Electrochemical Evaluation of Nucleoside Analogue Lamivudine in Pharmaceutical Dosage Forms and...

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Electrochemical Evaluation of Nucleoside Analogue Lamivudine inPharmaceutical Dosage Forms and Human Serum

Burcu Dogan,a Bengi Uslu,a Sibel Suzen,b Sibel A. Ozkan*a

a Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey*e-mail: [email protected] Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey

Received: February 11, 2005Accepted: April 13, 2005

AbstractLamivudine (LAM) is a synthetic nucleoside analogue with activity against human immunodeficiency virus-type 1(HIV-1) and Hepatitis B virus (HBV). The aim of this study was to determine LAM levels in serum andpharmaceutical formulations, by means of electrochemical methods using hanging mercury drop electrode (HMDE).On this electrode, LAM undergoes irreversible reduction at the peak potential near Ep� 1.26 V (vs. Ag/AgCl/3 MKCl). Reduction LAM signals were measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV) andsquare-wave voltammetry (OSW). DPV and OSW techniques for the determination of LAM in acetate buffer atpH 4.5, which allows quantitation over the 4� 10�6 to 1� 10�4 M range in supporting electrolyte for both methods,were proposed. The linear response was obtained in acetate buffer in the ranges of 2� 10�6 to 2� 10�4 M for spikedserum samples at pH 4.5 for both techniques. The repeatability and reproducibility of the methods for all media weredetermined. The standard addition method was used in serum. Precision and accuracy were also checked in all media.No electroactive interferences from the endogenous substances were found in serum. With respect to side effects ofhigh doses and short half-life of LAM, a fast and simple detection method is described in this study.

Keywords: Lamivudine, Zalcitabine, Voltammetry, Reduction, Serum, Pharmaceutical formulations

DOI: 10.1002/elan.200503307

1. Introduction

Lamivudine (LAM) (Scheme 1), a synthetic nucleosideanalogue with activity against human immunodeficiencyvirus-1 (HIV-1) and hepatitis B virus (HBV). It is anucleoside reverse transcriptase inhibitor structurally re-lated to cytosine with activity against retroviruses includingHIV. It is used, usually in combination with other antire-troviral, in the treatment of HIV infection. LAM isconverted intracellularly in stages to the triphosphate. Thistriphosphate halts the DNA synthesis of retroviruses,including HIV, through competitive inhibition of reversetranscriptase and incorporation into viral DNA. Following

oral administration LAM is rapidly absorbed and peakplasma concentrations are achieved in about 1 hour.Bioavailability is between 80 and 87%. Binding to plasmaprotein is reported to be less than 36% [1, 2].A survey of the literature revealed that there have been

very few methods for its determination in biological fluids.The analysis of LAM in human plasma, serum and urineusing LC [3], LC-MS [4], HPLC [5 – 10] and spectrophoto-metric [5] methods has been reported but there were noelectrochemical study on hanging mercury drop or by solidcarbon based electrodes published yet.Electrochemical techniques in perspective by introduc-

ing the most promising methods and evaluating theirrelative merits for specific applications [11 – 14]. Due tothe existing resemblance between electrochemical andbiological reactions can be assumed that the oxidation/reduction mechanisms taking place at the electrode and inthe body share similar principles. Biologically importantmolecules can be investigated electroanalytically by vol-tammetry in order to determine the molecule in differentways [15 – 16].This work was directed to study the voltammetric

behavior of LAM, owing to the high sensitivity andsimplicity of the voltammetric techniques, and lack ofliterature data on the electrochemical behavior of LAM.This paper described a fully validated, simple, rapid andmore sensitive developed procedure for the determinationScheme 1. Chemical structure of LAM and ZAL

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Electroanalysis 17, 2005, No. 20, 1886 – 1894 F 2005 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

of LAM in pharmaceutical formulations and serum employ-ing differential pulse and square wave voltammetry at thehanging mercury drop electrode. As an advantage thedetermination procedure did not require sample pretreat-ment or any time-consuming extraction step prior to thedrug assay.

