Chem. Anal. (Warsaw), 53, 967 (2008)

Electrochemical Determination of Carvedilol, Sildenafiland Paracetamol Using Glassy Carbon Electrode

by Irena Baranowska*, Marta Koper and Piotr Markowski

Department of Analytical and General Chemistry, Faculty of Chemistry,Silesian University of Technology,

ul. Strzody 7, 44-100 Gliwice, Poland

Keywords: Paracetamol; Carvedilol; Sildenafil; DPV; CV

A new and sensitive electroanalytical method for simultaneous determination of selecteddrugs has been developed and validated. The analysed drugs: paracetamol (PAR), carvedilol(CAR), and sildenafil (SIL) belong to three different therapeutic groups (analgesic,β/α1-adrenoceptor blockers, and inhibitors of type 5 phosphodiesterase). Cyclic voltammetry(CV) was used to study the electrochemical behavior of the drugs, while differential pulsevoltammetry (DPV) was used to determine CAR, SIL, and PAR. A glassy carbon electrode(GCE) was used as the working electrode, a Ag/AgCl/KCl(sat.) electrode served as the refe-rence electrode, and a platinum wire – as the auxiliary electrode. Determination of all drugswas performed in Britton–Robinson (BR) buffers of different pH. Determination was per-formed using the standard addition method. The calibration plots were linear in the concen-tration ranges 2.5–22.5 µg mL–1 for CAR and PAR, and 5.0–45.0 µg mL–1 for SIL. Preli-minary studies indicate that CAR and SIL can be determined simultaneously with PAR.The proposed method has been applied for the determination of PAR, CAR and SIL in urinesamples.

Opracowano i zwalidowano now¹, czu³¹ elektroanalityczn¹ metodê oznaczania wybranychleków. Przedmiotem badañ by³y: paracetamol (PAR), karwedilol (CAR) i sildenafil (SIL),nale¿¹ce do trzech ró¿nych grup terapeutycznych (przeciwbólowe, leki β/α1-adrenolitycznei inhibitory fosfodiesterazy typu 5). Metodê woltamperometrii cyklicznej (CV) zastosowanodo zbadania elektrodowych reakcji leków, podczas gdy metodê pulsowej woltamperometriiró¿nicowej (DPV) wykorzystano do oznaczenia iloœciowego CAR, SIL i PAR. Elektrod¹pracuj¹c¹ by³a elektroda z wêgla szklistego (GCE), elektrod¹ odniesienia – elektrodachlorosrebrowa (Ag/AgCl/KCl(nas.)), natomiast elektrod¹ pomocnicz¹ – drut platynowy. Jakoelektrolit podstawowy w badaniu wszystkich leków zastosowano bufor Brittona–Robinsona(BR) o ró¿nym pH. Oznaczanie przeprowadzono metod¹ wielokrotnego dodatku wzorca.

* Corresponding author. E-mail: irena.baranowska@polsl.pl; Fax: +48 32 237 1205Dedicated to Professor Rajmund Dybczyñski on the occasion of his 75th birthday.

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968 I. Baranowska, M. Koper and P. Markowski

In the clinical treatment of patients, various drugs belonging to different thera-peutic groups are often used simultaneously. Therefore, it is very important to deve-lop a new method for simultaneous determination of selected medicines. There aremany articles on drug determination but the reported studies are related to the singledrug determination. It is also possible to find papers on the simultaneous determina-tion of drugs belonging to the same therapeutic group. In this study two of the mostpopular drugs used in clinical practice in the treatment of heart failure – carvedilol(CAR) and sildenafil (SIL) – have been determined in combination with the com-monly used analgesic – paracetamol (PAR). PAR can be administered in heart failuretherapy as an antipyretic agent and pain killer, therefore the combination of CAR andSIL with PAR can be found together in biological samples (urine, plasma, blood).

