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Edge plane pyrolytic graphite as a sensing surface for the determination of
fluvoxamine in urine samples of obsessive-compulsive disorder patients
Sunita Bishnoi, Ashutosh Sharma, Rahul Singhal, Rajendra N. Goyal
PII:
S0956-5663(20)30482-6
DOI:
https://doi.org/10.1016/j.bios.2020.112489
Reference:
BIOS 112489
To appear in:
Biosensors and Bioelectronics
Received Date: 21 May 2020
Revised Date:
27 July 2020
Accepted Date: 30 July 2020
Please cite this article as: Bishnoi, S., Sharma, A., Singhal, R., Goyal, R.N., Edge plane pyrolytic
graphite as a sensing surface for the determination of fluvoxamine in urine samples of obsessivecompulsive disorder patients, Biosensors and Bioelectronics (2020), doi: https://doi.org/10.1016/
j.bios.2020.112489.
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© 2020 Published by Elsevier B.V.
Credit Authorship Contribution Statement
Sunita Bishnoi: Conceptualization, Investigation, Resources, Writing Original Draft, Writing Review & Editing
Ashutosh Sharma: Formal Analysis, Validation, Resources
Rahul Singhal: Data Curation, Resources
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Rajendra N. Goyal: Data Curation, Resources
Edge plane pyrolytic graphite as a sensing surface for the determination of fluvoxamine in
urine samples of obsessive-compulsive disorder patients
*Sunita Bishnoia, Ashutosh Sharmab, Rahul Singhalc, and Rajendra N. Goyald
a,b Department of Chemistry, Vivekananda Global University, Jaipur, 303012, India
Department of Physics, Malaviya National Institute of Technology, Jaipur, 302017 India
d
Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India
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c
*Corresponding author: E-mails:
[email protected]
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[email protected]
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ABSTRACT
There is an increasing demand for fast and sensitive determination of antidepressants in
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human body fluids because of the present scenario of rising depression cases at the global level.
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A simple and sensitive voltammetric method using edge plane pyrolytic graphite electrode
(EPPGE) as a novel sensor is presented for the determination of antidepressant fluvoxamine in
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urine and blood plasma samples of obsessive-compulsive disorder (OCD) patients. EPPGE is
delineated the first time for this determination. EPPGE exhibited strong electrocatalytic activity
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and enhanced reduction signal towards the sensing of fluvoxamine. Fluvoxamine gave a well-
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defined reduction peak at ~ – 670 mV using EPPGE. The fluvoxamine reduction peak current
was linear to its concentration in the range 5.00× 10–9 – 0.1 × 10–6 mol L−1 and the limit of
detection was found to be 3.5×10–9 mol L–1. The pre-eminence of EPPGE over mercury
electrodes has been proved in terms of sensitivity and imperative analytical parameters. The pH
study reveals the involvement of an equal number of electrons and protons in the reduction
reaction mechanism. The frequency study indicated the adsorption controlled irreversible
reaction mechanism. The stability and reproducibility of the offered sensor were also found most
favorable. The interference study confirmed the optimum selectivity of the proposed sensor. The
edge plane pyrolytic graphite sensing platform is recommended as a potential contender for the
accurate and fast determination of fluvoxamine in depression medications as well as biological
specimens of OCD patients.
Key Words: Fluvoxamine; Voltammetry; Edge Plane Pyrolytic Graphite Electrode; ObsessiveCompulsive Disorder Patients; Urine; Depression Medications
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1. Introduction
Depression is a universal disorder that influences the chemistry and function of the brain.
Depression is a major public health problem in developing as well as developed countries that
contributes to significant morbidity, disability as well as mortality along with significant
socioeconomic losses (Liu et al., 2019). Obsessive-compulsive disorder (OCD) is a type of
depression and the main symptoms of OCD include repetitive, distressing, and intrusive thoughts
and actions. The patient is unable to control these thoughts and actions. The development of
OCD is a chemical imbalance of serotonin levels in the brain (Brock and Hany, 2020).
