A Simply Fabricated Electrochemically Pretreated Glassy Carbon Electrode for Highly Sensitive Determination of Clonazepam by Adsorptive Cathodic Stripping Voltammetry

Salinee Khoka; Kritsada Samoson; Janjira Yodrak; Adul Thiagchanya; Apichai Phonchai; Warakorn Limbut

doi:10.1149/1945-7111/abfe45

Clonazepam is a sedative and anticonvulsant drug of the benzodiazepine group which has been widely prescribed as a remedy for anxiety, certain types of migraines, seizures, panic disorders, sleep disturbance and the treatment of status epilepsy in both children and adults.^1,2 Common side effects of clonazepam include fatigue, lethargy and drowsiness. High clonazepam dosages can cause dizziness, blurred vision, impaired motor coordination, euphoria, mood swings, slurred speech and vertigo as well as hostile behavior in certain cases.³ Clonazepam has been used illegally due to its widespread availability and side effects^4–6 and has also been used in cases of sexual assault.⁷ Therefore, a simple, fast and sensitive method of determining clonazepam is extremely important.

Developed analytical techniques for the measurement of clonazepam include gas chromatography,⁸ high-performance liquid chromatography,⁹ immunoassays,¹⁰ chemiluminescence,¹¹ spectrophotometry¹² and electrochemical techniques.^13–15 The electrochemical method is an efficient approach due to its simplicity, fast response, low cost and high sensitivity.^16,17 Adsorptive stripping voltammetry (ASV) is a particularly attractive electrochemical technique that combines two steps: preconcentration and stripping. In the preconcentration step, the target analyte accumulates on the electrode surface, which increases sensitivity and lowers the detection limit.^18,19 The various working electrodes used in recent electrochemical studies have included boron-doped diamond electrodes, gold electrodes, carbon paste electrodes, platinum electrodes and glassy carbon electrodes. In particular, the glassy carbon electrode (GCE) is extensively used as a working electrode due to its wide potential window, low background current, hardness, low cost and easily modified surface.^20,21 However, GCEs have some limitations. Their surface area is small and electron transfer can be sluggish, leading to poor sensitivity.^17,21

To improve the performance of GCEs, pretreatment is the most interesting option. Several pretreatment methods have been proposed during the last decade, including in situ laser irradiation,²² radio frequency plasma²³ and electrochemical pretreatment.^24,25 One of the most common activation techniques is electrochemical pretreatment. It is environment-friendly, fast, simple, and inexpensive. Furthermore, electrochemical pretreatment roughens the surface of the electrode and generates the functional groups of graphite oxide, which contain oxygen.^24–27 During the preconcentration step of ASV, these functional groups can enhance the adsorption of clonazepam on the surface of the electrode via π–π interaction and hydrogen bonding. The improved adsorption of the analyte can improve the detection limit and sensitivity of the sensor.

This work is the first report of an easily prepared electrochemically pretreated glassy carbon electrode (EPGCE) for highly sensitive determination of clonazepam. The EPGCE functioned as an adsorbent and a working electrode for the detection of clonazepam by adsorptive cathodic stripping voltammetry (AdCSV). The surface morphology and electrochemical properties of the GCE and EPGCE were studied and compared. The analytical determination of clonazepam was carried out by AdCSV. The operational conditions were optimized and applied during the measurement of clonazepam. The repeatability of the sensor for detection of clonazepam in beverage samples was evaluated.

Materials and Methods

Materials

Clonazepam was from clonaril®. Perchloric acid and potassium persulfate were from Sigma-Aldrich (St. Louis, USA). Sulfuric acid and sodium hydroxide were from Merck KGaA (Darmstadt Germany). Dipotassium hydrogen phosphate and potassium dihydrogen phosphate were from Ajax Finechem. Deionized water (18.2 MΩ cm⁻¹) (Barnstead^TM Easy Pure^TM II water purification system, Thermo Scientific^TM, USA) was used in the preparation of all solutions.

Apparatus

The surface morphology of the GCE and the EPGCE were studied by energy dispersive X-ray spectroscopy (EDX) (Quanta 400, FEI, USA). All electrochemical techniques were evaluated using the Autolab 910 PSTAT Mini controlled by the PSTAT software. The conventional three-electrode system comprised a platinum wire auxiliary electrode, an Ag/AgCl reference electrode and a fabricated EPGCE as the working electrode.

