This article was downloaded by: [Ankara Universitesi]On: 14 March 2013, At: 04:43Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UK

Analytical LettersPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lanl20

Electrochemical Investigationand Determination of theAntibacterial Loracarbef byVoltammetric MethodsBurcu Dogan Topal a , Aysegul Golcu b & Sibel A.Ozkan aa Ankara University, Faculty of Pharmacy,Department of Analytical Chemistry, Ankara, Turkeyb Kahramanmaras Sutcu Imam University, Facultyof Science and Arts, Department of Chemistry,Kahramanmaras, TurkeyVersion of record first published: 19 Mar 2009.

To cite this article: Burcu Dogan Topal , Aysegul Golcu & Sibel A. Ozkan (2009):Electrochemical Investigation and Determination of the Antibacterial Loracarbef byVoltammetric Methods, Analytical Letters, 42:4, 689-705

To link to this article: http://dx.doi.org/10.1080/00032710802678637

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes.Any substantial or systematic reproduction, redistribution, reselling, loan,sub-licensing, systematic supply, or distribution in any form to anyone isexpressly forbidden.

The publisher does not give any warranty express or implied or make anyrepresentation that the contents will be complete or accurate or up todate. The accuracy of any instructions, formulae, and drug doses should be

independently verified with primary sources. The publisher shall not be liablefor any loss, actions, claims, proceedings, demand, or costs or damageswhatsoever or howsoever caused arising directly or indirectly in connectionwith or arising out of the use of this material.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

ELECTROCHEMISTY

Electrochemical Investigation and Determinationof the Antibacterial Loracarbef by

Voltammetric Methods

Burcu Dogan Topal,1 Aysegul Golcu,2 and Sibel A. Ozkan1

1Ankara University, Faculty of Pharmacy, Department of AnalyticalChemistry, Ankara, Turkey

2Kahramanmaras Sutcu Imam University, Faculty of Science and Arts,Department of Chemistry, Kahramanmaras, Turkey

Abstract: Loracarbef has antibacterial activity and is oxidizable at the glassycarbon electrode. The electrochemical oxidation of Loracarbef was investigatedusing cyclic, linear sweep, differential pulse, and square wave voltammetrictechniques. The results obtained from cyclic voltammetry indicate that the oxida-tion process of Loracarbef is irreversible and diffusion controlled on glassycarbon electrode. The dependence of peak currents and potentials on pH, concen-tration, scan rate, and nature of the buffer was investigated. According to thelinear relation between the peak current and the concentration, differential pulseand square wave voltammetric methods (DPV and SWV) for Loracarbef quanti-tative determination were developed. Different parameters were tested to opti-mize the conditions for the determination of Loracarbef. The quantitativedetermination of Loracarbef was proposed in 0.1 M H2SO4, which allows quan-titation over the 6� 10�6 to 2� 10�4 M range. Precision, accuracy, reproducibil-ity, sensitivity, and selectivity were checked. The methods were proposed for thedetermination of Loracarbef in pharmaceutical dosage forms, adopting both

Received 7 November 2008; accepted 9 December 2008.This research was supported by a TUBITAK grant (No. 105T371) for

Associate Professor Aysegul Golcu.Address correspondence to Aysegul Golcu, Faculty of Science and

Arts, Department of Chemistry, Kahramanmaras Sutcu Imam University,Kahramanmaras, Turkey. E-mail: ag518@ksu.edu.tr

Analytical Letters, 42: 689–705, 2009Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print=1532-236X onlineDOI: 10.1080/00032710802678637

689

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

DPV and SWV modes. Both methods were fully validated. No electroactiveinterferences from the excipients were found in its dosage forms.

Keywords: Determination, electro-oxidation, loracarbef, pharmaceuticals,voltammetry

INTRODUCTION

Loracarbef (LOR) is a synthetic b-lactam antibiotic of the carbacephemclass for oral administration. The carbacephems are closely related tothe cephalosporins, but replacement of the sulfur atom in the 7-amino-cephalosporanic acid nucleus by a methylene group is said to enhancestability. LOR is used similarly to cefaclor in the treatment of susceptibleinfections of the respiratory and urinary tracts and of skin and softtissue (Sweetman 2002; http://www.rxlist.com/cgi/generic/loracarb.htm; Chamber 2007). LOR is well absorbed from the gastrointestinaltract with a bioavailability of 90%. A plasma half-life of about 1 h hasbeen reported, which is prolonged in renal impairment. About 25% isbound to plasma proteins (Sweetman 2002; http://www.rxlist.com/cgi/generic/loracarb.htm; Chamber 2007).

