1. INTRODUCTION:

Ciprofloxacin. HCL (CFX) {1-cyclopropyl- 6-fluoro- 4-oxo-7-(piperazin-1-yl) -1,4 dihydroquinoline-3-carboxylic acid hydrochloride} (Fig. 1) is a synthetic bactericidal from the 2nd generation of fluoroquinolones widely used in the treatment of urinary and respiratory tract infections caused by susceptible organisms. Exerting its bactericidal effect by obstructing the bacterial DNA gyrase, which cause inhibiting of the DNA synthesis and preventing the growth bacterial cell [1,2].

Tinidazole (TZL) {1-(2-ethylsulfonylethyl)-2-methyl-5-nitroimidazole}(Fig. 1) is a nitroimidazole alkylating agent that is used as an antitrichomonal agent against trichomonas vaginalis, entamoeba histolytica, and giardia lamblia infections. It also acts as an antibacterial agent for the treatment of bacterial vaginosis and anaerobic bacterial infections. The nitro- group of tinidazole is reduced by cell extracts of Trichomonas. The free nitro- radical generated as a result of this reduction may be responsible for the antiprotozoal activity. Chemically reduced tinidazole was shown to release nitrites and cause damage to purified bacterial DNA in vitro. Additionally, the drug caused DNA base changes in bacterial cells and DNA strand breakage in mammalian cells. The mechanism by which tinidazole exhibits activity against Giardia and Entamoeba species is not known yet [2,3].

Fig. 1: Chemical Structure of Ciprofloxacin.HCL and Tinidazole

The determination of CFX has been made by various analytical methods including HPLC and RP-HPLC [4], UV spectrophotometry [5,6,7,8], Derivative UV-Spectrophotometric [9,10], Spectroflourimetric [11], Rayleigh light scattering [12], Electrical Micro-Titration [13], Capillary Zone Electrophoresis [14], potentiometry [15,16]. While TZL determination has been made by various analytical methods including RP-HPLC [17], Spectrophotometric [18,19], polarographic method [20], Electrochemical Determination [21]. Some analytical methods were state for the simultaneous determination of Ciprofloxacin HCL and Tinidazole including RP-HPLC [22], Differential Pulse Polarography [23], to the best of our knowledge no simple method such as Potentiometric method using ion-selective electrodes (ISEs) was reveal to analyse both CFX and TZL simultaneously in there bulk solutions and pharmaceutical formulations.

In previous works we found ISEs which uses PVC as a matrix or a trap for ion pair useful for the determination of a single drug providing fast result, simple analysis procedures, and over that offering high selectivity towards the drug in the presence of various pharmaceutical excipients [24,25,26,27] also we succeed inventing a selective electrode for the Simultaneous determination of two drugs ciprofloxacine and metronidazole by using a PVC membrane containing two ion pairs [16].

In this work we are proposing a new drug selective polystyrene membrane for the Simultaneous potentiometric Determination of Ciprofloxacin and Tinidazole in their Pure Form solutions and Pharmaceutical Formulations.

2. MATERIALS AND METHODS:

2.1 Apparatus:

The electrochemical measurements were taken with two IONcheck 10 pH/mV meter- Radiometer analytical S.A., France, with CFX-phosphomolybdic acid (PMA), TZL- phosphomolybdic acid (PMA), or CFX-PMA+TZL-PMA –polystyrene (PST)– di-n-butyl phthalate (DBP) plasticizer membrane electrodes in conjunction with Ag/AgCl wire as an external reference electrode. Crison-GLP 21/EU pH-meter for pH adjustment. All potentiometric measurements made at room temperature 25±1°C with steadfast stirring using hot-plate magnetic stirrer MS 300 Bante, China. All weights were taken by analytical balance (BP 221S Sartorius, Germany) with accuracy ±0.1mg. Conductivity meter (inoLab-cond 720, Germany) was used for bi-distill water quality. Oven (WTB binder-78532 Tuttlingen, Germany).

