Scholarly article on topic 'Electrochemical determination of an anti-hyperlipidimic drug pitavastatin at electrochemically pre-treated polymer film based electrochemical sensor'

Electrochemical determination of an anti-hyperlipidimic drug pitavastatin at electrochemically pre-treated polymer film based electrochemical sensor Academic research paper on "Chemical sciences"

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{Pitavastatin / "Electrochemical sensor" / "Adsorptive stripping voltammetry" / "Biological fluids" / "Pharmaceutical formulations"}

Abstract of research paper on Chemical sciences, author of scientific article — Umar J. Pandit, Gowhar A. Naikoo, Mehraj Ud Din Sheikh, Gulzar A. Khan, K.K. Raj, et al.

Abstract An electrochemically pretreated silver macroporous (Ag MP) multiwalled carbon nanotube modified glassy carbon electrode (PAN-Ag MP-MWCNT-GCE) was fabricated for the selective determination of an anti-hyperlipidimic drug, pitavastatin (PST). The fabricated electrochemical sensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The fabricated electrode was employed in quantifying and determining PST through differential pulse adsorptive stripping voltammetry (DPAdSV) and CV. The electrode fabrication proceeded with remarkable sensitivity to the determination of PST. The effect of various optimized parameters such as pH, scan rate (ν), accumulation time (tacc), accumulation potential (Uacc) and loading volumes of Ag MP-MWCNT suspension were investigated to evaluate the performance of synthesized electrochemical sensor and to propose a simple, accurate, rapid and economical procedure for the quantification of PST in pharmaceutical formulations and biological fluids. A linear response of PST concentration in the range 2.0×10−7–1.6×10−6 M with low detection (LOD) and quantification (LOQ) limits of 9.66±0.04nM and 32.25±0.07nM, respectively, were obtained under these optimized conditions.

Academic research paper on topic "Electrochemical determination of an anti-hyperlipidimic drug pitavastatin at electrochemically pre-treated polymer film based electrochemical sensor"

Author's Accepted Manuscript

Electrochemical determination of an anti-hyperlipidimic drug pitavastatin at electrochemically pre-treated polymer film based electrochemical sensor

Umar J. Pandit, Gowhar A. Naikoo, Mehraj Ud Din Sheikh, Gulzar A. Khan, K.K. Raj, S.N. Limaye

www.elsevier.com'locate/jpa

PII: S2095-1779(17)30020-5

DOI: http ://dx. doi. org/ 10.1016/j .jpha.2017.03.002

Reference: JPHA351

To appear in: Journal of Pharmaceutical Analysis

Received date: 27 June 2016 Revised date: 5 March 2017 Accepted date: 12 March 2017

Cite this article as: Umar J. Pandit, Gowhar A. Naikoo, Mehraj Ud Din Sheikh Gulzar A. Khan, K.K. Raj and S.N. Limaye, Electrochemical determination o an anti-hyperlipidimic drug pitavastatin at electrochemically pre-treated polyme film based electrochemical sensor, Journal of Pharmaceutical Analysis http://dx.doi.org/10.1016/jjpha.2017.03.002

This is a PDF file of an unedited manuscript that has been accepted fo publication. As a service to our customers we are providing this early version o the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form Please note that during the production process errors may be discovered whic could affect the content, and all legal disclaimers that apply to the journal pertain

Electrochemical determination of an anti-hyperlipidimic drug pitavastatin at electrochemically pre-treated polymer film based electrochemical sensor

Umar J. Pandita*, Gowhar A. Naikoob, Mehraj Ud Din Sheikha, Gulzar A. Khana, K.K. Raja, S.N. Limayea

aDepartment of Chemistry, Dr. Harisingh Gour University, Sagar (M.P.)

