Rifampic in : E lectrochemical Ef fect on Blood Component by Cyclic Voltammetry Using Nano-Sensor

Rifampicin (RIF) compound was analyzed by electrochemical study using cyclic voltammetric method to characterize the electrochemical properties in blood medium. Glassy carbon electrode (GCE) was modified with carbon nanotubes (CNT) as a high sensitive sensor for using in the electro-analysis of RIF in blood medium. It was found that oxidation and reduction current peaks of RIF in blood medium were at the potential of 0.5 and -0.5 V, respectively. Different concentrations, pH, scan rates, reliability and stability of RIF in blood medium were studied. The diffusion coefficient of oxidation and reduction was determined using the Randles-Seveik equation. The result showed the average value of oxidation and reduction were 2.66 × 10 and 8.72 × 10 cms, respectively.


Introduction
The important study for the most famous types of the treatment tuberculosis (Tb) disease in terms of its effects on blood components is namely rifampicin (RIF).Our study focused on the effect the oxidation and reduction of RIF of on blood medium by electrochemical analysis using highly accurse nanosensors.In the recent fifteenth years, scientists have been focusing on the oxidative effect of the medicines in blood medium using oxidation-reduction method by cyclic voltammetric technique [1][2][3][4][5][6][7].Rifampicin, also known as rifampin, is an antibiotic used to treat several types of bacterial infections, including tuberculosis.
The structure of RIF compound is illustrated in Fig. 1 [8].Britton Robinson buffer solutions.The oxidation current peak for rifampicin showed a quasi-reversible reaction and an irreversible reaction for the isoniazid [9].Glassy carbon electrode (GCE) was modified with iron oxide nanoparticles (Fe 3 O 4 NPs) and multiwalled carbon nanotubes (MWCNTs) composite to determine RIF by cyclic voltammetric technique.The new sensors were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD) studies.The method was successfully applied for the determination of RIF in pharmaceutical tablets without any sample pre-treatment [10].The technique of differential pulse voltammetry for the determination of RIF as a drug in the associated form was evaluated in the quality of medicine distribution.The potential cyclic voltammetric determination of RIF was achieved at the modified electrode, presenting two oxidation peaks for RIF at potentials approximately 0.09 V (peak 1) and 0.70 V (peak 2) in phosphate buffer solution with a linear range of 0.08-15.00mM and a detection limit of 0.03 mM.The developed sensor was stable, reproducible and feasible for long-term use in biological samples with satisfactory results for RIF detection [11].Adsorptive stripping voltammetry of antibiotics of RIF was investigated by cyclic voltammetry and differential pulse voltammetry.

The electrochemical oxidation of RIF and isoniazid was studied by cyclic voltammetric technique in
The results showed that the determination of highly sensitive oxidation peak current was the basis of a simple, pharmaceutical formulations and biological fluids by differential pulse adsorptive stripping voltammetry.The limits of detection for the determination of RIF in bulk forms were 6.0 × 10 -8 mol/L and 1.3 × 10 -8 mol/L, respectively [12].
In this work, GCE was modified with CNT for using in cyclic vltammetric technique.RIF compound was studied to find the oxidation-reduction current peaks in blood medium as application study.

Experimental Materials and chemical reagents
RIF was received from Ajanta Pharma Limited, India.Normal saline was from ADWIC Pharmaceutical Division, Egypt.Carbon nanotubes (purity 99%) was supplied from Fluka Company, Germany.Other chemicals and solvents were of analytical grade and used as received from the manufacturer.Healthy human blood samples were received from Iraqi Blood Bank in Baghdad City of Medicine.Deionized water was used for the preparation of aqueous solutions.

Instrumentation
The EZstat series (potentiostat/glvanostat) provided from NuVant Systems Inc.Pioneering Electrochemical Technologies, USA was used in the present work.Electrochemical workstations of Bioanalytical system with potetiostate driven by electroanalytical measuring softwares were connected to a personal computer to perform cyclic voltammogram.Ag/AgCl (3 M NaCl) and platinum wire (1 mm diameter) was used as a reference and counter electrode, respectively.