2.2 Reagents and Materials:

Ciprofloxacin (CFX•HCl) 99.0% (Sigma-Aldrich), Tinidazole (TZL) 99.0% (Sigma-Aldrich), high molecular weight Polystyrene (PST) (Sigma-Aldrich), phosphomolybdic Acid (PMA) 99% (Sigma-Aldrich), d di-n-butyl phthalate (DBP) 99.0%, tetrahydrofuran (THF) 97.0%, hydrochloric acid, sodium hydroxide, potassium chloride (guarantee reagent grade, Merck, Germany) were used. Bi-distilled water (conductivity≤10 µS/cm), silver wire (Φ=1 mm, Swiss, 99.99%) was used.

2.3 Standard Drug Solutions:

Stock standard solutions (0.01 M) CFX•HCl (Mw=367.805 g.mol^-1), (0.01M) TZL (Mw=247.269 g.mol^-1) were prepared by dissolving accurate weight in 1 M KCL, this solutions were stable for several weeks if kept in the dark at 4°C. Working solutions ranging 0.1-10000µM were prepared by serial dilution of the previous stock solutions with 1 M KCL. These solutions are stable for 1 week if stored in a cool and dark place. Britton-Robinson universal buffers 0.2M were used [28,29].

3. Ion Selective Electrodes:

3.1 Preparation of Ion Pairs:

The ion pairs (IPs) were prepared by mixing equal volumes of 10mM CFX solution with 20mM phosphomolybdic acid (PMA) solution to form the first ion pair (CFX-PMA, IP-1), and equal volumes of 10mM TZL solution with 20mM phosphomolybdic acid (PMA) solution to form the second ion pair (TZL-PMA, IP-2). Each mixture was stirred for about 30 min, left in the dark for over-night to settling down. The resulting precipitates were filtered, washed with bi-distilled water several times until the conductivity of the washed water is close to the conductivity of the used bi-distilled water. After that, the precipitate was dried at room temperature over the night away from light and dust. Ion pairs were grounded into a fine powder with an agate mortar, then dried in the oven at 60°C until the weight was stable.

Ion pairs were stored in will-closed dark glass bottles at 4°C. The molecular ratios of the complexes were found to be 1:1 for CFX-PMA (IP-1), 1:1 for TZL-PTA (IP-2).

3.2 Casting of Ion selective Membranes:

The membranes were prepared by dissolving equal weights of matrix PST and the plasticizer (DBP), and the suitable weight of the ion pair (IP-1, IP-2 or IP-1+IP-2) to have the target composition of ion selective membrane. The mixture was dissolved by minimum volume of THF. The resulting solution was poured into a 9cm glass Petri dish and covered with a filter paper, avoided from air movement, dust and direct sunshine. The solvent was allowed to evaporate slowly at room temperature, leaving the casted ion selective membrane that represents the electro-active part of ion selective electrode (ISE). Membranes were stored between two aluminum foils, in will-closed container at 4°C.

3.3 Construction of Ion Selective Electrode (ISE):

Circular cut from casted membrane was glued to a polished polyethylene tube. The result bucket was attached to the end of a suitable glass tube. This body of the ISE was filled with internal reference solution consisting of 1 mM of CFX or 1 mM of TZL in 1M potassium chloride (KCl) solution. Ag/AgCl wire electrode (lab. assembly) was used as an internal reference electrode [30,31]. The indicator electrode conditioned by soaking it in a 1 mM aqueous CFX or TZL solution for 30 min.

3.4 Assembling of Ion Selective Electrode Cell:

Each cell was assembled by attaching the above ISE in conjunction with Ag/AgCl wire as an external reference electrode. The circuit was closed by attaching the cell and the outer reference electrode to temp./pH/mV-meter. And so we accomplished The following electro-chemical cells [32]:

(Sensor-1) SE_CFX-PMA:

Ag/AgCl-KCl (1M) + CFX (1mM) || CFX–PMA–DBP–PST membrane || Test solution-KCl (1M) || Ag/AgCl

(Sensor-2) SE_TZL-PMA:

Ag/AgCl-KCl (1M) + TZL (1mM) || TZL–PMA–DBP–PST membrane || Test solution-KCl (1M) || Ag/AgCl

(Sensor-3 '' Combined Sensor '') SE_CFX+TZL-PMA:

Ag/AgCl-KCl (1M) + CFX or TZL (1mM) || CFX–PMA + TZL-PMA–DBP–PST membrane || Test solution-KCl (1M) || Ag/AgCl

3.5 Electrodes Calibration:

A sample of standard solutions 0.1-10000 µM of CFX, TZL in 1 M KCL were transferred into a fit compartment held in fixed temperature chamber, and the cell assembled from membrane electrode in conjunction with Ag/AgCl reference electrode was immersed in the test solution. The measured potential was plotted against the minus logarithm of drug concentration (pC_CFX, pC_TZL). Between measurements the electrode was washed with bi-distilled water and wiped with tissue paper.

