron transfer-chemical reaction (EC) or elec-
tron transfer–chemical reaction–electron transfer (ECE) mechanism
which is evident from the shape of the voltammogram [56,57].
Tramadol shows very weak irreversible oxidation behaviour at the
bare GCE. But on the surface of the PNBMGCE, a well-defined oxidation Fig. 5. (A) Effect of pH on the anodic peak current and (B) Effect of pH on the anodic peak
peak is obtained (Fig. 4B).The results show that, in comparison to bare potential of 0.1 mM each of ACOP, TRA and CAF at PNBMGCE in PB, pH range of 2.0–9.0 at a
GCE, the anodic peak potential shifts negatively from 1.0 to ~ 0.9 V, sweep rate of 0.05 V/s.
98 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103

Scheme 3. Probable mechanisms for the electrochemical oxidation of PAR, TRA and CAF.

defined oxidation peak at almost all pH values except that an extra peak 0.015 V/pH for ACOP, TRA and CAF respectively and expressed by the
appeared at higher pH values which started to merge giving a broader following regression equations,
peak. The anodic peak current values of CAF initially increased from
pH 2.0 to 3.0 and were almost stable till pH 8.0 and decreases

ACOP : Epa ðVÞ ¼ 0:756−0:045 pH r2 ¼ 0:993 ð1Þ
afterwards.
The anodic peak potential (Epa) of ACOP, TRA and CAF was found
to be dependent on pH and shifted to less positive potential with in-

TRA : Epa ðVÞ ¼ 1:5−0:084 pH r2 ¼ 0:995 ð2Þ
creasing pH, suggesting the involvement of protons in the oxidation re-
action of ACOP, TRA and CAF at PNBMGCE. From the plot of Epa vs. pH
(Fig. 5B), it is clear that the oxidation peak potential varies linearly

with pH and is shifted to more negative by 0.045, 0.084 and CAF : Epa ðVÞ ¼ 1:49−0:015 pH r2 ¼ 0:990 : ð3Þ
 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103 99

deprotonation or the adsorption of oxidation products blocking the

electrode surface at this pH range as explained by Brett et al. [8,61,62].
However, there is no proof that the electrochemical oxidation follows
the same mechanism in this pH range.
For pH values from 2.0 to 7.0 the oxidation of CAF follows the mech-
anism as given in Scheme 3. Briefly the reaction proceeds by an overall
4e−, 4 H+ process, whereas for the oxidation of TRA the probable reac-
tion mechanism may follow the proposed route given in Scheme 3 [37].
From these studies and also keeping in mind the physiological pH,
pH 7.0 was adopted for further studies.

3.4. Effect of scan rate

The effect of varying scan rates (ν) on the oxidative peak currents of
0.1 mM each of ACOP, TRA and CAF at the surface of PNBMGCE in
0.2 M PB (pH 7.0) was studied. Figs. 6A, 7A and 8A shows the cyclic
voltammetric curves of ACOP, TRA and CAF obtained in the range of
0.05 to 1.0 V/s, 0.01 to 0.25 V/s and 0.01 to 1.2 V/s respectively. The ox-
idation peak currents were observed to increase with scan rate. In order
to examine whether or not the oxidative behaviours of ACOP, TRA and
CAF was due to diffusion or adsorption, plot of logarithm of oxidation
peak current of ACOP, TRA and CAF was obtained in the above given
range. A straight line was obtained for ACOP with a slope of 0.46
(Fig. 6B), which is comparable with theoretical slope of 0.5 for diffusion
controlled process, whereas TRA and CAF showed a mixed behaviour i.e.
both adsorption controlled and diffusion controlled process with a slope

Fig. 6. (A) CVs of ACOP at different sweep rates: 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45,
0.5, 0.6, 0.7, 0.8, 0.9, and 1 V/s (i → xv) and (B) dependence of the logarithm of oxidation
peak current on the logarithm of the sweep rate in PB (pH 7.0).

