Chemically Modified Carbon Sensors Mixed or Single for the Determination of Cardiovascular Drug Nafronyl Oxalate in Bulk, Praxilene and Human Fluids

Three novel chemically modified Carbon Paste Sensors (CMCPs) were proposed for the determination of nafronyl potentiometric in bulk, pharmaceutical dosage form; human plasma/urine. The sensors were based on an ion-pair associates of nafronyl silicotungstic acid (Nf-St) (sensor 1), nafronyl silicomolybdic acid (Nf-SM) (sensor 2), a mixture of (Nf-St)+(Nf-SM) (sensor 3). The modified sensors showed Nernstian slopes ranging from 58.5 ± 0.5-60.7 ± 0.5 mV over the concentration ranged from 1.0x10-7-1.0x10-2 M and pH 2.0-6.0 with detection limit 0.1 nM. The sensors exhibited good selectivity for nafronyl with respect to inorganic/organic cations, sugars and amino acids. The calibration curve, standard addition and potentiometric titration methods were applied for the determination of nafronyl ion in its bulk powder, pharmaceutical dosage form, and human fluids plasma/urine taken from a healthy volunteer and for the monitoring Praxilene tablets in vitro dissolution rates. Sensor 3 had been the best sensitivity so was successfully used for the determination the solubility products of ion-pair associates. The results were excellent and satisfactory recovery comparable to those obtained with the British Pharmacopoeia.

A Chemically Modified Sensor (CMEs) is one of the electrochemical methods which have been widely used as selective and sensitive analytical methods for the detection of the trace amounts of biologically important compounds. CMEs have important properties of their ability to catalyze the sensor process via significant decreasing of over the potential with respect to unmodified sensor. These sensors are capable of considerably enhancing the selectivity in the electroanalytical methods with respect to relatively selective interaction of the electron mediator with the target analyte in a coordination fashion.

Preparation of ion-pair:
The ion-exchangers, naftidrofuryl silicotungstate (Nf-ST), naftidrofuryl silicomolybdate (Nf-SM) were prepared by addition of 50 ml of 1.0x10 -2 M naftidrofuryl oxalate Nf-(COOH) 2 solution to 50 ml of 2.5x10 -3 M STA and SMA. The resulting precipitates were left in contact with their mother liquor overnight to assure complete coagulation. The precipitates were washed thoroughly with distilled water, then filtered and dried at room temperature and ground to fine powder. The resulting precipitates were left in contact with their mother liquor overnight to assure complete coagulation. The formation and purity of the ion-pairs and ion-associates, chemical compositions of the precipitates were checked by elemental analysis for carbon, hydrogen and nitrogen at the Micro Analytical Center, Faculty of Science, Cairo University.
Preparation of carbon paste sensors: Chemically modified carbon paste sensors (CMCPS-Nf) were prepared as previously described [21]. The sensor was used directly for potentiometric measurements without preconditioning requirements. A fresh surface of the paste was obtained by squeezing more out. The surplus paste was wiped out and the freshly exposed surface was polished on a paper until the surface showed shiny appearance.
Effect of pH on the sensor potential: The effect of pH of the test solution in batch measurements, on the potential values of the sensor system in solutions of different concentrations (1.0x10 -3 , 1.0x10 -4 and 1.0x10 -5 M) of the Nf-(COOH) 2 was studied. Aliquots of the drug solution (50 ml) were transferred to 100 ml titration cell and the tested ion-selective sensor in conjunction with the calomel reference sensor, and a combined glass sensor were immersed in the same solution. The mV and pH readings were simultaneously recorded. The pH of the solution was varied over the range of 1.0-10.0 by addition of very small volumes of 2 M HCl and/or (0.1-1.0 M) NaOH solution. The mV-readings were plotted against the pH-values for the different concentrations.
Temperature effect on the sensor: The study of thermal stability of the sensors, calibration graphs were constructed at different test solution-temperatures covering the range 25-50˚C. The slope, the standard sensor potentials (E o ), usable concentration ranges, and response time of the sensors corresponding to each temperature were reported.