^Department of Mathematics and Sciences, College of Arts and Applied Sciences, Dofar University, Salalah, Oman

umarche@gmail.com gowhar@du.edu.om

Corresponding author. Umar Jan Pandit

Abstract

An electrochemically pretreated silver macroporous (Ag MP) multiwalled carbon nanotube modified glassy carbon electrode (PAN-Ag MP-MWCNT-GCE) was fabricated for the selective determination of an anti-hyperlipidimic drug Pitavastatin (PST). The fabricated electrochemical sensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The fabricated electrode was employed in quantifying and determining PST through differential pulse adsorptive stripping voltammetry (DPAdSV) and CV. The electrode fabrication proceeded with remarkable sensitivity towards the determination of PST. The effect of various optimized parameters such as pH, scan rate (v), accumulation time (tacc), accumulation potential (Uacc) and loading volumes of Ag MP-MWCNT suspension were investigated to evaluate the performance of synthesized electrochemical sensor and to propose a simple, accurate, rapid and economical procedure for the quantification of PST in pharmaceutical formulations and biological fluids. A linear response of PST concentration in the range 2.0 x 10-7 M to 1.6 x 10-6 M with low detection (LOD) and quantification (LOQ) limits of 9.66±0.04 nM and 32.25±0.07 nM, respectively were obtained under these optimized conditions.

Keyword: Pitavastatin; Electrochemical sensor; Adsorptive stripping voltammetry; Biological fluids; Pharmaceutical formulations

1. Introduction

In recent years, the improvement in electrochemical techniques have led these techniques to be used in the field of pharmaceutical, environmental and biological sample analysis predominantly because of their high sensitivity, low instrumentation cost with relatively shorter analysis time and removal of tedious extraction procedures, as compared with other analytical techniques [1]. Electrochemical techniques have proven to be advantageous for developing very sensitive and selective methods for the determination of pharmaceutical, organic molecules and metal ions in samples of diverse origin [2]. Electrochemical techniques have the advantage of determining the biomolecular interactions and electrode mechanism of pharmaceuticals which provide an insight of the metabolic fate of drug molecules [3]. Electrochemical methods, such as differential pulse voltammetry (DPV), stripping analysis and square wave voltammetry (SWV), have higher sensitivity which made possible to trace analytes as low as Pictogram level with shorter analysis time as compared to the time consuming chromatographic methods [4]. The features that prove the dominance of DPV over other electroanalytical techniques are the rapid speed of analysis, lower consumption of electroactive species and less problems with fouling of the electrode surface [5].

The electro-analytical techniques have gained more importance with the discovery of carbon nanotubes (CNTs) [6]. The use of CNTs as electrochemical sensors in view of their unique geometrical, mechanical, electronic and chemical properties has gained considerable attention [7]. The advantage of CNTs based sensors has been the target of a large variety of applications, predominantly for solid-state chemical and biological sensors [8, 9]. A number of pharmaceutical molecules have been determined and quantified at electrode surfaces modified with CNTs [10-15]. The use of CNTs as electrode material has resulted in low detection limit, high sensitivity, reduction of overpotential and resistance to surface blockage [16].

Another advancement in electrochemical techniques have been the use of chemically modified electrodes (CMEs) that has been widely considered as a sensitive and selective analytical sensors for determination of trace amounts of biologically important and environmentally toxic compounds [17-19]. CMEs are bestowed with the ability to catalyze the electrode process by significantly decreasing the needed overpotential. These electrodes are capable of enhancing the selectivity in the electrochemical techniques by selective interaction of electron mediator with the analyte in a coordinating fashion [20-22].

Pitavastatin, 7-[2-Cyclopropyl-4-(4-fluoro-phenyl)-quinolin-3-yl]-3,5-dihydroxy-hept-6-enoic acid is an anti-hyperlipidemic drug that work by inhibiting conversion of HMG-CoA to mevalonic acid in the hepatocyte, through competitive blockade of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis [23, 24]. The quantification of pitavastatin has been reported by many analytical techniques [25-29]; however these methods are either costlier or time consuming. The present study was focused on developing a simple, economical and highly sensitive method for the quantification of pitavastatin in pharmaceutical formulations and biological samples.

2. Experimental

2.1 Apparatus and Reagents

All the electrochemical measurements were performed at ambient temperature of 298 K (250C) on a computer controlled NOVA software version 1.10.1.9 Metrohm Autolab B.V. PGSTAT128N equipped with a conventional three electrode system consisting of an Ag/AgCl (saturated KCl) reference electrode, platinum wire as counter electrode and bare and modified glassy carbon electrode (GCE) as working electrode. pH measurements were performed with Systronic digital ^pH meter model-361.

