INTRODUCTION:

Clopidogrelbisulphate (CLP) chemically is: methyl (+)-(s)-α-(o-chlorophenyl)6,7-dihydrothieno (3, 2-c) pyridine- 5(4H)- acetate bisulphate (Figure 1), is a new antiplatelet aggregation by selective preventing binding adenosine diphosphate (ADP) to its platelet receptor [1]. It prevent ischemic stroke, mycordil infraction and vascular disease [2].

Fig .1 : Chemical structure of CLP

Literature showed some analytical methods for determination of Clopidogrelbisulphate such as HPLC [3-5], HPTLC[6, 7], UV Spectrophotometric [8-12], H-NMR[13], GC-MS[14], Capillary electrophoresis [15,16], Voltammetry and potentiometric methods [17,18].

Ion selective electrode is one of the most effective, accurate, economic analytical methods for determination of organic and inorganic substances[19].The aim of this work was to develop a sensitive, selective and validated ISE_sfor the determination of Clopidogrelbisulphate in bulk and pharmaceutical formulations.

MATERIALS AND METHODS:

Apparatus:

The electrochemical measurements were made with IONcheck 10 pH/mvmeter–Radiometer Analytical S.A.(CEDEX-France) with electrodes consist of [Clopidogrel-Nessler reagent–poly phenyl chloride (PVC)–di butyl phthalate (DBP)] membrane in conjunction with double junction Ag/Ag Cl electrode as external reference electrode ( lab. assembly ), containing 1M KCl in the outer compartment. The pH measurements were made with Crison model Glp 21/ Eu (Spain). Ultrasonic processor model power sonic 405 (Korea). All potentiometric measurements were made at 25±1 C with constant stirring using hot-plate magnetic stirrer MS 300 BANTE (China). All weights were taken by analytical balance SARTORIUS model 2474 (Germany) with accuracy ±0.1mg.

Reagent and Materials.:

Analytical grade Clopidogrelbisulphate (Glenmarak Generics Ltd- India), high molecular weight PVC (SABC, KSA), di–n-butyl phthalate (DBP) 99%, Tetrahydroforan 97%, HCl 37%, NaOH 99.8%, KCl (GR grade), Mercury iodide Hg₂I₂, Potassium iodide KI, from (MERCK-Germany), Bi-distilled water (conductivity ≤10 μS/cm) were used.

Preparation of NESSLER's Reagent:

THOULET'S solution KHgI₃ was prepared by mixing 1 mmol hot aqueous suspension of mercury (II) iodide with 2 mmol solution of potassium iodide. To have NESSLER's reagent K₂HgI₄, 2 mmol potassium hydroxide was added. Any precipitate was filtered. Then, the filtrate brought to room temperature and diluted to the required concentration [20].

Standard Drug Solutions :

Stock standard (0.01 M) Clopidogrelbisulphate was prepared by dissolving accurate weight in Bi–distilled water using the ultrasonic bath. Working solution ranging (0.1-1000) µM were prepared by serial dilutions of the stock solution. Britton –Robinson universal buffer 0.2 M was used [21].

Ion Selective Electrodes:

Preparation of Ion-Pair(IP):

The ion pairs was prepared by mixing 50 mL of 1mM CLP with 50 mL of Nessler reagent. The precipitates Clopidogrel–Nessler reagent (yellowish powder) wasfiltered and washed thoroughly with distilled water until the conductivity of the washed water is close to the conductivity of bi –distilled water and dried at room temperature for 24 hrs. Ion pairwas stored in will – closed amber glass bottles. The molecular ratio was found to 1:2 with Nessler reagent.

Membranes Composition:

The membranes were prepared by dissolving equal weights of matrix PVC and the plasticizer (DBP) and suitable weight of ion pair in a minimum volume of tetrahydrofuran (THF). The solutions were poured into a petri –dish, covered with a filter paper and the solvent was allowed to evaporate slowly at room temperature to get the membranes that represent the electro-active part of ion selective electrode [22].

Construction of ISE_s:

A punched circular membrane was attached to a poly-ethylene tube in an electrode configuration and attached to the end of a glass tube. This electrode was filled with an internal solution consisting of 1mM CLP in 1 M KCl. Ag/AgCl wire electrode was used as an internal reference electrode. Then this electrode attached to double junction Ag/AgCl electrode as the external reference electrode, containing 1 M KCl in the outer compartment. The circuit closed by attaching the cell to pH/mvmeter [23, 24].

Electrode Calibration :

25 mL aliquots of (0.1-100000) µM Clopidogrel standard solutions were transferred into 50 mL beaker and the electrode in conjunction with Ag/AgCl reference electrode was immersed in the solution. All potentiometric measurements were performed using the cell:

Ag/AgCl –KCl (1M)+CLP (1mM)llCLP-NES –DBP –PVC membrane ll test solutionll Ag/AgCl –KCl (1M)

The measured potential was plotted against the minus logarithm of CLP concentration. The electrodewas washed with Bi–distilled water and dried with tissue paper between measurements.

Effect of Ion Pair Percentage on Electrode Potential :

We have prepared a set of membranes containing a different percentage of ion pair ranging between
(2-8)%, then we studied the effect of this percentage on electrode potential and we selected the percentage that showed the closet slope to Nernstian slope [22].