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Antidepressants correct the dysfunction of the brain by altering the chemicals that pass signals
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along nerve routes to the brain. Selective serotonin reuptake inhibitors (SSRIs) are the most
common antidepressants usually prescribed by doctors to treat depression. These are considered
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relatively safe and cause fewer side effects than other kinds of medications used to treat
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depression (Fasipe, 2018). Fluvoxamine (I) is one of the approved SSRIs by food and drug
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administration to treat OCD, depression, anxiety, and other mood disorders. Fluvoxamine is the
only monocyclic SSRI that belongs to 2-aminoethyl oxime ether of aralkyl ketones having
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antidepressant, anti-obsessive-compulsive, and anxiolytic properties (Westenberg and Sandner,
2006). Monocyclic SSRIs have a superior cardiovascular safety profile as compared to tricyclic
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antidepressants for patients with cardiovascular disease. Hence, being a monocyclic SSRI,
fluvoxamine is safe for cardiac and aged patients (Pang and Gudi, 2019). Fluvoxamine is
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chemically designated as 5-methoxy-4´-(trifluoromethyl) valerophenone-(E)-O- (2-aminoethyl)
oxime. Fluvoxamine does not exist as optical isomers due to the absence of asymmetric carbon
in its structure. Therefore, the potentially confusing issue of stereoisomerism does not happen
with fluvoxamine (Budau et al., 2017). The drug is excreted in the urine, although predominantly
as metabolites, with a small amount of the active parent compound. As the drug is excreted in the
urine with a small amount hence, it is challenging to detect its concentration in a complex
biological media; urine and blood plasma (Schatzberg and Nemeroff, 2017). Urine remains to be
the preferred drug-screening medium, although alternate specimens like oral fluid, sweat, hair
provide unique advantages in specific testing situations (Qriouet et al., 2019). The advantages of
urinalysis include non-invasive collection, the stability of specimens, and longer detection period
for most of the drugs, allowing detection and quantization with relatively inexpensive
instrumentations (Anand et al., 2018).
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Several methods, procedures, and approaches have been developed to detect fluvoxamine
such as flow-injection chemiluminescence (Yang et al., 2017; Hassanzadeh and Amjadi 2015),
capillary electrophoresis (Lin et al., 2016), spectrophotometry (Kishore et al., 2011; Devarajan et
al., 2015), high-performance liquid chromatography (Ulu, 2006; Comission, 2009; Ohkubo et al.,
2003), amperometry (Ajayi et al., 2016), fluorimetry (Darwish et al., 2009), gas
chromatography/mass detection (Wille et al., 2007) and gas chromatography/flame ionization
detection (Berzas Nevado et al., 2000). The main drawbacks of these techniques are the uses of
tedious and time-consuming sample preparation, extraction, extract cleanup, derivatization, and
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extract storage steps. The requirement of a large amount of solvent and sample along with
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expensive types of equipment and chemicals makes these techniques not suitable for routine
analysis. Electrochemistry based voltammetric methods are an economical, rapid, and simple
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alternate for on-site determination of various organic and inorganic species (Honeychurch,
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2019). Furthermore, the voltammetric method can be used without tedious and time taking
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sample preparation, derivatization, and extraction steps which are essential for conventional
methods. These assets make voltammetric techniques suitable and favorable tools for the
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analysis of electrochemically active compounds.
However, for voltammetric techniques, the fabrication of a sensitive, selective, stable,
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and reproducible sensor is still a major challenge, especially in matrix complexity. There are
only three publications available in the literature to date for the determination of fluvoxamine by
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voltammetry. All the reported methods used mercury as a sensing surface (Nevado et al., 2000;
Nouws et al., 2005; Madrakian et al., 2015). Although mercury electrodes are good for reduction
studies due to the advantages that they offer (Vyskocil and Barek, 2009). However, the toxicity
and handling problem of these electrodes cannot be denied. It is well known that mercury in the
lowest level of its concentration is hazardous for human health owing to its bioaccumulation in
the body. The practical utility of mercury electrodes is limited by their low robustness, tough
handling, and changeable surface area of mercury drop (Chooto, 2017). Further, mercury has
limited applications in the analysis of a more positive potential range (Harvey, 2020). Edge plane
pyrolytic graphite electrode (EPPGE) has a broad linear dynamic range and improved limits of
detection and sensitivities (Yuan et al., 2013). It exhibits a lower signal-to-noise ratio, a large
potential window for use in trace analysis, and reasonable selectivity, especially in complex
biological media. Further, this electrode offers simple fabrication, easy surface renewal process,
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fast response, and flexible applications (Banks and Compton, 2005). The main objective of the
proposed study is to explore EPPGE as a sensor for the determination of fluvoxamine which has
not been reported to date. Hence, because of the increasing demand and the drawbacks of earlier
reported methods; a simple, economical, non-toxic, handy, and fast method is proposed for the
determination of fluvoxamine in depression medications and body fluids of OCD patients.