Pitavastatin (PST) was purchased from Genetix Biotech Asia (P) Limited (New Delhi, India)

and used without further purification. Standard stock solution of PST (5 x 10-3 mol/L) was prepared in methanol and stored under refrigeration. MWCNTs (surface area < 200 m /g) were purchased from Sigma and used without further pre-treatment. 0.1 M phosphate buffer of

varying pH was prepared by dissolving appropriate volumes of 0.1 M each Na2HPO4 and NaH2PO4. The pH adjustments were achieved by 0.1 M HCl for lower pH and 0.1 M NaOH for higher pH. Pitava containing 2 mg PST each tablet (Zydus Cadila, India) and Flovas having 1 mg PST strength per tablet (IPCA Laboratories Ltd., India) were purchased from local pharmacy. All other chemicals and reagents used were of analytical grade. Double distilled water was used in all experimental measurements.

2.2 Preparation of modified electrodes

Silver macroporous monolith was synthesized via a reported [30] sol-gel method (supplementary). An equal weight of this synthesized Ag MP material and MWCNTs (5 mg each) were dissolved in 10 mL DMF and sonicated for 30 minutes to get a homogenized suspension. Prior to the electrode modification, glassy carbon electrode (GCE) was mechanically polished to mirror like appearance by polishing with alumina powder (Al2O3, 0.05 ^m and 0.3 ^m) and cleaned by ultrasonicating in 1:1 mixture of 0.1 M HCl and HNO3 for 10 minutes and dried at room temperature. 15 ^L of the prepared Ag MP and MWCNT suspension was cast onto the pre-cleaned surface of GCE and allowing the solvent to evaporate at room temperature. The electrode was washed with distilled water and designated as Ag MP-MWCNT-GCE. Electrochemical pretreatment of the as fabricated Ag MP-MWCNT-GCE was achieved by performing 20 consecutive cycles in 0.5 mM polyaniline solution by cyclic voltammetry in potential range of -0.5 to +2.0 V at a sweep rate of 50 mV/s. The electrode was washed with distilled water to remove any unadsorbed material and the electrode was designated as PAN-Ag MP-MWCNT-GCE. After the electropolymerization, the modified electrode was rinsed thoroughly with distilled water and then dried in air at room temperature.

2.3 Real sample preparation

Ten tablets from each brand (Pitava and Flovas) where homogenized to fine powder and appropriate weight (50 mg of Pitava and 100 mg of Flovas) of this powder was dissolved in 10 mL of methanol. The resulting solutions were first sonicated in an ultrasonicator bath for 10 min

and then centrifuged for 15 min at 3000 rpm. The clear supernatant was pipetted out and diluted with appropriate volumes of supporting electrolyte and stored until assay.

Blood and urine samples were collected from healthy volunteers after acquiring their formal consents. Blood samples were allowed to stand for 30 min to coagulate at room temperature. After coagulation of blood, samples were centrifuged for 20 min at 2000 rpm for serum separation. The supernatant serum generated was carefully separated using clean pipette. Urine samples were added with 1.5 mL acetonitrile for protein removal and vortexed for 60 s. The mixture was centrifuged for 15 minutes at 2000 rpm and supernatant was taken out. Both serum and urine samples were diluted 10 times with 0.1 M phosphate buffer and stored in refrigeration for further analysis.

2.4 Analytical procedure

5 mL of the supporting electrolyte (0.1 M phosphate buffer, pH 6.5±0.2) was taken in voltammetric cell and deoxygenated by purging nitrogen gas for 300 s. Suitable aliquots of standard PST solution was added to the supporting electrolyte immediately after recording the voltammogram of blank. The solution was again deoxygenated with purified nitrogen for 120 s. All voltammograms were scanned and recorded in negative potential from -0.7 to -1.8 V at a scan rate of 150 mV/s. The recording of voltammogram was repeated until a stable peak height was achieved.