LOR has been studied and determined only by three liquid chromato-graphic techniques (Alangary 1995; Kovach, Lantz, and Brier 1991;Klancke 1993). The reported methods either contain tedious and time-consuming procedures or require highly sophisticated instrumentation.Furthermore, no analytical methods for the determination of LOR, eitherin pharmaceutical dosage forms or bulk form, appear to have been reportedto date.

Because of the ever-increasing need for analytical methods with lowlimits of detection and determination and low maintenance costs, newmethodologies are constantly being developed. The voltammetric techni-ques offered the possibility for the sensitive estimation of LOR. To thebest of our knowledge, no electroanalytical assay has been performedon LOR.

Scheme 1. Chemical structure of LOR.

690 B. D. Topal et al.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

In past decades, modern voltammetric techniques have been used torealize the determination of organic compounds in diverse types ofsamples, especially in the pharmaceutical field (Ozkan, Uslu, andAboul-Enein 2003; Kissinger and Heinemann 1996; Wang 2006; Nouwset al. 2006; Uslu and Ozkan 2007; Smyth and Vos 1992). Modern electro-analytical techniques, such as differential pulse voltammetry (DPV) andsquare wave voltammetry SWV, have been used for the sensitive andquick determination of a wide range of drug substances with theadvantages that there is in most instances no need for derivatization ortime-consuming extraction steps and that these methods are less sensitiveto matrix effects than other analytical techniques (Ozkan, Uslu, andAboul-Enein 2003; Kissinger and Heinemann 1996; Wang 2006; Nouwset al. 2006; Uslu and Ozkan 2007; Smyth and Vos 1992; Kauffmannand Vire 1993; Dogan-Topal et al. 2008).

The aim of this study was to establish the experimental conditions toinvestigate the voltammetric behavior of LOR on glassy carbon electrodeusing cyclic, linear sweep, SWV, and DPV techniques. This articledescribes validated, simple, rapid, selective, and sensitive proceduresfor the determination of LOR in its dosage forms employing DPV andSWV at the glassy carbon electrode. The procedures did not require sam-ple pretreatment or any time-consuming extraction and evaporation stepprior to drug assay. The proposed methods might be alternatives to thehigh-performance liquid chromatography (HPLC) techniques in thera-peutic drug monitoring or the experimental data might be used for thedevelopment LC-EC detection method.

EXPERIMENTAL

Apparatus

Electrochemical measurements were performed using a BAS 100-Welectrochemical analyzer (Bioanalytical System, USA). A conventionalthree-electrode cell system incorporating the glassy carbon disc electrodeas a working electrode (/: 3 mm, diameter; BAS), an Ag=AgCl (3 M KCl;BAS) reference electrode, and a platinum wire auxiliary electrode wereused. The working electrode was polished manually with aqueous slurryof alumina powder (/: 0.01 mm) on a damp, smooth polishing cloth (BASvelvet polishing pad) before each measurement. All experiments wererealized at room temperature.

The pH was measured using a pH meter model 538, WTW (Austria)with a combined electrode (glass-reference electrode) with an accuracy of�0.05 pH.

Electrochemical Oxidation of Loracarbef 691

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

Operating conditions for DPV were pulse amplitude, 50 mV;pulse width, 50 ms; and scan rate, 20 mVs�1 and for SWV were pulseamplitude, 25 mV; frequency, 15 Hz; and potential step, 4 mV.

For the comparison, the ultraviolet (UV) spectrophotometric methodwas proposed using Perkin-Elmer Lambda 45 UV-vis double beamspectrophotometer with a slit width of 2 nm. The absorbance values weremeasured at 262 nm using 1-cm quartz cells.

Reagents

LOR and its two different type of dosage forms (Lorabid) capsule(400 mg) and oral suspension (200 mg=5 mL) were kindly provided byFako-Actavis Pharmaceutical Co. (Istanbul, Turkey). All chemicals forpreparation of buffers and supporting electrolytes such as H2SO4,H3PO4, NaH2PO4, Na2HPO4, H3BO3, CH3COOH, and NaOH werereagent grade (Merck or Sigma).