Additionally, the comparative study of the voltammetric response of edge plane pyrolytic
graphite electrode and basal plane pyrolytic graphite electrode (BPPGE) for fluvoxamine sensing
2.1 Instruments
(I)
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2. Experimental Methods
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has also been performed.
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Voltammetric experiments were performed using a voltammetric analyzer (Bioanalytical
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System) West Lafayette, IN, USA) Epsilon EC-USB attached with a three-electrode cell system.
Edge and basal plane pyrolytic graphite electrodes were used as working electrodes. Ag/AgCl
(3M NaCl) (BAS Model MF-2052 RB-5B) and platinum wire served as reference and auxiliary
electrode, respectively. All potentials are referred to the Ag/AgCl reference electrode at an
ambient temperature of 25±2 ◦C. The pH of buffer solutions was measured by Century India Ltd.
Digital pH-meter (model CP – 901) after standardization. The surface morphologies of basal and
edge plane electrodes surfaces were examined by recording SEM images using a Quanta 200 FESEM (FEI Company) instrument.
2.2 Chemicals and Analytical Procedure
Fluvoxamine (> 99.0% purity), buffer tablets of different pH, and methanol were purchased
from Sigma-Aldrich. Fluvoxamine tablets were purchased from local market medical shops of
Jaipur, India. All samples were used as received without any treatment. All reagents used for
determination were of analytical grade. Double distilled water was used for all experimental
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work. The stock solution of fluvoxamine (1.0 mM) was prepared with methanol-water (20-80
v/v) because of the low solubility of fluvoxamine in water. Phosphate buffer solutions (PBS)
[0.1M] of different pH were prepared using buffer tablets according to the reported method
(Ogawa et al., 2013). Further dilutions (calibration range/pH range) were made by diluting the
stock solution with PBS of required pH. Before recording cyclic voltammograms high-purity
nitrogen was passed in the solution for 12–15 minutes to deoxygenate the solutions. Cyclic
voltammograms were recorded applying a scan rate of 100 mVs−1 with an initial sweep to
negative potentials. The optimized parameters selected for square wave voltammetry were: initial
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E: −500 mV; final E: − 1000 mV; square wave amplitude (Esw): 25 mV; potential step (E): 4
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mV; square wave frequency (f): 15 Hz.
2.3 Fabrication of BPPGE and EPPGE
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Edge and basal plane pyrolytic graphite pieces (1 × 1 × 3 mm3) were obtained from Pfizer
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Inc. New York, U.S.A. Electrodes edge and basal plane pyrolytic graphite were prepared in the
laboratory according to the reported method (Moore et al., 2004). A pyrex glass tube of suitable
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length and diameter was cleaned and then dried. Epoxy resin was filled in glass tubes from one
end (Araldite, Ciba Geigy) up to a height of about 2 cm, with the help of a thin glass rod. Edge
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and basal plane pyrolytic graphite pieces were then placed in two different glass tubes cautiously
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from the other open end of tubes with the help of wire till 3/4th portion of the pieces gets covered
with epoxy resin to shun any air bubble between the tubes and the graphite pieces. The
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electrodes were then allowed to stand for 24 h until resin solidified. The glass tubes were rubbed
on an emery paper till the graphite become visible at the resin end. The electrodes surfaces were
washed several times with distilled water to remove fine powder sticks on the surface of the
electrodes. After filling mercury in the glass tubes copper wires were inserted to make contact of
electrodes with the outer circuit. The electrodes surfaces were finally cleaned by rubbing on
emery paper followed by washing with distilled water before using for experimental purposes.
