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 2^nd generation fluoroquinolone that is widely used in the therapy of mild-to-moderate urinary and respiratory tract infections caused by susceptible organisms.

Ciprofloxacin exerts its bactericidal effect by interfering with the bacterial DNA gyrase, thereby inhibiting the DNA synthesis and preventing bacterial cell growth^1,2.

Metronidazole (MZL) {2-(2-methyl-5-nitroimidazol-1-yl)ethanol} (Fig.1) is a synthetic nitroimidazole derivative with antiprotozoal and antibacterial activities used For the treatment of anaerobic infections and mixed infections, surgical prophylaxis requiring anaerobic coverage, Clostridium difficile-associated diarrhea and colitis, Helicobacter pylori infection and duodenal ulcer disease, bacterial vaginosis, Giardia lamblia gastro-enteritis, amebiasis caused by Entamoeba histolytica, and Trichomonas infections. Reduced form of metronidazole causes DNA strand breaks, thereby inhibiting DNA synthesis and bacterial cell growth ^2,3.

Fig.1: Chemical Structure of Ciprofloxacin. HCl and Metronidazole

The determination of CFX has been made by various analytical methods including HPLC and RP-HPLC⁴, UV spectrophotometry⁵, Derivative UV-Spectrophotometric⁶, Spectroflourimetric^7,8, Rayleigh light scattering⁹, Electrical Micro-Titration¹⁰, Capillary Zone Electrophoresis¹¹, potentiometry¹². While MZL determination has been made by various analytical methods including HPLC¹³, Spectrophotometric^14,15, cyclic voltammetric (CV) and differential pulse voltammetric (DPV)^16-18, Electrochemical Determination¹⁹. Some analytical methods were state for the simultaneous determination of Ciprofloxacin HCL and Metronidazole including HPLC and UPLC ⁴,²⁰ HPLC and TLC-Densitometric²¹, Spectrophotometric²², but no simple method such as Potentiometric method using ion-selective electrodes (ISEs) was reveal to analyse both CFX and MZL simultaneously in there bulk solutions and pharmaceutical formulations.

Since we found ISEs which uses PVC as a matrix or a trap for one 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^23-26, we consider putting two IP_s in the same membrane to achieve a selective electrode for the determination of either CFX or MZL according to the electrode filling solution. For that we arrange to make two ISE_s with the same membrane containing CFX-PTA (IP-1)+ MZL-PTA (IP-2) for each of them and differ in the filling solution, and according to this filling solution the electrode was selective for either of the two drugs CFX or MZL.

2. MATERIALS AND METHODS:

2.1 Apparatus:

All electrochemical measurements made with two IONcheck 10 pH/mV meter-Radiometer analytical S.A., France, with CFX-phosphotungstic Acid (PTA), MZL-phosphotungstic Acid (PTA), or CFX-PTA+MZL-PTA – poly(vinyl chloride) (PVC) – 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 was used for pH adjustment for all pH measurements. All potentiometric measurements made at 25±1°C with constant 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), Metronidazole (MZL) 99.0% (Sigma-Aldrich), high molecular weight poly vinyl chloride (PVC) (SABC, KSA), phosphotungstic Acid (PTA) 99% (BDH Laboratory, England), 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%) were used.

2.3 Standard Drug Solutions:

Stock standard solutions (0.01 M) CFX•HCl (Mw=367.805 g.mol^-1), (0.01 M) MZL (Mw=171.156 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 0.1-10000 µM 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 0.2 M were used²⁷.

3. ION SELECTIVE ELECTRODES:

3.1 Preparation of Ion Pairs:

The ion-pairs were prepared by mixing equal volumes of 10 mM CFX, 10 mM MZL solutions with 20 mM phosphotungstic Acid (PTA) to form the two ion pairs (CFX-PTA, IP-1), (MZL-PTA, IP-2) respectively. Each mixture was stirred for 30 min and 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-night away from light and dust. Using an agate mortar ion pairs were grounded into a fine powder, then dried in the oven at 60°C until the weight was stable.

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

3.2 Casting of Ion Selective Membranes:

The membranes were prepared by dissolving equal weights of matrix PVC 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 9 cm 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 MZL in 1M potassium chloride (KCl) solution. Ag/AgCl wire electrode (lab. assembly) was used as an internal reference electrode²⁸. The indicator electrode conditioned by soaking it in a 1 mM aqueous CFX or MZL solution for 30 min.