Stock solutions of LOR (1� 10�2 or 1� 10�3 M) were prepared indoubly distilled water and kept in the dark in a refrigerator. Differenttypes of supporting electrolyte and buffers were used in this study. Forthe supporting electrolyte, 0.1 M and 0.5 M H2SO4, 0.2 M phosphatebuffer at pH 2.0–7.0, 0.2 M acetate buffer at pH 3.70–5.70, and 0.04 MBritton-Robinson buffer at pH 2.0–11.0 were used. Standard solutionswere prepared by dilution of the stock solution with selected supportingelectrolyte to give solutions containing LOR in the concentration rangeof 6� 10�6 to 2� 10�4 M. The calibration equation for DPV and SWVwas constructed by plotting the peak current against LOR concentration.

Standard solutions were prepared by dilution of the stock solutionwith distilled water to give solutions containing LOR in the concentra-tion range 2� 10�5 to 9� 10�5 M for UV-spectrophotometric method.The calibration plot was constructed by plotting the absorbance againstthe compound concentration. All validation parameters were also calcu-lated for the comparison of the UV-spectrophotometric study.

Validation of the Proposed Methods

The ruggedness and precision of the methods were checked at differentdays, and results were given as repeatability (within day) and reproduci-bility (between days). Relative standard deviation (RSD%) values werecalculated to check the ruggedness and precision of the method (Rileyand Rosanske 1996; Swartz and Krull 1997; Ermer and McMiller 2005;De Bievre and Gunzler 2005).

692 B. D. Topal et al.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

The accuracy of the method is the degree of the nearness to the realvalue of the observed analysis results. The accuracy of the analysis wasdetermined by calculating the percentage relative error between themeasured mean concentrations and added concentrations in dosage formanalysis and in recovery studies. The precision and accuracy of theanalytical methods are described in a quantitative fashion by the use ofrelative errors (Bias %) also. One example of relative error is accuracy,which describes the deviation from the expected results.

The selectivity of the proposed methods for the determination ofLOR was examined in the presence of the pharmaceutical dosage formexcipients.

All solutions were kept in the dark and were used within 24 h toavoid decomposition. However, voltammograms of the sample solutionsrecorded over a week after preparation did not show any appreciablechange in assay values.

Analysis in Pharmaceutical Dosage Forms

LOR determination was performed on commercially available capsuleand oral suspension dosage forms.

Ten capsules were accurately weighed and emptied as completely aspossible. The empty capsules were weighed again, and the differenceswere given as the total amount of the 10 capsules’ contents. The com-bined contents of the capsules were thoroughly ground to a fine powder.A sufficient amount of this powder for preparing a stock solution of1� 10�3 M was accurately weighed and transferred into a 25-mL cali-brated flask, and and the volume was completed with selected supportingelectrolyte. The contents of the flask were sonicated for 10 min to providecomplete dissolution. The sample from the clear supernatant liquor waswithdrawn and quantitatively diluted with the selected supporting elec-trolyte. This solution was then transferred to a voltammetric cell, andDP and SW voltammograms were recorded. The drug content per cap-sule was determined referring to the related regression equations. Inactiveingredients contained in the capsules are indigocarmine, FeO (red,yellow, and black), and titanium dioxide.

In the case of oral suspension dosage form, first of all the suspensionwas prepared and well shaken after addition of the necessary amount ofwater. The required volume (accurately pipetted) of the suspensionsample, equivalent to a stock solution of concentration ca. 1� 10�3 M,was transferred to a 25-mL calibrated flask and completed to the volumewith selected supporting electrolyte. The contents were ultrasonicated for10 min to effect complete dissolution. Aliquots from the upper clear layer

Electrochemical Oxidation of Loracarbef 693

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

were taken and diluted with the selected supporting electrolyte. Oralsuspension samples were then transferred to a voltammetric cell, andvoltammograms were recorded. The drug content in per 5 mL oral sus-pension was determined by referring to the related regression equations.Inactive ingredients contained in the oral suspensions are strawberryflavor, erythrosine, sucrose, methyl paraben, and propyl paraben.Because other components of the matrix of capsule or oral suspensiondosage forms may interfere with the analysis or accurate quantitationof the analyte, potential effects from matrix components must beinvestigated. Recovery experiments were performed in the presence ofthe matrix (Riley and Rosanske 1996; Swartz and Krull 1997; Ermerand McMiller 2005; De Bievre and Gunzler 2005). As described in theprevious section, to study the accuracy and to check the interference fromthe excipients used in the formulations of these techniques, recoveryexperiments were carried out using the standard addition method. Toknow whether the excipients show any interference with the analysis,known amounts of pure LOR were added to the pre-analyzed capsuleand oral suspension dosage forms. These studies show no excipient inter-ferences; thus the procedures can be use to determine the amount of LORin the presence of the excipients of both dosage forms.