2. Experimental

2.1. Instrumentation

Electrochemical measurements were performed using aBAS 100 W electrochemical analyzer (Bioanalytical Sys-tem, USA). Voltammetric measurements utilized a hang-ing mercury drop electrode (HMDE). The three electrodesystem was completed by means of a platinum wireauxiliary electrode and on Ag/AgCl (3 M KCl) referenceelectrode.All the results in the figures are presented in respect to the

Ag/AgCl, 3 M KCl reference electrodes.The pH value of the solutions was measured using a

Model 538 pH meter (WTW, Austria) and calibrated withstandard buffers (FIXANAL, Riedel-de Haen, Germany).All measurements were carried out at ambient temper-

ature of the laboratory (23 – 27 8C).

2.2. Reagents and Solutions

LAM and its dosage forms were kindly provided by GlaxoSmith Kline Pharm. Ind.Each film-coated tablet (Epivir) was contained 150 mg of

lamivudine and the inactive ingredients hydroxypropylmethylcellulose, magnesium stearate, microcrystalline cel-lulose, polethlene glycol, polysorbate 80, sodium starchglycolate and titanium dioxide.Each onemilliliter of Epivir oral solution contained 10mg

of lamivudine (10 mg/mL) in an aqueous solution and theinactive ingredients artificial strawberry and banana flavors,citric acid (anhydrous), methylparaben, propylene glycol,propylparaben, sodium citrate (dehydrate), and sucrose [2].Model compound (zalcitabine) ZAL was supplied from

Roche Pharm. Ind.All chemicals used were of reagent grade quality (Merck,

Sigma or Riedel) and they were employed without furtherpurification. The standard stock solutions of LAM (1�10�3 M) were prepared daily by direct dissolution in water.Theworking solutionswere prepared by serial dilution of thestock solution with selected supporting electrolytes. Severalsupporting electrolytes were used such as sulfuric acid(0.2 M), phosphate buffer (0.2 M, pH 2.0 –11.0), acetatebuffer (0.2 M, pH: 3.50–5.50) and Britton –Robinson buffer(0.04 M, pH: 2.32–12). All solutions were freshly prepared,protected from light and were used within 24 h to avoiddecomposition.All thedrug freehumanblood serumsampleswere frozen and then thawed immediately before use.

2.3. Voltammetric Measurements

Dissolved oxygen was removed by passing pure nitrogen(99.9%) through the solution for 3.0 min prior to electro-chemical analyses. Differential pulse voltammetry (DPV)conditions were: Pulse amplitude: 50 mV; pulse width:50 ms; sample width: 17 ms; pulse period: 200 ms; scanrate: 20 mVs�1; and square wave voltammetry (OSW)conditions were: pulse amplitude: 25 mV; frequency:15 Hz;potential step: 4 mV.

2.4. Tablet and Suspension Treatment

Ten tablets of Epivir, each containing 150 mg of lamivudine,were powdered and the amounts corresponding to 1�10�3 M of LAM were weighed and dissolved in water. Thecontents of the flask were sonicated for 15 min to effectcomplete dissolution. After the excipients have settleddown, an aliquot of the clear supernatant was transferredquantitatively into a calibrated flask and diluted to a finalvolume of 10 mL with selected supporting electrolyte. Thenthe voltammogram was recorded following the voltammet-ric procedure.1.15 mL of Epivir oral solution, claim to contain 10 mg

LAMper 1.0 mL solution, was dissolved in 50 mLwater.Analiquot of this solution was transferred in to a 10.0 mLvolumetric flask and diluted to mark with supportingelectrolyte and the voltammogram was recorded.The nominal content of tablets and oral solution are

determined from corresponding regression equations.

2.4.1. Recovery Studies from Tablets and Oral Suspensions

For the accuracy and reproducibility of the proposedmethods, recovery experiments were carried out using thestandard addition method. For this procedure, knownamounts of the pure drugwere added to the earlier analyzedtablet and oral suspension formulations of LAM. Therecovery of the drugs was calculated by comparing theconcentration obtained from the spikedmixtures with thoseof the pure drug.