2.4 Urine Sample Preparation
The urine samples of three OCD patients (male: 38 years, height 162 cm, 66 kg; female: 50
years, height 152 cm, 56 kg; female: 42 years, height 158 cm, 62 kg) were collected after four
hours of administration of a single dose of 100 mg fluvoxamine tablet. These samples were
collected from the Psychiatric Centre, SMS medical college, and hospital, Jaipur, India after
getting clearance from the Ethics Committee. Urine samples were collected for three regular
days from each patient. Patients were under doctor's observation and admitted to the hospital for
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treatment. The amount of 100 ml of a urine sample from each patient was acidified with 60.0 µL
hydrochloric acid to disturb the protein binding of the drug. These solutions were centrifuged
and 2 ml of supernatant from each sample was used for analysis after 100 times dilution with 0.1
M phosphate buffer solution of pH 2 to minimize matrix complexity. A urine sample obtained
from a healthy volunteer (laboratory personnel) was used as control after 100 times dilution with
0.1 M PBS of pH 2.
2.5 Pharmaceuticals Sample Preparation
Four tablets of fluvoxamine (100 mg) were purchased from pharmacy shops, the samples
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were taken with their packing and receipt. Four tablets from various companies viz. FREXT-100
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(Intas Pharmaceuticals Ltd. Bhagey Khola, Rangpo East Sikkim-737132, India, Mfg. Lic. No.:
M/517/09), Uvox 100-Abbott (Mepro Pharmaceuticals Pvt. Ltd. Unit-II Q Road, Phase IV,
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G.I.D.C. Wadhwan City – 363035 Dist- Surendranagar, Gujarat, Mfg. Lic. N0. : G/1663),
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Fluvoxin 100 (M.L.: 374/DR/Mfg/2013 Sunpharma Laboratories Ltd. Vil. Kokjhar, Mirza
Palashbari Road, P.O. Palashbari, Distt. Kamrup, Assam -781128) and Voxamin-100 (Mfg. Lic.
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No. : 300, Micro Labs Limited, 92, Sipot Industrial Complex, Hosur – 635-126, T.N. were
analyzed. After determining the average weight of each tablet, one tablet from each sample was
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trodden by pestle and mortar. Fluvoxamine powder equivalent to the average weight of one
tablet was correctly weighed for each sample and placed into different volumetric flasks of 100
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mL. After labeling all the four samples, 20.0 mL of methanol was added to each volumetric
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flask. Then to dissolve drug powder perfectly, the flask content was sonicated for ten minutes
followed by adding distilled water up to 100 mL measuring mark. The mixture of each flask was
again centrifuged for five minutes and this mixture was then filtered into fresh beakers. The
apposite volume of the filtrate was transferred into a voltammetric cell and diluted with 0.1 M
phosphate buffer solution of pH 2.0 for required concentrations.
3. Results and Discussion
3.1 Cyclic Voltammetry
Cyclic voltammetry (CV) is a versatile method for scientific exploration and advances.
Firstly, 0.1 M blank PBS of pH 2 was placed in a voltammetric cell and after purging nitrogen
the cyclic voltammogram was then recorded with a scan rate of 100 mV/s using EPPGE. The
cyclic voltammogram of blank PBS is presented as a dashed line curve in Fig. 1, which shows no
peak in the entire potential region. To prepare the required concentration of fluvoxamine (15
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µM), the requisite amounts of fluvoxamine standard solution and 0.1 M PBS of pH 2.0 were
mixed in a voltammetric cell. These solutions were purged with nitrogen for five minutes.
EPPGE was then placed into the test solution and cyclic voltammograms were recorded in the
potential range of − 1.00 V to + 1.00 V vs. Ag/AgCl with a scan rate of 100 mV/s. A sharp
reduction peak at potential ~ − 700 mV (Fig. 1, solid line curve) with adequate peak current was
observed using EPPGE. The absence of any peak on the reverse sweep indicates that
fluvoxamine reduced irreversibly at EPPGE. Although cyclic voltammetry produces imperative
information about electron transfer and reversibility of reaction but considering the relatively
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enhanced sensitivity of square wave voltammetry (SWV) for analytical purposes, SWV was used
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for the detailed analysis of fluvoxamine in PBS as well as human body fluids (complex media).
