Materials Science and Engineering C 31 (2011) 230–237

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Materials Science and Engineering C

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Voltammetric behaviour of nitroxazepine in solubilized system and biological fluids

Rajeev Jain ⁎, Jahangir Ahmad Rather, Ashish DwivediSchool of Studies in Chemistry, Jiwaji University, Gwalior-474011, India

⁎ Corresponding author. Tel.: +91 751 2442766; fax:E-mail address: rajeevjain54@yahoo.co.in (R. Jain).

Scheme 1.

0928-4931/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.msec.2010.09.001

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 April 2010Received in revised form 27 July 2010Accepted 1 September 2010Available online 7 September 2010

Keywords:Nitroxazepine (Nitrox)Solubilized systemAdsorptive stripping voltammetryHMDEPharmaceutical formulationBiological fluids

This study reports the development and validation of sensitive and selective assay method for thedetermination of the antidepressant drug in solubilized system and biological fluids. Solubilized system ofdifferent surfactants including cationic, anionic and non-ionic influences the electrochemical response of drug.Addition of cationic surfactant cetrimide to the solution containing drug enhances the peak current signalwhile anionic and non-ionic showed an opposite effect. The current signal due to reduction process wasfunction of concentration of nitroxazepine, pH, type of surfactant and preconcentration time at the electrodesurface. The reduction process is irreversible and adsorption controlled at HMDE. Various chemical andinstrumental parameters affecting the monitored electroanalytical response were investigated and optimizedfor niroxazepine hydrochloride determination. The proposed SWCAdSV and DPCAdSVmethods are linear overthe concentration range 2.0×10-7– 5.0×10-9 mol/L and 6.1×10-7– 1.0×10-8 mol/L with detection limit of1.62×10-10 mo/L and 1.4×10-9 mo/L respectively. The method shows good sensitivity, selectivity, accuracyandprecision thatmakes it very suitable for determination of nitroxazepine in pharmaceutical formulation andbiological fluids.

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1. Introduction

Nitroxazepine hydrochloride (scheme 1) is a new psychoactivetricyclic antidepressant drug commonly used for treatment ofdepression and nocturnal enuresis. The antidepressant activity ofnitroxazepine hydrochloride is similar to that of other tricyclicantidepressants like imipramine. The mechanism of action involvesinhibition of both exogenously administered and endogenouslyreleased noradrenaline [1] (Scheme 1).

Themonitoring of such compounds is of great importance for qualitycontrol and clinical laboratories. To date only few analytical methodshave been reported for the estimation of nitroxazepine hydrochloride;they include polarographic analysis [2] and high-performance liquidchromatography [3]. The chromatographic methods are althoughaccurate; however they are time consuming, expensive, solvent usageinterference and require skilled personals and therefore are unsuitablefor online or field monitoring. New pharmaceutical preparations andbiosample analysis requires fast and specific method for the determi-nation of nitroxazepine hydrochloride. Therefore a novel technique forthe determination of nitroxazepine hydrochloride is still needed to bedeveloped. The versatility of electrochemical techniques and the lowdetection limits as well as its low acquitisition costs made them widelyapplied in various fields especially in the determination of biological/chemical materials which posses electroactive groups. Electrochemicalmethods have been proved to be sensitive and reliable for thedetermination of numerous electroactive drug components [4–9]. Theelectroanalytical techniques proved to be useful both for the analysis ofpharmaceutical formulation of drugs and in biological fluids. Theadvantages of electrochemical techniques in the analysis of drugs aretheir simplicity, low cost and relatively short analysis time as comparedto other routine analytical techniques.

Reviewing the literature neither adsorptive stripping nor squar-ewave voltammetric method for the assay and quantification ofnitroxazepine hydrochloride are reported. Hence the current electro-analytical research aimed to study the voltammetric behaviour ofnitroxazepine and its interfacial adsorptive accumulation onto thehanging mercury dropping electrode (HMDE). Based on the results

Table 1Electrochemical parameters of nitroxazepine hydrochloride in solubilized Systems.

Electrolyte (Buffer pH 10.0) aEpc /V (vs. Ag/AgCl) bIpc/μA

1.2×10-7 mol/L Nitroxazepine hydrochloride+8.14×10-4 mol/L Tween 20

0.41 0.41

1.2×10-7 mol/L Nitroxazepine hydrochloride+2.43×10-3 mol/L Cetrimide

0.52 0.46

1.2×10-7 mol/L Nitroxazepine hydrochloride+3.46×10-3 mol/ L Sodium Dodecyl Sulphate

0.48 0.28

1.2×10-7 mol/L Nitroxazepine hydrochloride+1.36×10-2 mol/L DMF

0.49 0.30

a Cathodic peak potential.b Cathodic peak current.

231R. Jain et al. / Materials Science and Engineering C 31 (2011) 230–237

obtained a sensitive, simple and low cost SWandDPV-AdSV procedureswere developed in solubilized system for the determination ofnitroxazepine hydrochloride in pharmaceutical formulation and inbiological fluids. There are certain advantages associated with thismethod such as more dissolution, high selectivity and no interferencefrom other active compounds present in commercial dosage form. Thedevelopment of meaningful dissolution procedure for drug productswith limitedwater solubilityhas beena great challenge to analyst [10]. Ithas been seen that surfactant play a very important role in electrodereactions not only in solubilizing organic compounds, but also providingspecific orientation of the molecules at the electrode interface [11,12].

