Investigation of the Voltammetric Behavior of Methyldopa at Poly (p-Aminobenzene Sulfonic Acid) Modified Sensor

¹Department Of Chemistry, Faculty Of Arts And Sciences, İnönü University, 44280, Malatya-turkey
²Division Of Analytical Chemistry, Department Of Basic Pharmaceutical Sciences, Faculty Of Pharmacy, İnönü University, 44280 Malatya, Turkey

INTRODUCTION: Methyldopa, is a catecholamine which is known by its chemical name 2-Methyl-3-(3,4-dihydroxyphenyl)-DL-alanine (Figure 1), and it is widely used to lower blood pressure. MD is a centrally acting adrenal-receptor that reduces high blood pressure and sympathetic tone.1 In adrenergic nerve terminals, it is converted to α-methyl noradrenaline, and its antihypertensive effect seems to be owing to this agent stimulate the central adrenoreceptors.2

Figure 1. Molecular structure of methyldopa.

In general, high-performance liquid chromatography with UV detection3, polarographic4, potentiometry5 and ultra viole visible spectrophotometry.6 Quantitative analysis of various catecholamine moleculas was performed electrochemically by flow injection technique.7,8 However, many of these techniques are require expensive equipment and time consuming. In addition, since this catecholamins are electrochemically active, it is also possible to determine the nature of the molecules that provide neurotransmission by electrochemical methods. Therefore, it is important to detection of MD in the presence of AA by a reliable method which has good selectivity and sensitivity.

Ascorbic acid (vitamin C) is a biologically and industrially important substance. Especially in food and pharmacological industries, it is very important to analyze this substance which is mostly used in the shortest time and with the most sensitive techniques).9 The coexistence of ascorbic acid, MD and other catecholamines with very close oxidation potentials leads to the response obtained by electrochemical techniques. For this reason, the increased sensitivity and selectivity of the new sensors produced to the MD has long been the subject of researchers working on. Using the polymer modified electrodes solve this problem. Electrochemical behavior of MD was studied at various polypyrrole electrodes.10-14

However, some disadvantages exist in the previously reported modified electrodes. AA is present as an anion in physiological pH (7.4), whereas MD is present as a cation. There are high electron density sulfo groups and electron-rich N atoms in the structure of p-(ABSA). For this reason, a negatively charged polymer film is required to eliminate the interference of AA in the determination of MD. The p-aminobenzene sulfonic acid molecule has a high electron density sulfo groups, and p-(ABSA) films are negatively charged. Due to the electrostatic repulsion between the negatively charged sulfo groups and the ascorbic acid anions in the modified sensor, the ascorbic acid shifts to a more negative potential and the dopaminic acid can be easily separated from the ascorbic acid. The p-(ABSA) modified sensor can show high selectivity against MD.15
In this study, electroanalytical methods were developed to detect methyl dopa in drug samples rapidly, reliably and sensitively using electrode modified with poly (p-ABSA). It has been determined that the modified sensor can be utilized to the MD determination even in the presence of ascorbic acid at the same time.

The analytical determination parameters such as the LOD, the quantitative determination LOQ and the concentration range were determined, and the amount of MD in the drug tablets and blood serum was found. To test the accuracy of the applied voltammetric method, MD recovery studies were performed in real samples.

METHODS: Materials
All chemicals were of analytical purity and were procured from Merck (Darmstadt, Germany) or Sigma Chemical Company. Prior to the polymerization, the solutions of monomer were held in the nitrogen gas atmosphere for about 10 minutes, and during the electropolymerization, the environment was covered with nitrogen gas. Voltammetric experiments were carried out in the phosphate buffer solution (pH 3.0). Methyl dopa and AA solutions were freshly prepared before the experiments. All solutions were made up with ultra-pure water.

Instrumentation
In voltammetry experiments, BAS (Bioanalytical Systems, Inc.) 100BW electrochemical analyzer was used. This analyzer is connected to a personal computer and the device is controlled, data stored and processed by means of software loaded and running under MS-Windows. This electrode system consisting of an Ag/AgCl reference electrode (CHI), a glassy carbon disc working electrode (geometric area: 6.85 mm2, CHI) and a Pt wire coil auxiliary electrode (CHI) was used.
Modification of Poly (p-ABSA) Sensor
Before modification, the working glassy carbon electrode (GCE) was cleared up using 0.3 and 0.05 µm Al2O3 slurry on polishing materials. After polished GCE was sonicated in 1: 1 nitric acid solution for 10 min and washed with ultra-pure water. Afterward, GCE was electrochemically cleaned by 20-times cycling in the potential range of −0.7 to 1.7 V with a scan rate of 100 mV/s in 0.5 M H2SO4. After that, the electrode was plunged into 0.1 M KCl solution containing 5.0 mM p-ABSA and modification procedure was performed by cyclic sweeping from -1.5 to 2.5 V for 14 cycles at 50 mV/s. Then, the modified sensor was conditioned by cyclic voltammetry in the potential range of -0.5-0.5 V with 100 mV/s in pH 3.0 PBS and was stored in PBS (pH 3.0).