CAR, (±)-1-(carbazol-4-yloxy)-3-((2-(o-methoxy-phenoxy)ethyl)o)-2-propanolis a noncardioselective β-adrenergic blocking agent with α1-blocking activity. It hasbeen proved to be an effective agent for the treatment of mild and moderate conges-tive heart failure. CAR is used as an adjunct in the conventional treatment.

SIL, 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo-[4,3d]pyrimidin-5-yl) phenylsulfonyl]-4-methylpiperazine citrate is a medicine used in thetreatment of pulmonary hypertension and impotence (erectile dysfunction).

PAR, N-acetyl-p-aminophenol, is an active metabolite of phenacetin. It is one ofthe most popular analgesic and antipyretic medicines. PAR is commonly used for thetreatment of fever and minor aches and pains.

Many various methods for determination of CAR, SIL, and PAR in pharmaceu-tical preparations and biological samples have been described. The most popularmethod is high performance liquid chromatography for determination of CAR [1–3],PAR [4–8] and SIL [8–15]. Other reported methods for determination of PARin pharmaceutical preparations or biological fluids are capillary electrophoresis [16,17], spectrophotometry [18–21] and voltammetry [22, 23]. Spectrophotometric [24]and voltammetric [25–27] determinations of SIL have been also performed. Applica-tion of voltammetric methods in determination of CAR have been described as well[28]. All reported electrochemical methods have been applied to determination ofsingle drugs only. PAR has been determined at C60-modified glassy carbon electrode(GCE) [22] and at nanogold-modified indium tin oxide electrode [23]. Determina-tion of CAR in a tablet form has been carried out using a carbon paste electrode [28].SIL has been determined on GCE [25] in a tablet form, as well as on a hangingmercury drop electrode [26, 27] in biological fluids and pharmaceuticals.

Liniowoœæ krzywych kalibracji badanych leków uzyskano dla zakresu stê¿eñ 2.5–22.5 µg mL–1 CAR i PAR oraz 5.0–45.0 µg mL–1 SIL. Badania wstêpne wykaza³y, i¿ CARi SIL mog¹ byæ oznaczane równoczeœnie z PAR w jednej próbce. Zaproponowane metodyzastosowano do oznaczania CAR, PAR i SIL w próbkach moczu.

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969Electrochemical determination of carvedilol, sildenafil and paracetamol

The aim of this study was to develop a rapid, simple, and sensitive method forsimultaneous determination of CAR, SIL, and PAR in model solutions and urinesamples.

EXPERIMENTAL

Chemicals and reagents

PAR was purchased from Fluka (Darmstadt, Germany). SIL was obtained from Pfizer Pharmaceuticals(New York, USA). CAR was bought from Toronto Research Chemicals Inc. (Toronto, Canada).

O-boric acid (H3BO3), acetic acid (CH3COOH), o-phosphoric acid (H3PO4), sodium hydroxide (NaOH),potassium phosphate monobasic (KH2PO4), sodium phosphate dibasic (Na2HPO4), methanol, ethanol, andacetonitrile, all of analytical grade, were purchased from POCH (Gliwice, Poland).

Human urine was obtained from healthy volunteers of different sex and age. Urine samples were analysedimmediately or they were stored at –18°C until analysis.

Apparatus

All voltammetric measurements were carried out using a µAUTOLAB potentiostat Type III (Eco-Chemie,The Netherlands) and three-electrode cell comprising a GCE as the working electrode, an Ag/AgCl/KCl(sat.)

as the reference electrode, and a platinum wire as the auxiliary electrode. Voltammetric measurements werecarried out in a 3 mL glass electrochemical cell. Argon stream was used for removal of oxygen from theinvestigated solutions. pH of the solutions was measured using an ELMETRON pH meter, Model CP–401(Zabrze, Poland).

Stock solutions

Separate stock solutions (1 mg mL–1) of the analysed drugs (PAR, CAR, and SIL) were prepared in10 mL volumetric flasks by dissolving an appropriate amount of the reference substance in methanol. Stocksolutions were prepared at the beginning of the study and were stored at 4°C. All prepared stock solutionswere stable for at least one month.