The aggregates of surfactants such as micelles, liquid crystallinevesicles etc.,which could enhance the stabilized content and the control ofrelease behaviour of drugs are widely studied as drug delivery systems[13,14]. Theuseof surfactants asdrugcarriersmakenecessary to study theinteraction of drugs with micellar systems. Micellar effects may be ofmany kinds including electrostatic, surface interactions, hydrophobicforces and partition between themicelle and thewater phase. In additionmicellar systems are considered to be primitive model systems forbiological membranes [15]. Effects of surfactant on solubility anddissolution rate of poorly soluble drugs are well characterized [16].Surfactants heavily influence the electrochemical processes of electro-active species [17,18] and thus are widely used in electronalyticalchemistry to improve the sensitivity and selectivity [19–22]. Adsorptionof surfactants on electrodes and solubilization of electroactive compoundsin micellar aggregates might significantly change the redox potential,charge transfer coefficients and diffusion coefficients that enhances theelectroanalytical response and hence lowers the detection limit [23,24].

In the present study, the effect of the changing the charge of thesurfactant used namely anionic, non-ionic and cationic, its concentrationswith the solution pH and concentration of analyte on the voltammetricresponseof thisdrughasbeenstudiedwith improvedsensitivity atHMDE.Thus the main objective of the present work is to develop anelectrochemical method for the determination of nitroxazepine hydro-chloride utilizing enhancement effect of solubilized system.

2. Experimental

2.1. Materials and methods

Nitroxazepine hydrochloride (99%purity)was obtained fromNovartisIndia Ltd. Mumbai, India and was used as received. Tablets containingnitroxazepine hydrochloride (Sintamil) labeled 75 mgwas obtained fromcommercial sources. KCl (0.1 mol/L) solution was prepared in doubledistilled water and used as supporting electrolyte. A stock solution ofnitroxazepine hydrochloride (1.0×0-4 mol/L) was prepared in DMF(Dimethylformamide), cetrimide, sodium dodecyl sulphate (SDS) and inTween 20. The solutions for recording voltammogramswere prepared bymixing appropriate volumeof stock solution, buffers and0.1 mol/LKCl. Allchemicals used are of analytical reagent grade quality andwere employedwithout further purification.

2.2. Procedure

2.2.1. Sintamil tablet solutionTen Tablets were weighed and the average mass per tablet was

determined.Aportionof thefinelygroundedmaterial equivalent to75 mgof nitroxazepine hydrochloride was accurately weighed and transferredinto the 100 mL calibrated flask containing 70 mL surfactant solution. Thecontent of theflaskwas sonicated for about 15 minutes and thenmadeupto the volumewith the surfactant solution. An aliquot of the solutionwasthen analyzed according to the proposed voltammetric procedure.

2.2.2. Serum and plasma analysisDrug-free human blood obtained from healthy volunteers (after

having obtained their written consent) was centrifuged at 5000 rpm for

30min at room temperature and separated serum and plasma sampleswere stored under refrigeration until assay. Then separated serum andplasma were treated with 1.0 mL of acetonitrile as protein denaturingand precipitating agent. After vortexing for 30 s, the mixture was thencentrifuged for 10 min at 5000 rpm in order to eliminate serum andplasmaprotein residues and supernatantwas taken carefully. An aliquotof serum and plasma sample was fortified with nitroxazepinehydrochloride dissolved in cetrimide to achieve a final concentrationof 1.0×10-4 mol/L. Appropriate volumes of this supernatant weretransferred into the volumetric flask and diluted up to the volumewith phosphate buffer of pH 10.0. Then the protein-free spiked serumand plasma containing drug were analyzed according to the proposedstripping voltammetric procedure.

2.3. Instrumentation

Electrochemical measurements were performed using a μ-AUTO-LAB TYPE III (Eco- Chemie B.V., Utrecht, The Netherlands) potentio-stat-galvanostat with 757VA computrace software. The utilizedelectrodes were hanging mercury drop electrode (HMDE) as workingelectrode, Ag/AgCl (3.0 mol/L KCl) as reference electrode and agraphite rod as auxiliary electrode. Controlled potential coulometricexperiments were carried out using an electrochemical cell i.e.,Autolab Potentiostat/Galvanostat PGSTAT Metrohm 663 VA standwith GPES 4.2 (General Purpose Electrochemical Software) software.Coulometric experiments were performed by the potentiostatic modeusing Pt foil with large surface area as working electrode and a Pt wireas the counter electrode. All the solutions examined by electrochem-ical technique and were purged for 10 min with purified nitrogen gasafter which a continuous stream of nitrogen was passed over thesolutions during the measurements. All pH-metric measurementswere made on a Decible DB-1011 digital pH meter fitted with a glasselectrode and a saturated calomel electrode as reference, which waspreviously standardized with buffers of known pH.

3. Results and Discussion

The electrochemical behaviour of nitroxazepine hydrochloride onHMDE was studied by using cyclic voltammetry (CV), differentialpulse cathodic adsorptive stripping voltammetry (DPCAdSV) andsquarewave cathodic adsorptive stripping voltammetry (SWCAdSV).In all electrochemical methods nitroxazepine hydrochloride gave onewell defined reduction peak in DMF and cetrimide which is attributedto the reduction of –NO2 group at mercury electrode.