RESULTS: Electropolymerization of p-Aminobenzene Sulfonic Acid
Figure 2 shows that the electropolymerization of p-ABSA at the GCE surface. the electropolymerization was performed in 0.1 M KCl solution containing 5.0 mM p-ABSA at a GCE by cyclic voltammetry technique in the potential range of -1.5-2.5 V. In the first cycle, two reduction peaks were obtained at 0.452 V (peak A) and 0.449 V (peak B), which might be related to the reduction of p-ABSA. Again in the first cycle, an oxidation peak observed at 0.824V (peak C). In the next and subsequent cycles, following the continuous scan, broader peaks were monitored that proved that the polymer film was constantly growing. It could be observed that the film growth was faster for the first five cycles than for the other cycles and also, the next cycles are no longer stable. From these, it could be said that p-ABSA was coated on the GCE surface by electrodeposition. A brown polymer was formed that was properly bonded on the GCE surface.

Figure 2. CVs of 5 mM p-aminobenzene sulfonic acid in 0,1 M KCl at GCE, Scan rate: 50 mV/s, 14 cycle.

The Effect of Film Thickness on MD Response
The film thickness, which is the number of cycles of electropolymerization, is one of the most important factors determining the polymer film selectivity property. By altering the amount of charge consumed during electropolymerization, it is possible to obtain poly (p-ABSA) films at desired thicknesses. Different film thicknesses were obtained by varying the cycles of the cyclic voltammetry parameter. The selectivity characteristics of poly (p-ABSA) sensors prepared in the range of 6–20 cycles to MD and AA were systematically examined. From the DPV results of MD, it was observed that the regular and repetitive responses could be obtained at 14 cycles film.
Electrochemical behavior of MD at poly(p-ABSA) modified sensor
The voltamograms achieved by the cyclic voltammetry technique of MD show a reduction potential of ~ 200 mV potential and an oxidation peak of ~ 220 mV potential (Figure 3). As seen from the cyclic voltamograms, the anodic and cathodic peak currents differ from each other, indicating that the MD molecule exhibits a semi-reversible reaction.

Figure 3. CVs of 0.01 mM MD in 0.1 mM PBS (pH 3.0) (A) GCE (B) at poly(p-ABSA) modified sensor. Scan rate: 50 mV/s.

Electrolyte Type Effect on Voltammetric Behavior of Methyl Dopa

By selecting an appropriate support electrolyte solution, it creates a conductive environment between the submerged electrodes. Apart from this basic purpose, the support electrolyte is doped in the polymer coming into the solution.

Figure 4. Electrolyte effect on voltammetric analysis of 0.01 mM Methyl Dopa on poly (p-ABSA) sensor. A) PBS (pH 7,0), B) NaClO4 C) KCl, D) NaCl, E) NaNO3 and F) Na2SO4.

The choice of support electrolyte depends on its resolution, dissociation degree and nucleophilic character. For this purpose, voltammograms of MD in Na2SO4, PBS (pH 7,0), NaNO3, NaClO4, NaCl and KCl electrolytes were taken (Figure 4). While taking a voltamogram at pH 7.0 in PBS, voltamograms were taken at the native pH of the other electrolyte species.

pH effect on the peak potential and current of MD
To observe how methyl dopa behaves in relation to pH in PBS, the potential-current relationships at pH 2-11 are plotted. Besides, Figure 5 indicates the relation of maximum peak potential and current on pH of electrolyte. As the pH increases for methyl dopa, the peak potentials are shifted to lower potentials.

Figure 5. pH effect on (A) current and (B) potential of 0.01 mM methyl dopa using DPV technique. Scan rate, 50 mVs-1

Determination of MD in the presence of AscorbicAcid
Determination of MD in poly (p-ABSA) was done with differential pulse voltammetry. Differential pulse voltamograms of different concentrations of MD on poly (p-ABSA) are shown in Figure 6. The results obtained showed that the anodic peak currents of MD were linear at a concentration range of 1 μM to 300 μM. The calibration equation for MD is calculated as Ipa (μA) = 0,56x + 48,42 C (μM). Calibration chart was drawn in the concentration range of 3.2 x 10-8 - 4.7 x 10-7 M using DPV technique. LOD and LOQ were detected as 5.0x10-9 nM and 10.6 nM (S/N=3), respectively.

Figure 6. A) Differential pulse voltammograms and B) calibration graphs in increasing concentration of MD at poly (p-ABSA) modified sensor. MD concentrations (μM) were in the range of 0,0 - 300,0 in 0.1 M PBS (pH 3.0).

Figure 6A shows the peak of increasing concentrations of MD in the presence of 0.5 mM constant ascorbic acid. MD concentrations were 0,0, 1.0, 8.0, 10.0, 20.0, 30.0, 40.0, 50.0, 75.0, 100.0, 125.0, 150.0, 200.0, 250,0 and 300,0 μM. As can be seen, the ascorbic acid peaks remain the same while the methyl dopa peaks increase proportionately. The fact that methyl dopamine gives this result in the presence of ascorbic acid at pH 3.0 reveals that methyl dopa can be determined by this method in real samples, for example in samples taken from human beings or animals.

