Amperometric dot-sensors based on zinc porphyrins for sildenafil citrate determination

Electrochimica Acta 58 (2011) 290–295

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Amperometric dot-sensors based on zinc porphyrins for sildenafil citrate determination Simona-Cornelia Balasoiu a,b , Raluca-Ioana Stefan-van Staden a,∗,1 , Jacobus Frederick van Staden a , Rodica-Mariana Ion c,25d , Gabriel-Lucian Radu b , Hassan Y. Aboul-Enein 26e a Laboratory of Electrochemistry and PATLAB Bucharest, National Institute of Research for Electrochemistry and Condensed Matter, 202 Splaiul Independentei Str., 060021 Bucharest, Romania b Department of Analytical Chemistry and Instrumental Analysis, Politehnica University of Bucharest, 1-7 Gh. Polizu Str., Bucharest, Romania c Department of Analysis, National Research & Development Institute for Chemistry and Petrochemistry ICECHIM, Spl. Independentei, 202, Sect. 6, Bucharest, Romania d Valahia University, Targoviste, 18-20 Blvd. Unirii., Targoviste, Romania e Pharmaceutical and Medicinal Chemistry Department, The Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12311, Egypt

a r t i c l e

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Article history: Received 20 August 2011 Received in revised form 18 September 2011 Accepted 18 September 2011 Available online 24 September 2011 Keywords: Dot-sensors Sildenafil citrate Zinc porphyrins

a b s t r a c t Three types of zinc porphyrins have been used for the design of carbon paste or diamond paste based amperometric dot-sensors. Sildenafil citrate was determined using these sensors by employing differential pulse voltammetry. The linear concentration range for the proposed electrodes was between 10−11 and 10−4 mol L−1 , with the lowest detection limit (2.46 pmol L−1 ) obtained using the diamond paste dot-sensor modified with zinc-5,10,15,20-tetra(4-sulfophenyl)porphyrin. Sildenafil citrate was determined reliably from its pharmaceutical formulations, namely Viagra with recovery values higher than 93%. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Sildenafil citrate, 1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl1-H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]4methylpiperazine citrate (Fig. 1) is the active ingredient of Viagra, which is mainly used as an oral drug for erectile dysfunctions, where its action involves the release of nitric oxide by inhibiting cyclic guanosine monophosphate, the specific monophosphodiesterase-5. The nitric oxide can also affect the regulation of blood pressure, immune defense, digestion, vision and smell [1–3]. Several analytical methods such as HPLC [4–6], micellar electrokinetic chromatography [7], extractive spectroscopy [8], resonance Rayleigh-scattering [9], and a FIA/UV spectrophotometric system [10] have been proposed for the detection of

∗ Corresponding author. Tel.: +40 751507779; fax: +40 213163113. E-mail address: [email protected] (R.-I. Stefan-van Staden). 1 ISE member. 0013-4686/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2011.09.040

sildenafil citrate. The need of procedures with a faster response, higher accuracy and better sensitivity led to the development of electrochemical methods as reliable analytical methods for the determination of sildenafil citrate in pharmaceutical formulations. Potentiometric sensors based on ion-association complexes of sidenafil citrate have been proposed, with limits of detection in the micromolars magnitude order [11,12]. The study of electrochemical oxidation of sildenafil citrate on carbon-based electrodes led to the development of amperometric sensors with even better characteristics [13,14]. The modification of a glassy carbon electrode with carbon nanotubes gave a performance of picomolar detection of the analyte [15]. Several studies proved that diamond could be used reliably as electrode material in the determination of sidenafil citrate [16], due to its unique electrochemical proprieties such as low background current, lack of adsorption and wide potential range. Furthermore, the use of diamond paste based electrodes is even suitable for its assay at picomolar magnitude orders [17]. In this paper six amperometric dot-sensors based on diamond paste and carbon paste modified with three types of zinc porphyrines, namely zinc-tetraphenylporphyrin (ZnTPP), zinctetranaphthaloporphyrin (ZnTNP) and zinc-5,10,15,20-tetra(4sulfophenyl)porphyrin (ZnTSPP) (Fig. 2) are described for the assay of sildenafil citrate.

