Bioelectrochemistry 88 (2012) 36–41

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Bioelectrochemistry

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Voltammetric determination of penicillin V in pharmaceutical formulations andhuman urine using a boron-doped diamond electrode

Ľubomír Švorc a,⁎, Jozef Sochr a, Miroslav Rievaj a, Peter Tomčík b, Dušan Bustin a

a Institute of Analytical Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, Bratislava, SK-812 37, Slovak Republicb Department of Chemistry, Faculty of Education, Catholic University in Ružomberok, Hrabovská cesta 1, Ružomberok, SK-034 01, Slovak Republic

⁎ Corresponding author.E-mail address: lubomir.svorc@stuba.sk (Ľ. Švorc).

1567-5394/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.bioelechem.2012.04.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 January 2012Received in revised form 10 April 2012Accepted 12 April 2012Available online 24 April 2012

Keywords:Penicillin VBoron-doped diamondDifferential pulse voltammetryPharmaceutical formulationHuman urine

Simple, sensitive and selective differential pulse voltammetric method for determination of penicillin V on abare (unmodified) boron-doped diamond electrode has been developed. Penicillin V provided highly repro-ducible and well-defined irreversible oxidation peak at very positive potential of +1.6 V (vs. Ag/AgCl). Theoptimum experimental conditions for oxidation of penicillin V were achieved in acetate buffer solution(pH 4.0). The modulation amplitude of 0.1 V, modulation time of 0.05 s and scan rate of 0.05 V s−1 were se-lected as optimum instrumental parameters for differential pulse voltammetry. Linear response of peak cur-rent on the concentration in the range from 0.5 to 40 μM with coefficient of determination of 0.999, goodrepeatability (RSD of 1.5%) and detection limit of 0.25 μMwere observed without any chemical modificationsand electrochemical surface pretreatment. The effect of possible interferents such as stearic acid, glucose,urea, uric acid and ascorbic acid appeared to be negligible which evidently proved the good selectivity ofmethod. The practical analytical utility of proposed method was demonstrated by determination of penicillinV in pharmaceutical formulations (tablets) and human urine samples with satisfactory recoveries (from 98 to101% for tablets and 97 to 103% for human urine).

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Penicillins belong to the most familiar group of β-lactam antibi-otics which have been used extensively in both veterinary andhuman medicine practices to prevent the infections of bacteria andfungi [1–3]. Despite the positive effects of penicillins, their occurrencein food or in aquatic environment after consumption presents a seri-ous health hazard [4]. Additionally, the presence of residues of peni-cillins in animal feedstuff may signify a serious problem because oftheir eventual spreading to the human food production chain (milk,meat) exerting a negative impact on public health such as severe al-lergic responses. Thus the analysis of penicillins plays a significantrole in drugs chemistry.

Many analytical techniques for determination of penicillins invarious matrices such as biological samples or pharmaceutical formu-lations have been published. High-performance liquid chromatogra-phy with different modes of detection, such as ultraviolet detection[5–7] and mass spectrometry [8–11] as well as spectrophotometric[12–15] techniques are among the most popular analytical methodsfor detection and quantification of penicillins. Generally, these methodsbelong to the most sensitive and selective, but on the other hand

rights reserved.

they are long lasting, expensive and often too laborious, when someprocedures such as derivatization, extraction and purification are in-cluded [16,17]. The electrochemical methods have many inherentadvantages such as simplicity, low-cost and possibility of miniaturiza-tion that eliminate limitations of other analytical methods [18,19]. Onthe basis of literature survey only a few papers dealing with an electro-analysis of penicillins were reported in comparison with other drugs.This is probably because oxidation of penicillins usually occurs as notclearly defined waves at a very high positive potential and thus maybe affected with electrochemical reactions limiting potential windowfrom the anodic side [20–22]. Some reports dealing with determinationof penicillins are based on pulsed amperometric technique [23,24], flowinjection analysis [25] or biosensors [26,27]. Stred'anský et al. [28] pub-lished a concise report dealing with amperometric pH-sensing biosen-sors for urea, penicillin and oxalacetate.

Boron-doped diamond (BDD) is a modern electrode materialwhich opens new possibilities of electrochemical investigations dueto its excellent features, such as the low background current, wide po-tential window in aqueous solutions, long-term stability of response,good resistance to surface fouling due to weak adsorption and lowsensitivity to dissolved oxygen [29–31]. The versatility of this elec-trode material has also been utilized for the development of sensorsand biosensors [32].

