Potentiometric Determination of Enrofloxacin Using PVC and Coated Graphite Sensors

Modern ion selective electrodes are based on membranes, across which material transport occurs, this material transport, includes both neutral and charged complex species or simple ions and electrons and leads to electrostatic potential differences across membranes. These so called membrane potentials reflect both composition and activities of ions in the exterior phase. The ion-selective electrode is capable of measuring selectivity and activity of a given ion regardless of other ions present in solution 1-4 .


INTRODUCTION
The ion-selective electrode is defined as an electrode that is capable of generating a difference in electrical potential between itself and a reference electrode, the output potential is proportional to the amount or concentration of the selected ion in the solution. [1][2] Modern ion -selective electrodes are based on membranes, across which material transport occurs, this material transport, includes both neutral and charged complex species or simple ions and electrons and leads to electrostatic potential differences across membranes. These so called membrane potentials reflect both composition and activities of ions in the exterior phase. The ion-selective electrode is capable of measuring selectivity and activity of a given ion regardless of other ions present in solution [1][2][3][4] .
Ion-selective electrodes can be classified according to the type and composition of the responsive membrane into glass electrodes, solid-state electrodes, liquid membrane electrodes, coated wire electrodes, gas sensing electrodes and enzyme substrate electrodes. 5 . Potentiometric detection based on ion selective electrodes offers the advantages of speed and ease of preparation, fast response time, reasonable selectivity, wide linear dynamic range, online measurement and low cost. 6 The most widely used solid membrane electrodes are the plasticized poly (vinyl chloride), (PVC) electrodes which are based on the formation of ion -associates between drugs and counter ions, then the formed ion-associate was used together with PVC and a suitable plasticizer in preparation of the membrane electrode. On the other hand, liquid membranes are formed from immiscible liquids that selectively bond certain ions.
The liquid ion-exchanger may be retained in a porous inert solid support which separates the liquid electrode inner solution from the test solution [7][8][9] .
Coated graphite sensor, in which the membrane is cast onto solid-like graphite, can be used as long as the matrix of the membrane which does not react with the internal wire. In the classical coated graphite design, a conductor is directly coated with an appropriate ion selective polymer membrane usually poly (vinyl chloride), poly (vinyl benzyl chloride) or poly (acrylic acid) to form an electrode system that is sensitive to electrolyte concentrations. The advantage of using coated graphite sensor is that it can be used in small volume of sample, simple design (absence of internal solution), mechanical flexibility and the possibility of miniaturization and micro fabrication. 10 Ion-selective electrodes have been extensively used in the detection of pharmaceutical compounds. [11][12][13][14][15][16][17] Enrofloxacin Figure (1) is 1-Cyclopropyl-7-(4ethyl-1-piperazinyl)-6-fluoro-1, 4-dihydro4oxo-3-quinolinecarboxylic acid. It is pale yellow crystals, slightly sol in water at pH 7. 18

Fig1. Structure formula of Enrofloxacin
Enrofloxacin is second generation fluoroquinolone antibacterial drugs that act by inhibition of deoxyribonucleic acid (DNA) gyrase, thus inhibiting both DNA and ribonucleic acid (RNA) synthesis.
It has activity against some Gram-positive aerobes such as staphylococci, and a wide range of Gram-negative bacilli and cocci, and other organisms such as mycoplasma, and chlamydia. It has been used in the treatment of osteomyelitis, sinus infections, otitis, difficult soft tissue infections, peritonitis, and pleuritis or pneumonia. 19 The literature review revealed that, several analytical methods have been reported for determination of ENRinpure form and pharmaceutical preparation using spectrophotometry [20][21][22][23][24][25][26][27] , spectrofluorimetry 27-32 , electrochemical [33][34][35] , Capillary electrophoresis 36 , andHPLC. [37][38][39][40][41] So the main aim of the present work is to design and prepare two ion selective electrodes, ENR-PVC sensor and ENR-Coated graphite sensor for determination of ENR in bulk powder and pharmaceutical preparation.

Standard Solutions
A stock standard solution of ENR (10 -2 M) was prepared by dissolving 0.359 g of the drug powder in 1mL HCl then add49 mL of water and completed to 100 mL with water. Different working solutions of varying strengths ranging from (10 -6 to 10 -3 M) were prepared by suitable dilution from the stock standard solution with water.

Pharmaceutical Sample Preparation
Content of one vial (100 mL) of Enro-flox 10% ® sterile solution for veterinary injection; labelled to contain 100 mg mL -1 were transferred to 100 mL volumetric flask. A volume equivalent to 0.359gm of ENR were transferred into 100-ml volumetric flask and completed to volume with the water to obtain a solution labelled to contain 10 -2 M of ENR.