3.4 Assembling of Ion Selective Electrode Cell:

The cell assembled by attaching the above ISE in conjunction with Ag/AgCl wire as external reference electrode. The circuit closed by attaching the cell and outer reference electrode to temp./pH/mV-meter. The following electro-chemical cells were accomplished²⁹:

SE_CFX-PTA: Ag/AgCl-KCl (1M) + CFX (1mM) || CFX–PTA–DBP–PVC membrane || Test solution-KCl (1M) || Ag/AgCl

SE_MZL-PTA: Ag/AgCl-KCl (1M) + MZL (1mM) || MZL–PTA–DBP–PVC membrane || Test solution-KCl (1M) || Ag/AgCl

SE_CFX+MZL-PTA: Ag/AgCl-KCl (1M) + CFX or MZL (1mM) || CFX–PTA + MZL-PTA–DBP–PVC membrane || Test solution-KCl (1M) || Ag/AgCl

3.5 Electrodes Calibration:

A suitable sample of 0.1-10000 µM standard solutions of CFX, MZL in 1 M KCL were transferred into a fit compartment held in stable temperature jacket, and the membrane electrode in conjunction with Ag/AgCl reference electrode was immersed in the test solution. All potentiometric measurements were performed using the cells assembly mentioned above. The measured potential was plotted against the minus logarithm of drug concentration (pC_CFX, pC_MZL). The electrode was washed with bi-distilled water and wiped with tissue paper between measurements.

3.6 Standard Addition Method:

The electrode was immersed into sample of 50 mL with unknown concentration and the equilibrium potential E₁ was recorded. Then 0.1 mL of 0.1 M of standard drug solution 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.

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3.7 Electrodes Selectivity:

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Selectivity coefficients K_CFX,B, K_MZL,B of the sensors towards different inorganic cations and some pharmacologically related compounds were determined according to IUPAC guidelines using the mixed solution method (MSM)^30,31. 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 identical conditions, and the following equations applied:

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K_CFX,B=(a'_CFX–a_CFX)/a_B

K_MZL,B=(a'_MZL–a_MZL)/a_B

At first, a known activity (a'_CFX), (a'_MZL) of the primary ion solution is added into a reference solution that contains a fixed activity (a_CFX), (a_MZL) 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) recorded again³².

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, MZL solutions. The pH of this solutions was adjusted between 1.0-8.0 using suitable amounts of 0.1 M KOH or HCl 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, MZL in Pharmaceutical Dosage Forms

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

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

Flagyl (tablets, OUBARI pharma, Syria): Each tablet contain 500 mg of MZL.

Ciprodiazole (tablets, MINAPHARM pharmaceutical, Egypt): Each tablet contain 500 mg of CFX and 500 mg of MZL.

Avilox M (Suspension, AVALANCHE Pharmaceuticals, India): Each 5 mL contain 125 mg of CFX and 100 mg of MZL.

Ten tablets weighed and ground into a fine powder. A quantity equivalent to one tablet was weighed and dissolved in 50 mL KCL (1M) with shaking for 5 min., transferred to 100 mL volumetric flask and diluted to the mark with KCL (1M), 10 mL of the solution was transferred to 100 mL volumetric flask and diluted to the mark with KCL (1M). Otherwise, 5 mL of the oral suspension was diluted with KCL (1M) into 50 mL volumetric flask, 10 mL of the solution was transferred to 100 mL 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 HPLC³³.

3.10 Effect of Ion Pair Percentage on Electrode Potential:

Three groups of electrodes containing 2-10% IP were constructed. The potentiometric response characteristics of the CFX, MZL and CFX+MZL sensors based on the use of CFX-PTA (IP-1) "group 1", MZL-PTA (IP-2) "group 2", or CFX-PTA+MZL-PTA (IP-1+IP-2) "group 3" ion pairs in plasticized PVC matrixes evaluated according to IUPAC recommendations³⁴. The graphs plotted for relation E(mV)=f(pC_CFX) or E(mV)=f(Pc_MZL).