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Atomic force microscopy (AFM) experiments were performed using an Agilent Technologies 5500 Scanning Probe Microscope (Agilent Technologies Inc., Santa Clara, CA) in air at room temperature with a 90 ␮m multipurpose scanner. Images of the modified carbon/diamond layers were acquired in the tapping mode (AAC mode AFM) using a silicon cantilever (PointProbe Plus Force Modulation) with tip radius <10 nm (length 227 ␮m, a force constant 1.8 N/m, and a resonance frequency of 69 kHz; Nanosensors) at scan rates from 0.5 to 1 line/s, the images being recorded with 512 × 512 pixels resolution. The imaging and analysis Software used was PicoView 1.6 (Agilent Technologies, Chandler, AZ). For additional image processing (profiles extractions from surfaces, filtering, line correction, analytical studies, and 2D and 3D parameters) a Pico Image software was used.

2.3. Design of carbon paste based dot sensors

Fig. 1. Sildenafil citrate.

2. Experimental 2.1. Reagents and materials Diamond powder (1 ␮m, synthetic), graphite powder (1–2 ␮m, synthetic) and Indigo Carmine were supplied by Aldrich. Paraffin oil was supplied by Fluka (Buchs, Switzerland). HClO4 and NaClO4 were purchased from Merck. All the solutions were prepared with deionised water obtained from a Direct-Q® 3Water Purification system (Millipore Corporation, Molsheim Cedex, France). The Zn complexes namely Zn-tetranaphthaloporphyrin (ZnTNP) [18], Zn-5,10,15,20-tetra(4-sulfophenyl)porphyrin (ZnTSPP) [19], and Zn-tetraphenyl porphyrin (ZnTPP) [20] were synthesized in our laboratory. 2.2. Instrumentation Amperometric measurements were recorded using a PGSTAT 302N potentiostat/galvanostat connected to a three-electrode cell, and linked to a computer via an Eco Chemie (Utretch, The Netherlands) software version 4.9. An Ag/AgCl (0.1 mol L−1 KCl) electrode served as reference electrode and a platinum electrode served as auxiliary electrode. The measurements were made within a potential range from −400 to 400 mV (vs. Ag/AgCl).

100 mg of paste (obtained by mixing paraffin oil and graphite powder in a ratio of 1:4 (w/w)) was modified by the addition of the modifier–ZnTPP/ZnTNP solution (10−3 mol L−1 , prepared in THF) or ZnTSPP solution (10−3 mol L−1 prepared in distilled water). The resulting paste was pressed into a plastic tube and the diameter of the active surface of the sensor was 300 ␮m. Electrical contact was obtained with an Ag/AgCl wire inserted into the carbon paste (CP). The surface of the electrode can be renewed by polishing with alumina paper. The electrode can be used for more than 6 months, between measurements when stored in a dry state, away from day light, at room temperature.

2.4. Design of diamond paste based dot sensors The modified diamond paste (DP) was prepared from 200 mg of synthetic monocrystalline diamond powder mixed with 20 ␮L paraffin oil and 100 ␮L solution of ZnTPP/ZnTNP (10−3 mol L−1 , prepared in THF) or ZnTSPP (10−3 mol L−1 prepared in distilled water). The modified paste was placed into a plastic tube with the diameter of the active surface of 300 ␮m. An Ag/AgCl wire was used as electric contact. The surface of the electrode can be renewed by polishing with alumina paper. The electrode can be used for more than 6 months, between measurements when stored in a dry state, away from day light, at room temperature.

Fig. 2. The structure of the proposed Zn porphyrins.

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Table 1 Response characteristics of the amperometric dot sensors. Dot sensor based on

Matrix

Equation of calibrationa

Linear conc. range (mol L−1 )

Limit of detection (mol L−1 )

ZnTNP

CP DP

H = 6.00(±0.7) × 10−10 + 4.79(±0.20) × 10−5 × cR = 0.995 H = 4.30(±0.5) × 10−10 + 9.16(±0.15) × 10−6 × cR = 0.980

10−8 to 10−5 10−11 to 10−5

2.62 × 10−9 1.43 × 10−12

ZnTPP

CP DP

H = 2.3(±0.8)4 × 10−9 + 3.12(±0.19) × 10−4 × c R = 0.960 H = 4.85(±0.1) × 10−10 + 5.15(±0.11) × 10−2 × cR = 0.969

