Novel azapropazone voltammetric sensors based on zinc oxide nanostructure

Azapropazone (AZA) is anti-inflammatory drug for treatment of arthritic conditions with low side effect. The construction and electroanalytical uses of brand-new AZA voltammetric sensor are demonstrated. Working carbon paste electrodes integrated with 5.0% ZnONPs exhibited proper catalytic effect toward oxidation of AZA molecule. At pH 5.0, AZA recorded single irreversible anodic oxidation at 0.462 V. Based on CV registered at variable conditions, oxidation of AZA molecule by one proton and two electrons of N16 with de-localized lone pair of electrons. Calibration graphs showed good linearity within AZA concentration from 19.5 to 1040.75 ng mL−1 with LOD and LOQ values of 1.67 and 5.6 ng mL−1. The presented sensor showed improved LOQ and LOD values. Also, electrooxidation mechanism of AZA molecule and EIS of sensors were reported. The proposed analytical approach was examined for sensitive voltammetric determination of azapropazone in pharmaceutical formulations and biological fluids with agreeable recoveries.


Introduction
One of the first examples of a somewhat effective anti-inflammatory is azapropazone of nonsteroidal antiinflammatory drugs class with some unique properties as successful medication in the treatment of various arthritic conditions and the gastrointestinal side effects' comparatively low frequency [1,2].The great antiinflammatory function of azapropazone takes place through its inhibitory effect on the production of tissuedestructive oxygen radicals, production of interleukin-1 by synovial tissue, accumulation and possibly degranulation of leukocytes, and release of the autolytic enzymes from lysosomal bodies [3].Azapropazone is converted by the hepatic metabolism into its 8-hydroxy (Mi307) with the same order of reactivity as that of the parent compound.
There is a growing interest in using pharmaceutical substances for the treatment of diseases and improvement of the body functions.Huge numbers of new drugs are introduced annually, and up to date, more than 100,000 dosage forms and 10,000 medicinal substances are registered worldwide [4][5][6][7].Monitoring the drug residues in pharmaceutical formulations and their metabolites of them in bodily fluids is of utmost importance for many research areas [8].Chromatographic techniques come first for monitoring medicinal substances followed by spectrophotometric ones [9][10][11].Among few analytical approaches for AZA quantification, the high-performance liquid chromatographic method [12][13][14][15] and thin-layer chromatography [16] are the most common.Oxidation of azapropazone with N-halouccinimide with the formation of a red-colored complex with an absorption maximum at 488 nm represents the sole spectrophotometric method for the determination of azapropazone in bulk powder and capsules [17].One of the main drawbacks of spectrophotometric methods is the lack of sensitivity and specificity for the analysis of biological samples.Meanwhile, chromatographic approaches offer improved sensitivity and selectivity; they operate only using sophisticated expensive instruments by expert personnel with a tedious treatment and handling of the biological samples.The stringent measuring conditions of chromatographic protocols obstacles to their application for routine analysis and onsite monitoring.Consequently, the development and validation of newly simple, sensitive and economic analytical methods for azapropazone are required.
The present work demonstrates the construction of novel carbon paste working sensors integrated with zinc oxide nanostructures for sensitive differential pulse voltammetric (DPV) determination of azapropazone in biological fluids and pharmaceutical formulations.The impact of the electroanalytical parameters on the sensor performance was illustrated elaborately and the oxidation mechanism of AZA molecule at the electrode surface was postulated with the aid of molecular orbital calculations.

Authentic drug solution
The standard azapropazone authentic [5- 300.362 g mol −1 ] was kindly provided by Delta Pharmaceutical Industries Delta Pharma Co, 10 th of Ramadan city, Egypt.The assigned purity was 99.8 ± 1.1% according to the nonaqueous potentiometric titration [46].The stock azapropazone solution was obtained via dissolving the appropriate weight of the standard AZA in water and kept in a refrigerator at 4°C.The working carbon paste sensors were constructed utilizing synthetic graphite powder and analytical grad paraffin oil (Sigma-Aldrich).Nanostructured zinc oxide ( < 50 nm, Sigma-Aldrich) was applied as an electrode modifier.BR buffer with concentration 4 × 10 −2 mol L −1 covering the pH range from 2 to 8 was adjusted with 2.0 × 10 −1 mol L −1 sodium hydroxide solution (Merck).