3.6 Standard Addition Method:

Each electrode was immersed into a sample of 50mL with unknown concentration and the equilibrium potential (E₁) was recorded. Then 0.1mL of standard drug solution 0.1M was added into the testing solution and the potential (E₂) was recorded. The concentration of the testing sample was calculated from the change of potential ΔE=E₂-E₁.

pot

pot

3.7 Electrodes Selectivity:

Selectivity coefficients K_CFX,B, K_TZL,B of the sensors towards different inorganic ions and some Excipient were determined according to IUPAC guidelines using the mixed solution method (MSM) [33,34]. The selectivity coefficient by mixed solution method was defined as the activity ratio of primary and interfering ions that give the same potential change under the same conditions, and the following equations applied:

K_CFX,B=(a'_CFX–a_CFX)/a_B

K_TZL,B=(a'_TZL–a_TZL)/a_B

At first, a known activity (a'_CFX), (a'_TZL) of the primary ion solution is added into a reference solution that contains a fixed activity (a_CFX), (a_TZL) of primary ions, and the corresponding potential change (ΔE) is recorded. Next, a solution of an interfering ion is added to the reference solution until the same potential change (ΔE) is recorded [35].

3.8 Effect of pH:

The effect of pH on the potential response of the prepared electrodes was studied using 0.01 and 0.001 M CFX, TZL solutions. The pH of these solutions was adjusted between 1.0-8.0 using suitable amounts of HCl or KOH solution. The potential readings corresponding to different pH values were recorded and plotted using the proposed electrodes. On other hand, the study was repeated using 0.005 M Britton-Robinson universal buffers.

3.9 Determination of CFX, TZL in Pharmaceutical Dosage Forms

The following formulations were used for the analysis of CFX, TZL and CFX+TZL combination by direct potentiometric determinations:

Ciproflex (Tablets, ALPHA pharmaceutical, Syria): Each tablet contain 500mg of CFX.

Fasigyn* (Tablets, Universal pharmaceutical Industries "UNIPHARMA", Syria. Under license from PFIZER inc. USA): Each tablet contain 500mg of TZL.

Ciplox-TZ (Tablets, CIPLA LTD, India): Each tablet contain 500mg of CFX and 600mg of TZL.

Ten tablets were weighed and grounded into a fine powder. A quantity equivalent to one tablet was weighed and dissolved in 50mL KCL (1M) with shaking for 5 min., transferred to 100mL volumetric flask and diluted to the mark with KCL (1M), 10mL of the solution was transferred to 100mL volumetric flask and diluted to the mark with KCL (1M). Each of the final solutions was analyzed as described under electrode calibration and standard addition methods. The results obtained were compared to those obtained from HPLC [36].

3.10 Effect of Ion Pair Percentage on Electrode Potential”

Three groups of electrodes containing 4-10% IP were constructed. The potentiometric response characteristics of the CFX, TZL and CFX+TZL sensors based on the use of CFX-PMA (IP-1) "group 1", TZL-PMA (IP-2) "group 2", or CFX-PMA+TZL-PMA (IP-1+IP-2) "group 3" ion pairs in plasticized PST matrixes were evaluated according to IUPAC recommendations [37]. The graphs plotted for relation:

E (mV) = f (pC_CFX)

E (mV) = f (pC_TZL)

4. RESULTS AND DISCUSSIONS:

4.1 Calibration Graph and Effect of Ion Pair Percentage on Electrode Potential

The linear part of the calibration graph was taken as the analytical range of the potentiometric sensor (quantitative part) and found to be 10-10000 µM. Where the total measuring range (TMR) which can be considered as qualitative part and including the linear part of the graph plus the lower curved part of the calibration graph. TMR was 5.62-17783 µM (Fig 2). In TMR the response of the electrode to changing concentration becomes gradually less as the concentration decreases. In order to measure Samples in this lower range we need to put in mind that more closely-spaced calibration points are needed to define the curve accurately, error % per mV will be increasingly higher as the slope reduces on the calculated concentration.