Fig. 1 Cyclic voltammograms of blank PBS (dashed line) and 15 µM fluvoxamine (solid line) at
pH 2 using EPPGE at 100 mV/s
3.2 Surface Area
The surface areas of both substrates BPPGE and EPPGE have been determined given the
imperative role of the surface area in better sensing. 1 mM K3Fe (CN)6 containing 0.1 M KCl
was used as a redox solution for the estimation of surface area. The cyclic voltammograms for
this solution were recorded at different scan rates using both BPPGE and EPPGE. A redox
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couple at both the electrodes was observed due to the Fe+3/Fe+2 redox process. The surface area
of the electrodes is calculated based on the equation;
ip = 0.4463 (F3 /RT) 1/2A n 3/2 DR1/2 C0 v1/2
(i)
where ip is peak current (Ampere), n is the number of electrons involved in overall electrode
reaction (n=1), F is faraday’s constant (96485 C/mol), A is the surface area of electrodes (cm2),
R is the universal gas constant (8.314 J/mol K), T is the absolute temperature (238 K), v is scan
rate (Vs-1), DR is diffusion coefficient (7.6 x 10-6 cm2/s), C0 is the bulk concentration of K3Fe
(CN)6 (Bard and Faulkner, 2000). In the above equation on putting the values of ip and v and the
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values of constants; the surface areas of BPPGE and EPPGE were calculated as 0.22 and 0.20
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cm2, respectively. The slopes of ip versus v1/2 plots were used to calculate the surface areas. The
surface areas for BPPGE and EPPGE are almost the same and within experimental limits. FE-
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SEM images of BPPGE and EPPGE are given in Fig. 2 which shows that the basal plane is
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rough as compared to the edge plane at which the edges of layers can be seen clearly. The SEM
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imaging also showed that EPPGE had a more porous structure than BPPGE.
Fig. 2 FE-SEM images of (a) basal plane pyrolytic graphite electrode and (b) edge plane
pyrolytic graphite electrode
3.3 Voltammetric Behavior of Fluvoxamine at BPPGE and EPPGE
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The electrochemical behavior of fluvoxamine was studied by square wave voltammetry
using both basal and edge plane pyrolytic graphite electrodes. Fig. 3 describes the square wave
voltammograms of 50 × l0−9 M fluvoxamine under optimum parameters in phosphate buffer of
pH 2 at BPPGE and EPPGE. Fluvoxamine gets reduced at ~ −740 mV (curve a) at the basal
plane whereas, at the edge plane the reduction peak potential shifts to ~ − 670 mV (curve b) with
noticeable enhancement in peak current. The peak current (peak b) at the edge plane is around
6.0 c-folds larger than the corresponding peak (peak a) at the basal plane. The decrement in
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cathodic peak potential and increment in peak current indicates that the edge plane efficiently
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electro-catalyzes fluvoxamine reduction by accelerating the electron transfer and thus improves
the electrochemical response. Edge plane pyrolytic graphite surface undergoes faster electron
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transfer than the basal plane because of the larger local density of states available for electron
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transfer. As per density functional theory calculations the HOMO and LUMO energies are
accumulated in the area of edge plane sites rather than the basal plane area. The electrochemical
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accessibility of reactive edge plane sites with high edge plane content results in increased
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electronic states density and heterogeneous electron transfer rate. This favors the current
increment and over-potential decrement in the electrochemical determinations (Kaplan et al.,
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2017). Additionally, impurities at edge planes, especially oxygen-containing functional groups
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that impetuously form in aerobic conditions, make a big impact on the local density of states
accessible for electron transfer (Goyal et al., 2010). Hence, it is concluded that the edge plane of
pyrolytic graphite is a better substrate as compared to the basal plane for the voltammetric
reduction of fluvoxamine.
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A comparison of square wave voltammograms of 50 nM fluvoxamine using BPPGE (a)
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Fig. 3
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and EPPGE (b) at pH 2. The background is shown by the dashed line using EPPGE.