Sensors selectivity:
The matched potential method is unique in that it depends neither on the Nicolsky-Eisenman equation nor on any of its modifications. This method was recommended in 1995 by IUPAC as a method that gives analytically relevant practical selectivity coefficient values. To determine the selectivity coefficients of different interfering ions for the studied sensors, a reference solution (a A ) is added an amount of the drug to give a final concentration of (a -A ), the shift in potential change (ΔΕ) is thus measured. To a reference solution containing the same concentration (a A ), a certain amount of interference ion that cause the same (ΔΕ) value is thus determined (A j ). The following equation is used to calculate the selectivity values of pot J Nf, z K log + [22,23]. Where: is the initial concentration of drug, a drug is the activity of the added drug and a j is the activity of the added interfering ion producing the same increase in potential.
In addition, the separate solutions method (SSM) was used. Two values of potential were determined for the same drug concentration and the interferents. The selectivity values of pot J Nf, z K log + are calculated using the following equation [24] Where: E 1 and E 2 are the sensor potentials of 10 -3 M solution of each of the Nf drug and interfering cation, J z+ , respectively and S is the slope of the calibration graph.

Determination by Standard addition method
In bulk powder: The potentiometric determination by the standard additions method was applied, in which a known incremental change is made through the addition of standard solution of the sample. This was achieved by adding known volumes of standard Nf-(COOH) 2 drug solution to 50 ml water containing different amounts of the investigated drug in its pure state. The change in mV reading was recorded for each increment and used to calculate the concentration of the drug in sample solution using the following equation [24]: Where C x is the concentration to be determined, V x is the volume of the original sample solution, V s and C s are the volume and concentration of the standard solution added to the sample to be analyzed, respectively, ΔΕ is the change in potential after addition of certain volume of standard solution, and S is the slope of the calibration graph.
In dosage form: For analysis of tablets of drug, the contents of five coated (Praxilene tablets, 200 mg/tablet) were powdered and an accurately weighed (200-250 mg) portion from each was mixed with 50 ml doubly distilled water, shaken in a mechanical shaker for about 30 min and filtered into a 100 ml volumetric flask, the solution was completed to the mark with doubly distilled water and shaken. Different volumes of the solution (1.0-10 ml) were taken and subjected to the standard additions technique of the potentiometric determination.
In human fluids: In spiked human plasma three of centrifugation 20 ml stoppered shaking tubes containing 0.5 ml Nf-(COOH) 2 drug solution from the concentration 1x10 -3 , 1x10 -4 and 1x10 -5 M and spiked with 4.5 ml plasma separately to each tube. An acetate buffer pH 4 was added to the plasma until adjusted the pH. The modified sensor was immersed in conjunction with the calomel electrode in these solutions and then washed with water between measurements. The emf produced for each solution was measured by the proposed sensor, and the concentration of Nf-(COOH) 2 drug was determined from the corresponding sensor calibration and standard addition methods.
In spiked urine five ml urine were putted to a 100 ml volumetric flask and spiked with different quantities of the concentrations from 1.0x10 -6 to 5.0x10 -4 M drug Nf-(COOH) 2 drug then left stirred for 5 min, completed to the mark with doubly bidistilled water. A small volume of 0.01 M HCl (0.1-2.0 ml) was added to adjust the pH ranging from 4 to 5. This spiked urine was subjected to the standard addition method for drug determination.

Determination by potentiometric titrations
An aliquot of the Nf-(COOH) 2 drug containing different weights ranging from 4.73-47.36 mg was dissolved into 50 ml by bidistilled water, the resulting solutions were titrated against 0.0025 M SMA and 0.0025 M STA using the corresponding sensor(s). The end points were determined from the conventional S-shaped curves by the first and second derivative plots. The same procedure was applied for the tablets.