2.5. Human Serum Treatment

Drug free human blood, obtained from healthy volunteers,was centrifuged (5000 rpm) in 30 min at room temperatureand the obtained serum samples were stored frozen untilthe assay. An aliquot volume of serum sample was fortifiedwith LAM dissolved in bidistilled water to achieve finalconcentration of 10�3 M LAM. This solution containedacetonitrile as serum precipitating agent. Acetonitrileremoves serum proteins more effectively, as the additionof 1 volume to 1.5 volumes of serum. After vortexing for30 s, the mixture was then centrifuged for 10 min at5000 rpm for getting rid of serum protein residues andsupernatant was taken. Appropriate volumes of this super-

1887Nucleoside Analogue Lamivudine in Pharmaceuticals

Electroanalysis 17, 2005, No. 20, 1886 – 1894 www.electroanalysis.wiley-vch.de F 2005 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

natant were transferred to a 100 mL volumetric flask anddiluted to mark with supporting electrolyte and thevoltammogram was recorded following the voltammetricprocedure as described above.Quantifications were performed by means of the calibra-

tion curve method.

3. Results and Discussion

3.1. Electrochemical Behavior of LAM at HMDE

In order to understand the electrochemical process occur-ring on HMDE cyclic voltammetry was carried out. The pHdependence reduction of LAM was studied systematicallyin the pH range between 1.8 and 12.0. The voltammetricresponse was markedly dependent on pH. According to thisstudy LAM response was obtained until pH 10.0. AfterpH 10.0 LAM did not give a response. Cyclic voltammo-gram of 4� 10�5 M solution of LAM, at a sweep rate of100 mVs�1, exhibited a single well-defined, irreversiblereduction peak at �1.26 V (2.0�pH� 10.0) (Fig. 1a – d).

No peak was observed on the reverse scan corresponding tothe main cathodic peak. Hence, this observation confirmedthe irreversibility of the reduction process. Besides, thecyclic voltammetric behavior of more concentrated LAMsuch as 2� 10�4 M yielded onewell-defined peak in stronglyacidic solution such as 0.2 M sulfuric acid and pH 2.0Britton –Robinson or phosphate buffer (Fig. 2a). As the pHincreased, this one wave was split into two peaks (Fig. 2b).At above pH 6.0 the two reduction peaks clearly mergedinto a single peak (Fig. 2c).At pH 11.0 the peak disappearedfor all concentrations.In Figure 3a the peak potential vs. pHplot is presented for

LAM. Peak potential is linear with pH and two differentzones can be observed, with break at pH 4.5 and this can beassociated with the pKa of cytosine that are equal to 4.5 [17].This break could be due to a change in protonation – de-protonation process of the electroactive molecule.For LAM the slope of the Ep-pH plot is �37.08 mV per

unit over the pH 2.0 – 4.5 and is�99.11 mVper unit over thepH 4.5 – 10.0, which suggests that the number of proton andelectrons involved in the reduction mechanism. Changes inslope of the Ep-pH plot was found at about pH 4.5. For pH

Fig. 1. Cyclic voltammograms of 4� 10�5 M LAM in Britton –Robinson buffer at pH 2.32 (a); in acetate buffer at pH 4.5 (b); inphosphate buffer at pH 5.00 (c); in Britton –Robinson buffer at pH 8.0 (d). Scan rate 100 mVs�1.

1888 B. Dogan et al.


values lower than 4.5 the slope is �37.08 mV per pH unitand in the more alkaline region dEp/dpH is �99.11 mV perpH unit. This indicates that the ratio of the number ofprotons and transferred electrons involved in the changetransfer changed from 1 to 2 for 4.5� pH� 10.0. This couldbe explained if, a product of reduction undergoes deproto-nation at pH> 4.5 while the protolytic state of the eductsremain, as it was lower pH. The reduction of LAM itselfmechanism involving the total gain of 3 electrons.The cathodic behavior of LAMwas comparedwith that of