The effect of varied fluvoxamine concentrations on its reduction peak current was studied
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using both edge and basal plane pyrolytic graphite electrodes. Different concentrations of
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fluvoxamine were prepared using the required amount of standard solution and 0.1 M PBS of pH
2.0 within the concentration range 5.0 × 10−9 to 0.1 × 10−6 molL−1 (EPPGE) and 30.0 × 10−9 to
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100 × 10−9 molL−1 (BPPGE) and then square wave voltammograms were recorded. As the
concentration increases the reduction peak current (ip) linearly increases at both BPPGE and
EPPGE as shown by calibration curves (a) and (b) respectively, in Fig 4. The relation between
peak current and concentration can be given by the following equations;
ip / µA = 0.122 ([fluvoxamine]/nM) + 0.03
at EPPGE
(ii)
ip / µA = 0.021 ([fluvoxamine]/nM) + 0.006
at BPPGE
(iii)
The correlation coefficients for the expressions were 0.995 and 0.982 for EPPGE and
BPPGE, respectively. The limit of detection (LOD) was calculated using the relation 3σ / b,
where σ is the standard deviation and b is the slope of the calibration curve. The obtained LODs
were 3.5 nM and 20 nM for EPPGE and BPPGE, respectively. The sensitivities found at BPPGE
and EPPGE are 0.021 and 0.122 μA nM-1, respectively as shown by the above-mentioned
regression equations. These equations indicate that the detection sensitivity at EPPGE is around
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six times higher as compared to BPPGE. In view of decreased reduction potential, wide
concentration range, low detection limit, and improved sensitivity, EPPGE has been utilized for
the detailed analysis of fluvoxamine in depression medications as well as body fluids of OCD
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patients.
(a)
(b)
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Fig. 4 Calibration plots for fluvoxamine with 5% error bar (n =3) at BPPGE (a), and EPPGE (b).
3.4 Proposed Method vs. Reported Method
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The proposed method has been compared with the earlier reported voltammetric methods in
terms of important analytical parameters. There are only three publications available in the
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literature to date for the determination of fluvoxamine by voltammetry. All the reported methods
used mercury as a sensing surface (Nevado et al., 2000; Nouws et al., 2005; Madrakian et al.,
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2015). The results of the offered method using EPPGE are noticeably better (Table 1) than the
results of the reported methods which used very expensive mercury nanoparticles-multi walled
carbon nanotubes coated glassy carbon electrode (HgNPs/MWCNTs/GCE), hanging mercury
drop electrode (HMDE) and static mercury drop electrode (SMDE) systems. The comparative
study indicates that EPPGE remarkably improves the reduction of fluvoxamine, which leads to
significant amelioration of peak current with the shift of peak potential to lower reduction values.
Consequently, a considerable improvement in sensitivity and limit of detection has been
observed. The proposed sensor also exhibited enhanced performance in terms of wide calibration
range, optimum selectivity in complex media, easy electrode preparation with surface renewal,
good stability, and reproducibility towards the sensing of fluvoxamine. Further, the offered
sensor is completely free from the changeable surface area, low robustness, toxicity and tough
handling problems of the earlier reported mercury electrodes.
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Table 1 A comparison of EPPGE with reported electrodes for the determination of fluvoxamine
HMDE
(Nevado
et al., 2000)
SMDE
(Nouws
et al., 2005)
BPPGE
(Present
Paper)
30 ×10–9 –
100 ×10–9
HgNPs/MWC
NTs/GCE
(Madrakian
et al., 2014)
0.020 × 10–6 –
1.750× 10–6
Calibration
Range
(mol L–1)
Limit
of
Detection
(mol L–1)
Limit of
Quantification
(mol L–1)
Sensitivity
(µA/ nM)
Peak Potential
(mV)
2× 10–8 –
3×10–6
6.00× 10–9 –
2.80 ×10–7
5.00× 10–9 –
0.1 × 10–6
7 ×10–9
4.7×10–9
20 ×10–9
10.0 ×10–9
3.5×10–9
2.4×10–8
1.6 ×10–8
66.6 ×10–9
0.002
0.001
0.021
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–
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760
33.3×10–9
– 740
0.024
–
EPPGE
(Present
Paper)
11.66×10–9
0.122
725
–
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Parameters
3.5 Effect of pH and Square Wave Frequency
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The square wave voltammograms were recorded in the pH range 1.5 to 4.5 to examine the
effect of pH on the electrochemical reduction of fluvoxamine. The concentration of protons will
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be decreased by increasing pH and protons will not be easily available for reduction therefore;
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the reduction will be difficult at higher pH values. The linear shift of peak potential towards
more negative values was observed with an increase in pH for the reduction of fluvoxamine at
EPPGE. The slope of the linear Ep vs. pH curve was ~ 59 mV/pH suggests the involvement of an
equal number of protons and electrons in the electrode reaction (Nosheen et al., 2012). The linear
relationship between Ep and pH can be expressed by the following equation with a correlation
coefficient of 0.994.