Electrode preparation

Prior to pretreatment, the GCE was polished carefully to a mirror finish with a sequence of 1.5, 0.5, and 0.05 μm alumina slurries. The polished GCE was ultrasonically washed with double deionized water for 5 min and dried with nitrogen gas. The polished GCE was electrochemically pretreated for 300 s in 5.00 mmol l⁻¹ potassium persulfate at a constant oxidative potential of 1.90 V (Fig. 1).

**Figure 1.** The schematic of the EPGCE preparation.
Download figure:
Standard image High-resolution image

Electrochemical measurements

The electrochemical properties of the GCE and EPGCE were determined in 0.10 mol l⁻¹ phosphate buffer solution (PB) at pH 7 using cyclic voltammetry (CV) from −0.90 to 0.40 V vs Ag/AgCl at a scan rate of 0.10 V s⁻¹. Determination by AdCSV was carried out in 0.10 mol l⁻¹ PB at pH 7 using two procedures: preconcentration and stripping. During preconcentration, clonazepam molecules were adsorbed for a set time at a set potential on the electrode surface. The preconcentration potential and time were optimized. During stripping, clonazepam molecules were stripped from the surface of the working electrode and the cathodic current response of clonazepam was recorded using linear sweep voltammetry from −0.20 to −0.90 V vs Ag/AgCl at a scan rate of 0.10 V s⁻¹.

Real sample analysis

Clonazepam solutions were prepared using three beverage samples from a local supermarket. For electrochemical detection, each beverage sample containing clonazepam was spiked into the electrochemical cell, which contained 0.10 mol l⁻¹ PB at pH 7 to obtain final clonazepam concentrations of 0.10, 0.25, 0.50, 0.75 and 1.00 mg l⁻¹. To determine the matrix effects, the sensitivity of the spiked curves and standard curves were compared using two-way ANOVA.

Result and Discussion

Characterization

The presence of oxygen-containing functional groups on the EPGCE surface was confirmed by EDX. EDX spectra of the GCE (Fig. 2A) and EPGCE (Fig. 2B) showed that the oxygen signal of the EPGCE was stronger, and therefore oxygen-containing functional groups had been successfully produced on the EPGCE surface.

**Figure 2.** EDX spectra of (A) GCE and (B) EPGCE: Cyclic voltammograms of the GCE and the EPGCE at a scan rate of 0.10 V s⁻¹ in 0.10 mol l⁻¹ PB pH 7 (C) without clonazepam and (D) with 5.00 mg l⁻¹ clonazepam: (E) Cyclic voltammograms of the electrochemical reduction of 5.00 mg l⁻¹ clonazepam at different scan rates (0.020–0.20 V s⁻¹): (F) Plot of cathodic currents vs scan rates using the EPGCE in 0.10 mol l⁻¹ PB pH 7 containing 5.00 mg l⁻¹ clonazepam.
Download figure:
Standard image High-resolution image

Electrochemical behavior

The electrochemical properties of the GCE and the EPGCE were determined by CV in 0.10 mol l⁻¹ PB at pH 7. The EPGCE (Fig. 2C (trace b)) produced a larger background current than the GCE (Fig. 2C(trace a)) because the EPGCE had a larger surface area due to the rough surface created during electrochemical pretreatment.²⁸ When 5.00 mg l⁻¹ of clonazepam were introduced into the 0.10 mol l⁻¹ PB, the GCE (Fig. 2D (trace a)) showed no current peak response from clonazepam. This was likely because clonazepam was not adsorbed on the GCE surface. However, when the EPGCE (Fig. 2D (trace b)) was tested, three peak currents were clearly observed. These responses included the cathodic peak of the reduction of the 7-nitro group (NO₂) of clonazepam to a hydroxylamine group (NHOH) with an associated loss of water at -0.56 V (peak I); the anodic peak of the oxidation of the hydroxylamine group to a nitroso group at around -0.05 V (peak II); and the cathodic peak of the reduction of the nitroso group back to a hydroxylamine group at −0.11 V (peak III). The electrochemical mechanism of clonazepam reduction has been widely proposed by several reports.^29,30 However, only the cathodic peak at −0.56 V was used in the determination of clonazepam by AdCSV because it is responsible for the reduction of the 7-nitro group of clonazepam, which is highly reproducible and sensitive.¹⁴