3. Results and discussion

3.1 Electrochemical characterization of modified electrodes

The modified and bare electrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques employing F e ( C N ) \ ^4 probe. CV measurements on 1.0 x 10-3 M KsFe(CN)6 probe in 0.1 M KCl produced a reversible couple with increased peak currents at modified electrodes compared to bare GCE. The electro-active surface area of all the electrodes were calculated employing Randles-Sevick [31] equation

/F3\1/2

Ipa = 0.4463 (-) n3 /2 A0D0/2Cu 1 2

Where is anodic peak current, n is number of electrons transferred, is surface area of electrode in cm2, v is scan rate (mV/s), is diffusion coefficient and concentration of

probe, respectively and all other terms have their usual meanings. For 1.0 mM K3Fe(CN)6 in 0.1

M KCl at T = 298 K, n = 1 and D 0 = 7.6 x 10- cm /s. The surface area was calculated from the slope of plot of I p aversus u 1 / 2 and was found as 0.0278 cm2, 0.0517 cm2, 0.0874 cm2 and 0.1053 cm2 for GCE, MWCNT-GCE, Ag MP-MWCNT-GCE and PAN-Ag MP-MWCNT-GCE, respectively.

EIS is an effective method for probing the changes in the surface of the modified electrodes. Fig. 1 shows Nyquist plot of GCE, MWCNT-GCE, Ag MP-MWCNT-GCE and PAN-Ag MP-

MWCNT-GCE in 5 x 10-3 M F e ( C N ) 3 "/4 " in 0.1 M KCl. The diameter of semicircle domains at high frequency region in the Nyquist plot provide the electron transfer resistance (Rct) at the electrode surface and are used to define the interface properties of the electrode [32]. The semicircle in high-frequency regions obtained for modified electrodes was smaller compared to that of bare GCE indicating decreased impedance at modified electrodes. Rct at bare GCE, MWCNT-GCE, Ag MP-MWCNT-GCE and PAN-Ag MP-MWCNT-GCE obtained are 47.06 K^, 31.28 K^, 26.71 K^ and 16.67 K^, respectively. The decreased impedance at modified electrodes is a consequence of very low Rct resulting in increased peak currents [33].

3.2 Electrochemical behaviour of drug at modified electrode

The electrochemical behavior of PST at bare and modified electrodes was investigated by cyclic voltammetry (CV) and differential pulse adsorptive stripping voltammetry (DPAdSV) in 0.1 M phosphate buffer (pH = 6.5±0.2). PST produces well-defined reduction peak in 0.1 M phosphate buffer of 6.5±0.2 pH at the surface of modified electrode. To elucidate the uniqueness and sensitivity of modified electrode, the electrochemical behaviour of 4.0 x

10-7 M PST was

investigated at four different working electrodes. Fig. 2 shows the resulted cyclic voltammograms of PST at bare GCE, MWCNT-GCE, Ag MP-MWCNT-GCE and PAN-Ag MP-MWCNT-GCE in phosphate buffer supporting electrolyte. The increase in peak current at PAN-Ag MP-MWCNT-GCE indicates strong accumulation of PST with increased electron transfer between PST and the electrode surface. This can be explained by the fact that multi-walled carbon nanotubes and porous silver increases the surface area of electrode and polyaniline enhanced the electron transfer process. The reduction peak at -1.31 V vs. Ag/AgCl for PST was an irreversible one, as on reversing the potential no anodic peak corresponding to the reduction process was observed even at positive potential. The irreversible nature of the electrode process was further established by increasing scan rates which resulted in shift of peak potential to negative values with increased peak currents [34].