RESULTS AND DISCUSSION

LOR was found as oxidizable compound on the glassy carbon electrode.No previous electrochemical study was available concerning the electro-oxidative behavior of LOR. The electrochemical behavior of LOR atglassy carbon electrode was studied in different supporting electrolyteand various buffer solutions between pH 0.3 and 11.0. LOR is manifestedon current–voltage curves recorded by CV on a glassy carbon electrodeby one main anodic peak. This peak sharpness is related with the mediumpH. Maximum peak currents and better peak shape were obtained inacidic pH values (Fig. 1a–c). Cyclic voltammetric measurements showedan irreversible nature of the oxidation process on glassy carbon electrode.The sweep was started at –0.25 V in the positive direction. At acidic pHs,LOR oxidation did not occur until about 1.30 V. The sweep was reversedat about 1.70 V. No reduction peak or wave corresponding to the mainoxidation peak was observed on the cathodic branch (Fig. 1). TheLOR wave decreased to the second or higher cycles in all media andpH values (Fig. 1a–d). This event may be partly attributed to the con-sumption of adsorbed LOR on the glassy carbon electrode surface.

Scan rate studies were carried out to investigate whether the processat the working electrode was under diffusion or adsorption control. CV

694 B. D. Topal et al.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

studies were obtained at different scan rates (5–1000 mVs�1). It wasfound that anodic peak potential shifted 74 mV more positive potentialvalue between 5 and 1000 mVs�1 scan rate studies, a further evidencefor the irreversible nature of the electrode reaction. A linear relationshipon the oxidation peak current with the square root of the scan rateshowed the diffusion control process. The equation is noted in 0.1 MH2SO4:

ipðmAÞ ¼ 0:197 v1=2ðmVs�1Þ þ 0:305 ðr; 0:998; n ¼ 10Þ

The effect of scan rate on peak current was also examined underthese conditions with a plot of logarithm of peak current (log i) versuslogarithm of scan rate (log v), giving a straight line within the same scanrate range. According the slope value (0.44) of next equation, the

Figure 1. Multisweep voltammograms of 2� 10�4 M LOR in different buffersolutions and supporting electrolyte: (a) 0.1 M H2SO4; (b) Britton–Robinson buf-fer at pH 2.0; (c) Britton–Robinson buffer at pH 4.0; (d) acetate buffer at pH5.67. Scan rate 100 mVs�1.

Electrochemical Oxidation of Loracarbef 695

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

diffusion controlled reaction was confirmed:

log ipðmAÞ ¼ 0:44 log v ðmVs�1Þ � 0:511 ðr; 0:999; n ¼ 10Þ

A straight line with a slope of 0.44 obtained from the logarithm ofpeak current versus the logarithm of scan rate values is found to closethe theoretical value of 0.5. This confirms an ideal reaction of solutionspecies (Laviron, Roullier, and Degrand 1980), so in this case the processhad a diffusive component. These results indicate that glassy carbonelectrode had been proposed for many electroanalytical applications,such as pharmaceutical and biological applications, because of the lowadsorption of organic compounds. We tried to show that the glassycarbon electrode can be used for the sensitive, rapid, and accurate deter-mination of LOR, as an alternative method to the published time-consuming separation techniques.

Because of the poorly resolved signal obtained by cyclic voltamme-try, the effect of pH on peak intensity and peak potential were studiedusing also DPV and SWV techniques between pH 1.0 and 9.0. Theobtained graphs from DPV and SWV were similar; the only SWV graphfor the oxidation potential and oxidation peak current is given in Fig. 2.The peak potential of the anodic response moved to less positive poten-tial values, and the oxidation peak was ill defined and disappeared afterpH 9.0. The plot of the peak potential versus pH showed two straightlines between 1.0 and 3.0 and between 3.0 and 8.0. These two linearsegments can be expressed by the following equations (Fig. 2a):

EpðmVÞ ¼ 1315:33� 3:5 pH; r ¼ 0:997 ðbetween pH 1.0 and 3.0Þ

EpðmVÞ ¼ 1356:48� 16:57 pH; r ¼ 0:998 ðbetween pH 3.0 and 8.0Þ

The intersection observed in the plot between pH 1.0 and 3.0 andbetween pH 3.0 and 8.0 can be explained by changes in protonation ofthe acid–base functions in the molecule. The intersection points of thecurve are close to the pKa values of LOR, which are described as pKa1