Figure 7. The increasing concentration of MD (0.01, 0.015, 0.020, 0.025, 0.030, 0.035 mM) with 0.5 mM ascorbic acid at poly (p-ABSA) modified sensor in 0.1 M PBS (pH 3.0).

In Figure 7, peak currents of ascorbic acid in increasing concentrations are observed in the voltammogram, as well as MDs with a constant concentration of 0.01 mM in Figure 8. The higher the ascorbic acid concentration is increased, the more the MD peaks are not deviated. This suggests that the method can be applied even in samples containing high concentrations of ascorbic acid.

Figure 8. The increasing concentration of ascorbic acid (0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mM) beside 0.01 mM methyl dopa at poly (p-ABSA) in 0.1 M PBS (pH 3.0).

Analytical applications
Five Alfamet tablets containing 250 mg MD in each tablet were directly weighed and powdered into the mortar. The amount of the calculated amount corresponding to the 100 mM concentration stock solution was weighed and transferred to a 10 mL volumetric flask and the volume was supplemented with ultra-pure water. The contents of the flask were subjected to centrifugation at 5000 rpm for 15 minutes to effect complete dissolution. Insoluble auxiliary substances dissolved. After then, the clear solution was taken up in the appropriate amount and the water was diluted to deionized water. The amount of MD in MD tablets was calculated with reference to the appropriate calibration graphs. Furthermore, the proposed techniques were tested by performing recovery tests. The obtained results are given in Tables 1 and 2. The proposed techniques can be successfully applied to MD analysis on tablets without any interference.

The quantity of MD in the tablets was computed from the suitable calibration charts. Furthermore, the validity of the proposed techniques was checked by carrying out recovery studies. Recovery results and the results obtained from the calibration chart can be seen in Tables 1 and 2. The proposed method was successfully performed to real samples beside interferences.

Table 1. Application of poly (p-ABSA) sensor to Methyl Dopa
Parameters Slope, mA/µM Intercept, µA Correlation coefficient, r
Proposed method 0.56 48.42 0.982

Table 2. Detection of MD in commercial tablets
Parameters Labeled, mM Found, mM RSD*, % Recovery, %
Proposed method 1 0.979 0.14 97.9
Blood Serum 1 0.764 0.22 76.4
* RSD: relative standard deviation.

DISCUSSION AND CONCLUSION: The results show that the proposed method can be easily used in the determination of catecholamines in drug samples and clinical analyzes. It has been observed that this method may be used to identify catecholamines in the blood serum.

Keywords:

Poli (p-Aminobenzen Sülfonik Asit) Modifiye Sensör ile Metildopanın Voltametrik Davranışının İncelenmesi

¹İnönü Üniversitesi, Fen Edebiyat Fakültesi, Kimya Bölümü, 44280 Malatya-türkiye
²İnönü Üniversitesi,temel Eczacılık Bilimleri, Analitik Kimya Anabilim Dalı, 44280, Malatya-türkiye

GİRİŞ ve AMAÇ: Poli (p-aminobenzen sülfonik asit) modifiye sensörün karbon elektrotlar kullanılarak metil dopa (MD) ve askorbik asitin duyarlı ve güvenilir şekilde tespit edilmesi
YÖNTEM ve GEREÇLER: Elektropolimerizasyon, 0.1 M KCl çözeltisi içerisinde çevrimli voltametri (CV) ile gerçekleştirildi. Modifiye sensör, 2-12 pH aralığında diferansiyel pulse voltametri (DPV) teknikleri ile ortaya çıkan metil dopanın oksidasyonu için yüksek elektrokatalitik etkiye sahiptir.
BULGULAR: MD'nin voltametrik tayini için, en iyi sonuçlar fosfat tampon çözeltisi (PBS) (pH 3) DPV tekniği ile elde edildi. MD için konsantrasyon aralığı, DPV ile 3.2 x 10-8 ila 4.7 x 10-7 M arasındaydı. Duyarlılık çalışmalarında, kantifikasyon sınırı (LOQ) ve tespit limiti (LOD) sırasıyla 10.6 nM ve 5.0 nM idi. Değiştirilen sensör, bazı gerçek numunelerde eş zamanlı olarak askorbik asit (AA) gibi MD ve parazitlerin belirlenmesi için kullanılmıştır. Elde edilen sonuçlar, modifiye edilmiş elektrot ve önerilen yöntemin iyi duyarlılık, tekrarlanabilirlik, tekrarlanabilirlik ve stabiliteye sahip olduğunu ortaya çıkardı.
TARTIŞMA ve SONUÇ: Elde edilen sonuçlar, modifiye edilmiş elektrot ve önerilen yöntemin iyi duyarlılık, tekrarlanabilirlik, tekrarlanabilirlik ve stabiliteye sahip olduğunu ortaya çıkardı.

Anahtar Kelimeler:

Gamze Erdoğdu, Şevket Zişan Yağcı, Ebru Kuyumcu Savan. Investigation of the Voltammetric Behavior of Methyldopa at Poly (p-Aminobenzene Sulfonic Acid) Modified Sensor. . 2019; 16(4): 450-456

Corresponding Author: Gamze Erdoğdu, Türkiye