Effect of scan rate

The electrochemical behavior of a solution of 5.00 mg l⁻¹ of clonazepam in 0.10 mol l⁻¹ PB pH 7 was investigated at the EPGCE by CV at different scan rates from 0.020 to 0.20 V s^{− 1} (Fig. 2E). The cathodic peak current for the reduction of clonazepam was found to be linearly correlated with the scan rate (v). The linear regression equation was I_pc (μA) = (271 ± 6) v (V s⁻¹) + (4 ± 1) (μA) and the correlation coefficient = 0.9978 (Fig. 2F). The results confirm that the electrochemical reduction of clonazepam at the EPGCE was an adsorption-controlled process.^6,13,30

Optimization

In order to achieve the optimal electrochemical reduction of clonazepam at the EPGCE, the operational conditions of the system were optimized. All parameters were evaluated with 0.10, 0.25, 0.50, 0.75 and 1.00 mg l⁻¹ clonazepam standards in 0.10 mmol l⁻¹ PB at pH 7. Parameters that provided the highest sensitivity were selected as the optimal conditions.

The electrochemical pretreatment

Substances.—The effect of the medium used in electrochemical pretreatment was evaluated by determining electrode sensitivity toward clonazepam after pretreatment of the electrode with 0.10 mol l⁻¹ of each chemical pretreatment substances (i.e., sodium hydroxide (NaOH), nitric acid (HNO₃), perchloric acid (HClO₄), potassium persulfate (K₂S₂O₈), and sulfuric acid (H₂SO₄)) at a constant oxidative pretreatment potential at 1.80 V for 300 s. The results demonstrated that electrochemical pretreatment with potassium persulfate provided the highest sensitivity (Fig. 3A). This was because potassium persulfate can produce of the oxygen-containing functional groups of graphite oxide on electrode surface, which confirm by EDX results as shown in Supporting Material Fig. S1 (available online at stacks.iop.org/JES/168/057513/mmedia). Therefore, the electrodes pretreated with potassium persulfate were used in subsequent experiments.

**Figure 3.** The sensitivity (0.10–1.00 mg l⁻¹ clonazepam) of (A) sensors electrochemically pretreated for 300 s with 10.00 mmol l⁻¹ concentrations of different substances (oxidative pretreatment potential, 1.80 V; preconcentration potential, −0.40 V; preconcentration time, 180 s): (B) of sensor pretreated with 10.00 mmol l⁻¹ potassium persulfate for 300 s at different oxidative pretreatment potentials (preconcentration potential, −0.40 V; preconcentration time, 180 s): (C) of sensor pretreated with 10.00 mmol l⁻¹ potassium persulfate at 1.90 V for different pretreatment times (preconcentration potential, −0.40 V; preconcentration time, 180 s): (D) of sensor pretreated at 1.90 V for 300 s with different concentrations of potassium persulfate (preconcentration potential, −0.40 V; preconcentration time, 180 s): (E) sensitivity (0.10–1.00 mg l⁻¹ clonazepam) of sensor pretreated at 1.90 V for 300 s with 5.00 mmol l⁻¹ potassium persulfate at different preconcentration potentials (preconcentration time, 180 s) and (F) at different preconcentration times (preconcentration potential, −0.30 V).
Download figure:
Standard image High-resolution image

Oxidative pretreatment potential.—The oxidative potential used to electrochemically pretreat the GCE was an important factor affecting the adsorption and electrochemical reduction of clonazepam because it affected the surface roughening of the electrode. The effect of oxidative pretreatment potential was investigated at 1.70, 1.80, 1.90, and 2.00 V. The sensitivity increased simultaneously and then gradually decreased above 1.90 V (Fig. 3B). This because the optimal oxidative pretreatment potential of persulfate can be affected the surface roughening of the electrode, which can indicate by the SEM images of the electrode surface before (Supporting Material Fig. S2A) and after the pretreatment (Supporting Material Fig. S2B). From SEM images, the GCE surface appeared as a large rough surface area after pretreatment with persulfate. Moreover, to evaluate the electroactive surface area of the electrodes before and after the pretreatment, the CV information in Supporting Material Fig. S3 was applied to calculate the electroactive surface area via the Randle-Sevcik equation. The electroactive surface area of 0.10 cm² and 0.16 cm² was calculated for the unpretreated electrode and the pretreated electrode, respectively, indicating the increasing electrode surface area after persulfate pretreatment. Therefore, an oxidative potential of 1.90 V was utilized for subsequent experiments.