Preparing the modification of glassy carbon electrode with carbon nanotube (CNT/GCE)
The mechanical attachment technical method to prepare the CNT/GCE working electrode was employed for preparation of the nano-sensor [13,14].The method of the modification of GCE included abrasive application of the multiwall carbon nanotubes (MWCNT) on the clean surface of GCE, forming an array of MWCNT as modified working electrode MWCNT/GCE and replaced in 10 mL of the diluted blood with normal saline ratio of 1 : 9 in the cyclic voltammetric cell; then all the electrodes (working electrode, reference electrode and counter electrode) were connected with the potentiostat.Procedure 10 mL of the sample of blood with normal saline was replaced in cyclic voltammetry cell; then, each of modified working electrode (MWCNT/GCE), reference and counter electrodes was immersed in the blood sample and connected in the potentiostat and in turn with a personal computer to discuss the cyclic voltammogram of the compound in the cell.Afterwards, 1 uL of the 0.1 M RIF solution was added to every spike by micropipette in the blood medium to determine effect of the redox process of RIF on the blood component.

Results and Discussion
The current study was concerned with the electrochemical behavior of the RIF compound in blood medium which included different concentrations, pH, scan rate, and stability study of CNTs on the GCE as a high sensitive sensor.

Effect of different concentrations
Different concentrations of RIF compound in blood medium were studied using nano-sensor (CNT/GCE) to determine the detection limit and the sensitivity of the method.Fig. 2 shows the cyclic voltammogram of RIF at different concentrations which enhanced the oxidation-reduction current peaks against the increasing concentrations of RIF in blood medium.Fig. 3 and 4 show the calibration curve of RIF compound in different concentrations of 0.5-4 mM in blood medium.Linearity of the plot of RIF concentration of up to 0.5 mM with a current sensitivity of close to 40.0 μA/ mM was observed with curvature being detected at a concentration of greater than 0.5 mM.The calibration plots were performed at the CNT/GCE in RIF with blood medium; there also showed a good linearity of cathodic current versus different concentrations of RIF as described by y = 0.8842 x + 19.477, R 2 = 0.8925.The detection limit of the method based on CNT modified GCE for the determination RIF was found to be 0.5 mM.

Effect of different scan rates
Fig. 5 illustrates the cyclic voltammogram of RIF in blood medium at different scan rates from 0.01-0.1 V/sec.A good relationship between the oxidation and reduction current peaks and scan rates was found, as shown in Fig. 6 and 7, respectively.
A reasonably linear dependence of oxidationreduction current peaks of RIF in blood medium on scan rate is described by y = 0.5701 x + 2.1399, R 2 = 0.9829, and y = 0.4745 x + 1.7706, R 2 = 0.9966, respectively.The slope of graph log plot (Fig. 6 and 7) is 0.47, which is quite comparable with the theoretical slope of 0.5 for diffusion controlled process [15].

Determination of diffusion coefficient from scan rate
Diffusion coefficient value was determined from the Randles-Sevcik equation which describes it as   reversible redox couple peaks [16,17]: where I p is the current peak (μA), n is the number of moles of electrons transferred in the reaction, A is the area of the electrode (cm 2 ), D f is the diffusion coefficient (cm 2 /sec), and V is the scan rate of the applied potential (V/sec).
The diffusion coefficient values of oxidationreduction reaction (D fa , D fc ) for RIF in blood medium was determined from equation (1), as shown in Table 1.The average values of oxidation and reduction were 2.66 × 10 -5 and 8.72 × 10 -5 cm 2 /sec, respectively.Increasing of scan rate Fig. 6 Plot log I pc against log V of rifampicin compound in blood medium using CNT/GCE as working electrode versus Ag/ AgCl as reference electrode at different scan rates from 0.001-0.1 V/sec.Fig. 7 Plot log I pa against to log V of rifampicin compound in blood medium using CNT/GCE as working electrode versus Ag/ AgCl as reference electrode at different scan rates from 0.001-0.1 V/sec.Diffusion coefficient of oxidation-reduction for RIF in blood medium was determined through cyclic voltammetric technique and compared with the values in literature.The contribution of the fractal surface of the modified electrode (CNT/GCE), and the possibility of involving of subsequent chemical reaction to the redox current peak affecting the diffusion coefficient were analyzed [18].