Zalcitabine (ZAL) as a model compound to support theworking hypothesis that was the cytosine moiety in LAMthat undergoes the reduction. ZAL and LAM have similarchemical formula. They both contains cytosine ring(Scheme 1). To explain the reduction mechanism of LAM,all the necessary experiments such as pH, scan rate etc. tookplace with ZAL. ZAL was reduced over most of the pHrange in a single peak in low concentrations such as 4�10�5 M (Fig. 4a). As a similar behavior like LAM, moreconcentrated ZAL such as 2� 10�4 M, showed one well-defined peak in acidic solution than<pH 4.0 (Fig. 4b). Alsoas the pH increased, this one wave was split into two peaksfor high concentrations (Fig. 4c). At above pH 6.0, the two

reduction peaks clearly merged into a single peak (Fig. 4d).At pH 11.0 the peak disappeared for all concentrations. Allthe obtained potentials from the investigated pH rangesobserved about 0.04 V less negative than that main peak ofLAM.InFigure 5a thepeakpotential versus pHplot is presented

for ZAL. Peak potential is linear with pH and the twodifferent zones can be observed, with break at pH 4.6 closeto lamivudine break and cytosine pKa value (pKa: 4.5) [17].ForZAL the slope of theEp-pHplot is�45.41 mVper pH

unit over the pH 1.5 – 4.6 and is �104.86 mV per unit overthe pH 4.6 – 9.0 and then becomes pH independent, whichsuggests that the number of proton and electrons involved inthe reductionmechanism. This indicates that the ratio of thenumber of protons and transferred electrons involved in thechange transfer changed from 1 to 2 for 4.6� pH� 9.0. Thereduction mechanism of ZAL involves the total loss of 3electrons like LAM (Scheme 2) [18, 19].From the results obtained by studying the pHdependence

of the reduction potential and peak current of LAMoptimum pH values (Fig. 3b) were selected for furthercharacterization by CV. Analyzing the evolution of peakcurrent (Fig. 3b) it is possible to observe that this parameter

Fig. 2. Cyclic voltammograms of 2� 10�4 M LAM in phosphate buffer at pH 2.0 (a); in acetate buffer at pH 4.5 (b); in phosphate bufferat pH 6.00 (c); in phosphate buffer at pH 8.0 (d). Scan rate 100 mVs�1.



is affected by the pH value with a clear change at aboutpH 4.0 and 4.5, corresponding to maximum pHs for theprocess. However, acetate buffer at pH 4.5 was chosen withrespect to sharp response and better peak shape for thecalibration curve for pharmaceutical dosage formand serumsamples. This pH value was chosen as the working pH forfurther studies.Dependence of peak currents on pH (Fig. 5b) presents a

more complex situation: whereas in Britton –Robinsonbuffers the peak current decreases gradually from 2.0 to 6.0,in simple acetate and phosphate buffers the current reachesa limiting value at about pH 3.5 and 2.0, respectively andslowly increases with further increase in pH (Fig. 5b). Thepeak current was decreased again at pH 10.0.

No evidence for reversibility was seen for the peak in therange 5 – 1000 mV s�1. When the scan rate was varied from5 – 1000 mVs�1 in 8� 10�5 M solution of LAM, a lineardependence of the peak intensity ip (mA) upon the squareroot of the scan rate v1/2 (mVs�1)was found, demonstrating adiffusional behavior. The equation is noted below in acetatebuffer at 4.5:

ip(mA)¼ 0.184v1/2 (mV s�1)� 0.54 r¼ 0.993 (n¼ 10)

It follows from the variation of the logarithm of the peakcurrent as a function of the logarithm of the sweep rate inthe range of 5 – 1000 mV s�1 that the process is dif-fusion controlled because the value of the straight linelog ip¼ f(log v) is equal to 0.68. This value is very close to thetheoretical value of 0.5, which is the expected for an idealreaction of solution species [20]. This showed that theprocess had a diffusive component. The equation obtainedis:

log ip(mA)¼0.678(log v)(mV s�1)�1.30 r¼0.999 (n¼10)

When the scan rate was varied from 5 – 1000mVs�1 in 4�10�5 M solution of model compound ZAL, a linear depend-ence of the peak intensity ip (mA)upon the square root of thescan rate v1/2 (mV s�1) was also found, demonstrating adiffusional behavior. The equation obtained is:

ip (mA)¼ 0.1182v1/2 (mV s�1)� 0.2552 r¼ 0.996 (n¼ 10)

It follows from the variation of the logarithm of the peakcurrent of ZAL as a function of the logarithm of the sweeprate in the same range that the process is diffusion controlledbecause the value of the straight line log ip¼ f (log v) is equalto 0.60 [19]. The equation is also noted below:

log ip (mA)¼0.595(logv)(mVs�1)�1.25 r¼0.999 (n¼10)

The linear segment can be expressed by the followingequation in pH 4 Britton –Robinson buffer using CVtechnique for ZAL.