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 includes the linear part of the graph plus the lower curved part of the calibration graph. TMRs were 3.16-100000 µM for CFX-PTA, and 5.62-17783 µM for MZL-PTA (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 incrementally 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 -59.46 mV.decade^-1 at 6% CFX-PTA (sensor-1), -59.1 mV.decade^-1 at 7% MZL-PTA (sensor-2), and -59.50 mV.decade^-1 for CFX, -58.97 mV.decade^-1 for MZL (IP-1+IP-2) in the combined sensor (sensor-3). At percentages of ion pair higher than those previously mentioned a decline in the electrode response, range and slope of the liner area was resoluted due to the kinetic of the ion pair inside the membrane (Fig. 3). Table 1 summarized the least squares equations data.

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

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

	Sensor-1 CFX-PTA					Sensor-2 MZL-PTA					(Combined Sensor) Sensor-3 CFX MZL
IP %	4	5	6	7	8	4	5	6	7	8	6	7
S, mV. decade^-1	-52.67	-56.54	-59.46	-66.25	-73.81	-39.46	-48.72	-53.94	-59.10	-52.66	-59.50	-58.97
b, mV. decade^-1	262.27	251.19	285.41	311.95	345.71	819.56	865.42	909.19	940.55	967.56	286.20	941.87
*r^{2 }**	0.9846	0.9859	0.9995	0.9996	0.9978	0.9726	0.995	0.999	0.9965	0.9985	0.9995	0.9965

* Correlation coefficient

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

Interfering, B	K_Drug,B
Interfering, B	Sensor-1 CFX-PTA	Sensor-2 MZL-PTA	Sensor-3 CFX MZL
Sodium chloride	6.7×10⁻³	5.7×10⁻³	6.6×10⁻³	5.8×10⁻³
Potassium chloride	3.5×10⁻³	3.6×10⁻³	3.6×10⁻³	3.7×10⁻³
Calcium chloride	7.6×10⁻³	6.4×10⁻³	7.7×10⁻³	6.3×10⁻³
Magnesium chloride	4.1×10⁻³	5.1×10⁻³	4.0×10⁻³	5.0×10⁻³
Magnesium stearate	5.6×10⁻³	4.9×10⁻³	5.6×10⁻³	4.8×10⁻³
Microcrystalline Cellulose	3.1×10⁻³	3.5×10⁻³	3.2×10⁻³	3.4×10⁻³
Glucose	3.2×10⁻³	2.2×10⁻³	3.1×10⁻³	2.3×10⁻³
Starch	3.5×10⁻³	3.1×10⁻³	3.4×10⁻³	3.2×10⁻³
Lactose monohydrate	2.1×10⁻³	1.9×10⁻³	2.2×10⁻³	2.1×10⁻³
Ciprofloxacin. HCL	----	1.6×10⁻⁴	----	1.7×10⁻⁴
Metronidazole	1.3×10⁻⁴	----	1.4×10⁻⁴	----

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)

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4.2 Electrodes Selectivity:

The acquire selectivity coefficients K_CFX,B, K_MZL,B of the sensors regarding different inorganic interrupting, some pharmaceutically related compounds, and the other drug for each electrode are given in Table 2. The result shows a reasonable selectivity for CFX and MZL in the presence of many related interferences.

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-PTA sensor, and 2.0-5.0 for MZL-PTA 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-PTA sensor, and 2.0-6.0 for MZL-PTA sensor (Fig. 4).

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

When pH was decreased under 2.0, the potential measured with the electrode declined as a result of the decampment of H⁺ ions out of membrane. At pH values higher than 6.5 the potential also declined 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^35,36.

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

Parameter	CFX-PTA	MZL-PTA	(Combined Sensor) Sensor-3 CFX MZL
IP%	6%	7%	6%	7%
Slope, mV. decade^-1	-59.46 ± 0.11	-59.10 ± 0.07	-59.5 ± 0.13	-58.97 ± 0.012
Intercept, mV. decade^-1	285.41	940.55	286.20	941.87
Correlation coefficient (R²)	0.9995	0.9965	0.9995	0.9965
Linear range, µM	10-10000	10-10000	10-10000	10-10000
TMR, µM	3.16-100000	5.62- 17783	3.16-100000	5.62-17783
LOD, µM	0.122	0.308	0.114	0.309
LOQ, µM	0.370	0.933	0.346	0.936
Response time for 1mM, sec	10 ± 3	12 ± 2	12 ± 2	14 ± 2
Life time, day	15	13	13 (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^**

^aFive replicate measurement. *Without buffer. **Using Britton-Robinson universal buffer