3.3 Optimization of experimental parameters

3.3.1 Optimization of varying Ag MP-MWCNT dosages

For acquiring maximum sensitivity of PAN-Ag MP-MWCNT modified GCE towards the determination of PST, varying volumes of Ag MP-MWCNT suspension were applied on GCE prior to the electrochemical pretreatment of modified electrode. For this purpose several electrodes were designed and varying dosages of Ag MP-MWCNT suspension was directly caste onto the clean and polished surface of GCE followed by its electrochemical pretreatment in 0.5 M polyaniline solution. Fig. 3 shows variation of peak current of 4.0 x 10-7 M PST with increasing volumes of Ag MP-MWCNT suspension. Initially the peak current jumps rapidly, reaching its maximum when 15 ^L suspension was used and thereafter the peak current stabilized, however at higher volumes, a decrease in peak current was observed. This may be a consequence of increasing thickness of nanoparticle film resulting in decreasing electron transfer rate. Hence for maximal sensitivity 15 ^L Ag MP-MWCNT suspension was loaded on GCE surface.

3.3.2 Influence of accumulation time and potential

When considering the adsorptive property of drug it becomes important to study the effect of both accumulation time (tacc) and accumulation potential (Uacc). Fig. 4 shows adsorptive stripping voltammograms of 4.0 x 10-7 M PST at different accumulation time ranging from 0 to 180 s at PAN-Ag MP MWCNT-GCE while the inset (Fig. 4) plot represents the variation of peak current with accumulation time. The initial increase in peak current upto 60 s indicates much of the drug is adsorbed at the surface of electrode and thereafter the peak current tends to be almost stable indicating the amount of drug adsorbed at electrode surface tends to a limiting value. Moreover the impact of accumulation potential on voltammetric peak current was studied over the range -1.0 V to +0.5 V. Considerable increments of peak currents were

observed for 4.0 x 10- M PST towards positive potential and acquired maximum peak current value at -0.1 V and afterwards peak current decreased sharply. Hence for maximum analytical sensitivity, tacc of 60 s and Uacc of -0.1 V was applied in further investigations.

3.3.3 Influence of pH

The pH of supporting electrolyte exerts a substantial effect on the electrochemical redox property of an analyte, moreover important information regarding the involvement of protons and electrons participating in the electrode process can be acquired by studying the drug under different pH conditions. Taking into account, the above facts the electrochemical reduction process of PST was studied in terms of varying pH (Fig. 5) of supporting electrolyte (0.1 M phosphate buffer). It was observed that peak potential varied linearly with pH of supporting electrolyte, this dependence of peak potential on pH indicates involvement of protons in the electrode reaction process [16]. Fig. 6 shows shift of peak potential to more negative potential with increasing pH, while peak current intensity increases only upto pH 6.5±0.2, and decreases at higher pH. The slope of 54.77 mV/pH obtained for the plot in Fig. 5 lies close to the theoretical value (59 mV/pH) expected for a redox process involving equal number of protons and electrons. Thus all electrochemical measurements of PST were performed an optimal pH of 6.5±0.2.

3.3.4 Influence of scan rate

Scan rate studies are helpful in drawing very useful information such as electrochemical reaction mechanism, diffusion or adsorption controlled nature, the reversible or irreversible nature of electrode process. The influence of scan rate on reduction peak of 4.0 x 10-7 M PST at PAN-Ag MP-MWCNT-GCE was studied over the range 25-200 mV/s. As depicted in Fig. 7 the increase in scan rates leads to increment in peak current (Ip) with shift of peak potential (Ep) to more negative potentials indicating irreversible nature of electrode process [35]. The scan rate of 150 mV/s was selected as suitable for determination of PST as it sufficiently rapid for routine analysis, produces sharper and well defined voltammetric responses. Linear relationships were also observed for log Ip vs. log v (Fig. 8a) with a linear equation slope of 0.89 close to theoretical value of 1.0 for a typical adsorption controlled process [36-38]. Linear relationship between Ep and log v (Fig. 8b) was observed signifying that electron transfer is not fast [39]. The adsorption controlled nature of the electrode process was further verified by obtaining linear graph between Ip and potential scan rate (v) (Fig. 8c).