3.1. Nitroxazepine behaviour in solubilized system

The influence of different solubilized system of surfactants includingcationic (cetrimide), anionic (SDS) and neutral (Tween-20) on thereduction of nitroxazepine hydrochloride were explored by theproposed voltammetric procedure and the specific values of Ipc andEpc were summarized in Table 1. On comparing the voltammetric

Fig. 1. Square-wave voltammogram of 4.5×10-5 mol/L nitroxazepine hydrochloridesolution (C) blank (B) in presence of cetrimide and (A) in DMF.

232 R. Jain et al. / Materials Science and Engineering C 31 (2011) 230–237

behaviour of nitroxazepine hydrochloride inDMF and in the presence ofsurfactants (a neutral, a cationic and an anionic type), it is observed thatnitroxazepine hydrochloride shows substantial increase in peak currentand the limit of detection is found to be lower in cationic surfactantcetrimide (Fig. 1), while neutral and anionic surfactants showed anopposite effect. The other reason for increase in peak current is due tothe complex formation which could effectively promote the voltam-metric response of nitroxazepine hydrochloride. Electrochemical para-meters of nitroxazepine hydrochloride determined at HMDE insolubilized systems are shown in Table 1.

The cationic surfactant cetrimidehas a hydrophobic head onone sideand a long hydrophobic tail on the other side and had been widely usedin electrochemistry and electroanalytical chemistry field [25]. Surfac-tants could not only endow the electrode/solution interface withdifferent electrical properties, but also adsorb on the electrode /solutioninterface with different electrical properties or aggregate into supra-molecular structures to change the electrochemical processes [26,27].

3.2. Effect of cetrimide concentration

The effect of cetrimide concentration on the cathodic peak current ofnitroxazepine hydrochloride is shown in Fig. 2. The cathodic peakcurrent increases steadily in the beginning with increase in concentra-tion of cetrimide and reaches amaximum at 2.43×10-3 mol/L. Itmay beinterpreted that the adsorption behaviour of cetrimide changes frommonomer adsorption to monolayer adsorption with increase in

Fig. 2. Effect of cetrimide concentration on squarewave cathodic peak current of1.2×10-6 mol/L nitroxazepine at HMDE. The error bars represents standard deviationfrom three separate experiments.

concentration of cetrimide at the electrode surface. The cetrimideconcentration of 2.43×10-3 mol/Lmay have reached the critical micelleconcentration (CMC), but the electrode process is cooperated by theadsorption and accumulation of cetrimide. However peak currentdecrease as further increase in cetrimide concentration, it may becausedby themicelle effect i.e., electron transfer betweennitroxazepinehydrochloride and electrode surface would be inhibited by aggregates ofmicelles. Also the increase of hydrophobicity of the possible cetrimidemicellesmight decrease the electron transfer rate constant [28] and resultin the decline of peak current at rather high cetrimide concentration. Tosum up, the cetrimide concentration of 2.43×10-3 mol/L can furthestenhances the electrochemical signal of nitroxazepine hydrochloride.

3.3. Effect of pH

For controlling pH various electrolytes such as Britton Robinsonbuffer, acetate buffer, borate buffer, citrate buffer and phosphate bufferwereused. Thebest resultswith respect to sensitivity accompaniedwithsharper responsewereobtainedwithphosphate buffers. Thus studywasmade in the pH range 2.0 – 11.0 in phosphate buffers at a targetconcentration of 1.4×10-6 mol/L nitroxazepine hydrochloride solution.With the rise in pH the peak potential shifted towards more negativepotential which indicated the existence of a protonation reactioncoupled with the nitroxazepine hydrochloride reduction process.

The relation between peak potential Ep of the wave and pH of themedium over the range 2.2–11.0 is expressed by the following equations:

SWCAdSV; pH 2:2−11:0 : Ep =mV vs:Ag= AgClð Þ

= 58:29 pH + 758:5 pH; r2 = 0:996

DPCAdSV; pH 2:2−11:0 : Ep =mV vs:Ag= AgClð Þ

= 56:54 pH + 791:4 pH; r2 = 0:991

The obtained ∂Ep/∂pH values 58.29 and 56.54 mV/pH are close to59 mV/pH at 250C (298 K) clearly indicate that equal number ofelectrons and protons are involved in electroreduction of niroxaze-pine hydrochloride at HMDE [29].

As shown in Fig. 3, the peak height steadily increases as the pHincreases and reaches a maximum at pH 10.0. Therefore, pH 10.0 waschosen as the optimum one for the determination of nitroxazepinehydrochloride.

3.4. Optimization of operational parameters

Adsorptive Stripping voltammetry is important analytical techniquefor determination of wide range of electroactive pharmaceutical

Fig. 3. Influence of pH on the squarewave cathodic adsorptive peak current response for1.4×10-6 mol/L nitroxazepine hydrochloride in phosphate buffer (pH 2.0 –11) after180 s pre-concentration time; frequency (f)=80 Hz, Δs=10 mV and pulse amplitu-de=50 mV at Eacc=- 0.5 V. The error bars represents standard deviation from threeseparate experiments.

Table 2Analytical parameters for voltammetric determination of nitroxazepine hydrochlorideusing SWCAdSV and DPCAdSV modes.