It is important to remark that there were no reports for determi-nation of penicillins on BDD electrode published yet in literature.

S

N

O

HO

CH3

CH3

O

NH

O

O

Scheme 1. Structural formula of penicillin V.

Fig. 1. Cyclic voltammograms of (a) 0 M and (b) 10 μM PEN in ABS at pH 4.0 on bareBDD electrode with scan rate of 0.05 V s−1.

37Ľ. Švorc et al. / Bioelectrochemistry 88 (2012) 36–41

This paper demonstrates the application of bare BDD electrode as asensitive electrochemical sensor for the voltammetric determinationof penicillin V (Scheme 1) without any chemical modifications and/or electrochemical pretreatment of electrode surface. This simple,low-cost and practical analytical approach is illustrated on quantifica-tion of penicillin V in pharmaceutical formulations (tablets) andhuman urine samples.

2. Experimental

2.1. Chemicals

Penicillin V (PEN), sugars (glucose, fructose, sucrose, lactose, cel-lulose and starch) urea, uric acid, stearic acid and ascorbic acid wereobtained from Zentiva (Hlohovec, Slovak Republic) and used as re-ceived without any further purification. Various supporting electro-lytes such as phosphate buffer, Britton-Robinson buffer, acetatebuffer, sulfuric acid, sodium hydroxide and acetic acid were of Lac-hema (Brno, Czech Republic) production. All reagents were of analyt-ical grade. All aqueous solutions were made with double-distilleddeionized water with resistivity above 18 MΩ cm. Standard calibra-tion solutions were prepared by appropriate dilution of stock solutionwith supporting electrolyte.

2.2. Apparatus

All electrochemical experiments were performed in the three-electrode single compartment glass cell. This cell consisted of Ag/AgCl (3 M) reference electrode, platinum wire as counter electrodeand bare BDD electrode inserted in an inert polyether ether ketone(PEEK) body with an inner diameter of 3 mm (Windsor ScientificLtd, United Kingdom) used as working electrode. All potentialsreported in this paper were obtained vs. Ag/AgCl electrode at labora-tory temperature of 25±1 °C. Voltammetric measurements were car-ried out using AUTOLAB PGSTAT-302N (EcoChemie, The Netherlands)potentiostat/galvanostat controlled with the NOVA 1.7 software. AllpH values were measured with pH meter Model 215 (Denver Instru-ment, USA), which was calibrated with standard buffer solutions.

3

1.50

1.55

1.60

1.65

EP /

Va

pH4 5 6

Fig. 2. Effect of pH of supporting electrolyte (ABS) on the peak potential of 10 μM PENon bare BDD electrode (error bars are constructed for six measurements).

2.3. Measurement procedures

20 mL of supporting electrolyte containing an appropriate amountof PEN were added to the electrochemical cell. Cyclic voltammetry(CV) and differential pulse voltammetry (DPV) were employed with-out deaeration since oxygen did not influence the oxidation of PEN.Five CV voltammograms were obtained for each measurement, andthe last scan was considered for evaluation. The calibration curvewas constructed from the average of six consecutive DPV measure-ments for each calibration standard solution. The detection limit(LOD) was calculated as three times the standard deviation for theblank solution divided by the slope of the calibration curve. The stan-dard addition method was used for analysis of pharmaceutical formu-lations and human urine spiked with aliquot amounts of PEN.

3. Results and discussion

3.1. Cyclic voltammetry

It is well-known that determination of biologically active com-pounds on bare electrodes is sometimes difficult due to the absenceof electrocatalytic behavior of bare electrodes [19]. Cyclic voltam-metry was applied to study the electrochemical behavior of PEN onbare BDD electrodes. All necessary factors influencing the currentresponse of PEN were carefully checked to reach the conditions atwhich the best analytical performance was achieved.

3.1.1. Effects of supporting electrolyte and pHIn order to find the appropriate medium for oxidation of PEN var-

ious supporting electrolytes such as sulfuric acid, acetic acid, sodiumhydroxide, phosphate buffer solution (PBS), Britton-Robinson buffersolution (BRS) and acetate buffer solution (ABS) were tested. Theuse of sulfuric acid, acetic acid and sodium hydroxide was inconve-nient due to the less sensitive, bad shaped and reproducible currentresponses (results not shown). The oxidation peak was very well-defined in PBS, BRS and ABS, but the best results were obtained inABS in which the magnitude of peak current was found to be the2–3 times higher than in PBS and BRS. Following these facts ABSwas chosen in further experiments.