3.4. Chronocoulometric behaviour and plausible redox mechanism

In view of adsorption controlled nature of electro-reduction of PST, the chronocoulometric behaviour of PST was examined to determine number of electrons (n) transferred in the electrode process and to determine the surface coverage (P) of PST at the electrode surfaces.. The number of electrons transferred 'n' can be estimated by performing controlled potential coulometry from the charge consumed at desired concentration of PST. The electrolysis was performed at the optimized pH for three concentrations of PST (15 ^g/mL, 25 ^g/mL and 40 ^g/mL) by placing the solution in voltammetric cell and continuously stirring and purging with nitrogen gas during electrolysis against Ag/AgCl reference electrode. Number of electrons transferred 'n' was

calculated using the equation Q = n F N , where Q is charge consumed in coulombs, F is Faraday's constant and N is number of moles of analyte. 'n' for three different concentration was calculated to be 2.07, 1.96 and 2.03, indicating the reduction of PST at PAN-Ag MP-MWCNT-GCE is a two electron process. Based on the results obtained in CV, DPAdSV and controlled potential electrolysis, the probable electrochemical reduction mechanism of PST is shown in scheme 1.

Chronocoulometry was further employed for the determination of diffusion coefficient and

Qads of PST at the electrode surfaces from Q vs. t plots employing Anson equation [40]. The plot of Q vs. t1/2 in absence and presence of PST showed linear relationships and were almost parallel which further supports the adsorptive controlled nature of electrode process. Surface coverage of PST for all the electrodes was calculated by using the equation

Qa ds = nFAr 0

The calculated values of diffusion coefficient and surface coverage of PST at different electrode surfaces are listed in Table 1. It was observed that PAN Ag MP-MWCNT-GCE showed maximum surface coverage for PST which is a collective effect of Ag MP, MWCNT and polyaniline increasing both surface area and electron transfer rate thus favoring kinetics of PST reduction.

3.5. Analytical applications

3.5.1 Calibration curve

In order to examine the feasibility of proposed method as analytical tool for the trace determination of PST, DPAdS voltammograms were recorded at least 5 times under the optimized conditions at the modified electrode surface. The reduction peak currents were found proportional to the concentrations of PST over the range 2 x

10-7 M to 1.6 x 10-6 M in 0.1 M phosphate buffer of pH 6.5±0.2. Deviation from linearity was observed both at higher as well as lower concentrations of PST; this may be due to adsorption of PST or its reduction product at the electrode surface. Fig. 9 shows DPAdS voltammograms of different concentrations of PST under the optimized method with Fig. 9 inset representing the calibration plot of the same. The regression data of calibration plot obtained by the developed electrochemical method for the

determination of PST are presented in Table 2. The limit of detection (LOD) and limit of

3s 10s

quantification (LOQ) were calculated using the equations [34] L 0 D = — ; L 0 Q = —, where 's'

is standard deviation and 'm' is slope of the calibration plot. The calculated LOD was found to be 9.66±0.04 nM with LOQ was amounted to be 32.25±0.07 nM.

For practical application and validation of the proposed method intraday (over a single day)

and interday (after 5 days) recovery measurements were performed with three (6.0 x 10-/ mol/L, 8.0 x 10-7 mol/L and 1.0 x 10-6 mol/L) different concentrations of PST. DPAdSV method was employed for recovery measurements and standard addition method was used on each concentration with five replicates to achieve better accuracy and precision. The recovery results vary between 97.50 to 99.00 for intra-day and 96.66 to 99.00 for inter-day recovery measurements. The results of recovery measurements are tabulated in Table 3.

3.5.2 Reproducibility and Stability of modified electrode

The reproducibility and stability of modified PAN-Ag MP-MWCNT-GCE was examined by performing ten successive cyclic voltammograms of 4.0 x 10-7 M PST solution. The relative standard deviation of peak current was noticed to be 2.98%. For long-term stability, the modified electrodes where stored under refrigeration for two weeks. It was observed that the electrode retained almost 96.08% of its initial response. The results indicated an acceptable reproducibility and stability of the modified electrode for the determination of PST.

3.5.3 Interference studies

The possible interference of some common excipients was investigated to access the sensitivity of the proposed method. The tolerance limit was defined as the maximum concentration of interferent which causes an error less than ±5 %. The adsorptive stripping voltammograms of 4.0 x 10-7 M PST was recorded in presence of various interferents. It was noticed that glucose, sucrose and cellulose (up to 60-fold excess), Na+, K+, Ca++ and Cl- (up to 80-fold excess) and talc, starch and dextrose (up to 100 fold excess) did not interfere in the

determination of PST. Thus we suggest the proposed method offered good sensitivity for the determination of PST.