We found that increasing IP percentage in the membrane of the selective electrode increasing the response of the electrode and the stability of potentiometric readings besides increasing the slope of the liner area for equation curve E = f(Pc_Drug) reaching -59.76 mV.decade^-1 at 7% CFX-PMA (sensor-1), -59.01 mV.decade^-1 at 7% TZL-PMA (sensor-2), and -59.28 mV.decade^-1 for CFX, -59.29 mV.decade^-1 for TZL (IP-1+IP-2) in the combined sensor (sensor-3). At percentages of ion pair higher than 7% a decrease in the electrode response, range and slope of the liner area was resulted due to the kinetic of the ion pair inside the membrane (Fig. 3). Table 1 summarize the least squares equations data.

Table 1: The least squares equations data obtained from the liner equation

	Sensor-1 CFX-PMA					Sensor-2 TZL-PMA					(Combined Sensor) Sensor-3 CFX TZL
IP %	5	6	7	8	9	5	6	7	8	9	6	7
S, mV.decade^-1	-48.58	-54.60	-59.76	-56.53	-53.30	-48.27	-54.67	-59.01	-45.80	-33.671	-59.28	-59.29
b, mV.decade^-1	241.18	261.45	283.91	269.73	253.15	641.77	653.07	673.96	588.55	534.43	281.93	674.84
*R^{2 }**	0.9953	0.9989	0.9998	0.9968	0.9991	0.9984	0.9939	0.9991	0.9957	0.9763	0.9997	0.9991

*Correlation coefficient

Fig. 2: Effect of IP content on CFX, TZL calibration curves

Fig. 3: Effect of IP percentage in the ion selective membrane on the slope of the liner area for equation curve: E = f(Pc_Drug)

Pot

pot

4.2 Electrodes Selectivity

Pot

The acquire selectivity coefficients K_CFX,B, K_TZL,B of the sensors regarding different inorganic interruptings, some Excipients, and the other drug for each electrode are given in Table 2. The result shows a credible selectivity for CFX and TZL in the presence of many related interferences.

Comparing selectivity coefficientts K_CFX,B for both the presently used polystyrene and PVC membrane in our previous work [16] we found better selectivity for strongly interfering lipophilic anions, in our case chloride versus Ciprofloxacin, and that can be attributed to the structure of the polystyrene membrane and the chemical properties of the polystyrene matrix which prefer the kinetic discrimination process in comparison to PVC membrane [38].

Table 2: Selectivity coefficient of some interfering ions by suggested ISEs

Interfering, B	K_Drug,B
Interfering, B	Sensor-1 CFX-PMA	Sensor-2 TZL-PMA	Sensor-3 CFX TZL
Sodium chloride	4.3×10⁻³	5.3×10⁻³	4.4×10⁻³	5.5×10⁻³
Potassium chloride	2.1×10⁻³	3.1×10⁻³	2.1×10⁻³	3.2×10⁻³
Calcium chloride	3.5×10⁻³	5.5×10⁻³	3.6×10⁻³	5.4×10⁻³
Magnesium chloride	2.2×10⁻³	4.7×10⁻³	2.1×10⁻³	5.9×10⁻³
Magnesium stearate	4.7×10⁻³	4.5×10⁻³	4.8×10⁻³	4.9×10⁻³
Microcrystalline Cellulose	3.3×10⁻³	3.4×10⁻³	3.4×10⁻³	3.4×10⁻³
Glucose	3.3×10⁻³	2.6×10⁻³	3.1×10⁻³	3.2×10⁻³
Starch	3.4×10⁻³	3.6×10⁻³	3.5×10⁻³	3.2×10⁻³
Lactose monohydrate	2.7×10⁻³	2.3×10⁻³	2.5×10⁻³	2.6×10⁻³
Ciprofloxacin. HCL	----	1.5×10⁻⁴	----	1.6×10⁻⁴
Tinidazole	1.1×10⁻⁴	----	1.2×10⁻⁴	----

4.3 Effect of pH on response

We found that the potential remained constant in spite of the pH change in the ranges of 2.0-6.0 for CFX-PMA sensor, and 2.0-5.0 for TZL-PMA sensor, which suggest the applicability of the developed electrodes in the pH described ranges. When Britton-Robinson universal buffer was used a fixed potential was obtained in the ranges of 2.0-6.5 for CFX-PMA sensor, and 2.0-6.0 for TZL-PMA sensor (Fig. 4).