Ep (mV)¼ 39.78 log vþ 1206.26 r¼ 0.9948 (n¼ 10)

The linear segment can be expressed by the followingequation in pH 4.5 acetate buffer using CV technique forLAM.

Fig. 3. Effects of pH on LAM cathodic peak potential (a) andpeak current (b); LAM concentration 4� 10�5 M. (*) 0.04 MBritton –Robinson; (^) 0.2 M acetate and (~) 0.2 M phosphatebuffers.

Scheme 2. Possible reduction mechanism of LAM (Adapted from [18])



Ep (mV)¼ 28.85 log vþ 1216.72 r¼ 0.997 (n¼ 10)

The slope of value of the straight lineEp¼ f (log v) for LAMand ZAL are in agreement with a fast electron transferfollowed by a first order chemical reactionwhich is probablyloss of ammonia before further electron transfer task place.Variation the logarithm of the peak potential of LAM in

pH 4.5 acetate buffer solution as a function of the logarithmof concentration can be expressed by the following equationfor DPV:

Ep(mV)¼ 0.02 log Cþ 1330.31 r¼ 0.70 (n¼ 10)(4� 10�6 – 2� 10�4 M)

For ZAL the logarithm of the peak potential in pH 4.0Britton Robinson buffer solutions as a function of thelogarithm of concentration can be expressed by the follow-ing equation for DPV:

Ep (mV)¼ 5.355 log Cþ 1250.52 r¼ 0.943 (n¼ 9)(2� 10�6� 4� 10�5 M)

As it can be seen the above equations peak potential isindependent on the LAM and ZAL concentrations. Inaddition, the peak potential shifts negatively for about66 mV and 100 mV, respectively for LAM and ZAL whenincreasing for the scan rate, which is, confirmed theirreversibility of the reduction process.The Tafel plots (log i vs.E) were obtained with a scan rate

of 5 mV s�1 beginning from a steady-state potential inacetate buffer at pH 4.5 for LAM. The acn value of cathodicreaction from the slope of the linear part of the Tafel plotwas found to be as 0.34. The exchange current density (I0) is9.33� 10�14 A cm�2 for this system. These values togetherwith the absence of anodic waves in cyclic voltammetryindicated the irreversibility of the reduction reaction. [21,22]. These values forZAL,were found asacn¼ 0.32 and I0¼1.995� 10�13 A cm�2.Considering the above results and bearing in mind the

electrochemical behavior of cytosine, the parent base ofLAM at HMDE [18], we may assume that the reductionprocess is located on the cytosine moiety on the molecule.When the reductive behavior of cytosine was compared to

Fig. 4. Cyclic voltammograms of 4� 10�5 M ZAL in Britton –Robinson buffer at pH 4.0 (a); 2� 10�4 M ZAL in Britton –Robinsonbuffer at pH 3.0 (b); 2� 10�4 M ZAL in Britton –Robinson buffer at pH 5.00 (c); 2� 10�4 M ZAL in Britton –Robinson buffer at pH 7.0(d). Scan rate 100 mVs�1.



LAM and ZAL reduction as a function of pH, similarelectrochemical behavior and conclusions were found [18].It was demonstrated that cytosine exhibited a 3e� voltam-metric wave, which occur at potentials more negative thanpyrimidine waves. This was due to the three successivereduction processes show in literatures [18, 19]; the initialreaction was similar to that for cytosine in that the twoelectrons were added across the 3,4-azomethine bond. Thiswas followed by first order chemical reaction with loss ofammonia before further electron transfer took place(Scheme 2). Our results are in good agreement with theliterature results [18, 19].