SWCAdSV

OperationalParameters

Tablet Serum Plasma

Linearity range(mol/L)

2.0×10-7-5.0×10-9 7.1×10-7 - 9.2×10-8 7.1×10-7- 9.2×10-8

Slope (μA/ mol/L) 3.2×106 9.0×106 5.0×106

Intercept (μA) 0.2202 0.0295 0.0095asa 1.73×10-4 1.4×10-4 7.1×10-4bsb 1.32×10-4 1.22×10-4 5.2×10-4

LOD(mol/L) 1.62×10-10 4.6×10-10 6.26×10-9

LOQ(mol/L) 5.4×10-10 10.4×10-10 14.2×10-9

DPCAdSV

OperationalParameters

Tablet Serum Plasma

Linearity range(mol/L)

6.1×10-7-1.0×10-8

1.0×10-7 –

7.7×10-71.0×10-7 –

7.7×10-7

Slope (μA/mol/L) 2.7×105 1.0×106 1.0×106

Intercept (μA) 0.3674 0.0001 0.0293asa 1.26×10-4 2.3×10-3 1.4×10-3bsb 1.1×10-4 1.83×10-3 1.14×10-3

LOD (mol/L) 1.4×10-9 4.9×10-9 8.2×10-9

LOQ (mol/L) 4.6×10-9 11.0×10-9 16.0×10-9

a Standard deviation of intercept of regression line.b Standard deviation of the slope of regression line.

233R. Jain et al. / Materials Science and Engineering C 31 (2011) 230–237

compounds which can be adsorbed onto the HMDE. Besides mercury is avery attractive choice of electrode materials because it has high overvoltage that greatly extends the cathodic potential window (compared tosolid electrode materials) and posses a highly reproducible, smooth andreadily renewable surface. The variation of the stripping voltammetricpeak current of nitroxazepine hydrochloride in cetrimide at HMDE wasinvestigated using squarewave and differential pulse modes. Both thetechniques gave good results, but squarewave cathodic adsorptivestripping voltammetry has been chosen for optimizing the operationalparameters. The important instrumental variables such as preconcentra-tion potential (Eacc), preconcentration time (tacc), pulse amplitude (ΔEsw),scan increment (Δs) and frequency (f) were examined.

3.4.1. Influence of preconcentration timeThe effect of preconcentration time for 1.5×10-7 mol/L nitrox-

azepine hydrochloride was investigated from 10 to 300 s. A linearrelationship is observed in preconcentration time range from 0 to180 s. Above 180 s, saturation coverage of the electrode occurs. Thusfor this work preconcentration time of 180 s was chosen foradsorptive stripping voltammetric determination of nitroxazepinehydrochloride.

3.4.2. Influence of preconcentration potentialThe influence of preconcentration potential (Eacc) on the cathodic

peak current (Ip) of nitroxazepine hydrochloride was also examinedover the potential range -0.1 V to -1.2 V and the maximum peakcurrent was achieved at - 0.5 V. Hence preconcentration potential of -0.5 V was used throughout the present study. At more cathodic valuesa decrease in peak current was observed.

3.4.3. Influence of frequencyFrequency was varied from 10 to 140 Hz using a scan increment of

10 mV, pulse amplitude of 50 mV and 180 s preconcentration time. Alinear relationship was obtained between the peak current andfrequency of the signal up to 80 Hz. Hence the frequency of 80 Hz waschosen for entire analysis.

3.4.4. Influence of scan increment and pulse amplitudeThe effect of scan increment on adsorptive cathodic peak current

of the drug in cetrimide revealed that the peak current enhanced uponthe increase of scan increment (2–10 mV). A scan increment of 10 mVwas used in the present study. At pulse amplitude 50 mV, the peakcurrent was found be maximum. Several instrumental parameterswhich affect the voltammetric response were also optimized for e.g.,mercury drop size, stirring rate and the rest period. The workingconditions decided upon were: drop size 4 cm2 and 2000 rpm. Thestripping current is not significantly affected when varying the restperiod, since it was observed that 10 s was sufficient for the formationof a uniform concentration of the reactant onto the mercury drop.

3.5. Validation of method

According to international conference on Harmonization (ICH)guidelines [30], the analytical method was validated with respect toparameters such as limit of quantitation (LOQ), limit of detection(LOD), precision, accuracy, robustness and ruggedness.

3.5.1. Detection limit and quantitation limitDetection limit is calculated by equation LOD=3 S.D./b, where S.D.

is standard deviation of intercept and b is slope of the regression line.LOD for standard solution is 1.62×10-10 mo/L.

The quantitation limit is examined by the equation LOQ=10 S.D./b.The lower limit of quantitation for the standard solution is 5.4×10-10.Analytical parameters for voltammetric determination of nitroxazepinehydrochloride using differential pulse and square wave voltammetricmethods were tabulated in Table 2.

3.5.2. SpecificitySpecificity is the ability of method to measure analytical response in

presence of all potential impurities that often accompany the drug invarious pharmaceutical preparations. The specificity is examined inpresence of some common excipients (e.g., cellulose, lactose, talc andmagnesium stearate) added in the same ratio as in pharmaceuticalpreparation [31,32]. In order to evaluate the effect of presence of theexcipients on the proposed methods, the standard addition method wasapplied. For this reason appropriate volume of nitroxazepine hydrochlo-ride tablet solution was added to the supporting electrolyte. After thevoltammograms were recorded, known volumes of standard solution ofniroxazepine hydrochloride were added and voltammograms wererecorded. The regression equations of standard addition methods werefound to be Ip/μA=3.2×106 [Nitrox]/μA/ (mol/L)+0.2202; (r2=0.994);n=3 and Ip/μA=2.9×106 [Nitrox]/μA/ (mol/L)+0.2102; (r2=0.992);n=3 for SWCAdSV method respectively. There was no differencebetween the slopes of two methods calibration curve and standardaddition method. The data showed that there was no interaction ofexcipients in the analysis of nitroxazepine hydrochloride in phar-maceutical formulation by the proposed method. Therefore thecalibration curve method which is easier and quicker than thestandard addition method was used in quantitative analysis ofnitroxazepine hydrochloride. The amount found of nitroxazepinehydrochloride in pharmaceutical formulation (sintamil 75 mg) is74.4 mg i.e., 99.2% recovery.