3.24 and pKa2 6.84 (SciFinder ScholarTM 2006 software). These pKavalues were obtained from SciFinder Scholar. These were calculated byAdvanced Chemistry Development (ACD=Labs) software V. 8.14 pro-gram. The linearity was observed in the pH range of 1.0 to 3.0, givinga negative slope of approximately 3.5 mV per pH unit and 16.57 mVper pH unit between pH 3.0 and 8.0 (Fig. 2a). According to the slopeof the second linearity range, one electron was involved in the rate-determining step with some effect of the protons. By analyzing the

696 B. D. Topal et al.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

evolution of peak current (Fig. 2b), it is possible to observe to that thisparameter is affected by the pH value and nature of the buffer. The peakcurrent reached the highest value in 0.1 M H2SO4 supporting electrolyte.Hence, 0.1 M H2SO4 was selected for further work because it not onlygave the highest peak current but also gave the best peak shape withDPV and SWV techniques for the determination of LOR.

Of the different functional groups of LOR, the nitrogen atom in theaza-bicyclo ring may be more easily oxidizable than the other possible

Figure 2. Effects of pH on LOR anodic peak potential (a) and peak current (b);LOR concentration 2� 10�4 M; 0.1 M H2SO4 (�); 0.04 M Britton–Robinsonbuffer (D); 0.2 M acetate buffer (�); and 0.2 M phosphate buffer (&).

Electrochemical Oxidation of Loracarbef 697

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

oxidizable center. The anodic behavior of LOR was also compared to theother cephalosporin group antibiotics such as cefixime, cefotaxime, cefo-perazone, and cefepime (Golcu, Dogan, and Ozkan 2005; Dogan-Topalet al. 2008a, 2008b; Ozkan, Uslu, and Zuman 2002) to identify the oxida-tive center of LOR. Because of the differences of the molecular structurebetween LOR and other cephalosporins, we may assume that the nitrogenatom in the azo-bicyclo ring is protonated with one electron oxidationprocess.

Analytical Application and Validation

SWV and DPV techniques are effective, rapid, and sensitive electroanaly-tical methods with well-established advantages, including good discrimina-tion against background currents and low detection and determinationlimits (Ozkan, Uslu, and Aboul-Enein 2003; Kissinger and Heinemann1996; Wang 2006). These techniques have been shown to be highly suitablefor measuring organic drug active compounds. Different supporting elec-trolytes and pH were examined for obtaining the best response for thedetermination of LOR. For this, sulfuric acid, Britton–Robinson acetate,and phosphate buffer in different pH values were investigated. The bestresults with respect to signal enhancement and peak shape accompaniedby sharper response was obtained with 0.1 M H2SO4 (Fig. 3a,b). Valida-tion of the proposed methods for the quantitative determination of LORwas examined via evaluation of linearity range limit of detection (LOD),limit of quantification (LOQ), repeatability, reproducibility, precision,accuracy, specificity, robustness, stability, and recovery.

The quantitative evaluation is based on the dependence of the peakcurrent on LOR concentration (Fig. 3). Two calibration plots from thestandard solution of LOR according to the procedures described pre-viously were constructed using DPV and SWV. The plot of peak currentversus the concentration of LOR was found to be linear in the concentra-tion range between 6� 10�6 and 2� 10�4 M by using both techniques,indicating that the response was diffusion controlled within this concen-tration range. Above the upper limit of the concentration range (3� 10�4

M), a loss of linearity may be due to the adsorption of LOR on theelectrode surface. Calibration graphs and related parameters wereconstructed using data from DPV and SWV measurements, and least-squares were evaluated using the linear regression method. In Table 1,the analytical characteristics are summarized for both techniques. Thedeveloped methods were validated according to the standard procedures(Riley and Rosanske 1996; Swartz and Krull 1997; Ermer and McMiller2005; De Bievre and Gunzler 2005). Accuracy, precision, specificity,

698 B. D. Topal et al.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

repeatability, reproducibility, LOD, and LOQ of the proposed techniqueswere assessed by performing replicate analysis of the standard solutionsin supporting electrolyte within calibration curves.