4.4 Lifetime Study:

We estimated the lifetime of the electrodes from the calibration curves, for that daily-periodical tests of standard CFX and MZL 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 MZL solution. The slopes of the calibration curves were -59.45 mV. decade^-1 for CFX-PTA (sensor-1), -59.30 mV.decade^-1 for MZL-PTA (sensor-2), and -59.47 mV.decade^-1 for CFX-PTA, -58.95 mV.decade^-1 for MZL-PTA (sensor-3) at 25°C. The electrodes were continuously soaked in 1mM solution of CFX or MZL for about 20 days. The calibration plot slopes declined delicately from day to day reaching -53.51 mV.decade^-1 for CFX-PTA (sensor-1), -53.37 mV.decade^-1 for MZL-PTA (sensor-2), and -53.52 mV.decade^-1 for CFX-PTA, -53.06 mV.decade^-1 for MZL-PTA (sensor-3) after 15 days for CFX-PTA and 13 days for MZL-PTA sensors, so the lifetime for the combined sensor (sensor-3) is limited to 13 days. This demonstrate that soaking sensors in the drug solution for a long time has a ruinous 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).

4.6 Quantification of CFX, MZL:

The examined sensors were useful in the potentiometric determination of CFX and MZL in pure solutions by calibration graph and standard addition method as well as in direct determinations of CFX and MZL in both pure solutions (Table 4) and pharmaceutical preparations (Table 5). The results obtained for pharmaceutical preparations were compared with a reference HPLC method [28], the X̄ ± SD (R%) values were 503.0 ± 1.87 mg (100.60%), 504.0±1.92 mg (100.96`%), and 126.40 ±0.55 mg (101.12%) for Ciproflex, Ciprodiazole, and Avilox M respectively using sensor-1 (CFX-sensor), 508.0±1.87 mg (101.60%), 504.2 ± 1.92 mg (100.84%), 101.80±0.84 mg (101.80%) for Flagyl, Ciprodiazole, and Avilx M respectively using sensor-2 (MZL-sensor). while the X̄±SD (R%) values using the sensor-3 (combined sensor) were as the follow: 502.6±1.67 mg (100.52%) for CFX in Ciproflex, 508.0±1.58 mg (101.60%) for MZL in Flagyl, 503.2±3.35 mg (100.64%), 503.8±3.77 mg (100.76%) for CFX, MZL respectively in Ciprodiazole, 125.88±0.76 mg (100.70%), 101.62±1.11 mg (101.62%) for CFX, MZL respectively in Avilox M.

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

Taken C_CFX•HCl		Sensor-1*			Taken C_MZL		Sensor-2*
(μg/mL)	mol/L	R%	SD	RSD%	(μg/mL)	mol/L	R%	SD	RSD%
0.3678	1×10^-6	99.95	0.0017	0.46	0.1712	1×10^-6	99.95	0.0013	0.74
3.678	1×10^-5	100.16	0.0114	0.31	1.712	1×10^-5	99.89	0.0158	0.92
36.78	1×10^-4	99.51	0.4062	1.11	17.12	1×10^-4	99.98	0.0483	0.28
367.8	1×10^-3	99.96	0.1949	0.05	171.2	1×10^-3	99.93	0.3033	0.18
3678	1×10^-2	99.93	3.3615	0.09	1712	1×10^-2	100.01	4.9699	0.29

Taken C_CFX•HCl		Sensor-3*			Taken C_MZL		Sensor-3*
(μg/mL)	mol/L	R%	SD	RSD%	(μg/mL)	mol/L	R%	SD	RSD%
0.3678	1×10^-6	99.02	0.0031	0.86	0.1712	1×10^-6	99.53	0.0021	1.22
3.678	1×10^-5	99.08	0.0378	1.04	1.712	1×10^-5	99.77	0.0164	0.96
36.78	1×10^-4	99.24	0.3536	0.97	17.12	1×10^-4	99.63	0.0472	0.28
367.8	1×10^-3	99.83	0.2168	0.06	171.2	1×10^-3	99.88	0.3162	0.18
3678	1×10^-2	99.92	4.3244	0.12	1712	1×10^-2	99.75	4.8166	0.28

*Average of five replicates.