Samples of pharmaceutical and biological classes
Pharmaceutical formulation, Prolixan (600 mg/tablet, Delta Pharmaceutical Industries Delta Pharma Co), was obtained from local markets.Four capsules were weighed, ground and an amount equivalent to one capsule was dissolved in water, filtered and diluted to the appropriate AZA concentration.
Fresh human plasma, given by healthy volunteers, was spiked with standard azapropazone solution, treated with acetonitrile (2:1 ratio) to precipitate the sample protein, diluted up to 10 mL with water and centrifuged at 10,000 rpm.Spiked urine samples were mixed with methanol to remove protein and the AZA content in the clear supernatants of both samples was assayed voltammetrically in comparison with pharmacopoeial methods [46].

Acid and base degradation of azapropazone
The stock AZA standard solution was treated with 1 mL of 2N HCl solution followed by refluxing for 30 min at 60°C.The resultant solution was neutralized and diluted to the desired AZA concentration.The alkaline degradation process was carried out through refluxing of the stock AZA solution with 1.0 N NaOH solution for 30 min at 60°C.The resultant solution was neutralized as usual.

Electrode fabrication and measuring apparatus
The synthetic graphite powder was mixed with 10 mg ZnONPs and blended carefully with 80 μL of paraffin oil for 15 min.The electrode body was filled with the homogeneous carbon paste in accordance with other locations' detailed designs [32].A newly fresh surface was achieved simply through polishing with a wet filter paper.Voltammetric measurements were performed using 797 VA station (Metrohm, Switzerland) including the traditional three electrodes cell composed of silver/silver chloride, platinum wire (Metrohm) and homemade carbon paste as a reference, auxiliary and the working electrode, respectively.

Analytical procedures
Various increments of the AZA stock solution were added to the measuring cell at pH 5, and the DPVs were monitored at the optimized electrochemical parameters as follows: pulse amplitude 50 mV, pulse duration 40 ms, pulse width 100 ms, scan rate 40 V s −1 , voltage step 6 mV and voltage step time 0.15 s.The recorded peak heights were plotted against the AZA concentration.

Molecular orbital calculations
Molecular orbital calculations were performed to sustain the proposed AZA electrooxidation mechanism at the ZnO-integrated carbon paste surface using Gaussian 09 suite programs [47].

Results and discussion
Electroanalytical approaches ranked third following the chromatographic and spectrophotometric methods for the drug quality control and monitoring of the pharmaceutically active compound and their metabolites in biological fluids [18][19][20][21].Custom electrochemical sensors provide adequate overall performance for analyzing pharmaceutical compounds using simple instrumentation and sample pretreatment steps based on their improved sensitivity and selectivity.Nanostructured metal oxides are cable to catalyze electrooxidation of pharmaceutically and biologically active molecules; therefore, they are usually introduced as electrode modifiers to enhance the performance of the analytical method [33][34][35][36][37].Among different metal oxides, ZnO was reported as a common efficient electrode modifier for voltammetric sensors [42,[48][49][50][51][52].Thus, the working CPEs bulk integrated with ZnONPs were characterized and utilized for DPV determination of azapropazone in pharmaceutical formulations and biological fluids.

Electrooxidation of AZA at ZnONPs/CPEs
The electrochemical attitude of azapropazone was explored at the surface of CPEs integrated with different amounts of ZnO nanostructures (Figure 1(a)).On the bare CPEs, AZA molecule registered a single irreversible anodic oxidation peak at 0.512 V. Upon modification of the working electrode with 1.0% ZnONPs, more than two folds amplification of the peak current was recorded which may be attributed to the electrocatalytic effect of ZnO towards the oxidation of azapropazone at the electrode surface and the enhancement of the electroactive surface area which facilitates the electron transfer process.Gradual improvement of the peak height was monitored to reach its maximum performance at 5.0% ZnO content (Figure 1(b)).Further increase in the modifier content resulted in higher background current and distorted oxidation peak.