The slope of these graphs should be equal to -59RT p/nF where p is

the number of protons involved in the electrode reaction, n is the num-
ber of electrons transferred and the rest are commonly known constants
[58,59]. In this study, the p/n values for ACOP, TRA and CAF were calcu-
lated as 0.86, 1.42 and 0.44 which infers that the exchange of electrons
is accompanied by exchange of protons in case of ACOP. On the basis of
this evidence and previous work on the oxidation of ACOP, the number
of electrons and protons involved is predicted to be two, a conclusion
that is consistent with that reported in the literature [28,31,56,60].
The − 45 mV/pH unit slope value of ACOP was biased from the
Nernstian two-electron, two-proton process (−59 mV/pH). This devia-
tion indicates that the electrode process is more complex and is caused
by the fact that small quantity of ACOP is oxidised to N-hydroxy acet-
aminophen [56] and the oxidation mechanism is given by Scheme 3.
In case of CAF and TRA an unequal number of protons and electrons
are involved in the oxidation process. In the case of tramadol and
caffeine, a slope of −84 or −15 mV per pH unit indicates that the elec-
trode process is more complex in the studied pH window and the ratio
of number of protons and transferred electrons is not equal to 1 and
involve more protons than electrons e.g., three protons and two elec-
trons in case of tramadol and involve less protons than electrons in
case of caffeine e.g., 4 electrons and one proton. Although the reasons
for this behaviour is unclear, this could be explained as the interference
due to uncertainties introduced by the close proximity of voltammetric Fig. 7. (A) CVs of TRA at different sweep rates: 0.01, 0.03, 0.05, 0.1, 0.15, 0.2 and 0.25 V/s
peak to the background discharge probably due to oxygen evolution as (i → vii) and (B) dependence of the logarithm of oxidation peak current on the
explained by Spatarua et al. or the product of oxidation undergoing logarithm of the sweep rate in PB (pH 7.0).
100 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103

same solution. For this purpose firstly the concentration of TRA and
CAF were kept constant while concentration of ACOP was varied from
0.2 to 16.2 μM as shown in Fig. 9A. Likewise the TRA concentration
was increased from 1.0 to 310 μM as shown in Fig. 9B in presence
of fixed concentration of ACOP and CAF and finally CAF concentration
was varied from 0.8 to 20 μM as shown in Fig. 9C in presence of fixed

Fig. 8. (A) CVs of CAF at different sweep rates: 0.01, 0.03, 0.05, 0.07, 0.1, 0.13, 0.16, 0.19,
0.25, 0.3 and 0.35 V/s (i → xi) and (B) dependence of the logarithm of oxidation peak
current on the logarithm of the sweep rate in the range 0.01 to 1.2 V/s in PB (pH 7.0).

of 0.83 and 0.81 respectively (Figs. 7B and 8B). The linear regression
equations are given by,


ACOP : logipa ¼ 0:46 logν þ 1:604 r2 ¼ 0:999 ð4Þ


TRA : logipa ¼ 0:83 logν þ 0:293 r2 ¼ 0:999 ð5Þ


CAF : logipa ¼ 0:81 logν þ 0:334 r2 ¼ 0:996 ð6Þ

Further, with increasing scan rates the anodic peak potentials of

ACOP and CAF shifts to the more positive value and cathodic peak po-
tential of ACOP shifts to more negative value, whereas the potential
shift of TRA was not linear at higher scan rates i.e. the oxidation process
becomes somewhat slow may be because of the bulkier groups being at
the closer proximity.

3.5. Determination of ACOP, TRA and CAF

The proposed method was employed for determination of ACOP, TRA

and CAF by differential pulse voltammetric method (DPV, pulse height -
0.05 V, pulse width - 0.06 s and pulse period – 0.5 s) in 0.1 M PB (pH 7.0)
at the surface of PNBMGCE at a sweep rate of 0.05 V/s. DPVs were record-
ed to evaluate the electrochemical response of different concentration of
ACOP, TRA and CAF when all the three substances were present in the
Fig. 9. (A) DPVs of ACOP at different concentrations from (i) 0.2 to (ix) 16.2 μM in the
presence of 0.1 mM each of TRA and CAF (B) DPVs of TRA at different concentrations
from (i) 1.0 to (vi) 310 μM in the presence of 0.1 mM each of ACOP and CAF and
(C) DPVs of CAF at different concentrations from (i) 0.8 to (x) 20 μM in the presence of
0.1 mM each of ACOP and TRA at PNBMGCE in pH 7.0 PB at a sweep rate of 0.05 V/s:
Inset are the respective calibration plots.
 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103 101