3.5.3. Accuracy and precisionThe accuracy of the analysis was determined by calculating the

percentage relative error between the measured mean concentrationand the nominal concentration [33,34]. The precision of the analysiswas determined by calculating the relative standard deviation (%RSD). The precision around the mean should not exceed 15% of theRSD in biological fluids. The intra-day studies were performed in oneday; inter–day studies were performed in six days over a period of oneweek (Table 3). The results obtained for inter–day and intra–dayaccuracy and precision indicates the high accuracy and precision ofthe proposed method.

Table 3Accuracy and Precision for the analysis of nitroxazepine hydrochloride in spikedplasma and serum by SWCAdSV (n=3).

Added Conc.(ng/mL)

Found Conc.(ng/mL)

Inter-day,RSD (%)

Intra-day,RSD (%)

aRelativeerror (%)

Plasma10 10.2 7.08 5.26 2.0020 19.2 2.17 2.06 -2.0650 49.1 2.53 1.92 -1.80

Serum10 10.11 7.08 6.30 1.0020 20.02 2.17 5.70 0.1050 50.05 2.31 2.20 1.00

a [(found−added)/added]×100.

Fig. 4. Cyclic voltammogramsof 1.5×10-6 mol/L nitroxazepinehydrochloride in2.43×10-3

mol/L cetrimide in phosphate buffer (pH 10.0) at a scan rate of 100 mV/s, equilibriumtime=10 s. (2) after preconcentration and (1) curve shows 0 s preconcentration.

Fig. 5. Cyclic voltammogramsof 1.5×10-6 mol/L nitroxazepinehydrochloride in2.43×10-3

mol/L cetrimide at different scan rates; (a) 100 mV/s (b) 200 mV/s (c) 300 mV/s(d) 400 mV/s (e) 500mV/s.

234 R. Jain et al. / Materials Science and Engineering C 31 (2011) 230–237

3.5.4. RobustnessThe robustness was examined by evaluating the influence of small

variation of some of themost important procedure variables includingpre-concentration potential (Eacc) and pre-concentration time (tacc).The obtained result provided an indication of the reliability of theproposed procedure for the assay of nitroxazepine hydrochloride andhence it can be considered robust. The obtained mean percentagerecoveries based on the average of five replicate measurements werenot significantly affected within the studied range of variations ofsome operational parameters and consequently the proposed proce-dure can be considered robust.

3.5.5. RuggednessThe ruggedness test [35] of the analytical assay method is defined

as degree of reproducibility of assay results obtained by the successfulapplications of the assay over time and multiple laboratories andanalysts. Two analysts analyzed the same standard with SWCAdSVand DPCAdSVmethods using the same instrument. Themethodswerefound to be rugged with the results of variation coefficients 0.9 and1.6% for SWCAdSV, 1.2 and 1.1% for DPCAdSV method for first andsecond analysts respectively. The results show no statistical differ-ences between different analysts.

3.6. Cyclic voltammetric behaviour

The reversibility of the reduction process was investigated byusing cyclic voltammetry. The cyclic voltammograms of 1.5×10-6 mol/L nitroxazepine hydrochloride with 2.43×10-3 mol/ L cetrimidein phosphate buffers (pH 2.2 – 11.0) at HMDE exhibits a single welldefined peak in the potential range - 0.42 to - 0.56 V at allconcentrations due to the reduction of –NO2 group. No oxidationpeak was observed in the positive scanning half cycle, indicating theirreversible nature of electrode process [36]. The peak potentialshifted to a more negative value on the increase of the scan rateconfirming the irreversible nature of the reduction process [37]. For atotally irreversible electrode reaction, the relationship between thepeak potential (Ep) and the scan rate (ν) is expressed as Ep=(2.303RTαnF) log (RT/αnF) – (2.303 RTαnF) log ν. A straight line is observedwhen the peak potential (Ep) is plotted against log ν (mV/s) at aparticular concentration in pH 10.0 and can be expressed by theequation:

Ep =mV = 21:682 log ν mV= sð Þ–3:18; r2 = 0:993

Where Ep is the peak potential and ν is the scan rate. From the slopeof the straight line (ΔE/logν(mv/s), the αn value is calculated by usingthe expressionΔE/log ν(mV/s)=−30 mV/αn, the αn value is found tobe 1.26. Fractionalα values confirms the irreversible nature of electrodeprocess.

For finding the adsorptive character of the drug at HMDE a cyclicvoltammogram (Fig. 4; curve 2) was recorded after 180 s preconcen-tration at – 0.5 V and with zero second (Fig. 4; curve 1) preconcen-tration time. The peak current (Ip) increases after preconcentration ofthe drug on the electrode surface for 180 s.