For the sensitivity and precision of the proposed methods, the LODand LOQ values were calculated as LOD¼ 3 s=m and LOQ¼ 10 s=m,

Figure 3. DP (a) and SW (b) voltammograms of different concentrations of LORin 0.1 M H2SO4. Experimental conditions were described in the text. (1) Support-ing electrolyte; (2) 4� 10�5M; (3) 8� 10�5M; (4) 2� 10�4M LOR in 0.1 M H2SO4.

Electrochemical Oxidation of Loracarbef 699

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

where s is the standard deviation of the peak currents (three runs) and mis the slope of the calibration equation (Riley et al. 1996; Swartz andKrull 1997; Ermer and McMiller 2005; De Bievre and Gunzler 2005].The obtained data are shown in Table 1. These limits indicate the highsensitivity of the proposed methods. The low values of SE of slope andintercept and greater than 0.999 correlation coefficient values establishedthe precision of the proposed techniques.

The repeatability (within day) and reproducibility (between days) ofpeak current and peak potentials were also tested for DPV and SWVmethods in the same day and different days. These values indicated that

Table 1. Characteristics and related validation data of LOR calibration equationsin 0.1 M H2SO4 for DPV, SWV, and UV-spectrophotometric techniques

Parameter DPV SWV UV-spectrophotometry

Measured potential(V)=absorbance(nm)

1.28 1.30 262

Linearity range (M) 6� 10�6 to2� 10�4

6� 10�6 to2� 10�4

2� 10�5 to9� 10�5

Slope (mA.M�1) 1.54� 104 1.66� 104 1.08� 104

Intercept (mA) 0.015 0.012 –0.027Correlation

coefficient0.999 0.999 0.998

SE of slope 7.80� 101 1.08� 102 2.89� 102

SE of intercept 6.48� 10�3 8.95� 10�3 1.72� 10�2

LOD (M) 2.44� 10�7 8.24� 10�7 1.50� 10�6

LOQ (M) 8.12� 10�7 2.75� 10�6 5.0� 10�6

Repeatability of peakcurrent=absorbance(RSD%)

0.60 0.46 0.75

Repeatabilityof peak potential=wavelength(RSD%)

0.14 0.14 0.02

Reproducibilityof peak current=absorbance(RSD%)

0.72 1.90 2.62

Reproducibilityof peak potential=wavelength(RSD%)

0.17 0.22 0.03

700 B. D. Topal et al.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

the proposed methods have high repeatability and precision for the LORanalysis. The related parameters were shown in Table 1 as RSD % values.

The stability of the reference substance and sample solutions waschecked by analyzing prepared standard LOR in supporting electrolyteaged at þ4�C in the dark against a fresh sample. Repetition of sampleanalysis for LOR after a weeklong period did not show any significantchange in results, indicating the stability of the drug.

The robustness of the proposed methods was examined by evaluatingthe influence of small variations of some of the most important procedurevariables such as pH, nature of the buffer, and scan rate effect. All theseparameters were checked during the method optimization step.

Table 1 demonstrates the good precision, accuracy, sensitivity, andreproducibility.

All these validation parameters were also checked for the proposedspectrophotometric method, and the results are presented in Table 1.

Analysis of LOR in Dosage Forms

For showing the applicability and selectivity of the proposed methods,the determination of LOR from its pharmaceutical dosage forms usingproposed methods were realized. The selectivity of the optimized meth-ods for the determination of LOR was examined in the presence of exci-pients, which are described in the experimental section (Riley et al. 1996;Swartz and Krull 1997; Ermer and McMiller 2005; De Bievre andGunzler 2005). This study was realized from real excipients of LOR dos-age forms. Hence, with these studies, the selectivity and the applicabilityof the proposed methods were shown. The obtained results are presentedin Table 2. These values showed no significant excipient interference;thus, the procedures were able to determine the amount of LOR in thepresence of excipients. Comparison of the DPV and SWV results ofLOR, raw material, and pharmaceutical dosage forms showed that thepeak current and peak potential of LOR did not change. According tothese obtained results, the proposed methods can be considered to beselective. On the basis of obtained results, both DPV and SWV methodswere applied to the direct determination of LOR pharmaceutical dosageforms, using related calibration equations without any sample prepara-tion, extraction, and filtration or evaporation steps other than anadequate dilution step. All these studies were realized using the spectro-photometric method also. The results are shown in Table 2.

The mean results for five determinations of all techniques are veryclose to the declared values. The results showed that the proposedmethods could be applied with great success to LOR determination incapsule and suspension dosage forms without any interference.