Content uniformity assay of Praxilene tablets:
A tablet of Praxilene, (200 mg/tablet) was immersed in the measuring flask for measuring each sensor was immediately putted in the sample solution three times and then washed between each individual measurement with distilled water to reach steady potential. The content uniformity was evaluated from the calibration graph by using the mean potential. For the spectrophotometric measurements by employing UV absorbance λ max 283 nm with the standard solution.
Determination of the solubility product constant: About 0.5 g portion of each ion-associate Nf 4 -ST and Nf 4 -SM was added to 50 ml distilled water. The solution were shaken for about 24 h and left to stand for a week to attain a stable equilibrium. Then, each saturated solution was decanted to a dry beaker and the equilibrium concentration of drug ion present was determined potentiometriclly using the corresponding sensors by the standard additions method, and hence the solubilities and the solubility product constants of the ion associates were calculated. The conductivity of these solutions were measured at 25 o C and the specific conductivities (λ o ), corrected for the effect of solvent, were calculated and used to obtain the equivalent conductivities (λ) of these solutions. Straight line plots of λ versus √c were constructed and , λ 0 STA and 0 SMA were determined from the intercept of the respective line with the λ-axis. The activity coefficients of the ions employed were taken as unity because all the solutions were sufficiently dilute. The values of λ 0 (Nf-ST) and λ 0 (Nf-SM) were calculated using Kohlrausch's law of independent migration of ions [25].
The solubility (S) and solubility product (K sp ) of a particular ion associate were calculated using the following equations: S=K s x1000 λ 0 (ion-associate) K sp =256 S 5 for 1:4 ion-associates Where K s is the specific conductivity of a saturated solution of the ion associate, determined at 25 o C and corrected for the effect of solvent. The saturated solution was made by stirring a suspension of the solid precipitate in distilled water for 60 min and then leaving it for 24 h at 25 o C before measuring the conductivities.
Dissolution: One tablet of Praxilene, (200 mg/tablet) was placed in one vessel of tablet dissolution instruments apparatus 2 (paddle method). In vitro release study, the testing liquids, prepared from gastric media permit the dissolution medium pH 1.2 (900 ml of 0.01M HCl) was maintained at 37 ± 0.5˚C for 2 h. The dissolution speed of the naftidrofuryl tablet is measured with gentle agitation (60 rpm), to come close to physiological conditions (coated tablet). At intervals time, the investigated sensor in conjunction with (SCE) reference sensor recorded the potential values and the amount of releasing was calculated from the calibration graph.
For the spectrophotometric measurements, 5.0 ml aliquots of the dissolution solution were withdrawn, filtered, diluted with 0.01M HCl and the concentration of samples was analyzed using UV spectrophotometer (1800, Shimadzu, Japan) and the absorbencies were measured at λ max 283 nm. A calibration graph was used for drug release calculation.

Result and Discussion
The nature of binder and the effect of composition It is well known that, the sensitivity and linearity for a given CMCPs depend significantly on the nature with the amount of the modifier in the paste and the ratios of ion-exchanger(s)/graphite/binder that significantly influence the sensitivity and selectivity of sensors. For this reason, the influence of the binder type and its quantity on the characteristics of the studied sensors was investigated by using five binder with different polarities including TCP, DOP, DBP, TBP and 2-NPPE as shown in Table 1. In addition, five pastes of varying nature and ratios of ion-exchanger(s)/graphite/binder were prepared for the systematic investigation of each paste composition as shown in the same table. As can be seen from the emf-pDs plots Figure 2 the sensor with 2-NPPE as a solvent mediator produced the Nernstian response, likely due to better dielectric characteristics of 2-NPPE comparing to other solvents, and the ability of 2-NPPE to extract naftidrofuryl ions from the aqueous solution to the organic paste phase. Trials are done to improve the performance characteristics of the sensors, as the presence of a mixture. When mixed different percentages of Nf-SM ion-exchanger, to Nf-ST, it is found that, mixed (2.5% Nf-* Five replicates

Reproducibility of the Sensor
The examination of the repeatability for the potential reading of the Nf-ST/CMCP sensor was taken by the subsequent measurements in 1.0x10 -3 M Nf-(COOH) 2 solution then followed by measuring the first set of solution at 1.0x10 -4 M Nf-(COOH) 2 . The standard deviation values for each sensor are given in Table 3 by measuring emf for five replicate measurements. The obtained values indicate the excellent repeatability of the potential response of the sensors. The slope of the calibration graph obtained by the sensors was found to decrease slightly after several times of use, which may be attributed to surface contaminations.