The oxidation peak at about 1.6 V (vs. Ag/AgCl) was registeredwithout any cathodic peak on the reverse scan which clearly indicat-ed that the charge transfer during electrode process is electrochemi-cally irreversible on bare BDD electrode (Fig. 1). It is also apparentthat background current appeared to be sufficiently low at higher po-tentials on bare BDD electrode.

In general, the pH value of supporting electrolyte is a significantfactor that affects electrochemical behavior of biologically active

3

16

20

24

pH

I P /

µA

4 5 6

Fig. 3. Effect of pH of supporting electrolyte (ABS) on the peak current of 10 μMPEN areBDD electrode (error bars are constructed for six measurements).

Fig. 4. Cyclic voltammograms of 10 μMPEN in ABS at pH 4.0 on bare BDD electrode for aseries of scan rates (v): (a) 0.01, (b) 0.025, (c) 0.05, (d) 0.1, (e) 0.2 and (f) 0.3 V s−1.The dependence between peak current and square root of the scan rate appears inthe inset.

0

20

40

60

80

I / µ

A

[PEN] / µM10 20 30 40

38 Ľ. Švorc et al. / Bioelectrochemistry 88 (2012) 36–41

molecules. The effect of the pH on peak potential was investigated inthe pH range of 3–6. As depicted in Fig. 2 the peak potential (EP)shifted towards less positive values as the pH of supporting electro-lyte was gradually increased. The relationship between peak potential(EP) and pH of ABS was linear and dependence can be presented asEq. (1):

EP=V ¼ 1:830� 0:005ð Þ=V− 0:056� 0:001ð ÞpH R2 ¼ 0:998� �

: ð1Þ

The slope of the equation is very close to the anticipated value of−0.059 V. This result revealed that an equal number of participatedprotons and transferred electrons are involved in the oxidation ofPEN on bare BDD electrode. The effect of pH on the peak currentwas also studied in the pH range of 3–6. The obtained results showedthe peak current (IP) of PEN increased slightly and reached the max-imum approximately at pH 4.0 and then decreased as shown in Fig. 3.Based on these observations and in order to obtain high selectivityand sensitivity ABS at pH 4.0 was chosen as optimum supporting elec-trolyte and used in further experiments.

3.1.2. Mechanism of oxidation of PENThe chemical oxidation of the penicillins has been widely studied,

but sometimes with different results in comparison with electro-chemical oxidation. We have assumed that in our case the site of ox-idation of PEN was within the β-lactam backbone and not in the sidechains in comparison with e.g. amoxicillin when a significantly differ-ent response would be expected because of the presence of a phenolgroup in the side chain. The sulfide moiety of the β-lactam ring mightbe oxidized into a sulfoxide derivative of PEN in participation ofwater, two protons and transfer of two electrons as depicted inScheme 2.

The dependence of peak potential EP vs. pH (Fig. 2) was used forcalculation of numbers of protons and confirmation of the mechanismof oxidation. On the basis of the calculated slope (0.056 V) from ex-perimentally measured dependence EP vs. pH, the number of protonsn taking part in oxidation of PEN was found to be 1.89 for z=2. Fromthis value it is obvious that approximately two protons are participat-ed in the oxidation reaction of PEN. The calculated number of protons

S

NO

HOO

NH

O

R S

NO

HO

CH3

CH3

CH3

CH3

O

NH

O

R

O

H2O

-2e- -2H+

Scheme 2. Reaction of electrochemical oxidation of penicillin V (R=phenoxymethyl).

is biased from the expected value (2.00) due to high irreversibility ofmass transfer during the oxidation process of PEN.

3.1.3. Effect of scan rateThe effect of scan rate (v) on the peak current was investigated in

order to characterize the mass transport in diffusion layer of BDDelectrode. Fig. 4 demonstrates that the anodic peak current of 10 μMPEN was increased with increasing of scan rate. From the inset ofFig. 4 it can also be seen that peak current is linearly proportional(R2=0.999) to the square root of the scan rate within the range of0.01–0.3 V s−1 indicating that the mass transport is controlled by dif-fusion thus proving that the rate-limiting adsorption and/or specificinteractions on bare BDD electrode surface are negligible [33]. Theslight shift of peak potential towards more positive potential was ob-served as the scan rate increased.

3.2. Determination of PEN

The DPV method has been applied to numerous biologically activecompounds, by which the low detection and quantification permitthe analysis of their trace amounts. In our case, the optimal instru-mental parameters of DPV were modulation amplitude 0.1 V, modu-lation time 0.05 s and scan rate 0.05 V s−1.