Effect of different pH
RIF compound had especial electrochemical behavior for oxidation-reduction current peaks at different pH from 4-10, which is illustrated in Fig. 8  and 9, respectively.The oxidation peak was enhanced in acidic medium and disappeared in alkaline medium, while the reduction peak remained in both media (acid and base).The relationship of oxidation-reduction current peaks of RIF in blood medium at acidic and alkaline pH is shown in Fig. 8 and 9, respectively [19].It is very important to look for the effect of pH on the blood component with RIF drug in treatment of patients.It was found that oxidation current peak of RIF enhanced at neutral pH of the blood medium as shown in Fig. 8, while the reduction current peak of RIF enhanced at neutral pH as shown in Fig. 9; hence, RIF functioned as a good treatment in pH 7 of blood medium.

Reliability and stability Study
RIF compound in blood medium was studied by nano-sensor (CNT/GCE) in cyclic voltammetry.To ensure that the results were reliable in this study, ten times of scanning was used for the voltammogram (Fig. 10), and the relative standard deviation (RSD) was determined for both oxidation-reduction current peaks of RIF as of ± 0.329% and ± 0.411%, respectively [20][21][22][23][24].A good nano-sensor was used in this study, i.e.CNT/GCE as working electrode with high sensitivity   with good stability of nanomaterials on the surface of GCE of low RSD at ten times recording in cyclic voltammetry as shown in Fig. 10.

Conclusions
The effects of rifampicin compound on blood components were studied by electrochemical analysis method using nano-sensor CNT/GCE in cyclic voltammetric technique.The oxidation current peak of rifampicin compound in blood medium appeared in the cyclic voltammogram at potential of +0.5 V which caused an oxidative stress of blood compound by producing free radicals in this medium, and so the pharmacokinetics study of effect of the rifampicin was induced in blood medium through using it as a drug in different metabolic actions of these chemical compounds in human body.However, in some patients the hypoglycemic effect of using rifampicin may be affected during concomitant treatment with rifampicin.

Fig. 3
Fig.3 Calibration curve of reduction current peak of rifampicin in blood medium against different concentrations.

Fig. 4
Fig.4 Calibration curve of oxidation current peak of rifampicin in blood medium against different concentrations.

Fig. 2
Fig. 2 Cyclic voltammogram of rifampicin at different concentrations in blood medium on CNT/GCE as working electrode versus Ag/AgCl as reference electrode at the scan rate of 100 mV/sec.

Fig. 5
Fig.5 Cyclic voltammogram of rifampicin at different scan rates from 0.01-0.1 V/sec in blood medium on CNT/GCE as working electrode versus Ag/AgCl as reference electrode.

Fig. 10
Fig. 10 Cyclic voltammogram of rifampicin in blood medium at tenth times recording.

Fig. 8
Fig. 8 Relationship between oxidation current peak and different pH of 4-10 in blood medium.

Fig. 9
Fig. 9 Relationship between reduction current peak and different pH of 4-10 in blood medium.

Table 1
Diffusion coefficient values at different scan rates of oxidation-reduction current peaks of rifampicin compound in blood medium

Table 2
Relative standard deviation of using modified working electrode (CNT/GCE) for oxidation current peak of rifampicin in blood medium

Table 3
Relative standard deviation of using modified working electrode (CNT/GCE) for reduction current peak of rifampicin in blood medium