Pretreatment time.—To study the production of a rough surface and oxygen-containing functional groups of graphite oxide on the electrode, the pretreatment time was varied from 0 to 600 s (Fig. 3C). The results indicated that sensitivity increased with pretreatment time and reached a maximum at 300 s. Increments of pretreatment time increased the roughness of the electrode surface and the number of oxygen-containing functional groups of graphite oxide on the electrode. During preconcentration, these changes could increase the amount of clonazepam adsorbed onto the EPGCE and therefore, signal sensitivity. The constant state of sensitivity at electrodes pretreated for longer than 300 s was likely because all the active sites on the GCE surface were already oxidized³¹ at 300 s. As a result, the surface roughness and the number of oxygen-containing functional groups of graphite oxide remained the same and adsorption of clonazepam during preconcentration was unchanged. Therefore, the pretreatment time of 300 s was chosen as the optimal condition.

Potassium persulfate concentration.—The effect on the GCE surface of electrochemical pretreatment with different potassium persulfate concentrations was determined from 0 to 50.00 mmol l⁻¹ (Fig. 3D). The sensitivity of the current response was highest at the EPGCE pretreated with 5.00 mmol l⁻¹ potassium persulfate and remained the same at higher concentrations. Therefore, 5.00 mmol l⁻¹ potassium persulfate was selected as the optimal condition for all subsequent experiments.

Preconcentration potential and preconcentration time

The preconcentration procedure is particularly important for electrochemical reduction in the AdCSV technique because it can improve the detection limit and sensitivity of the system by increasing the adsorption of target molecules on the electrode surface. The effect of preconcentration potential was evaluated by varying the voltage applied to the working electrode from −0.50 to −0.10 V (Fig. 3E). The highest sensitivity was produced at −0.30 V. The influence of preconcentration time was investigated between 180 and 540 s (Fig. 3F). The sensitivity of the signal increased as the preconcentration time increased from 180 to 360 s and then gradually decreased beyond this time. Therefore, the preconcentration potential of −0.30 V and preconcentration time of 360 s were used in further studies.

Analytical performance

Linearity, limit of detection and limit of quantification

Under the optimal conditions, the cathodic response increased linearly with the increasing concentration of clonazepam up to 1.50 mg l⁻¹. Linearity was in the range of 0.0250 to 1.50 mg l⁻¹ (Fig. 4A). Figure 4B shows the cathodic response to different concentrations of clonazepam. The sensitivity of the developed sensor against electrode surface area was 453 μA mg⁻¹ l cm⁻². The detection and quantitation limits were 19 and 63 μg l⁻¹, which were calculated based on 3 and 10 standard deviations of the intercept divided by the slope of the calibration curve, respectively. When compared with other electrode systems for the detection of clonazepam (Table I), the developed clonazepam sensor demonstrated a wide linearity and the lowest detection limit. The excellent analytical performances were most likely due to the electrochemical activation that produced a rough surface and oxygen-containing functional groups of graphite oxide on the electrode. The pretreatment increased clonazepam adsorption in the preconcentration step of AdCSV.

**Figure 4.** (A) The calibration curve of cathodic current vs clonazepam concentration: (B) Adsorptive cathodic stripping voltammograms at different concentrations of clonazepam: The repeatability of the EPGCE preparation for (C) the same electrode and (D) six different electrodes.
Download figure:
Standard image High-resolution image

Table I. Comparison of the analytical performances of the developed sensor and other electrode fabrications for the detection of clonazepam.

Modified electrode	Technique	Linear range (mg l⁻¹)	LOD (μg l⁻¹)	Sample	References
SFs−IL/GCE^{^a)}	DPV^{^g)}	0.0316–78.9	20.84	Pharmaceutical tablets, serum and urine samples	15
CNFs/CNPs/GCE^{^b)}	LSV^{^h)}	0.0316–3.16	25.26	Pharmaceutical tablet and human serum samples	32
MIP/CPE^{^c)}	Potentiometry	0.0316–31,572	230.47	Clonazepam tablets and synthetic serum	33
MWCNTs/ZnONPs/CPE^{^d)}	DPV	0.390–7.70	173	Human urine	14
SPCE^{^e)}	DPV	2.05–8.00	1960	Beverage and serum samples	6
EPGCE^{^f)}	^ⁱ)AdCSV	0.0250–1.50	19	Beverage samples	This work

^a)SFs−IL/GCE: Silver fibers and Ionic liquid composite modified glassy carbon electrode.^b)CNFs/CNPs/GCE: Cellulose nanofibers/carbon nanoparticles nanocomposite modified on glassy carbon electrode.^c)MIP/CPE: Molecular imprinted polymer modified carbon paste electrode.^d)MWCNTs/ZnONPs/CPE: Multi-walled carbon nano tubes immobilized with zinc oxide nano particles modified carbon paste electrode.^e)SPCE: Screen-printed carbon electrode.^f)EPGCE: Electrochemically pretreated glassy carbon electrode.^g)DPV: Differential pulse voltammetry.^h)LSV: Linear sweep voltammetry.ⁱ⁾AdCSV: Adsorptive cathodic stripping voltammetry.