Dynamic response time
The response time of the sensor is defined as the time between addition of the analyte to the sample solution and the time when a limiting potential has been reached. The dynamic response time [26] was obtained for the sensor by recording the time required to achieve a steady-state potential (within ± 1 mV) after subsequence immersions of the sensor in a series of drug solutions, each having a 10-fold increase in concentration from 1.0x10 -7 to 1.0x10 -2 M. The practical response time was examined by following Nf-(COOH) 2 concentration by up to 10-fold. The sensor reached steady potential within 5-7 sec. This is most probably due to the fast exchange kinetics of association-dissociation of naftidrofuryl ion with the ionophores at the solution-paste interface. The potential-time plot for the response of the sensor Nf-CMCPs is shown in Figure 3.

Effect of pH
In analytical determinations with CMCP sensors, studies were carried out to reach the optimum experimental conditions. The potential of the pH profile is one parameter that obtained in the studying Nf-CMCPs and the responses of the sensors are almost constant over the pH range 2.0-6.0. In this range the sensor can be safely used for the determination of the drug. Therefore, there is no need to adjust the pH or use buffer solution, as the drug solution is in the allowable range of the pH and the pH range from 2.0 to 6.0 was assumed to be the working pH range of the sensors. It can be seen from Figure 4 that at pH values lower than the 2.0 pH ranges, the potential readings slightly different which can be related to interference of hydronium ion while at pH values higher than pH 6, the potential readings decrease gradually due to the formation of free base of the drug and decrease of the protonated species in the test solutions.

Effect of temperature
The investigation of the thermal stability of CMCPs is of prime importance in characterization of their behavior. In case if the thermal coefficient is high, more than 1 mV/ o C, then it is important that

Sensors selectivity
In analytical applications, the selectivity for the analyte must be as high as possible, i.e. the selectivity for foreign substances must be very small, so that the sensor exhibits a Nernstian dependence on the primary ion over a wide concentration range. The response of the sensors towards different substances and ionic species such as inorganic/organic cations, amino acids, sugars that may be present in the pharmaceutical preparations was checked both by SSM and or MPM conditions and the values of selectivity coefficients were used to evaluate of their interference. The inorganic cations do not interfere due to their mobility and permeability is difference as compared to nafronyl cation. For amino acids and sugars the high selectivity is related to the difference in polarity and lipophilic nature of their molecules relative to nafronyl cation as shown in Table 5.

Analytical applications
Standard addition method: The analytical applications involve determination of the drug in its bulk powder, pharmaceutical dosage form, and human fluids plasma/urine by the standard addition method [24], which is frequently the most, applied one in using CMCPs. The method was proved successful for the determination of nafronyl ions. The data reflect the high accuracy and precision of the investigated sensors for determination of nafronyl ion and its statistical data treatments in comparison with official methods [27,28].

Potentiometric titration:
The potentiometric titration technique usually offers the advantage of high accuracy and precision. A further advantage is that the potential break at the titration end-point must be well defined. The titration process was carried in pure solution and/ or pharmaceutical dosage form containing from 2.37-47.36 mg Nf-(COOH) 2 . As shown in Figure 5 and Table 6 with its statistical data treatments in comparison with official methods [27,28].

Robustness and ruggedness:
The examination of the robustness method was checked by the replacement of aqueous solution with acetate buffer pH 4 ± 0.5 for the Nf-CMCPSs. All results are in a good agreement with those obtained from standard drug solution as shown in Table 1. In addition, the reproducibility or the ruggedness was examined by using another model type of pH-meter (Jenway, 3510) for each sensor (1, 2 and 3). Table 7 10    Precision: The precision of the Nf-CMCPS method was tested by repeating the proposed CMCPS method for analysis of the investigated nafronyl ions in intra-day (within the day) and inter-day (consecutive days) to five replicates measured as percentage relative standard deviation (% RDS). The percentage RSD values are less than 2%, indicating acceptable and good precision.

Determination of the solubility products of the ionexchangers
It is noteworthy to mention that the solubility of an ion-exchanger is one of the main factors controlling the sensitivity of the ion-selective sensor, which incorporate this ion-exchanger as electroactive material. This is confirmed by the values of solubilites whereas the solubilities have the lowest value the detection limits also have as given in Table   Vol X ± S.E: Recovery ± standard error F-tabulated is 6.39 at 95.0% confidence limit, t-tabulated at 99.0% confidence limit and 6 degrees of freedom (1) British Pharmacopoeia (Non-aqueous titration with 0.1 M perchloric acid) Table 6: Determination of Nf-CMCPs applying the standard addition method in bulk powder, Praxilene (200 mg) and spiked human plasma/urine and its statistical data treatments in comparison with official methods.