3.5.4 Determination of PST in real samples

To validate the practical applications of the optimized electrochemical procedure, it was successfully applied for the determination of PST in real samples (Pharmaceutical preparations and biological fluids). The unknown or spiked PST amount was calculated using standard addition method. The results of pharmaceutical analysis indicated excellent precision and accuracy towards the determination of PST under the optimized conditions. A mean recovery of 99.01 for Pitava brand and 97.72 for Flovas brand was achieved by the present work. The detailed results of the pharmaceutical analysis compared with some other reported analytical methods [26, 27] methods are presented in Table 4.

The proposed method was further subjected for accuracy measurements by analyzing drug free and spiked biological (serum and urine) fluids, in absence of drug no peak was observed corresponding to PST. The fluids were then spiked with known concentrations of PST and by using standard addition method, the amount of drug recovered was determined. The results were subjected to statistical analysis for reliability of data and are tabulated in Table 5. The recovery measurements were between 99.50% to 101.10% and 97.20% to 100.20% for serum and urine samples, respectively.

4. Conclusion

An efficient green synthetic sol-gel protocol was adopted for synthesizing silver macroporous (Ag MP) material and applied in fabricating an electrochemically pretreated glassy carbon electrode. The modified PAN-Ag MP-MWCNT-GCE exhibited several advantageous features such as simple preparation procedure, increased surface area, excellent electrocatalytic activity, better electron transfer rate and strong adsorption towards an anti-hyperlipidimic drug pitavastatin. Such features resulted in higher sensitivity and increased electrochemical activity

for determining PST in pharmaceutical and biological samples. Different experimental conditions and instrumental parameters where optimized for proposing a simple, highly sensitive and accurate method for determining PST as low as 9.66 nM concentration. The studies thus open an opportunity to adopt the present method for pharmacokinetic studies and quality control and assurances laboratories.

Acknowledgment

Authors are highly grateful to UGC-SAP, SIC and Department of Chemistry, Dr. H.S.G. (Central) University, Sagar for providing necessary lab facilities and required instrumentation.

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Fig. 1 Nyquist plots of EIS at bare (a) GCE, (b) MWCNT-GCE, (c) Ag MP- MWCNT-GCE and

(d) PAN-Ag MP-MWCNT-GCE in 5.0 x 10-3 M Fe(CN)63-/4- probe. Frequency range: 106 Hz to 10-1 Hz.

Fig. 2 Cyclic voltammograms of 4.0 x 10-7M PST at (a) bare GCE, (b) MWCNT-GCE, (c) Ag MP-MWCNT-GCE and (d) PAN-Ag MP-MWCNT-GCE in 0.1 M phosphate buffer with pH 6.5±0.2.

Fig. 3 Plot showing variation of peak current (Ip) of 4.0 x 10- M PST with different loading volumes of Ag MP-MWCNT on GCE.

Fig. 4 Adsorptive stripping voltammograms of 4.0 x 10-7 M PST at (1^10) (1) 0 s (2) 20 s (3) 40 s (4) 60 s (5) 80 s (6) 100 s (7) 120 s (8) 140 s (9) 160 s (10) 180 s accumulation time. Inset: Variation of peak current with accumulation time.

Fig. 5 Adsorptive stripping voltammograms of 4.0 x 10-7 M PST at (a) 4.5 (b) 5.5 (c) 6.5 (d) 7.5

(e) 8.5 (f) 9.5 pH in 0.1 M phosphate buffer.

Fig. 6 Dependence of peak potential (Ep) of 4.0 x 10- M PST on pH of supporting electrolyte. Inset: Variation of peak current (Ip) with pH.

Fig. 7 Cyclic voltammograms of 4 x 10-7 M PST at (a ^ h) 25, 50, 75, 100, 125, 150, 175 and 200 mVs-1 scan rate in 0.1 M phosphate buffer (pH 6.5).