Fig. 4: Effect of pH on the potential response of the CFX and TZL sensors using 10 mM, 1 mM drug solution, or 5 mM Britton-Robinson universal buffer solution.

The potential measured with the electrode declined at pH below 2.0 as a result of the emigration of H⁺ ions out of membrane. The potential also declined at pH values higher than 6.5 caused by the progressive increase in the concentration of the non-protonated drugs in the solutions, or due to the effect mobility of the ion pair inside the ion selective membrane [39,40].

4.4 Lifetime Study:

We estimated the lifetime of the electrodes from the calibration curves, for that daily-periodical tests of standard CFX and TZL solutions (1–10000 µM) were made and its response slopes were calculated. The calibration graphs were plotted after optimum soaking time of 6 hours in 1mM CFX or TZL solution. The slopes of the calibration curves were -59.76 mV.decade^-1 for CFX-PMA (sensor-1), -59.01 mV.decade^-1 for TZL-PMA (sensor-2), and -59.28 mV.decade^-1 for CFX-PMA, -59.29 mV.decade^-1 for TZL-PMA (sensor-3) at 25°C. The electrodes were continuously soaked in 1mM solution of CFX or TZL for 20 days. The calibration plot slopes declined delicately from day to day reaching -53.78 mV.decade^-1 for CFX-PMA (sensor-1), -53.11 mV.decade^-1 for TZL-PMA (sensor-2), and -53.35 mV.decade^-1 for CFX-PMA, -53.36 mV.decade^-1 for TZL-PMA (sensor-3) after 17 days for CFX-PMA and 16 days for TZL-PMA sensors, so the lifetime for the combined sensor (sensor-3) is limited to 16 days. This demonstrate that soaking sensors in the drug solution for a long time has a destructive effect on the response of membrane. The same effect appears after working with the sensors for a long time.

4.5 Response characteristics and Statistical Data:

The characteristics performance of the three suggested electrodes was determined and the results were summed up in Table 3. The three suggested sensors show near Nernestian response over the concentration range 10-10000 µM (pC_Drug = 2-5).

Table 3: Response characteristics of CFX, TZL, or CFX+TZL sensors^a.

Parameter	CFX-PMA	TZL-PMA	(Combined Sensor) Sensor-3 CFX TZL
IP%	7%	7%	7%	7%
Slope, mV.decade^-1	-59.76 ± 0.12	-59.01 ± 0.16	-59.28 ± 0.11	-59.29 ± 0.22
Intercept, mV.decade^-1	283.91	673.96	281.93	674.84
Correlation coefficient (R²)	0.9998	0.9991	0.9997	0.9991
Linear range, µM	10-10000	10-10000	10-10000	10-10000
TMR, µM	5.62-17783	5.62-17783	5.62-17783	5.62-17783
LOD, µM	0.072	0.154	0.072	0.154
LOQ, µM	0.218	0.465	0.218	0.465
Response time for 1 mM, sec	11 ± 1	13 ± 1	11 ± 1	13 ± 2
Life time, day	17	16	16 (the shorter life time)
Working pH range	2.0-6.0^* 2.0-6.5^**	2.0-5.0^* 2.0-6.0^**	2.0-6.0^* 2.0-6.5^**	2.0-5.0^* 2.0-6.0^**

^a Five replicate measurement.

* Without buffer.

** Using Britton-Robinson universal buffer

4.6 Quantification of CFX, TZL:

The examined sensors were useful in the potentiometric determination of CFX and TZL in pure solutions by calibration graph and standard addition method as well as in direct determinations of CFX and TZL in both pure solutions (Table 4) and pharmaceutical preparations (Table 5). The results obtained for pharmaceutical preparations were compared with a reference HPLC method [22]; the X̄ ± SD (R%) values were 502.8±1.48 mg (100.56%), 500.8±1.30 mg (100.16%) for Ciproflex and Ciplox-TZ respectively using sensor-1 (CFX-sensor), 504.8±0.84 mg (100.96%), 600.2±1.30 mg (101.03%) for Fasigyn and Ciplox-TZ respectively using sensor-2 (TZL-sensor). while the X̄ ± SD (R%) values using the sensor-3 (combined sensor) were as the follow: 503.0±1.22 mg (100.60%) for CFX in Ciproflex, 504.0±1.64 mg (100.84%) for TZL in Fasigyn, 502.2±2.39 mg (100.44%), 601.6±1.34 mg (100.27%) for CFX, TZL respectively in Ciplox-TZ.