10−8 to 10−6 10−11 to 10−9

2.93 × 10−9 4.26 × 10−12

312.00 5.15 × 104

−25 ± 3 180 ± 7

ZnTSPP

CP DP

H = 3.65(±0.4) × 10−9 + 1.99(±0.23) × 10−5 × c R = 0.915 H = 3.33(±0.65) × 10−10 + 0.15(±0.03) × c R = 0.988

10−8 to 10−4 10−11 to 10−9

6.63 × 10−9 2.46 × 10−12

19.90 1.50 × 105

−25 ± 4 160 ± 5

a

Sensitivity (␮A ␮mol−1 L) 47.9 9.16

E (mV)

−25 ± 7 180 ± 5

H = A; c = mol L−1 .

2.5. Differential pulse voltammetric method Differential pulse measurements were performed at room temperature. The applied pulse potential was 25 mV s−1 vs. Ag/AgCl. The modified carbon or diamond paste amperometric dot-sensor with the reference and counter electrodes were dipped into a cell containing a series of standard solutions in the range of 10−14 to 10−2 mol L−1 of sildenafil citrate prepared in a solution of 0.01 mol L−1 HClO4 and NaClO4 (pH 2). The peak heights were measured at the potential given in Table 1 and were plotted against the concentrations of standard solutions of sildenafil citrate. The unknown concentrations were calculated from the calibration graphs.

modifiers revealed the formation of smooth, highly ordonated surfaces in case of carbon paste, whereas in the case of diamond paste the modification with ZnTSPP gave the best surface characteristics. It can also be observed that the density of the active centers in this case is the highest. These results are correlated with the performances of the dot sensors based on diamond paste modified with ZnTSPP, for which the lowest detection limits was achieved (Table 1). The modification of the carbon paste using ZnTPP and ZnTNP led to the formation of surfaces with similar topographic

2.6. Uniformity content test of Viagra tablets Five tablets of Viagra containing 50 mg/tablet sildenafil citrate were dissolved separately and buffered with 0.01 mol L−1 HClO4 and NaClO4 (pH 2) solution and diluted to mark with deionised water in a 100 mL volumetric flask. The measurements were done using the prepared solutions, the current recorded, and plotted on the calibration graphs as described above. 2.7. AFM measurements The active surface of the proposed dot-sensors was analyzed using AFM. Alternating Current (AC) Mode AFM was used for all the measurements. In AC Mode AFM, or tapping mode, the AFM cantilever is driven at its fixed end with a piezoelectric actuator that is positioned under the substrate to which the cantilever is attached. An alternating current (AC) voltage is applied to this actuator, whose resulting motion is then amplified at the cantilever’s free end, where the tip is. The image data obtained were saved in bidimentional as well as tridimentional format. The images were processed by first order flattening in order to remove the background noise. All the data analysis was performed using PicoView 1.6.2 Software. The topography of the surfaces was investigated in a range of scan areas from 50 × 50 to 1 ␮m × 1 ␮m. Fig. 3 presents the tridimentional images of electrodes active surface obtained for a scan area of 1 ␮m × 1 ␮m. The data are displayed using a color mapping for heights, darker areas representing low height features and lighter areas standing for higher surfaces. 3. Results and discussion 3.1. Surface characterization for Dot-sensors Figs. 3 and 4 present the topography of modified carbon and diamond paste dot sensors using AFM. Generally, for all three modifiers used, the surface roughness of carbon paste based dot sensors is comparable with the active surface of diamond paste dot sensors, as shown in Table 2. The use of ZnTPP and ZnTNP as matrix

Fig. 3. AFM topography images of the active surface of the carbon paste modified dot-sensors with – ZnTPP (a), ZnTNP (b) and ZnTSPP (c) at a scan area of 1 ␮m × 1 ␮m.

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3 2

I, nA

2

1 1

0 -0.2

-0.1

0.0

0.1

E, mV Fig. 5. Typical differential pulse voltammograms obtained at carbon paste dotsensors modified with ZnTNP – 1, ZnTPP – 2, and ZnTSPP – 3 for a solution of 10−8 mol L−1 sildenafil citrate.