Electrochemical Oxidation of Loracarbef 701

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

As far as we know, no official method is described in pharma-copoeias related to the LOR dosage forms. For this reason, the UV-spectrophotometric method was also proposed and validated for thecomparison with proposed DPV and SWV method.

In comparison to the spectrophotometric method, the proposed DPVand SWV techniques are more sensitive, rapid, and easy. On the otherhand, as Table 2 shows, the proposed methods were compared with thespectrophotometric method using student’s t and F tests. All methodsshowed similar accuracy and precision. According to the student’s t andF tests, the calculated t and F values did not exceed the theoretical valuefor a significance level of 0.05. Statistical analysis of the results showedno significant difference between the performance of the compared UV-spectrophotometric and proposed DPV and SWV methods as regards tosimplicity. On the other hand, wide linearity range, low detection and deter-mination limits, rapidity, and better accuracy and precision were obtainedwith the proposed DPV and SWV methods, without any filtration steps.

For the accuracy of the methods, the recovery studies were realized.Recovery studies were carried out after addition of known amounts ofpure drug to various pre-analyzed dosage forms of LOR. To detect

Table 2. Comparative pharmaceutical dosage form assay and recovery studiesof LOR

Capsule Oral suspension

Parameter DPV SWV UV-spec. DPV SWV UV-spec.

Labeled claim(mg)

400.00 400.00 400.00 200.00 200.00 200.00

Amount found(mg)a

400.92 399.27 401.50 200.14 199.63 200.23

RSD (%) 0.22 0.43 0.006 0.13 0.45 0.27Bias (%) –0.23 0.18 –0.38 –0.07 0.19 �0.12tcalculated 0.21 0.021 ttheoretical:

2.310.72 0.23 ttheoretical:

2.31Fcalculated 0.20 0.019 Ftheoretical:

2.600.19 0.35 Ftheoretical:

2.60Added (mg) 20.00 20.00 20.00 20.00 20.00 20.00Found (mg)a 19.99 19.98 20.25 20.01 19.98 19.94Recovery (%) 99.98 99.92 100.40 100.03 99.88 99.70RDS (%) of

recovery0.22 0.08 1.20 0.12 0.29 1.15

Bias (%) 0.02 0.10 –1.25 –0.03 0.12 0.3

aEach value of the mean of five experiments.

702 B. D. Topal et al.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

interactions of excipients, the standard addition technique was applied.Recovery experiments using developed assay procedures furtherindicated the absence of interference from commonly encounteredpharmaceutical excipients used in the capsule and suspension dosageforms (Table 2). The results indicate the validity of proposed techniquesfor the determination of LOR in pharmaceutical dosage forms.

CONCLUSION

The electrochemical behavior and determination of LOR on glassycarbon electrode was established and studied for the first time. LOR isirreversibly oxidized at high positive potentials. Because of the highsensitivity and selectivity of electrochemical methods, the determinationof LOR has been subject of considerable interest, and time-consumingmethods have been developed for its determination. Glassy carbonelectrode showed perfect results for the electro-oxidative determinationof LOR. The diffusion control and an irreversible response were obtainedat all pH values and buffers. The proposed DPV and SWV techniques forthe determination of LOR in its dosage forms were found to bemore sensitive, simple, selective, and rapid than the proposed UV-spectrophotometric method. The principal advantages of the proposedvoltammetric methods over the UV-spectrophotometric one are theabsence of influence of matrix effects, no need the filtration or precipita-tion steps, and higher selectivity and sensitivity because of the possibilityof higher sample dilution. The proposed DPV and SWV techniques canbe applied directly to the analysis of capsule and suspension dosage formswithout the need for separation or filtration steps, because there was nointerference from the excipients. These proposed voltammetric methodsmay be preferred to reported LC methods for the determination ofLOR in pharmaceutical dosage forms. The experimental data might beused for the development LC-EC method for the determination ofLOR. These methods could be easily used in quality control laboratoriesfor the determination of LOR.

REFERENCES

Alangary, A.A. 1995. A simple high-performance liquid chromatographic assayfor Loracarbef in human plasma. Anal. Lett., 28: 1819–1831.

De Bievre, P. and Gunzler, H. (Eds.). 2005. Validation in Chemical Measurements.Berlin: Springer. Chamber, H. F. 2007. b-Lactam and other cell wall- andmembrane-active antibiotics. In Basic and Clinical Pharmacology, 10th ed.,ed. B.G. Katzung. McGraw Hill.