Fig. 8 (a) Plot of logarithm of peak current vs. logarithm of scan rate (mV/s) (b) Variation of peak potential with logarithmic of scan rate (c) Variation of peak current (Ip) with potential scan rate (mV/s)

Fig. 9 DPAdS voltammograms of a) 1.6 pM, b) 1.4 pM, c) 1.2 pM, d) 1.0 pM, e) 0.8 pM, f) 0.6

pM, g) 0.4 pM, h) 0.2 pM of PST in 0.1 M phosphate buffer of pH 6.5. Inset: Variation of peak currents with concentrations of PST.

Scheme 1 Probable electrode reaction mechanism of electro-reduction of PST at modified electrode surface

120 160

Z'(KQ ) Fig. 1

Potential (V)

Current ((jA)

Peak current (jiA)

Potential (V) Fig. 5

£ 0.7 s

£ 0-6

—i— 1.4

Potential (V) Fig. 7

—i— 1.6

—i— 1.8

log Scr

—i— 2.4

^ 1.25-

1.6 1.8 2.0 log Scan rate (mV/s)

l-1-1-1-1-r

20 40 60 80 100 120 140 160 180 200 220

Scan rate (mV/s)

30252015-

-1.0 -1.1 -1.2 -1.3 -1.4 -1.5 -1.6 Potential (V)

Fig. 9

Scheme 1

Table 1 Diffusion coefficient and surface coverage values of PST at different electrode surfaces.

Electrodes Diffusion Coefficient (10-6 cm2/s) Surface Coverage (10-11 mol/cm2)

GCE 4.17±0.05 0.53

MWCNT-GCE 5.12±0.07 1.26

Ag MP-MWCNT-GCE 5.98±0.08 3.73

PAN Ag MP=MWCNT-GCE 6.95±0.05 8.51

Table 2 Regression data of calibration plot for PST using DPAdSV.

Parameters DPAdSV

Peak potential Ep (V) -1.31

pH 6.5

Buffer type/strength (M) Phosphate/0.1

Uacc (V) -0.1

tacc (s) 60

Linearity range (^M) 0.2-1.6

Slope (jiA/jiM ) Intercept ( jA) Correlation coefficient LOD (nM)

LOQ (nM)_

4.60 0.992 9.66±0.04 32.25±0.07

Table 3 Analytical precision and recovery data for intra- and interday recovery measurements

Parameters

Intra-day

Inter-day

Addeda (|M) 0.6 0.8 1.0 0.6 0.8 1.0

Found (|M) 0.59 0.78 0.99 0.58 0.79 0.99

Recovery (%) 98.33 97.50 99.00 96.66 98.75 99.00

Standard deviation (SD) 0.005 0.015 0.006 0.007 0.005 0.009

RSD (%) 0.90 1.95 0.66 1.25 0.7 0.91

Average of five determinations

Table 4 Results of determination of PST in pharmaceutical samples.

Parameters

DPAdSVa Pitava Flovas

HPTLCD [26] Pitava Flovas

Spectrophotometry0 [27] Pitava

Label claim (mg) 2 1 1 1 1

Amount found (mg) 1.98d 0.984d 0.998 0.998 1.01

Recovery (%) 99.01 97.72 99.83 99.80 101.35±0.99e

RSD (%) 1.43 1.50 0.03 0.13 ND

LOD 9.66 nM 10 ng 0.298 mg/mL

LOQ 32.25 nM 30 ng ND

a Present Method; Average of four determinations; c Applied method B; Mean of five determinations; Average of six determinations: ND (Not determined)

Table 5 Precision and accuracy of assay of PST in spiked biological samples.

Sample Added conc. (^M) Found Conc. (^M) Recovery (%) % RSD

Serum 0.6 0.597 99.5 1.21

0.8 0.799 99.8 0.57

1.0 1.011 101.1 0.60

1.2 1.195 99.58 1.91

Urine 0.6 0.583 97.2 1.27

0.8 0.802 100.2 0.84

1.0 0.998 99.8 0.92

1.2 1.198 99.8 1.14