Preparation of the Ion Association Complex
The ion association complex, (ENR-TPB) was prepared by mixing of 50 ml of 10 -2 M of both ENR and sodium tetraphenylborate solutions. The resulting precipitate was left in contact with their mother liquor for 6h, then the precipitate was filtered and washed thoroughly with distilled water and left to dry at room temperature for 24h.

Preparation of the Membrane
In a glass petri dish (5-cm diameter), 90 mg of DOP was thoroughly mixed with 90 mg of PVC and 20mg of ENR-TPB. The mixture was dissolved in 7 mL of tetrahydrofuran. The petri dish was then covered with a Whitman No. 3 filter paper and left to stand overnight to allow for solvent evaporation at room temperature. A master membrane with a thickness of 0.1 mm was obtained.

Electrode Assembly
From the master membrane,an 8 mm diameter disk was cut out from the prepared membrane and glued using tetrahydrofuran to a transposable PVC tip that was clipped into the end of the electrode glass part.
The resulting electrode body was filled with equal portions of 10 -2 M KCl and 10 -2 M ENR. The prepared sensor was preconditioned by soaking in 10 -2 M drug solution for 4 h. When not in use, the sensor was stored in air.

Preparation of the Ion Association Complex
The ion association complex, enrofloxacin tetraphenylborate (ENR-TPB) was prepared by mixing of 50 ml of 10 -2 M of both ENR and sodium tetraphenylborate solutions.
The resulting precipitate was left in contact with their mother liquor for 6h, then the precipitate was filtered and washed thoroughly with distilled water and left to dry at room temperature for 24h.

Preparation of the Membrane
In a glass petri dish (5-cm diameter), 90 mg of DOP was thoroughly mixed with 90 mg of PVC and 20mg of ENR-TPB. The mixture was dissolved in 7 ml of tetrahydrofuran and homogenized thoroughly. The solvent was slowly evaporated at room temperature until oily concentrated mixture was obtained.

Electrode Assembly
It was prepared using commercial graphite bar (2.5 cm length an, 3 mm diameter). One end of the bar was used for connection, while the other was dipped in the electro active membrane mixture. The process was repeated several times until a layer of a proper thickness were formed covering the terminal end of graphite bar. The electrode was left standing at room temperature to dry. The uncoated end of the graphite rod was sealed in a poly tetra ethylene tube; the tube was filled with metallic mercury into which a copper wire was dipped. The prepared sensor was preconditioned by soaking in 10 -2 M drug solution for 6 h. When not in use, the sensor was stored in air.
 Potential measurement conditions of the proposed sensors The electrochemical system can be represented as following: The conditioned sensors were immersed in conjunction with Ag/AgCl reference electrode in the solutions of ENR in the range of 10 -6 to 10 -2 M. They were allowed to equilibrate while stirring until achieving constant reading of the potentiometer. Then, the electromotive force values were recorded within ± 1 mV.Calibration graphs were plotted that related the recorded electrode potential values versus the negative logarithmic value of the drug concentration.

RESULTS AND DISCUSSION
Electrochemical techniques are powerful and versatile analytical techniques that offer high sensitivity, accuracy, and precision as well as a large linear dynamic range, with relatively lowcost instrumentation. 2 In the present study two types of ion selective membrane electrodes, PVC membrane and coated graphite sensors have been constructed for determination of ENR. The methods are based on the fact that, ENR behave as a cation with an anionic type of ion exchanger such as tetraphenylborate to prepare water insoluble association complex using precipitation based technique. The resulting precipitates have low solubility product, suitable grain size and physically compatible with the matrix.

Characteristics of the Developed Sensors
The electrochemical performance of the investigated sensors was evaluated according to IUPAC recommendation data. 4 Calibrations were carried out by immersing the developed sensors in conjunction with Ag/AgCl reference electrode in solutions of ENR in the concentration range of 10 -6 to 10 -2 M. The potential displayed by the proposed sensors for constructive measurements of the standard drug solutions in the same day and from day to day did not vary by more than ± 1 mV. Calibration slopes did not change by more than ± 1 mV/decade concentration over a period of 2 weeks. The performance characteristics of the proposed sensors were summarized in Table  (1

).
The profile of the potential in mV versus negative logarithmic molar concentration of ENR for the investigated sensors was plotted.