Statistical analysis of the results obtained by the proposed and comparison methods using Student’s t-test and variance ratio F-test, showed no significant difference between the developed methods and the reference HPLC method regarding accuracy and precision, respectively [41].

5. METHOD VALIDATION:

5.1 The linearity, LOD, and LOQ

We measured CFX and TZL standard solutions of 0.1-10000 µM (Pc_Drug=1-6) using the three suggested ISEs in conjunction with Ag/AgCl reference electrode. Each of the different concentration of standard solution was tested five times. The obtained potentials of the five analyses were averaged at each concentration. The average potential was plotted versus Pc_CFXor Pc_TZL according to the straight-line equation: E = S×Pc_CFX + b, or E = S×Pc_TZL + b . The three suggested sensors exhibited a linear response all over the concentration range 10-10000 µM over a pH range of 2.0-6.0 for CFX determination and a pH range of 2.0-5.0 for TZL determination. The limit of detection (LOD) and the limit of quantification (LOQ) were determined according to the IUPAC recommendation [34]. LOD and LOQ values were 0.0720 µM, 0.2182 µM, respectively for ciprofloxacin in both sensor-1 (CFX-PMA) and sensor-3 (combined sensor), 0.1536 µM, 0.4654 µM, respectively for Tinidazole in sensor-2 (TZL-PMA) and sensor-3 (combined sensor). The results are listed in Table 3.

Table 4: Direct determinations of CFX and TZL in bulk solutions using proposed sensors

Taken C_CFX•HCl		Sensor-1*			Taken C_TZL		Sensor-2*
(μg/mL)	mol/L	R%	SD	RSD%	(μg/mL)	mol/L	R%	SD	RSD%
0.3678	1×10^-6	99.78	0.0021	0.58	0.2473	1×10^-6	99.59	0.0016	0.64
3.678	1×10^-5	100.05	0.0187	0.51	2.473	1×10^-5	100.16	0.0055	0.22
36.78	1×10^-4	99.78	0.1000	0.27	24.73	1×10^-4	100.08	0.0837	0.34
367.8	1×10^-3	99.97	0.1000	0.03	247.3	1×10^-3	100.02	0.1140	0.05
3678	1×10^-2	99.98	1.7889	0.05	2473	1×10^-2	99.99	1.3038	0.05

Taken C_CFX•HCl		Sensor-3*			Taken C_TZL		Sensor-3*
(μg/mL)	mol/L	R%	SD	RSD%	(μg/mL)	mol/L	R%	SD	RSD%
0.3678	1×10^-6	99.97	0.0004	0.09	0.2473	1×10^-6	99.97	0.0002	0.09
3.678	1×10^-5	99.70	0.0302	0.82	2.473	1×10^-5	99.90	0.0047	0.19
36.78	1×10^-4	99.11	0.3261	0.89	24.73	1×10^-4	99.94	0.0152	0.06
367.8	1×10^-3	99.93	0.4037	0.11	247.3	1×10^-3	99.97	0.0837	0.03
3678	1×10^-2	99.95	1.9235	0.05	2473	1×10^-2	99.98	1.3416	0.05

*Average of five replicates.

Table 5: Determinations of CFX, and TZL in pharmaceutical preparations using proposed combined sensor