The adsorptive character was also confirmed by studying theeffect of scan rate (ν) on stripping peak current (Ip) (Fig. 5). As thesweep rate was increased from 100 mV/s to 500 mV/s at a fixedconcentration of nitroxazepine hydrochloride (i) the peak potentialshifted cathodically (ii) the peak current increased steadily, and (iii)the peak current function, I/ACν1/2 exhibited near constancy. WhereA is the cross sectional area of electrode in cm2 and C is the con-centration of nitroxazepine in mol/L. A straight line is observedwhen Ip is plotted against ν1/2, which may be expressed by theequation:

Ip = μA = 4:498 ν =mV= sð Þ + 0:141; r2 = 0:998

235R. Jain et al. / Materials Science and Engineering C 31 (2011) 230–237

The adsorption effect was also identified by a plot of log Ip vs. log υgiving a straight line which can be expressed by the equation:

log Ip = μA� �

= 0:9622 log υ mV= sð Þ−2:415; r2 = 0:999

A slope of 0.96 which is close to the theoretical value of 1.0,indicating the adsorptive nature of the reduction process [38,39].

3.7. Controlled potential coulometric behaviour

By using controlled potential coulometry, the number of electronstransferred, n values were calculated from the charge consumed bythe desired concentration of nitroxazepine hydrochloride. The chargeconsumed was determined in basic medium. For this purpose 2.0 mLof 5 mg/mL solution of the electroactive species was placed in the celland electrolysis was carried out at a potential of - 0.3 to - 0.6 V againstAg/AgCl reference electrode. During the electrolysis, solutions werecontinuously stirred and purged with nitrogen. Number of electrons nwas calculated using the equation Q=nFN, where Q is charge incoulombs, F is Faraday's constant and N is number of moles of thenitroxazepine hydrochloride. The value is found to be four forcathodic peak of nitroxazepine hydrochloride in cetrimide.

On the basis of CV, DPCAdSV, SWCAdSV and coulometric studiesfollowing mechanism may be postulated for the reduction of nitrox-azepine hydrochloride (Scheme 2).

Scheme 2.

3.8. Analytical applications

The applicability of the proposed voltammetric method for thedetermination of nitroxazepine hydrochloride in pharmaceuticalformulation, serum and plasma was examined by measuring thestripping peak current as function of concentration.

Fig. 6. The dependence of the SWCAdS voltammetric current for nitroxazepinehydrochloride in 2.43×10-3 mol/L cetrimide at different concentrations; Eacc=-0.5 V,tacc=180 s, frequency f=80 Hz, pulse amplitude ΔEsw=50mV and scan incrementΔs=10 mV (a) Blank (b) 5×10-9 mol/L (c) 1.0×10-8 mol/L (d) 1.5×10-8 mol/L(e). 4.0×10-8 mol/L (f) 8.0×10-8 mol/L (g) 1.2×10-7 mol/L (h) 1.6×10-7 mol/L(i) 2.0×10-7 mol/L. The error bars represents standard deviation from three separateexperiments.

3.8.1. Nitroxazepine assay in pharmaceutical formulationOn the basis of electrochemical reduction of nitroxazepine

hydrochloride, an analytical method has been developed fordetermination of drug in pharmaceutical formulation. A linearrelationship between peak current and nitroxazepine hydrochlorideconcentration was observed over the concentration range 2.0×10-7

to 5.0×10-9 mol/L in 2.43×10-3mol/L cetrimide. The calibrationgraph was represented by the following equations:

SWCAdSV : Ip = μA = 3:2 × 106 Nitrox½ �= μA= mol = Lð Þ + 0:2202; r2

= 0:993; n = 3 ð1Þ

DPCAdSV : Ip = μA = 2:7 × 105 Nitrox½ �= μA= mol = Lð Þ−0:3674; r2

= 0:995; n = 3 ð2Þ

The current is mainly adsorption-controlled and proportional tothe concentration over a convenient range. Representative squar-ewave voltammograms are shown in Fig. 6.

Fig. 7. Square-wave obtained for determination of nitroxazepine hydrochloride inspiked (a) serum and (b) plasma samples, Eacc=-0.5 V, tacc=180 s, frequencyf=80 Hz, pulse amplitude ΔEsw=50 mV and scan increment Δs=10 mV (a) Blank(b) 7.16×10-7 mol/L (c) 5.21×10-7 mol/L (d) 3.17×10-7 mol/L (e) 2.12×10-7 mol/L(f) 9.2×10-8 mol/L.

236 R. Jain et al. / Materials Science and Engineering C 31 (2011) 230–237

3.8.2. Nitroxazepine assay in spiked biological samplesThe applicability of proposed procedure for the assay of nitrox-

azepine hydrochloride in spiked human biological fluids was tested.Fig. 7 illustrates the response of successive concentrations ofnitroxazepine hydrochloride in spiked serum and plasma followingits preconcentration onto the HMDE for 180 s. The variation of peakcurrent with nitroxazepine concentration was studied in both serumand plasma samples. The concentration of nitroxazepine was linearover the range of 9.2×10-8 to 7.1×10-7 mol/L by SWCAdSV and7.7×10-7 to 1.0×10-7 mol/L by DPCAdSV in plasma and serumsamples according to equations:

serum

DPCAdSV : Ip = μA = 1:0 × 106 Nitrox½ �= μA= mol= Lð Þ

+ 0:0001; r2 = 0:998; n = 3

ð3Þ

SWCAdSV Ip = μA = 9:0 × 106 Nitrox½ �= μA= mol= Lð Þ

+ 0:0295; r2 = 0:998; n = 3

ð4Þ

plasma

DPCAdSV : Ip = μA = 1:0 × 106 Nitrox½ �= μA = mol= Lð Þ

+ 0:0293; r2 = 0:999; n = 3

ð5Þ

SWCAdSV : Ip = μA = 5:0 × 106 Nitrox½ �= μA= mol = Lð Þ

+ 0:0095; r2 = 0:998; n = 3 ð6Þ

Quantifications were performed by means of the calibration curvemethod from the related calibration equation. The LOD and LOQ valueswere calculated based on peak current and were tabulated in Table 2.