The calibration curve was constructed by measuring the peak cur-rent with optimized DPV parameters. An average of six consecutivemeasurements was used for calibration curve construction in Fig. 5.The dependence of peak current on concentration of PEN shows agood linearity (R2=0.999) in the concentration range from 0.5 to

Fig. 5. Differential pulse voltammograms of various concentrations of PEN: (a) 0.5,(b) 1, (c) 2, (d) 4, (e) 6, (f) 10, (g) 15, (h) 20, (i) 30 and (j) 40 μM in ABS at pH 4.0on bare BDD electrode at optimized DPV parameters: modulation amplitude of 0.1 V,modulation time 0.05 s and scan rate 0.05 V s−1. The dependence between peakcurrent (μA) and concentrations of PEN (μM) appears in the inset (error bars areconstructed for six measurements).

Table 1Comparison of the proposed method with some previously reported electrochemicalmethods for determination of penicillins.

Electrode Method LR(μM)

LOD(μM)

Reference

PGA/GCE SWV 2–25 0.92 [20][VO(Salen)]/CPE DPV – 16.6 [21]Au electrode PAD – 0.4 [24]HMT/Pt electrode AD 4–2000 4 [28]BDD electrode DPV 0.5–40 0.25 This work

Abbreviations: LR: linear range; LOD: detection limit; PGA: polyglutamic acid; GCE:glass carbon electrode; CPE: carbon paste electrode; HMT: Hematein; DPV:differential pulse voltammetry; SWV: square-wave voltammetry; AD: amperometricdetection; PAD: pulse amperometric detection.

Table 2Determination results of PEN in pharmaceutical formulations (mg per tablet) by theDPV method (n=6).

Tablet Determined(mg)⁎

Added(mg)

Found after spiking(mg)⁎⁎

Recovery(%)

250 243±6 50 294±7 98500 505±7 100 607±10 101750 757±11 150 891±9 99

The confidence interval for 95% probability: x± tn−1,α SD/n1/2 (t5;0.05=2.0150).⁎ Results determined by the calibration curve.⁎⁎ Results determined by the standard addition method for spiked samples.

39Ľ. Švorc et al. / Bioelectrochemistry 88 (2012) 36–41

40 μM as depicted in the inset of Fig. 5. The linear regression equationwas expressed as Eq. (2):

Ip PENð Þj=μA ¼ 2:90� 0:12ð Þ=μAþ 2:01� 0:02ð Þ PEN½ �=μM: ð2Þ

The detection limit was found to be 0.25 μM. In order to examinethe repeatability, repeated DPV experiments were run in 10 μM PENin ABS at pH 4.0 on the bare BDD electrode. The DPV voltammogramswere evaluated by ten consecutive measurements under the sameoperating conditions over the short time interval with relative stan-dard deviation (RSD) of 1.5% revealing the good repeatability of theproposed method.

3.3. Comparison with other electrochemical methods

A comparison between the analytical performance of the pro-posed method on the bare BDD electrode and some reported electro-chemical methods for determination of penicillins are given inTable 1. As it was mentioned earlier, no official attempt for sensitivedetermination of penicillins on bare BDD electrode has been publi-shed to date in literature.

Concerning PEN the calculated detection limit is quite lower thanthose obtained by modified electrodes or biosensors [20,21,28].Moreover, the preparation of modified electrodes is a time consumingprocess that usually involves various steps in incorporation of the dif-ferent modifiers to the electrode surface leading to the not always re-producible results. Further, it is sometimes useless to modify theelectrode surface for selectivity and sensitivity improvement in apractical process of analysis. This fact may seem to be less important;however this observation decreases the number of operations in ana-lytical process (lower risk of measurement errors), reduces expensesand operational skills of analysts related to chemical modification.

Fig. 6. Differential pulse voltammograms of 10 μM PEN: (a) in the absence, (b) in thepresence of 0.5 mM of uric acid in ABS at pH 4.0 on bare BDD electrode. The conditionswere the same as those in previous measurements.

Good linear concentration range and low detection limit was reachedwith no electrochemical pretreatment or chemical modification ofBDD electrode in our experiments.