Electrochemical Oxidation of Loracarbef 703

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

Dogan-Topal, B., Golcu, A., Dolaz, M., and Ozkan, S.A. 2008a. Anodicoxidation of antibacterial drug cefotaxime sodium and its square wave anddifferential pulse voltammetric determination in pharmaceuticals and humanserum. Curr. Pharm. Anal., in press.

Dogan-Topal, B., Golcu, A., Dolaz, M., and Ozkan, S.A. 2008b. Electrochemicalbehaviour of the bactericidal cefoperazone and its selective voltammetricdetermination in pharmaceutical dosage forms and human serum. Curr.Pharm. Anal., in press.

Dogan-Topal, B., Uslu, B., Ozkan, S.A., and Zuman, P. 2008. Electrochemicaldetermination of HIV drug Abacavir based on its reduction. Anal. Chem.,80: 209–216.

Ermer, J. and McMiller, J.H. (Eds.). 2005. Method Validation in PharmaceuticalAnalysis. Weinheim: Wiley-VCH.

Golcu, A., Dogan, B. and Ozkan, S.A. 2005. Anodic voltammetric behavior anddetermination of Cefixime in pharmaceutical dosage forms and biologicalfluids. Talanta, 67: 703–712.

http://www.rxlist.com/cgi/generic/loracarb.htmKauffmann, J.M. and Vire, J.C. 1993. Pharmaceutical and biomedical applica-

tions of Electroanalysis: A critical review. Anal. Chim. Acta, 273: 329–334.Kissinger, P.T. and Heinemann, W.R. (Eds.). 1996. Laboratory Techniques in

Electroanalytical Chemistry. 2nd Ed. New York: Marcel Dekker.Klancke, J.W. 1993. Determination of monovalent and divalent cations and

chloride in the carbacephalosporin loracarbef by ion chromatography.J. Chromatogr., 637: 63–70.

Kovach, P.M., Lantz, R.J., and Brier, G. 1991. High-performance liquidchromatographic determination of loracarbef, a potential metabolite, cefaclor,and cephalexin in human plasma, serum, and urine. J. Chromatogr., B567: 129–139.

Laviron, E., Roullier, L., and Degrand, C. 1980. A multilayer model for the studyof space distributed redox modified electrodes, part II: theory and applicationof linear potential sweep voltammetry for a simple reaction. J. Electroanal.Chem., 112: 11–23.

Nouws, H.P.A., Delerue-Matos, C., Barros, A.A., and Rodrigues, J.A. 2006.Electroanalytical determination of paroxetine in pharmaceuticals. J. Pharm.Biomed. Anal., 42: 341–346.

Ozkan, S.A., Uslu, B., and Aboul-Enein, H.Y. 2003. Analysis of pharmaceuticalsand biological fluids using modern electroanalytical techniques. Crit. Rev.Anal. Chem., 33: 155–171.

Ozkan, S.A., Uslu, B., and Zuman, P. 2002. Electrochemical reduction andoxidation of the antibiotic cefepim on a carbon electrode. Anal. Chim. Acta,457: 265–274.

Riley, C.M. and Rosanske, T.W. (Eds.). 2008. Development and Validation ofAnalytical methods. New York: Elsevier Science.

SciFinder Scholar. 2006. Advanced Chemistry Development (ACD=Labs)Software, ver. 8.14 for Solaris (1994–2008). American Chemical Society.

704 B. D. Topal et al.

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3

Smyth, M.R., and Vos, J.G. (Eds.). 1992. Analytical Voltammetry. Amserdam:Elsevier.

Swartz, M.E. and Krull, I.S. (Eds.). 1997. Analytical Method Development andValidation. New York: Marcel Dekker.

Sweetman, S.C. 2007. The Complete Drug Reference. 33rd Ed. London:Pharmaceutical Press.

Uslu, B. and Ozkan, S.A. 2007. Solid electrodes in electroanalytical chemistry:Present applications and prospects for high-throughput screening of drugcompounds. Comb. Chem. High T. Scr., 10: 495–513.

Wang, J. (Ed.). 2006. Analytical Electrochemistry. 3rd Ed. NJ: Wiley-VCH.

Electrochemical Oxidation of Loracarbef 705

Dow

nloa

ded

by [

Ank

ara

Uni

vers

itesi

] at

04:

43 1

4 M

arch

201

3