Surface area calculation and impedimetric characterization of electrodes
The electrochemical impedance spectroscopy (EIS) is usually carried out for a better understanding of the mass transfer process at the electrode surface and charge transfer resistance.Information about the impedance variations at the electrolyte interface and electrode through the electrochemical progression can be monitored.Herein, CPEs integrated with different concentrations of ZnO were electrochemically characterized in ferricyanide (FCN) solution (Figure 2).Noticeable gradual improvement of the faradaic current readouts was recorded upon integration with ZnO nanostructure illustrating their electrocatalytic activity (Figure 2(a)).Cyclic voltammograms were performed at variable scan rates (from 0.02 to 0.200 Vs −1 ) to evaluate the active surface area.The peak current density showed a linear dependence with the square root values of scan rate indicating the diffusion-controlled process followed in [Fe (CN) 6 ] 3-/4-system at the electrode surface.Among different tested ZnO contents, high and well-defined redox peaks were monitored at 5.0% ZnOmodified electrode.The electroactive surface area was calculated for each electrode based on the peak currents achieved at different scan rates.The Randles-Sevik equation-based estimation of the electroactive area [53] was raised from 0.032 cm 2 for the blank CPEs to 0.105 cm 2 for 5.0% ZnONPs/CPE.The ratio between the electroactive surface area (A eff ) and the geometric area (A g ) enhanced from 1.019 in the case of a bare electrode to 3.344 for the AnONPs/CPE.
The Nyquist diagrams recorded in ferricyanide solution for CPEs incorporated with different ZnONPs were illustrated in Figure 2   semicircle portion indicates the electrochemical process of electron transfer.The estimated R ct value of the bare carbon paste was found to be 380 compared with 148 for the ZnONPs/CPE.These results demonstrate that the fast electron transfer kinetics of 5.0% ZnO-modified CPE which manifested the electrocatalytic activity of ZnO modified.

Electrochemical behavior of AZA at different pH
Azapropazone molecule showed pKa value of 6.5 as referring to the enolic dione system [2], therefore, its electrochemical behavior will be pH-dependent.Cyclic voltammograms recorded at various pH values ranging from 2.0 to 8.0 were illustrated in Figure 3(a).Low peak currents were recorded at lower pH values which improved to reach the maximum values around the pKa value of the drug (pH from 4 to 6).The recorded peak height was achieved at pH 5 which was diminished to its half value at pH 7. The selected pH value is near that reported for AZA at the GCE working electrode [30].
The oxidation potential was shifted towards the negative direction against the pH value (Figure 3 The low value of the intercept indicates that there is no side reactions while the non-Nernstian slope value assumed the non-equal number of electrons and protons that involved in the electrooxidation of azapropazone [56,57].This comes in contrary to that reported by GCE [30] in which typically the Nernstian slope value with equal numbers of electrons and protons were proposed.

Effect of the scan rate
Recording cyclic voltammograms for the target analyte at different sweep rates is represented an efficient tool to investigate the electrode reaction mechanism and to estimate the number of electrons that participate in the electrooxidation reaction [55].In the present study, cyclic voltammograms were monitored for azapropazone at different scan rates ranging from 0.020 to 0.200 Vs −1 (Figure 4(a)).Improvement of the peak height and shifting of the peak potential to more positive value was observed at higher scan rates.Moreover, the recorded peak current correlated linearly (r = 0.9930) with the square root value of the scan rate assuming the irreversibility of the electrooxidation of AZA molecule at the electrode surface (Figure 4(b)).Moreover, the logarithmic value of the peak current was correlated linearly with the logarithm value of the scan rate (Figure 4(c)) with a slope value of 0.28179 μA Vs −1 (which is near to the theoretical value 0-0.5) assuming the diffusion-controlled mechanism at the electrode surface [55,56,58].This was sustained through recording cyclic voltammograms at the accumulation potentials (S1), where the peak heights remain constant.Moreover, the peak potential values registered at variable sweep rates showed a linear relationship versus logarithmic value of the sweep rate (E (V) = 0.5419 + 0.0551 [log (υ/Vs −1 )], r = 0.9871, Figure 4(d)) suggesting the participation of 2.145 electron [59] in the electrooxidation of AZA molecule compared with one electron on the GCE [30].
The best that we can tell, the present work postulates for the first time the electrooxidation mechanism of azapropazone with the aid of molecular orbital calculations (S2).It is suggested that oxidation of AZA molecules takes place through the oxidation of the tertiary amine group (N16) with the un-localized lone pair of electrons (Scheme 1).Bonding of the amino group with aliphatic side chain containing two electron donating methyl groups sustains this postulation which is nearly fitting with molecular orbital calculation computed with MOPAC software using the MP7 method indicating the charge density for each atom of the structure compound.Moreover, there is no double bond resonance compared with other nitrogen atom in the compound.Thus, the following scheme postulates the oxidation process by loss of two electrons and one proton with the formation of an iminium ion intermediate.