0.6

2

3

I, nA

1

0.3

0.0

0.0

0.2

0.4

E, mV

Fig. 4. AFM topography images for the active surface of the diamond paste modified dot-sensors with – ZnTPP (a), ZnTNP (b) and ZnTSPP (c) at a scan area of 1 ␮m × 1 ␮m.

characteristics. ZnTPP and ZnTSPP had the same effect in the case of diamond paste modification. For the characterization of the surface of the dot sensors, Sq is defined (accordingly with ISO25178) as the root mean square height – standard deviation of the height distribution (RMS surface roughness). This value result by computing the standard deviation for the amplitudes of the surface. The surface roughness of the dot-sensors varies in the range of 11.4–3.01 nm, slight differences being observed between the two matrices (Table 2). 3.2. Electrocatalytic oxidation of Sildenafil citrate Figs. 5 and 6 present the typical differential pulse voltammograms obtained with carbon (Fig. 5) and diamond (Fig. 6) Table 2 AFM analysis of the dot-sensors active surface roughness for a scan area of 1 ␮m × 1 ␮m. Modifier

ZnTPP

Matrix

CP

DP

ZnTNP CP

DP

ZnTSPP CP

DP

Sq (nm)

3.26

6.04

11.4

3.01

5.20

3.92

Fig. 6. Typical differential pulse voltammograms obtained at diamond paste dotsensors modified with ZnTSPP – 1, ZnTNP – 2, and ZnTPP – 3 for a solution of 10−11 mol L−1 sildenafil citrate.

paste dot-sensors modified with zinc porphyrins at a scan rate of 25 mV s−1 . As observed in Fig. 5, the carbon paste based dot-sensors modified with zinc porphyrins present a signal, with well defined peak currents at a potential around −25 mV. The highest signal at concentration of 10−8 mol L−1 for the sildenafil citrate solution was achieved by using the carbon paste dot-sensor modified with ZnTSPP. Fig. 6 depicts the behaviour of sildenafil citrate at diamond paste modified electrodes. The dot-sensors modified with ZnTPP and ZnTNP presents a well defined peak at the potential around 180 mV, while the dot-sensor based on diamond paste modified with ZnTSPP presents a slightly lower signal for a concentration of sildenafil citrate of 10−11 mol L−1 .

Table 3 Amperometric selectivity coefficients for the proposed dot-sensors. amp

Modifier

Matrix

Ksel vs. IC

ZnTPP

CP DP

3.15 2.46

ZnTNP

CP DP

2.22 2.00

ZnTSPP

CP DP

3.65 3.32

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Table 4 Recovery of sildenafil citrate from pharmaceutical products. Modifier

Matrix

% Recovery of sildenafil citrate from Pharmaceutical formulation

Urine Bias %

ZnTPP ZnTNP ZnTSPP

CP DP CP DP CP DP

97.87 99.98 99.52 93.15 99.48 99.03

± ± ± ± ± ±

0.17 0.16 0.12 0.06 0.07 0.15

0.12 0.11 0.09 0.09 0.05 0.11

Bias % 95.51 98.75 95.42 97.31 99.33 96.41

± ± ± ± ± ±

0.08 0.05 0.17 0.10 0.12 0.08

0.05 0.03 0.11 0.08 0.08 0.03

N = 5.

3.3. Response characteristics of the dot sensors based on zinc porphyrins In the present study the influence of two matrices, namely carbon paste, and diamond paste have been investigated, comparing the influence of modification with three types of zinc porphyrins for each in order to find the best electrochemical dot-sensor for the assay of sildenafil citrate from pharmaceutical formulations and biological samples. The response characteristics of the studied amperometric dot-sensors are presented in Table 1. Sildenafil citrate was detected in a concentration range from 10−11 to 10−4 mol L−1 . The highest sensitivity was recorded for the dotsensor based on ZnTSPP and diamond paste. The lowest limit of detection was obtained by using the dot-sensor based on diamond paste modified with ZnTSPP. The wider concentration range was achieved by the utilization of a dot-senzor based on ZnTNP and diamond paste. Diamond paste based dot-sensors can be used for determination of sildenafil citrate at low concentrations. Comparing these results with those reported previously by Stefan-van Staden et al. [17], regarding diamond paste (synthetic-2) based sensors, the utilization of the modifier in sensors’ design made possible their utilization on a wider linear concentration range, the upper limit being 10−5 mol L−1 ; this extension will make possible the utilization of undiluted samples of Viagra for analysis. The response of the dot-sensors was stable for 7 weeks, with RSD values lower than 0.85%, recorded when the sensors were used every day continuously and stored over night at room temperature. 3.4. Selectivity studies Indigo carmine (IC) was tested as main interfering species using the mixed solution method in the determination of sildenafil citrate. IC is found together with sildenafil citrate in the pharmaceutical formulation, giving the well known blue color of the tablets. The concentration of the interferent in the solution was kept as 10 times higher than the concentration of the analyte. Amperometamp ric selectivity coefficients, Ksel , calculated accordingly with the method proposed by Wang [21], are presented in Table 3. All the measurements were performed at 25 ◦ C. It can be seen that the dotsensors based on carbon and diamond paste modified with ZnTSPP are less selective than the dot-sensors based on ZnTPP and ZnTNP. The best results were obtained by using ZnTNP as modifier; for this modifier the selectivity was not dependent on the type of matrix used for sensor design. 3.5. Analytical applications The electrochemical dot-sensors were used successfully in the determination of sildenafil citrate from biological samples as well as in the content uniformity test of Viagra tablets as shown in Table 4. The dot-sensors can be used reliably for the detection of sildenafil citrate in either pharmaceutical formulations