– Ep / mV = 550.6 + 59.65 pH vs. Ag/AgCl
at EPPGE
(iv)
The voltammograms were recorded for fluvoxamine at various frequencies using square
wave voltammetric conditions. Square wave voltammetry allows faster measurements with a
wide dynamic concentration range and increased sensitivity. It effectively eliminates the
background current from the measurements and allows voltammograms recording at higher scan
rates (Mirceski et al., 2013). The relation between reduction peak current of 10 nM fluvoxamine
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and square wave frequency (f) was studied in the frequency range 5 − 180 Hz at pH 2. The peak
current was found to increase linearly with square wave frequency and the linear relation
between ip and f can be given by the following equation having a correlation coefficient 0.992.
ip / 10-6A = 0.023 f (Hz) + 0.688
at EPPGE
(v)
The peak potential was found to shift towards more negative values with an increase in
square wave frequency. The plot of Ep versus logf was found to be linear and this relation can be
stated by the following equation with a correlation coefficient 0.990.
−Ep/mV = 144.0 log f + 506.3
at EPPGE
(vi)
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These results are following the characteristics of irreversible adsorption controlled
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electrochemical reaction (Maxakato, 2019; Massaroppi et al., 2003; Ardila et al., 2014). These
interpretations maintained the observations acquired by cyclic voltammetry studies.
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3.6 Urine Samples of OCD Patients
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The vital exploit of the proposed sensor is to determine fluvoxamine in urine samples of
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OCD patients undergoing treatment with fluvoxamine for depression. The square wave
voltammograms have been recorded for the urine samples of OCD patients, using the edge plane
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pyrolytic graphite electrode. A urine sample of a healthy volunteer was used as control. Fig. 5
shows square wave voltammogram recorded for the control urine sample exhibited no peak in
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the entire potential region (dashed line curve) and square wave voltammogram recorded for
OCD patient urine sample 1 exhibited a well-defined peak ~ − 670 mV corresponding to the
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reduction of fluvoxamine (solid line curve). The voltammograms were also recorded for the
urine samples of OCD patients, after spiking with fluvoxamine, to prove the peak at ~ − 670 mV
is due to the reduction of fluvoxamine. The peak height of the peak at ~ − 670 mV raises on
fluvoxamine spiking, thus verifying the peak at ~ − 670 mV is due to the reduction of
fluvoxamine. The concentration of fluvoxamine in the urine sample of OCD patients (Sample 1)
was calculated using the regression equation and found to be 55 nM. Three urine samples from
different patients (at similar medical prescriptions) have been analyzed to confirm the results.
Ten aliquots of a sample have been analyzed to examine the precision of the proposed method.
The relative standard deviation (RSD) was found to be less than 2.8 % that indicates the well
reproducible results.
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3.7 Depression Medications
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urine sample (solid line) at EPPGE
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Fig.5 Square wave voltammograms of a control urine sample (dashed line) and OCD patient
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The practical applicability of the proposed method was further examined by analyzing four
commercial medicinal samples having fluvoxamine. The required concentration range for all the
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samples was prepared to develop a calibration plot for fluvoxamine maleate present as the main
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content in tablet samples. The square wave voltammograms were recorded for each sample using
EPPGE at pH 2. Then by putting the peak current values in the regression equation, the quantity
of fluvoxamine in each tablet sample was calculated. Table 2 indicates that the content values
determined by the proposed method are almost the same as claimed on pharmaceutical samples.