Table 2 Table 4
Analytical parameters for the calibration curves of ACOP, TRA and CAF. Determination of ACOP, TRA and CAF in Ultracet and Sinarest tablets (n = 3).

Analyte Linear range Sensitivity R2 LOD Sample Sample Specified Spiked Found % Recovery % RSD
(M L−1) (μA cm−2 mM−1) (μM L−1) no amount quantity (mg)
(mg) (mg)
ACOP 2.0 × 10–7 to 1.62 × 10–4 1611 0.998 0.08
TRA 1.0 × 10–6 to 3.1 × 10–5 33 0.996 0.5 Acetaminophen 1 325.0 – 309.2 95.1 3.8
CAF 8.0 × 10–7 to 2.0 × 10–5 1360 0.999 0.1 (aUltracet) 2 20.0 332.4 96.3 3.2
3 40.0 347.0 95.0 3.9
Paracetamol 1 500.0 – 485.4 97.0 2.7
concentration of ACOP and TRA. The obtained results shows that the (bSinarest) 2 20.0 494.9 95.1 3.1
3 40.0 520.0 96.3 3.0
presence of other two compounds exhibits no observable interfer-
Tramadol 1 37.5 – 35.6 95.1 3.2
ence and the oxidation processes of ACOP, TRA and CAF at (aUltracet) 2 10.0 45.8 96.5 1.9
PNBMCPE surface are independent and therefore independent mea- 3 20.0 56.0 97.3 1.3
surement of the three analytes is possible without any interference. Caffeine 1 30.0 – 28.5 95.0 2.9
The detection limits of ACOP, TRA and CAF were found to be 0.08, 0.5 (bSinarest) 2 10.0 38.8 97.1 1.4
3 20.0 48.8 97.9 1.3
and 0.1 μM, respectively. The analytical parameters obtained for their
a
determination are summarised in Table 2. These results are compared Johnson & Johnson, USA, Acetaminophen IP 325 mg and Tramadol Hydrochloride
IP 37.5 mg.
with other modified electrodes reported in the literature [Table 3]. b
Centaur Pharmaceuticals Pvt Ltd, Paracetamol IP 500 mg and Caffeine IP 30 mg.

3.6. Stability and reproducibility

Various parameters such as repeatability, reproducibility and stability
were studied for 0.1 mM ACOP, TRA and CAF in a mixture. The relative
standard deviations obtained for five replicate measurements of ACOP,
TRA and CAF in a mixture were 1.4, 2.1 and 1.9% respectively. Reproduc-
ibility is obtained by measuring the oxidation current values for similar
fresh solutions. Compared to the obtained original oxidation current
values, discrepancies of only 3.2, 4.8 and 3.4%, respectively, are observed
in the measurements with the fresh solutions prepared. Also, stability
tests for PNBMGCE were performed by measuring the current response
of 0.1 mM each of ACOP, TRA and CAF mixture over a period of one
month, and it is found that the PNBMGCE maintains 95.8% of its initial ac-
tivity under proper storage conditions. This may be because when stock
solution of NB (85% Nile blue chloride) was prepared in double distilled
water some insoluble particles were present which was further filtered
to get clear solution which might have improved the quality of monomer
and its stability during the electro polymerisation process. The weight of
the insoluble particles was considered while preparing the concentration
of the monomer.
3.7. Analytical application
In order to further evaluate the validity of the proposed method, the
contents of ACOP, TRA and CAF in drug formulations were determined
in triplicate by addition and recovery experiments using the proposed
method. Specifically ACOP and TRA in Ultracet (Acetaminophen IP
325 mg/tablet and Tramadol IP 37.5 mg/tablet) and ACOP and CAF
in Sinarest (paracetamol IP 500 mg/tablet and caffeine IP 30 mg/tab-
let) was analysed by DPV technique exactly under identical condi-
tions that were employed while recording DPV for the analysis
purpose. Solutions obtained by dissolution of these tablets were sub-
sequently diluted so that the concentration lies in the range of cali-
bration plot. The signals of the sample solutions were confirmed for
ACOP, TRA and CAF by enhancement of the peaks when their stan-
dard solutions of different concentrations were added to the sample
solutions, respectively. The results obtained are summarised in
Table 4. It was found that the results obtained by the proposed meth-
od agree well with the labelled contents. Recovery tests were also
performed and the mean results of three analysis for added ACOP,
TRA and CAF ranged from 95.06 to 97.9% with a minimum RSD.
This shows the potential applicability of PNBMGCE for the deter-
mination of ACOP, TRA and CAF in real samples without any
interference.
In addition to the active ingredients of interest, the effects of some
possible interferents such as sodium carbonate, citric acid, acetylsalicylic
acid, benzoic acid, fructose and sucrose were also investigated by addition
of these substances to the solution containing 1.0 × 10–4 M each of ACOP,
TRA and CAF by the proposed method. According to the obtained results
presence of 100 fold excess of these compounds had no influence on
the peak current of active ingredients in the proposed method (signal
change below 5%).