or biological samples. Accordingly with the values presented in Table 4, the values of RSD(%) are lower than 0.1%, with bias lower than 1%. The best results were recorded for the dot sensor based on carbon paste and ZnTSPP, and for the dot-sensor based on diamond paste and ZnTPP. While carbon paste based sensors were used reliably for pharmaceutical samples, the diamond paste based sensors were the best when used for clinical analysis. 4. Conclusions The proposed amperometric dot-sensors provided a good response for the detection of sildenafil citrate from its pharmaceutical form, namely Viagra, as well as from biological samples. Their response characteristics made them useful for the detection of sildenafil citrate in a wide concentration range (10−11 to 10−4 mol L−1 ), with a picomolar limit of detection (2.46 × 10−12 mol L−1 by using a diamond paste dot-sensor modified with ZnTSPP). The dot-sensors present the advantages of a simple construction, robustness, low cost production, they can be reuse by simply polishing of the dot-sensor surface by alumina paper, and kept a long period while not in use in a dry and dark place. Another advantage with the use of the dot-sensors is the modifier, namely the zinc-porphyrins, which present a high degree of biocompatibility, making possible their utilization for in vivo tests. The use of the proposed method present the advantage of the direct determination with high precision, fast and stable response at a low consumption of sample and buffer. The dot-sensor based on diamond paste modified with ZnTSPP proved to achieve the best response characteristics. Acknowledgments The authors are grateful to Program Ideas 2009–2011, Contract 746/19.01.2009 for the financial support given for this project. S.C. Balasoiu is greateful to the project funded by the Sectoral Operational Programme Human Resources Development 2007-2013 of the Romanian Ministry of Labour, Family and Social Protection through the Financial Agreement POSDRU/6/1.5/S/19–ID 7713. References [1] S. Koneru, S.V. Penumathsa, M. Thirunavukkarasu, R. Vidavalur, L. Zhan, P.K. Singal, R.M. Engelman, D.K. Das, N. Maulik, J. Cell. Mol. Med. 12 (2008) 2651. [2] F. Montorsi, H. Padma-Nathan, S. Glina, Urology 68 (2006) 25. [3] A.R. McCullough, C.C. Carson, D. Hatzichristou, Urology 68 (2006) 38. [4] H.Y. Aboul-Enein, M.M. Hefnawy, J. Liq. Chromatogr. Related Technol. 26 (2003) 2897. [5] N. Daraghmeh, M. Al-Omari, A.A. Badwan, A.M.Y. Jaber, J. Pharm. Biomed. Anal. 25 (2001) 483. [6] A. Abd-Elbary, N.H. Foda, O.N. El-Gazayerly, Chromatographia 59 (2004) 561. [7] J.J. Berzas-Nevado, J. Rodriguez Flores, G. Castaneda Penalvo, N. Rodriguez Farinas, J. Chromatogr. A 953 (2002) 279. [8] N.D. Dinesh, P. Nagaraja, N.M. Made Gowda, K.S. Rangappa, Talanta 57 (2002) 757. [9] S.P. Liu, L. Fan, X.L. Hu, Z.F. Liu, S. Li, Anal. Sci. 22 (2006) 819.

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