The relative standard deviation was less than 3.8 % for three identical determinations. Thus, the
analysis of fluvoxamine in depression medications further verifies the practical utility of the
developed method.
Table 2
Sample
Frext
A comparison of experimental and labeled fluvoxamine concentration in depression
medications using EPPGE
Labelled concentration
(mg/tablet)
100
Experimental concentration
(mg/tablet)
97.5
Error (%)
− 2.5
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Uvox
100
98.2
− 1.8
Fluvoxin
100
95.6
− 4.4
Voxamin
100
96.4
− 3.6
The R.S.D. value for determination was less than 3.8 % for n=3.
3.8 Interference Study
The selectivity of a sensor plays an important role in the analytical determination of drugs
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coexisting with common excipients and matrix complexity. Interference of common species has
been tested regarding the tolerance limit for the determination of 100 nM fluvoxamine at
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EPPGE. The maximum concentration of the interfering species was taken as a tolerance limit
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that caused ±5% relative error in the determination of fluvoxamine. Interference by common
biological interferences such as glucose, lactose, arginine, carbonate, starch, K+, Na+, Mg+2, and
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Ca+2 has been examined. It was observed that there is no interfering effect of these commonly
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coexisting species of biological samples on the voltammetric response of fluvoxamine. The
results also indicated that no intervene was caused by similar antidepressants such as fluoxetine
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venlafaxine, citalopram, and sertraline in the determination of fluvoxamine. Therefore, this study
suggested that the offered sensor is selective for the determination of fluvoxamine in human
Conclusions
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body fluids and depression medications.
The unmodified edge plane pyrolytic graphite electrode is presented the first time as a newfangled sensor for the voltammetric determination of fluvoxamine in depression medications and
urine samples of OCD patients. The dominance of EPPGE over BPPGE has been proved towards
the electrochemical sensing of fluvoxamine. The accuracy of the proposed method has been
confirmed by a recovery experiment using the standard addition method. The comparative study
between EPPGE and reported mercury sensors, strongly suggested the preeminence of EPPGE in
terms of wide potential range, low limit of detection, high sensitivity, good stability, simple
preparation, easy surface renewal, and low cost. Moreover, the offered unmodified sensor is
completely free from the changeable surface area, low robustness, toxicity, and the tough
handling problem of earlier reported all three voltammetric sensing systems using HMDE
(Nevado et al., 2000), SMDE (Nouws et al., 2005) and HgNPs/MWCNTs/GCE (Madrakian et
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al., 2014). The limitation of the proposed method comprises the requirement of significant
electroactivity of the target compound. However, in view of the established dominance of
voltammetric techniques over conventional methods, the proposed sensor can be sturdily
recommended for the rapid and accurate determination of antidepressant fluvoxamine. Hence,
this sensor may have a colossal impact both on the clinical and pharmaceutical sectors. In the
future, the proposed sensor may also be used as an array to sense similar drugs in the body fluids
of different patients’ types.
Acknowledgments
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Author Dr. Sunita Bishnoi gratefully acknowledges doctors and staff of Psychiatric
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Centre, S.M.S Medical College & Hospital, Jaipur for their valuable suggestions and providing
medicines and patients’ biological samples for study. One of the authors, (AS) is thankful to
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Prof. (Dr.) Y.C Sharma, Dean R&D Cell, VGU, Jaipur, and Prof. (Dr.) S. Choudhary, VIT Jaipur
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for important scientific discussions. Authors are thankful to Material Research Centre, MNIT
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Jaipur for providing the FESEM facility and Mr. Anuj Bishnoi for his help in grammatical
corrections. Thanks are also due to Sr. Technician Mr. Ramesh Verma, VIT Jaipur for his help in
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Highlights
Edge plane pyrolytic graphite electrode as a sensor for fluvoxamine determination
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Fluvoxamine determination in urine samples of obsessive-compulsive disorder patient
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Electrochemical study of depression medications by square wave voltammetry at EPPGE
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The dominance of edge plane pyrolytic graphite electrode over mercury electrodes
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Low cost, fast, and accurate bio-detection method for electroactive antidepressants
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Declaration of competing interests: NONE
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The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.