Effect of Ion Association Complex Percentage
The ion association complex is the most important part of an ion selective sensor. It is the electro active ingredient which is responsible for the selective recognition of the ion in the developed sensor.

ENR-PVC membrane sensor
The main components of a PVC membrane sensor are ion association complex, PVC and plasticizer.
For the preparation of the membrane, the ion association complex, plasticizer and PVC should be taken in the appropriate percentageweight ratios to improve the performance of the developed sensor. ENR-TPB was prepared and tested as a modifier for the proposed sensor. It was studied by varying the percentages of the ion association complex, while keeping the percentages of the PVC and the plasticizer equal 1:1 as shown in Table (2). The sensor made of 10% (w/w) of ENR-TPB exhibited the exhibited a near Nernstian slope of 58.09mV/decade.

ENR-Coated graphite sensor
The ion association complex, ENR-TPB, was prepared and tested as a modifier for the proposed sensor. It was studied by varying the percentages of the ion association complex, while keeping the percentages of the PVC and the plasticizer equal 1:1 as shown in Table (

Effect of Soaking Time
Freshly prepared sensors must be soaked to activate the surface of the membrane to form an infinitesimally thin gel layer at which ion exchange occurs. The investigated sensors were soaked in 10 -2 M solution of the corresponding drug. Calibration graphs were constructed for the sensor after different time intervals (0, 2, 4, 6, 8 and 12 h) till the slope of the calibration graph deviated largely from the Nernstian value and the sensor. The results indicated that the optimum soaking time was 4h for ENR-PVC membrane sensor and 6 h for the ENR-Coated graphite sensor as shown in Table (3).
Fig2. Effect of pH on the response of enrofloxacin using PVC and coated graphite membrane sensors.

Sensors Selectivity
The influence of the related interfering compounds on the response of the investigated sensors towards the drug was investigated. The separate solution method (SSM) was applied based on measuring the potential of 10 -3 M Where E1 and E2 are the electrode potential of 10 -3 M solution of each of investigated drug and interfering ion [J +z ], respectively, and S is the slope of calibration curve. The interfering compounds were; potassium chloride, calcium chloride, magnesium chloride, sodium chloride, nickel chloride, glucose, urea, glycine and sucrose.
The results of the calculated selectivity coefficients indicated that the proposed sensors were highly selective towards the studied drugs as shown in Table (

Response Time of the Proposed Sensors
For analytical applications, the response time of the prepared sensor is of critical importance. The average time required for the electrode to reach a steady potential response within ±1 mV of the final equilibrium value after successive immersion of a series of the drug solutions, each having a 10-fold difference in concentration, was investigated. Stable responses were achieved within 40 s for ENR-PVC membrane sensor and 50 s for ENR-Coated graphite sensor.

Methods Validation 4, 44-45.
Linearity and range: Under the described experimental conditions, the calibration graph for each sensor was constructed by plotting the recorded sensor potential versus negative logarithmic value of the drug concentration. The regression plots were found to be linear over the range of 10 -5 -10 -2 M for both sensors, as shown in Figure (3).

Limit of detection (LOD):
Measured by interception of the extrapolated arms of Figure(3). It was found to be 8.2x10 -6 M for PVC membrane sensor and 8.8x10 -6 M for coated graphite sensor. The small values of LOD indicate good sensitivity of the described sensors. The results are given in Accuracy and Precision: Accuracy of the described methods, calculated as the mean percent recovery (%R), was assessed by applying the described procedure for triplicate determination of three concentration levels covering the linearity range of each drug (10 -2 , 10 -3 and 10 -4 M). The results in Table (1) indicated the accuracy of the proposed method.
Precision of the methods, calculated as the percent of relative standard deviation (%RSD), was assessed by triplicate determination of three concentration levels covering the linearity range of each drug (10 -2 , 10 -3 and 10 -4 M) within one day for repeatability and on three successive days for Intermediate precision. The small values of %RSD indicated high precision of the method as shown in Table (1).

Application of the Method to Pharmaceutical Sample
The proposed method was applied for the selective determination of enrofloxacin in presence of its acid induced degradate in Enro-Flox® 10% sterile solution for veterinary injection. Satisfactory results were obtained in good agreement with the label claim. The obtained results were statistically compared to those obtained by the reported method. 46 No significant differences were found by applying t-test and F-test at 95% confidence level, 47 indicating good accuracy and precision of the proposed methods for the analysis of the studied drugs in its pharmaceutical dosage form, as shown in Table ( The values in parenthesis are tabulated values of "t" and "F" at (P = 0.05).