Table 3
Comparison of the analytical parameters for the simultaneous detection of ACOP, TRA and CAF by DPV technique in 0.1 M PB (pH 7.0) with other modified electrodes.

Electrode Analyte Dynamic range (μM) Limit of detection (μM) Reference

SWCNT modified carbon-ceramic electrode ACOP 0.2–150 0.12 [26]

N-(3,4-dihydroxyphenethyl)-3,5-dinitrobenzamide modified MWCNT ACOP 5–270 10.0 [28]
Poly (AY)/nano-TiO2/GCE ACOP 12–120 2.0 [29]
Poly (3, 4-ethylenedioxythiophene) – modified screen-printed electrodes ACOP 4–400 1.39 [30]
Aluminium electrode modified by Thin layer of palladium ACOP 100–5000 5.0 [31]
(PANI–MWCNTs) composite modified electrode ACOP 1–100 0.25 [19]
PNBMGCE ACOP 0.2–16.2 0.08 This work
Carbon nanoparticles/GCE TRA 10–1000 1.0 [32]
Glassy carbon electrode TRA 15–75 2.2 [22]
PNBMGCE TRA 1.0–310 0.5 This work
Boron-doped diamond CAF 9.7–110 7.0 [33]
Nafion/MWCNTs/GCE CAF 2.9–377 0.23 [34]
Nafion/GCE CAF 0.99–10.6 0.79 [35]
Nafion/boron-doped diamond electrode CAF 0.2–12 0.1 [25]
Poly (safranine)/GCE CAF 0.3–100 0.1 [36]
Nafion-coated GCE CAF 100–2000 0.038 [27]
PNBMGCE CAF 0.8–19.7 0.1 This work
102 S. Chitravathi, N. Munichandraiah / Journal of Electroanalytical Chemistry 764 (2016) 93–103
4. Conclusions
PNBMGCE was prepared by the electropolymerisation of Nile blue
on GCE and characterised by CV, AFM and EIS techniques. This PNB-
film exhibited a strong catalytic effect towards the electro-oxidation
of ACOP, TRA and CAF and also used for the determination of these
drugs. This method is expected to find wide application, particularly in
the search for new materials and combinations in order to reduce
adverse drug reaction and to take less medication. Moreover, good sen-
sitivity, high selectivity with low detection limits and low cost of the
sensor, makes this method very able for accurate determinations in
pharmaceutical and clinical preparations.
Acknowledgements
The authors wish to acknowledge the financial support given by Dr.
D. S. Kothari Post-Doctoral Fellowship F. 4 - 2 / 2006 (BSR) / 13 - 987 /
2013 (BSR) scheme under UGC, INDIA.
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