Procedure for determination in model solutions

For the voltammetric studies, 2 mL of the supporting electrolyte (BR buffer of pH in the range1.81–7.24) were transferred into the voltammetric cell. To remove oxygen, the solution was purged with pureargon for 15 min and then for 30 s before each measurement. Subsequently, an appropriate amount ofthe drug (5 µg CAR, 10 µg SIL, 5 µg PAR) was added and voltammograms were recorded for differentconcentrations of standard solutions. Before the measurements, GCE was polished manually to the mirrorfinish using the alumina paste (0.3 µm particle size) and thoroughly rinsed with purified water and ethanol.Each measurement was repeated three times using fresh sample solutions to check reproducibility ofthe results. The experiments were performed at the ambient laboratory temperature. Scan rate in DPV was25 mV s–1.

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970 I. Baranowska, M. Koper and P. Markowski

Calibration plots

Linear regression equations for the calibration plots were evaluated by the least squares method andgiven together with the corresponding determination ranges. They were presented in the form: y = ax + b,where y indicates the peak current (A), a – the slope of the linear plot (A µg-1 mL), b - its intercept (A), andx - concentrations of the analysed drugs in model solutions. All measurements were repeated six times. Thepeak current magnitude vs. concentration dependence was recorded in the analyte’s concentration range 2.5– 60.0 µg mL-1. The peak current magnitude was measured as a distance between the baseline and the currentmaximum. LOQ was calculated as 3·LOD.

Determination procedure for urine samples

Urine samples were collected during 24 h from healthy volunteers and filtered through a 0.45 µm nylonBakerbond filter. To the 2 mL-in-volume urine aliquot, standard solutions were added (15 µg CAR and10 µg PAR, 20 µg CAR and 10 µg PAR, 20 µg SIL and 20 µg PAR). The matrices were precipitated with2 mL of acetonitrile and 2 mL of phosphate buffer (pH 6.88), and mixed. The samples were centrifuged(2500 rpm) for 10 min [29]. The supernatant was filtered through a 0.45 µm nylon Bakerbond filter andtransferred to an Oasis® MCX cartridge (150 mg, 6 mL). The SPE procedure comprised conditioning (3 mLof methanol, 3 mL of distilled water), sample loading, washing (3 mL of distilled water), drying, and elution(4 mL of dichloromethane : 2-propanol : ammonium, 78:20:2 v/v/v). The eluates were evaporated to drynessunder a stream of nitrogen. The residues were dissolved in 2 mL of BR buffer and transferred to thevoltammetric cell.

RESULTS AND DISCUSSION

Determination of drugs in model solutions

Using BR buffer as the supporting electrolyte, electrochemical responses of CAR,SIL and PAR were well-defined. The cyclic voltammograms show irreversible oxida-tion peaks of SIL and CAR.

The peak obtained in the voltammogram of SIL corresponded to the oxidation ofthe piperazine ring [25]. In the pH range 1.81–7.24 one anodic wave was observed.Cyclic voltammograms were recorded from –0.5 V to 1.6 V at six different pH values.Electrochemical behavior of SIL in BR buffer of different pH is illustrated in Figure1A. A well-defined peak at pH ≤ 4.15 was observed. At pH > 4.15, oxidation peak ofSIL was unclear. The largest well-shaped peak was observed in BR buffer of pH 3.26.For this reason, pH 3.26 of BR buffer was selected for DPV determination of SIL.DPV method was applied to the investigation of the pH dependence of the peakpotential. DP voltammograms were recorded in the pH range 1.81–7.24. As show inFigure 1B, the oxidation peak of SIL strongly depends on pH and shifts to lowervalues with the increasing pH. DP voltammograms of SIL were very well-definedand were used for its quantification (Fig. 2A). Average limits of detection (LOD) andquantification (LOQ) for SIL were 1.65 µg mL–1 and 5.0 µg mL–1, respectively.