Commercial Name	Composition	X̄ ± SD, mg^a	R%	t-value^b	F-value^c
		Sensor-1 CFX-PMA
Ciproflex	Ciprofloxacin	502.8 ± 1.48	100.56	1.2061	1.6923
Fasigyn	Tinidazole	----	----	----	----
Ciplox-TZ	Ciprofloxacin	500.8 ± 1.30	100.16	1.0289	2.1250
Ciplox-TZ	Tinidazole	----	----	----	----
		Sensor-2 TZL-PMA
Ciproflex	Ciprofloxacin	----	----	----	----
Fasigyn	Tinidazole	504.8 ± 0.84	100.96	2.1381	1.4000
Ciplox-TZ	Ciprofloxacin	----	----	----	----
Ciplox-TZ	Tinidazole	600.2 ± 1.30	101.03	0.3430	2.1250
		Sensor-3 CFX-PMA + TZL-PMA
Ciproflex	Ciprofloxacin	503.0 ± 1.22	100.60	1.0955	1.1538
Fasigyn	Tinidazole	504.0 ± 1.64	100.84	0.5443	3.8571
Ciplox-TZ	Ciprofloxacin	502.2 ± 2.39	100.44	0.3746	4.3846
Ciplox-TZ	Tinidazole	601.6 ± 1.34	100.27	0.6667	3.6000

^a Average of five replicates.

^b Tabulated t-value at 95% confidence level is 2.776.

^c Tabulated F-value at 95% confidence level is 6.39.

5.2 Recovery and Precision:

We calculate the recovery by comparing the potential of the found CFX or TZL concentration to direct added standard in Britton-Robinson universal buffer (pH=2-6). Precision reported as RSD %. Its values of inter-a-day (three replicates) and inter-day (three different days) studies for the repeated determination were less than 2% which indicating good precision (Table 4).

6. CONCLUSION:

We concluded that CFX-PMA-PST, TZL-PMA-PST, CFX-PMA+TZL-PMA-PST membrane ion selective sensors offers a precious technique for direct determination of CFX and TZL in pharmaceutical preparations as well as in pure form solutions. The construction of sensors is something simple, fast, and can be remade. The sensors show an excellent selectivity towards the drug in presence of various pharmaceutical excipients, and it can be used as indicator electrodes in potentiometric titrations of CFX and TZL. Also using the polystyrene membrane awarded better selectivity for strongly interfering lipophilic anions comparing to PVC membrane.

Two electro-active ion pairs of CFX and TZL with PMA were performed as three sensors for the determination of CFX and TZL. The three polystyrene membrane sensors showed good analytical performance. The sensors exhibit a rapid, steady, and near Nernestian response over a relative wide drug concentration range of 10-10000µM (pC_Drug=2-5).

Using CFX-PMA + TZL-PMA as a combined electro-active materials in the same polystyrene membrane we arrange to fabricate a novel electrode that is sensitive to either CFX or TZL according to the standard filling solution of the electrode, and in this way; when we use two of this combined electrodes (one filled with CFX standard solution "ciprofloxacin selective electrode" and the other filled with TZL standard solution "Tinidazole selective electrode" each in conjunction with Ag/AgCl external reference electrode) connected to two separate mV-meters, we can take two readings for CFX and TZL simultaneously and in this way we could determine the two drugs (CFX and TZL) in their combined solutions.

The suggested sensors accomplished LOD and LOQ values of 0.072µM, 0.218µM, respectively for ciprofloxacin in sensor-1 (CFX-PMA) and sensor-3 (combined sensor), 0.154µM, 0.465µM, respectively for Tinidazole in sensor-2 (TZL-PMA) and sensor-3 (combined sensor), with response time of 11±1 sec, 13±1 sec, 11±1 sec (CFX) or 13±2 sec (TZL) for sensor-1, sensor-2, sensor-3 respectively. The suggested sensors have a measurement pH ranges 2.0-6.0 for CFX, and 2.0-5.0 for TZL without using any buffer.

The direct determination of CFX and TZL showed an average recovery of 99.97%, 100.02% and 99.93% (CFX) or 99.97% (TZL) and a mean relative standard deviation of 0.03%, 0.05% and 0.11% (CFX) or 0.03% (TZL)% at 1mM (367.8μg/mL CFX, or 247.3μg/mL TZL) for sensor-1, sensor-2, sensor-3, respectively. The acquire results were within the acceptance range of less than 2.0% of RSD% for precision and more than 99.11 % of R % for the accuracy. The sensors were used as indicator electrodes for direct determination of CFX and TZL in there pharmaceutical preparations as well as in pure form solutions.

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Received on 14.11.2019 Modified on 04.01.2020

Research J. Pharm. and Tech. 2020; 13(12):5963-5971.

DOI: 10.5958/0974-360X.2020.01041.0