Repeatability

The repeatability of a single EPGCE and six different EPGCEs was evaluated by measuring the cathodic responses of clonazepam standards at 0.10, 0.25, 0.50, 0.75 and 1.00 mg l⁻¹. Using the single EPGCE, six sets of measurements were determined for each concentration (n = 3). The relative standard deviations (RSDs) of all concentrations ranged from 1.52% to 5.55%, which were acceptable results according to the AOAC guidelines of 15% and 11% for 0.1 mg l⁻¹ and 1 mg l⁻¹, respectively (Fig. 4C). When using six different EPGCEs for each concentration, the RSDs ranged from 3.00% and 6.98%, which were well within the acceptance limits (Fig. 4D). The results indicated that the developed clonazepam sensor had good repeatability.

Real sample analysis

Under the optimum conditions, the fabricated sensor was used to measure clonazepam in three beverages: Full Moon, Smirnoff and drinking water. Recoveries were evaluated by spiking beverage samples containing clonazepam into the detection medium (0.10 mol l⁻¹ PB pH 7). To determine the matrix effects, the sensitivity of the spiked curves and standard curves were compared using two-way ANOVA. Since the two slopes showed no significant difference (P > 0.05) (Supporting Material Fig. S4), there was no matrix effect and therefore the clonazepam concentration in beverage samples could be calculated from the standard calibration curve. The recovery values from the determination of clonazepam ranged from 98 ± 2 to 102 ± 4% (Table II). Since these values are acceptable according to AOAC recommendations, the developed sensor has the potential to be applied for the determination of clonazepam in beverages.

Table II. The measurement of clonazepam in three beverage samples and the recoveries.

Beverage	Clonazepam spike (mg l⁻¹)	Clonazepam measurement (mg l⁻¹)	%Recovery	%RSD
Full Moon	0.10	0.099 ± 0.003	99 ± 3	2.98
	0.50	0.50 ± 0.01	100 ± 2	2.27
	1.00	1.01 ± 0.02	101 ± 2	1.55
Smirnoff	0.10	0.099 ± 0.003	100 ± 3	2.61
	0.50	0.50 ± 0.02	101 ± 4	3.82
	1.00	0.98 ± 0.02	98 ± 2	2.46
Water	0.10	0.099 ± 0.004	100 ± 4	4.11
	0.50	0.51 ± 0.02	102 ± 4	4.25
	1.00	1.00 ± 0.02	100 ± 2	1.87

Conclusions

The simple fabrication of an electrochemically pretreated glassy carbon electrode for highly sensitive determination of clonazepam was successfully demonstrated. The electrochemically pretreated glassy carbon electrode was prepared by a simple method. The rough surface and oxygen-containing functional groups of the electrochemically pretreated glassy carbon electrode enhanced the adsorption of clonazepam in the preconcentration step of adsorptive cathodic stripping voltammetry. The enhanced absorption of the target analyte improved the sensor performance for the determination of clonazepam. Under optimal conditions, the electrochemically pretreated glassy carbon electrode achieved excellent adsorption and electrochemical reduction of clonazepam, demonstrating a wide linear range, high sensitivity (453 μA mg⁻¹ l cm⁻²), low limit of detection (19 μg l⁻¹), low limit of quantification (63 μg l⁻¹), good repeatability and good recovery . Furthermore, the sensor was successfully used for the measurement of clonazepam in beverage samples.

Acknowledgments

This project is funded by National Research Council of Thailand (SCI6305166e). Financial support from the Forensic innovation center, the Center of Excellence for Innovation in Chemistry (PERCH-CIC), the Office of the Higher Education Commission, the Center of Excellence for Trace Analysis and Biosensors (TAB-CoE), Division of Health and Applied Sciences, Graduate School and Faculty of Science, Prince of Songkla University, Hat Yai, Thailand are all gratefully acknowledged. Thanks also to Thomas Duncan Coyne, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand for assistance with the English.

A Simply Fabricated Electrochemically Pretreated Glassy Carbon Electrode for Highly Sensitive Determination of Clonazepam by Adsorptive Cathodic Stripping Voltammetry

Article metrics

Permissions

Author e-mails

Author affiliations

ORCID iDs

Dates

Abstract