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971Electrochemical determination of carvedilol, sildenafil and paracetamol

Figure 1. A – Cyclic voltammograms of 50 µg mL–1 SIL in BR buffer at different pH. B – Effect of pHon SIL peak potential

Calibration plot for SIL model solutions exhibited the dependence of the SILpeak current on its concentration in the range 5.0–45.0 µg mL–1 (Fig. 2B).

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972 I. Baranowska, M. Koper and P. Markowski

Figure 2. A – Differential pulse voltammograms for different concentrations of SIL in BR buffer at pH3.26; concentrations: a) 0 (base electrolyte), b) 5.0, c) 15.0, d) 25.0, e) 35.0 µg mL–1. B – Cali-bration plot for SIL was obtained using DPV results

Cyclic voltammograms of CAR show two oxidations peaks (Fig. 3A). The oxida-tion mechanism of CAR is determined by the presence of two rings in its molecule.The first peak Ip1 and the second peak Ip2 are related to the reaction within the carba-zole ring and the alkoxybenzene ring, respectively [28].

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973Electrochemical determination of carvedilol, sildenafil and paracetamol

Figure 3. A – Cyclic voltammograms of 25 µg mL–1 CAR in BR buffer at different pH. B – Effect of pHon CAR peak potential Ep1

Cyclic voltammograms were recorded in BR buffer in the pH range 1.81–7.24.Potential of the first peak depended on the pH of the supporting electrolyte and shiftedto lower values with the increasing pH (Fig. 3A). The corresponding plot illustratingthis dependence was obtained based on DPV results. The obtained cuve was ilustratedat Figure 3B. The best-shaped peaks were obtained at pH 2.09; therefore DP voltam-mograms and the corresponding calibration plots were recorded at this pH. DP voltam-mograms showed a broad second peak, inapplicable for analytical determination.The first peak was more distinct throughout the pH range examined. Therefore, itwas used in the analytical procedure (Fig. 4A).

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974 I. Baranowska, M. Koper and P. Markowski

Figure 4. A – Differential pulse voltammograms for different concentrations of CAR in BR buffer at pH2.09; concentrations: a) 0 (base electrolyte), b) 5.0, c) 10.0, d) 15.0, e) 20.0 µg mL–1. B – Cali-bration plot for CAR was obtained using DPV results

The calibration plot for the model solution of CAR in the concentration range2.5–22.5 µg mL–1 was obtained. LOD and LOQ were 0.85 µg mL–1 and 2.5 µg mL–1,respectively (Fig. 4B). Large intercept was addressed to the relatively small increaseof the peak current in the examined concentrations range.

Cyclic voltammograms of PAR showed a well-defined oxidation peak and a broadand indistinct reduction peak in the pH range 2.09–5.01. The reduction peak becamemore distinct at pH > 7.24. Electrochemical behavior of PAR was studied in the BRbuffer in the pH range 1.81–7.24. The mechanism of the oxidation process was rela-ted to the presence of the amine group in PAR molecule [22]. As shown in Figure 5,the position of the oxidation peak was strongly pH-dependent and shifted to lowerpotentials with the increasing pH. Cyclic voltammogram recorded in the BR buffer at

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975Electrochemical determination of carvedilol, sildenafil and paracetamol

pH 2.09 showed the oxidation peak at 0.687 V. The peak potential vs pH dependencewas determined by DPV. The results are illustrated in Figure 5B.

Figure 5. A – Cyclic voltammograms of 25µg mL–1 PAR in BR buffer at different pH. B – Effect of pHon PAR peak potential Ep

The Ep vs pH relation was linear. All measurements were done for model solu-tions. The oxidation peak of PAR was distinct throughout the investigated pH rangeof the BR buffer. Sharper and narrow peak was obtained in the acidic media. There-fore, analytical investigations were performed at pH 2.09. The calibration line wasplotted on the basis of DPV results obtained in the PAR concentration range 2.5–45.0µg mL–1 (Fig. 6). LOD and LOQ for PAR were 0.83 µg mL–1 and 2.5 µg mL–1, res-pectively.