3.2. Analytical Applications

3.2.1. Validation of the Procedure

Once the most ideal and suitable chemical conditions andinstrumental parameters for the voltammetric determina-tion were established, a calibration plot for the analyzeddrug was recorded to estimate the analytical characteristicsof the developed method.In order to develop a voltammetric procedure for

determination of a drug, DPV and OSW techniques wasselected since these techniques are effective and rapidelectroanalytical techniques with well-established advan-tages, including good discrimination against backgroundcurrents and low detection limits. Two calibration graphsfrom the standard solution of LAM according to theprocedures described above were constructed by usingDPVand OSW.For purpose of analysis the optimum pH and supporting

electrolyte for LAM was pH 4.5 acetate buffer where thepeak was sharp and reproducible and was preferred for theanalysis. Standard addition and calibration methods wereused for the estimation of the drugs in serum and pharma-ceutical formulations, respectively.

Fig. 5. Effects of pH on ZAL cathodic peak potential (a) andpeak current (b); ZAL concentration 4� 10�5 M. (&) 0.2 and0.5 M H2SO4; (*) 0.04 M Britton –Robinson; (^) 0.2 M acetateand (~) 0.2 M Phosphate buffers.

Table 1. Regression data of the calibration lines for quantitative determination of LAM by DPVand OSW in supporting electrolyte andhuman serum.

DPV OSW

Supporting electrolyte Serum Supporting electrolyte Serum

Measured potential (V) 1.224 1.300 1.252 1.320Linearity range (M) 4� 10�6 – 1� 10�4 2� 10�6 – 2� 10�4 4� 10�6 – 1� 10�4 2� 10�6 – 2� 10�4

Slope (mAM�1) 7.088� 103 4.77� 103 1.04� 104 6.61� 103

Intercept (mA) 0.0004 0.008 0.0096 0.0041Correlation coefficient 0.999 0.999 0.999 0.999SE of slope 1.38� 102 8.54� 101 1.96� 102 1.13� 102

SE of intercept 0.0069 0.0064 0.0097 0.0085LOD (M) 6.28� 10�8 8.65� 10�8 2.02� 10�8 6.36� 10�8

LOQ (M) 2.09� 10�7 2.88� 10�7 6.72� 10�8 2.12� 10�7

Repeatability of peak current (RSD%) 0.30 0.49 0.32 0.52Repeatability of peak potential (RSD%) 0.29 0.14 0.14 0.14Reproducibility of peak current (RSD%) 0.14 0.39 0.10 0.73Reproducibility of peak potential (RSD%) 0.15 0.17 0.14 0.17



Calibration curves were made for LAM showing thelinear dependence between peak current and concentrationin the range of 4� 10�6 – 2� 10�4 M. The characteristics ofthe calibration plots are summarized in Table 1.These relationships were used for the analysis of pharma-

ceutical dosage forms. The quantitative and statistic param-eters obtained for the validation of the methods arecollected in Table 1.The lowest detectable concentrations and the lowest

quantitative concentrations of LAM are shown in Table 1,which were estimated based on the following equations [23,24]:

LOD¼ 3 s/m LOQ¼ 10 s/m

Abbreviation s represents the standard deviation of thepeak currents (three runs) andm represents the slope of therelated calibration curve.Repeating five experiments on 6� 10�5 M LAM for both

techniques tested the repeatability (one day) and reprodu-cibility (over a week) of peak potential and peak currents.The results were shown also in Table 1.

3.2.2. Determination of LAM in PharmaceuticalFormulations

Both methods were successfully applied to the determina-tion of LAM in commercial tablet and oral solution dosageforms.There is no official method present in any pharmaco-

poeias related to pharmaceutical dosage forms or bulk drugsof LAM. For this reason, in order to check the accuracy,precision and selectivity of the developed method, arecovery study was carried out. This helped to prove theabsence of interferences by excipients. Moreover, in order

to know whether the excipients in the tablets and solutionsshow any interference with the analysis, the proposedmethods were evaluated by recovery tests after addition ofknown amounts of pure drug to various pre-analyzedformulations of LAM. For the recovery study standardaddition method was used.The percentage recoveries based on 5 separate determi-

nations are abridged in Table 2.The results were in good agreement with the label claim

and the proposed techniques were sufficiently accurate andprecise in order to be applied to pharmaceutical dosageforms.