Linearity
The performance characteristics of the fabricated carbon paste sensors integrated with zinc oxide nanoparticles  Obtained from an average of five experiments.
were evaluated under the optimized measuring conditions mentioned above.Different ascending increments of the standard azapropazone stock solutions were added to the supporting electrolytes and the estimated peak current of the recorded differential pulse voltammograms were plotted against AZA concentration in nanogram range (Figure 5 and Table 1).Calibration graphs with high correlation coefficient (r = 0.9996) were constructed within the azapropazone concentration range from 19.5 to 1040.75 ng mL −1 .The evaluated limit of detection (LOD) and limit of quantification (LOQ) values were 1.67 and 5.6 ng mL −1 , respectively [60].Bulk modification of the working CPEs offers prolonged operational lifetime reaching one month of continuous measurements and shelf lifetime about 4 months at 4°C with acceptable efficiency and measurement reproducibility.Regeneration of the electrode surface was carried out through polishing its surface with a wet filter paper to get a newly active surface and avoid the adsorption of the AZA molecule or its oxidized form at the electrode surface [32].
Comparing the performance of the presented sensor with the sole reported voltammetric GCE sensor for determination of azapropazone, ZnONPs/CPE showed improved LOQ and LOD values in addition to the prolonged operational lifetime and reproducibility of measurements.In addition more detailed discussion about the electrooxidation mechanism of AZA molecule was reported (S3).

Selectivity
Azapropazone is converted by the hepatic metabolism into its 8-hydroxy-AZP metabolite which was secreted in urine (20% of the dose) and plasma (up to 10% of the parent substance) [61].According to the postulated oxidation mechanism, electrooxidation of azapropazone molecule takes place through oxidation of the amino group (N16), therefore it is expected that both AZA and its 8-hydroxy-AZP metabolite will give similar oxidation behavior and the introduced sensor can be utilized for assaying of both compounds.Next, trials were performed for simultaneous DPV determination of azapropazone in the presence of its acid and base degradation products (S4).None of these two degradation products recorded an oxidation peak which may be attributed to the removal of the electrochemically active tertiary amine group (N16); therefore, azapropazone can be voltammetrically assayed freely from interference of its degradation products.
Moreover, the effect of variety of excipients and contaminates that are usually present in pharmaceutical preparations on the analytical signal of azapropazone was investigated.Differential pulse voltammograms for AZA were recorded in the presence of excipients that are usually added to pharmaceutical formulations (such as starch, citric acid, glucose, propylene glycol) and certain metal ions.The tolerance limit represents the interferent concentration that resulted in the relative error ±10%.DPV peaks for the standard AZA solution in the presence and absence of the interferent species were recorded and the average recoveries for AZA were calculated as a percentage of the two peaks.Acceptable recovery values with high tolerance limit were recorded in the presence of excipients and additives while metal ions such as Fe, Co, Ni or Cu showed negative interference which may be attributed to the possible complexation among these metal ions and azapropazone [62].

Sample analysis
The achieved linearity and sensitivity of the fabricated zinc oxide nanoparticle-based sensor recommended its utilization for DPV determination of AZA in both pharmaceutical formulations and biological samples.Real azapropazone samples were spiked with known concentrations of the stock solution and assayed voltammetrically in comparison to the official methods [46].The reported high recoveries with lower relative standard deviations demonstrate the applicability of the presented analysis protocol (Table 2).

Conclusion
Herein, novel azapropazone voltammetric carbon paste sensors integrated with zinc oxide nanoparticles were constructed and fully characterized for sensitive DPV determination of azapropazone in biological fluids and pharmaceutical formulations.With enhanced sensitivity, AZA molecule was oxidized at the electrode surface with diffusion-controlled electrode reaction through the participation of two electrons and one proton as assumed by the pH, scan rate and molecular orbital calculation studies.At the optimum measuring conditions, AZA can be assayed within the linear concentration range from 19.5 to 1040.75 ng mL −1 revealing LOD value of 1.6 ng mL −1 .The fabricated sensor exhibited improved performance and can be introduced as an efficient tool for sensitive and reliable voltammetric determination of azapropazone in pharmaceutical and biological samples.

Figure 3 .
Figure 3. (a) Voltammetric behaviour of 2.0 μg mL −1 AZA recorded at different pH values and (b) peak currents and peak potential recorded at different pH values.
(b)) postulating the involvement of the proton in the oxidation of azapropazone at the electrode surface [53-55].A linear relationship was illustrated between the oxidation peak potential and the pH value of the supporting electrolyte [E (V) = 0.6590-0.0358[pH], r = −0.9906].

Figure 5 .Scheme 1 .
Figure 5.The determined differential pulse voltammetry of AZA with ZnO/CPE based sensor at pH 5.0.

Table 2 .
Differential pulse voltammetric determination of azapropazone in pharmaceutical formulations and biological samples.
a n = 3 measurements of test.a n = 5 measurements of test.a n = 3 measurements of test.