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

Commercial Name	Composition	X̄ ± SD, mg^a	R%	t-value^b	F-value^c
		Sensor-1 CFX-PTA
Ciproflex	Ciprofloxacin	503.0 ± 1.87	100.60	0.7171	2.6923
Flagyl	Metronidazole	----	----	----	----
Ciprodiazole	Ciprofloxacin	504.0 ± 1.92	100.96	1.1624	0.3814
Ciprodiazole	Metronidazole	----	----	----	----
Avilox M	Ciprofloxacin	126.40 ± 0.55	101.12	1.6329	0.6000
Avilox M	Metronidazole	----	----	----	----
		Sensor-2 MZL-PTA
Ciproflex	Ciprofloxacin	----	----	----	----
Flagyl	Metronidazole	508.0 ± 1.87	101.6	0.4781	4.3750
Ciprodiazole	Ciprofloxacin	----	----	----	----
Ciprodiazole	Metronidazole	504.2 ± 1.92	100.84	1.3949	0.4353
Avilox M	Ciprofloxacin	----	----	----	----
Avilox M	Metronidazole	101.80 ± 0.84	101.80	1.1225	2.1739
		Sensor-3 CFX-PTA + MZL-PTA
Ciproflex	Ciprofloxacin	502.6 ± 1.67	100.52	1.3363	2.1538
Flagyl	Metronidazole	508.0 ± 1.58	101.60	0.5657	3.1250
Ciprodiazole	Ciprofloxacin	503.2 ± 3.35	100.64	0.4008	1.1546
Ciprodiazole	Metronidazole	503.8 ± 3.77	100.76	0.4747	1.6706
Avilox M	Ciprofloxacin	125.88± 0.76	100.70	0.3548	1.1444
Avilox M	Metronidazole	101.62 ± 1.11	101.62	0.4855	3.7950

^aAverage of five replicates.

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

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

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 them regarding accuracy and precision, respectively³⁷.

5. METHOD VALIDATION:

5.1 The linearity, LOD, and LOQ:

We measured CFX and MZL 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_MZL according to the straight-line equation: E =S × Pc_CFX + b, or E =S × Pc_MZL + 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 MZL determination. The limit of detection (LOD) and the limit of quantification (LOQ) were determined according to the IUPAC recommendation [32]. LOD and LOQ values were 0.122 µM, 0.370 µM, respectively for ciprofloxacin in sensor-1 (CFX-PTA), 0.308 µM, 0.933 µM, respectively for metronidazole in sensor-2 (MZL-PTA), 0.114 µM, 0.346 µM,respectively for ciprofloxacin in sensor-3, 0.309 µM, 0.936 µM, respectively for metronidazole in sensor-3 (combined sensor) (Table 3).

5.2 Recovery and Precision:

We calculate the recovery by comparing the potential of the found CFX or MZL 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-PTA-PVC, MZL-PTA-PVC, CFX-PTA+MZL-PTA-PVC membrane ion selective sensors offers a precious technique for direct determination of CFX and MZL 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 MZL.

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

Using CFX-PTA + MZL-PTA as a combined electro-active materials in the same membrane we could arrange a novel electrode that is sensitive to either CFX or MZL 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 MZL standard solution "Metronidazole 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 MZL simultaneously and in this way we could determine the two drugs (CFX and MZL) in their combined solutions.

The suggested sensors accomplished LOD and LOQ values of 0.122 µM, 0.370 µM, respectively for ciprofloxacin in sensor-1 (CFX-PTA), 0.308 µM, 0.933 µM, respectively for metronidazole in sensor-2 (MZL-PTA), 0.114 µM, 0.346 µM,respectively for ciprofloxacin in sensor-3, 0.309 µM, 0.936 µM, respectively for metronidazole in sensor-3 (combined sensor), with response time of 10 ± 3 sec, 12 ± 2 sec, 12 ± 2 sec (CFX) or 14 ± 2 sec (MZL) 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 MZL without using any buffer.

The direct determination of CFX and MZL showed an average recovery of 99.96, 99.93 and 99.83% (CFX) or 99.88% (MZL) and a mean relative standard deviation of 0.05%, 0.18% and 0.06% (CFX) or 0.18% (MZL)% at 1 mM (367.8 μg/mL CFX, or 171.2 μg/mL MZL) 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 99.02 % of R % for the accuracy. The sensors were used as indicator electrodes for direct determination of CFX and MZL in there pharmaceutical preparations as well as in pure form solutions.

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Received on 29.01.2019 Modified on 18.03.2019

Research J. Pharm. and Tech. 2019; 12(7): 3377-3384.

DOI: 10.5958/0974-360X.2019.00570.5