3.2.3. Determination of LAM in Spiked Human SerumSample

Figure 6 illustrates DP and OSW voltammograms obtainedfrom serum spiked at different concentration of LAMfollowing the optimized conditions.The peak current was linearly related to LAM concen-

tration over the range 2� 10�6� 2� 10�4 Maccording to theequation:

For DPV ip(mA)¼ 4.77� 103þ 0.008 C (M)

For OSW ip(mA)¼ 6.61� 103þ 0.0041 C (M)

The estimated detection limits for both methods are shownalso inTable 1. The amount ofLAM in serumwas calculatedfrom related linear regression equation for both methods.The precision and accuracy of LAM in serum were assessedfrom five replicates at 6� 10�5 M. Good recoveries of LAMwere achieved from serum (Table 3). As can be seen inFigure 6, in the potential range where the analytical peakappeared there were no reduction compounds and no extranoise peak found from biological material.Stability of serum samples kept in refrigerator (þ4 8C)

was tested by making five consecutive analyses of thesample over a period of approximately 8 h. There were nosignificant changes observed in the peak currents andpotentials between the first and last measurements.

4. Conclusions

The electrochemical behavior of LAM on HMDE wasestablished and studied for the first time. LAM is irrever-sibly reduced at negative potentials.This work shows that the LAM concentration in human

serum and in pharmaceutics can be determine by usingvoltammetric techniques on the basis of their reduction

Table 2. Application of the proposed voltammetric methods tothe analysis of commercial tablets and solutions.

Tablet (mg) Oral solution (mg/mL)

DPV OSW DPV OSW

Labeled claim 150 150 10 10Amount found [a] 149.18 149.30 9.998 9.996RSD (%) 0.17 0.11 0.07 0.07Bias (%) 0.55 0.47 0.02 0.04Added 20.00 20.00 5.00 5.00Found [a] 19.89 19.91 5.00 4.99Recovery (%) 99.45 99.53 99.98 99.96RSD % of recovery 0.37 0.51 0.26 0.055Bias (%) 0.55 0.45 0 0.2

[a] Each value is the mean of 5 experiments.

Table 3. Determination of LAM in human serum samples for DPV and OSW methods.

Technique LAM added (M) LAM found [a] (M) Average recovery (%) Bias (%)

DPV 6� 10�5 5.99� 10�5� 0.55 99.83 0.17OSW 6� 10�5 5.996� 10�5� 0.55 99.93 0.07

[a] Average value� SD of five determination.



process corresponding to the cytosine moiety over theHMDE. This behavior provides a useful tool for detectionand quantification of drugs at low levels of concentration inbiological fluids.The procedure showed clear advantages such as no

pretreatment or time-consuming extraction steps and couldbe adopted for the pharmacokinetic studies as well as forquality control laboratory studies.With this study it was not intend to show the pharmaco-

dynamic properties of LAM since only healthy volunteerswere used for the sample collection and results may be of nosignificance. It only shows that there is a great possibility ofmonitoring LAM makes the method useful for pharmaco-kinetic and pharmacodynamic purposes.

5. Acknowledgements

This work was supported by Ankara University ScientificResearch Project Foundation (Grant No: 20030803037 and20030803043) Turkey. The authors also gratefully acknowl-edge Glaxo-Smith-Kline Pharm. Ind. for supplying LAMandRochePharm. Ind. for supplyingZALstandard samplesand their pharmaceutical dosage forms.

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[24] M. E. Swatz, I. S. Krull, Analytical Method Development andValidation, Marcel Dekker, New York 1997.

Fig. 6. Differential pulse (a) and square wave (b) voltammo-grams obtained for the determination in spiked serum 1) blank; 2)2� 10�5 M; 3) 6� 10�5 M; 4) 1� 10�4 M LAM extract in acetatebuffer at pH 4.5.



Electrochemical Evaluation of Nucleoside Analogue Lamivudine in Pharmaceutical Dosage Forms and...

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