4. Conclusion

In the daily practice it is necessary to perform drug analyticalmonitoring in order to adequate the dose of these substances and toavoid toxic effects. The voltammetric reduction of nitroxazepinehydrochloride under the conditions described in this work is anirreversible process controlled by adsorption. The adsorptive techni-ques, DPCAdSV and SWCAdSV are effective and rapid with wellestablished advantages including good discrimination against back-ground current and lowdetection limits. Furthermore, determination ofdrug in solubilized system provides new medium for study ofinteraction of drugs with biological membranes, because surfactantsinteracts with adsorbing membranes enhancing permeability ofdissolved drugs. As applied to serum and plasma samples, thesemethods have the advantage that no prior extraction procedure isrequired before the analysis. The developed method with the detectionlimit of 1.62×10-10 in presence of surfactant is more sensitive thanalready reported analytical methods. The method also provides a rapidand simple approach for the determination of nitroxazepine hydro-chloride in pharmaceutical dosage forms and in biological fluids. Theabove stripping methods shows high percentage of recovery meanscompounds are almost completely extracted from pharmaceuticalformulation and thus above method can be used to quantify thenitoxazepine without interference from other ingredients.

References

[1] J. David, R.S. Grewal, Pharmacological properties of Sintamil, a new antidepressantagent, Indian J. Exp. Biol. 12 (1974) 225.

[2] A.K. Mishra, K.D. Gode, Polarographic determination of nitroxazepine hydrochlo-ride in Tablets, Analyst 110 (1985) 31.

[3] J. Joseph-Charles, M. Bertucat, Isocratic High Performance Liquid Chromatographyfor the Simultaneous Separation of Eleven Tricyclic Imipramine-Derived Com-pounds of Therapeutic Interest, J. Liq. Chrom. Rel. Technol. 2 (1998) 3047.

[4] B. Uslu, S.A. Ozkan, Anodic voltammetry of abacavir and its determination inpharmaceuticals and biological fluids, Electrochim. Acta 49 (2004) 4321.

[5] S.A. Ozkan, B. Uslu, P. Zuman, Electrochemical oxidation of Sildenafil Citrate(Viagra) on carbon, Anal. Chim. Acta 501 (2004) 227.

[6] S.A. Ozkan, B. Uslu, Z. Senturk, Electroanalytical Characteristics of Amisulpride andVoltammetric Determination of the Drug in Pharmaceuticals and BiologicalMedia, Electroanalysis 16 (2004) 231.

[7] S.A. Ozkan, B. Uslu, H. YAboul-Enein, Analysis of Pharmaceuticals and BiologicalFluids using Modern Electroanalytical Techniques, Cirt. Rev. Anal. Chem. 33(2003) 155.

[8] P.T. Kissinger, W.R. Heinenam (Eds.), Laboratory Techniques in Electroanalyticalchemistry, Second ed., Marcel Dekker, New York, 1996.

[9] J. Wang, Analytical Electrochemistry, Second ed.Wiley-VCH, New York, 2001.[10] R. Jain, N. Jadon, K. Radhapyari, Determination of antihelminthic drug pyrantel

pamoate in bulk and pharmaceutical formulations using electro-analyticalmethods, Talanta 70 (2006) 383.

[11] A.T. Florence, I.G. Tucker, K.A. Walters, Structure performance Relationship inSurfactants, in: M.J. Rosen (Ed.), ACS Symp Series 253, American Chemical Society,Washington DC, 1984.

[12] J. Fendler, E. Fendler, Catalysis in Miceller and Macromolecular Systems,Academic, NY, 1975.

[13] M.J. Rosen, Surfactants and Interfacial Phenomenon, Interscience, New York,2004.

[14] J.R. Crison, N.D. Weiner, G.L. Amidon, Dissolution media for in vitro testing ofwater-insoluble drugs: effect of surfactant purity and electrolyte on in vitrodissolution of carbamazepine in aqueous solutions of sodium lauryl sulfate, J.Pharm. Sci. 86 (1997) 348.

[15] J.F. Rusling, F.N. Alaa-Eldin, Enhanced electron transfer for myoglobin insurfactant film on electrodes, J. Am. Chem. Soc. 15 (1993) 11891.

[16] J.R. Crison, V.P. Sha, J.P. Skelly, G.L. Amidon, A theoretical basis for abiopharmaceuticals drug classification: the correlation of in vitro drug productdissolution and in vivo bioavailability, J. Pharm. Sci. 85 (1996) 1005.

[17] M. Plavšiæ, D. Krnaric, B. Osoviæ, The electrochemical processes of copper in thepresence of triton X-100, Electroanalysis 6 (1994) 469.

[18] J. Yang, N.F. Hu, J.F. Rusling, Enhanced electron transfer for hemoglobin in poly(ester sulfonic acid) films on pyrolytic electrodes, J. Electroanal. Chem. 463 (1999)53.