Pure solutions
Official ( 8. This is in agreement with what was stated by Pungor and Toth [29]. The reciprocal of the solubility product is approximately equal to the equilibrium constant (K) of the precipitation reaction employed in such titration.
A n-+ n D + ↔ D n A K=1/KSP A nis the counter anion and D + is the drug cation, number of experiments (N) =4.

Potentiometric monitoring of Plavix tablet dissolution
The changes in the electrochemical potential of the dissolution medium caused by the dissolving drug can be detecting by the sensor monitors to the dissolution of the tablet. These changes are converted into the concentration in % of dissolved drug via dedicated ("Potential measured in mV, to Concentration"). The results are compared to subsequent sample analysis using UV. It shows that nafronyl releases immediately after capsule was ruptured. Taking into account that the X ± S.E: Recovery ± standard error F-tabulated is 6.39 at 95.0% confidence limit, t-tabulated at 99.0% confidence limit and 6 degrees of freedom Table 7: Determination of Nf-CMCPs applying the potentiometric titration method in bulk powder and Praxilene (200 mg) with its statistical data treatments in comparison with official methods.

Pure solutions
Official (   releasing was achieved in three stages; the first during 15 min nearly 50% was released, the second more than 74% drug was released within 30 min and complete dissolution in the third stage within 45 min according to USP [19].
For the UV spectrophotometric assay, fixed volumes of the dissolution medium were withdrawn, diluted with 0.01 M HCl, measured at λ max 283 nm and compared with a calibration graph. Figure 6 shows the dissolution profiles of nafronyl tablet using both measurement techniques. The results obtained by spectrophotometric and potentiometry are almost identical but the use of the potentiometric method sensor has the advantage of more sensitive due to overcome the matrix effect.

Comparison of the nafronyl selective sensors
The earlier research described Nafronyl ion-selective membrane electrodes with either the nafronyl-dipicrylamine ion-pair complex in 1,2-dichloroethane or the nafronyl-dinonylnaphthalenesulphonic acid ion-pair complex in a PVC matrix as electroactive materials were used Ref. 9 . Membrane selective electrodes to determine Naftidrofuryl Oxalate (NFL) in presence of its alkaline degradate (I). The membrane selective electrodes include construction of water insoluble ionassociation complexes. These are NFL-tetraphenyl borate (NFL-TPB), NFL-reinikate (NFL-R). These complexes are used as electroactive materials, in poly-vinyl chloride (PVC) matrix membrane sensors, for the determination of NFL Ref. 10 . Sensors for the quantitative determination of NFT in pure form, pharmaceutical formulations, in the presence of its degradation products, in plasma and CSF. The use of sulphonated calix-8-arene as ionophores increased the membrane sensitivity Ref. 11 . But no method was found for the quantitative determination of NFT by chemical modified carbon paste sensor (CMCPs) as the present work.
For comparison, the performance characteristics of the choice sensor and the reported ISE method are showed in Table 9. It is noteworthy that the proposed sensor CMCPS is comparable with most of the reported sensors with regard to working concentration range, response time and low detection limit. The comparisons indicate that the present sensor is high selective than the previous methods in applications.

Conclusion
The preparation and investigation of the performance characteristics of CMCPS for the determination of cardiovascular drug naftidrofuryl ion. The proposed potentiometric methods based on the construction of different selective sensors with ion exchangers in analytical characteristics for the determination of Nf-(COOH) 2 in its pure solutions, pharmaceutical preparation and human fluids. The excellent recoveries and low relative standard deviations obtained reflect the high accuracy and precision of the proposed method. In addition, the method is easy to operate, high sensitivity, simple, fast static response; reasonable selectivity; long term stability and applicability over a wide concentration range with minimal sample pretreatment and inexpensive making it an excellent tool for the routine determination of Nf in quality control laboratories. The sensor developed is superior as compared with the naftidrofuryl selective sensor described in the literature [9][10][11].  Table 9: Comparison between the published and the current methods for determination of Nf-ISS.