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976 I. Baranowska, M. Koper and P. Markowski

Figure 6. A – Differential pulse voltammograms for different concentrations of PAR in BR buffer at pH2.09; concentrations: a) 0 (base electrolyte), b) 5.0, c) 10.0, d) 15.0, e) 20.0 µg mL–1. B – Cali-bration plot for PAR was obtained using DPV results

Determination of CAR and SIL in the presence of PAR

The results of preliminary studies of drugs were used to estimate optimum para-meters for simultaneous determination of SIL and PAR, and CAR and PAR. Theobtained calibration plots for the drugs determined in pairs have different slopes andintercepts than the calibration plots of single drugs. The corresponding linear calibra-tion equations are presented in Table 1.

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977Electrochemical determination of carvedilol, sildenafil and paracetamol

Table 1. Linear regression equations for the calibration plots obtained in quantitative determination ofCAR and SIL in combination with PAR in model solutions (n = 6)

y – intensity of current [A].x – concentration [μg mL–1].

Determination of drugs in urine samples

The described methods can be successfully applied to the determination of theexamined drugs in spiked urine samples. Urine samples were prepared as describedin the Experimental part. All measurements were repeated three times applying stan-dard addition method. The percentage recoveries for CAR with PAR and SIL withPAR are given in Table 2.

Table 2. Urine assay results and mean recoveries of CAR and SIL in combination with PAR in spikedurine samples (n = 3)

(Continuation on the next page)

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978 I. Baranowska, M. Koper and P. Markowski

Table 2. (Continuation)

a Standard deviation.b Coefficient of variation.c Standard deviation of the mean.d Confidence interval (α = 0.05; t = 4.303).

Figure 7. Differential pulse voltammograms for SIL with PAR (A), and for CAR with PAR (B). (Continu-ation on the next page)

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979Electrochemical determination of carvedilol, sildenafil and paracetamol

As shown in Figure 8B, the peaks of CAR and PAR overlap and to separate them thefirst derivative method has to be used. The voltammograms of urine samples spikedwith CAR–PAR and SIL–PAR pairs are presented at Figure 8.

Figure 8. A – Differential pulse voltammograms for SIL with PAR in urine samples (a); spiked with 10 µgSIL and 10 µg PAR (b); 20 µg SIL and 20 µg PAR (c). B – Differential pulse voltammograms forCAR with PAR in urine samples (a); spiked with 10 µg CAR and 10 µg PAR (b); 20 µg CARand 20 µg PAR (c)

CONCLUSIONS

We have developed a new method for determination of SIL, CAR, and PAR inbiological samples using glassy carbon electrode and DPV technique. This procedu-res have not been described in the literature yet. It can be used as an alternative for chro-

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980 I. Baranowska, M. Koper and P. Markowski

matography and other voltammetric methods involving mercury electrode [26, 27].The presented primary CV studies were aimed at the observation of electrode pro-cesses and determination of the potential-pH dependencies. A new method for simul-taneous determination of SIL with PAR and CAR with PAR makes possible the analysisof body fluids (urine). The results indicate that matrix interferences can be eliminatedapplying the described separation procedures. Satisfactory recoveries of the analytesfrom the real samples, precision, accuracy, and a good agreement between the stu-died concentrations ranges and the real ranges encountered in the urine samples collec-ted from patients treated with the investigated drugs make the developed methodapplicable in the clinical analysis.

Acknowledgement

This work was supported by the funds for scientific research in 2007–2009 in the frames of the ProjectNo. N20402532/0738.

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Received July 2008Revised September 2008Accepted October 2008

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