3.4. Interference study

The influence of interfering compounds commonly existing inpharmaceutical formulations and human urine was also examinedby DPV under the same experimental conditions. The commercialtablets of Penicillin V® usually consist of phenoxymethylpenicillin(PEN, active and substantial component of the tablet) and some adju-vants as lactose monohydrate, microcrystalline cellulose, stearic acid(and its magnesium salt) and silicon dioxide. The interference studywas realized by addition of each substance with varying concentra-tion to the solution containing fixed amount of 10 μM PEN in ABS atpH 4.0. It was found that 100-fold excess of the common ions usuallypresent both in tablets and urine such as Na+, K+, Mg2+, Zn2+, Fe3+,Cu2+, Al3+, Si4+, Cl−, NO3

−, PO43− and SO4

2− showed no effect on ox-idation peak of PEN. Glucose did not influence the peak current of PENeven up to 10-fold excess. Stearic acid, sugars (fructose, sucrose, lac-tose, cellulose and starch), urea, uric acid and ascorbic acid did notsignificantly interfere in 50-fold excess with oxidation of PEN(RSDb5%). The uric acid as an illustrated example is depicted inFig. 6. These results suggested that determination of PEN in pharma-ceutical formulations and human urine samples was not affected bythe most common interfering compounds and the proposed methodis sufficiently selective.

3.5. Real sample analysis

3.5.1. Determination of PEN in tabletsAt first, the developed method was applied to the pharmaceutical

formulations. Commercial tablets of Penicillin V®were obtained fromthe local pharmacy. The tablets were weighted, ground into powderand then dissolved in deionized water with intensive stirring of

Fig. 7. Differential pulse voltammograms of urine sample of volunteer: (a) in the ab-sence, (b) after spiking with 30 μM of PEN in ABS at pH 4.0 on bare BDD electrode.The conditions were the same as those in previous measurements.

Table 3Determination results of PEN in spiked human urine samples by the DPV method(n=6).

Sample Added(μM)

Found(μM)

RSD(%)

Recovery (%)

1 30 29±2 2.3 972 30 29±3 2.9 973 30 31±2 1.8 103

40 Ľ. Švorc et al. / Bioelectrochemistry 88 (2012) 36–41

magnetic stirrer for 30 min. The mixture was filtered and transferredinto a 25 mL volumetric flask. To obtain final concentrations in therange of calibration curve, the sample solutions were suitably dilutedwith ABS at pH 4.0 and then added in appropriate amount to the elec-trochemical cell for determination of PEN using DPV.

In order to estimate the accuracy of the proposed analytical tech-nique, the standard addition method was performed by spiking of al-iquot amounts of standard PEN. The results are summarized inTable 2. The average recovery values (from 98 to 101%) revealedgood accuracy of the presented method. This fact indicated that noimportant matrix effects were present for the samples analyzed bythe DPV method. The method is rapid and simple and thus it can berecommended for the analysis of PEN in pharmaceutical formulations.As it can be seen in Table 2, no significant differences were observedbetween the values found with the DPV method and those declaredby producer.

3.5.2. Determination of PEN in human urine samplesThe standard addition method was also used for analysis of spiked

human urine samples of three healthy volunteers as depicted in Fig. 7.Recovery tests were carried out by adding the aliquot amount of stan-dard of PEN to the urine samples which did not contain the examineddrug. Each 1.0 mL of fresh urine sample was taken and diluted to20 mL with ABS at pH 4.0 and then directly analyzed. The resultsare listed in Table 3. The recovery values for determination of PENadded to the urine samples were satisfactory (from 97 to 103%). Itcan be concluded that the proposed method is suitable also for deter-mination of PEN in the urine samples.

4. Conclusions

In this study a bare boron-doped diamond electrode was for thefirst time applied as an electrochemical sensor for the detection andquantification of trace amounts of PEN. The proposed analytical tech-nique is rapid, simple and low-cost in comparison with other analyt-ical methods used for the determination of penicillins. The detectionlimit is better than in the previously reported methods and isobtained as a consequence of very high S/N ratios without any chem-ical modification and electrochemical pretreatment of the BDD sur-face. The presented method offers selective and sensitive possibilityfor analysis of biological samples and pharmaceutical formulationscontaining penicillins with no special pretreatment of samples exceptdilution. This rapid, simple and oftentimes very effective way of sam-ple pretreatment before analysis decreases the number of operationsin whole analytical process and thereby reduces the risk of contami-nation and measurement errors. BDD electrode (bare or modified)can be strongly recommended as a drug sensor in clinical analysis ofvarious biological active compounds and in doping test in competitivegames due to rapidity, simplicity and accuracy.

Acknowledgments

This work was supported by the Grant Agency of the Slovak Re-public (grant nos. VEGA 1/0182/11 and 1/0008/12) and Program tosupport young researchers (no. 6406).

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