[19] R. Jain, M. Ritesh, A. Dwivedi, Effect of Surfactant on voltammetric behaviour ofOrnidazole, Colloids Surf. A: Physicochem. Eng. Aspects 337 (2009) 74.

[20] R. Jain, A. Dwivedi, R. Mishra, Voltammetric behaviour of cefdinir in solubilizedsystem, J. Colloid Interface Sci. 318 (2008) 296.

[21] A.P. Doe, C.R.T. Tarley, N. Maniasso, L.T. Kubota, Exploiting micellar environmentfor simultaneous electrochemical determination of ascorbic acid and dopamine,Talanta 67 (2005) 829.

[22] D. Attwood, A.T. Florence, Surfactant Systems, Their Chemistry, Pharmacy andBiology, Chapman and Hall, London, 1983.

[23] J.F. Rusling, F. Nassar Alaa Eldin, Enhanced electron transfer for myoglobin insurfactant films on electrodes, J. Am. Chem. Soc. 115 (1993) 11891.

[24] M. Plavšić, D. Krznarić, B. Ćosović, The electrochemical processes of copper in thepresence of triton X-100, Electroanalysis 6 (1994) 469.

[25] A.B. Mandal, B.U. Nair, Cyclic Voltammetric technique for the Determinationofthe critical micelle concentration of surfactants, self-diffusion Coefficient ofmicelles, and partition coefficient of electrochemical probe, J. Phys. Chem. 95(1991) 9008.

[26] S. Monika, K. Kurt, R. Georg, N. Christian, Anodic stripping voltammetricdetermination of titanium (IV) using carbon paste electrode modified withcetyltrimethylamoniumbromide, Talanta 43 (1996) 1915.

[27] S. Monika, K. Kurt, R. Georg, A newmethod for the voltammetric determination ofmolybdenum (VI) using carbon paste electrodes modified in situ withcetyltrimethylamonium Bromide, Anal. Chim. Acta 350 (1997) 319.

[28] A.P. Abbott, G. Gounili, J.M. Bbbitt, J.F. Rusling, Electron Transfer betweenAmphiphilic Ferrocenes and Electrodes in Cationic Micellar Solutions, J. Phys.Chem. 9 (1992) 11091.

[29] P. Zuman, The Education of Organic Electrode Process, Academic Press, New york,1969.

[30] International conference on Harmonisation, ICH Harmonised Tripartite GuidelineText on Validation of Analytical Procedure Fed Regist, 60, 1995.

[31] E.J. Laviron, A multilayer model for the study of space distributed redox modifiedelectrodes: Part II. Theory and application of linear potential sweep voltammetryfor simple reaction, J. Electroanal. Chem. 112 (1980) 11.

[32] E. Hamam, Reduced intensity preparative regimens for allogeneic hematopoieticstem cell transplantation: a single center experience, J. Pharm. Biomed. Anal. 30(2002) 651.

[33] O. Abdel Razak, A randomized, comparative study of two regimens of beta-artemether for the treatment of uncomplicated, Plasmodium falciparum malaria,in northern Ghana, J. Pharm. Biomed. Anal. 34 (2004) 433.

[34] S. Braggio, R.J. Barnaby, P. Grossi, M. Cugola, A strategy for validation ofbioanalytical methods, J. Pharm. Biomed. Anal. 14 (1996) 375.

[35] The United, States pharmacopoeia (USP), convention Inc 2003 Methods, J. Pharm.Biomed. Anal. 14 (1996) 375.

[36] M.M. Ghoniem, K.Y. El-Baradie, A. Tawfik, Electrochemical behaviour of theantituberculosis drug isoniazid and its square-wave adsorptive strippingvoltammetric estimation in bulk form, Tablets and biological fluids at a mercuryelectrode, J. Pharm. Biomed. Anal. 33 (2003) 673.

[37] S. Halverson, E. Jacobsen, Electroreduction and polarographic determination ofnitrazepam in serum, Anal. Chim. Acta 59 (1972) 127.

237R. Jain et al. / Materials Science and Engineering C 31 (2011) 230–237

[38] A.J. Bard, L.R. Faulker, Electrochemical Methods: Fundamentals and applications,John Wiley & Sons Inc., New York, 2002.

[39] E. Laviron, Adsorption autoinhibition and autocatalysis in polarography and linearpotential sweep voltammetry, J. Electroanal. Chem. 52 (1974) 355.

Dr. Rajeev Jain is Professor of chemistry at Jiwaji University Gwalior, Madhya PradeshIndia. He received Ph.D in analytical chemistry from university of Roorke, Roorke, India(Now Indian Institute of Technology Roorke, India). He was awarded D.Sc in Analyticalchemistry from Jiwaji University Gwalior, India in 1990. He is a Member ofElectrochemical Society, USA. His research interest is electroanalytical chemistry andsensors.

Jahangir Rather is a Ph.D. scholar in chemistry at Jiwaji University Gwalior, India. Hereceived B.Sc from University of Kashmir in 2006. He received M.Sc in chemistry fromJiwaji University Gwalior. His research interest is mainly focused on electrochemicalsensors.

Ashish Dwivedi received M.Sc in analytical chemistry from Jiwaji University Gwalior,India in 2005. He received Ph.D chemistry from Jiwaji University Gwalior, India in 2009.His research field includes electrochemical analysis of pharmaceuticals in solubilizedsystems.