Effect of pH:

The effect of pH on the potential response was investigated using (10^-2and10^-3) MClopidogrel solutions. The pH of the solutions was adjusted between 1-6 using suitable amounts of 1M KOH or HCl solution. The potential readings corresponding to different pH value were recorded. On the other hand, the study was repeated using Britton – Robinson universal buffer. The pH of the buffer solutions was adjusted between 1-6, and a specific volume of it was added to the drug solution.

Selctievity:

Selectivity coefficients K ^Pot_A,Bwere determined by the matched potential method [25] In this method, the potentiometric selectivity coefficient is defined as the activity ratio of primary and interfering ion that give the same potential change under the same conditions.

K=(à-a ) / a _B

At first, a known activity (à_CLP) of the primary ion solution is added into a reference solution that contains a fixed activity (à_CLP) of primary ions, and the corresponding potential change (ΔE) is recorded. Then, a solution of an interfering ion is added to the reference solution until the same potential change (ΔE) recorded again [26].

Determination of Clopidogrel in Pharmaceutical Dosage Forms:

Local manufacturing formulations, Copiral (75 mg film coated tablets, AVENZOR pharmaceutical industries), PLOFEXINE (75 mg film coated tablets, ASIA pharmaceutical industries) and Platgo (75 mg f.c.t, MEECYPHARMA pharmaceutical industries) were used for the analysis of CLP by direct potentiometric determination method. Ten tablets of each drug were finely powdered and accurate weight equivalent to one tablet was taken and dissolved using the same procedure used to dissolve the drug substance. The solution was filtered through a common filter paper and transferred to 100 ml volumetric flask and diluted to the mark with bi–distilled water, from this solution 2.14 mL was taken and diluted to50 mL to get 10^-4 M concentration. The final solution was analyzed as described under electrode calibration.

RESULTS:

Calibration Graphs and the Best Electrode

The analytical range of a potentiometric electrode was the linear part of the calibration graphs, it was
(10^-2-10^-5) M, pc=(2-5), see figure 2 Increasing IP percentage in the membrane was found to increase the electrode response the slope of the linear area reaching -28.73 m.v.decade^-1m.v.decade^-1 for electrode 6%ion pair ratio as showed in figure 3, but at percentage higher than that detective we found a decrease in electrode response and slope, that due to the kinetic of the ion pair inside the membrane.

Fig. 2: Effect of IP content on CLP-NES calibration curve.

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

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

CLP-NES
IP%	2	4	6	8
S,mv.decade^-1	21.67	23.17	28.73	27.06
b, mv	-8.28	78.97	104.88	80.51
r²	0.9899	0.9794	0.9898	0.9952

Effect of pH on potential:

The effect of pH of the CLP test solution on electrode potential was investigated. The results showed that the potential remained constant despite the pH change in the range between 1-3 and we found the same results when we used a Britton –Robinson universal buffer as shown in figures 4, that indicate the applicability of this electrode in the pH specified range.

Fig. 4: Effect of pH on the potential response of theCLP-NES electrode using 10 mMCLP solution, 1 mMCLP solution, in BRITTON-ROBINSON universal buffer solutions.

Selectivity of the Electrodes:

the results of the potentiometric selectivity coefficients of the prepared electrodes showed high selectivity toward Clopidogrel in presence of different inorganic cations and some pharmacologically related compounds.The results summarized in table 2.

Table 2: Selectivity coefficient of some interfering ions by CLP-NES

Interfering, B	K_MFX,B	Selectivity
Ca Cl₂	13x10^-3	76.9
NH₄Cl	15x10^-3	66.6
NaCl	4.4*10^-3	227.2
KCl	26*10^-3	38.4
dextrose	5.93*10^-3	168.6
CMC	7.20*10^-3	138.3
Mg stearate	9.36*10^-3	106.8

Life Time Study:

Clopidogrelselective electrode life time was estimated with a calibration curves periodical test of standard solutions (10^-1-10^-6) M and calculation of its response slope. The calibration curves were plotted after an optimum soaking time of 6 hrs in 1 mMCLP solution. The slope was-28.73 m.v.decade^-1 for electrode^-1at 25 °C. The electrode was continuously soaked in 1 mM solution of CLP for about 15 days. The calibration plot slopes decrease slightly to be -23.85 after 11 days .These indicate that soaking the electrode in drug solution for a long time has a negative effect on the response of membrane.

Response Characteristics and Linearity:

Linear range, correlation coefficient and detection limits, were given in Table3. The electrode show a nearly Nernestain response in the range between(10-10000) µM, PC (2-5).

Table 3: Response characteristics of CLP-NES electrode

Parameter	CLP-NES
IP%	6%
Slope, mV.decade^-1	-28.73
Intercept, mV.decade^-1	104.88
Correlation coefficient (R²)	0.9898
Linear range, μM	10-10000
LOD, μM	0.063
LOQ, μM	0.191
Response time for 1 mM, sec	18 ± 2
Life time, day	11
Working pH range	1-3

Quantification of Clopidogrel bisulphate:

The developed electrode found to be effective in the potentiometric determination of Clopidogel in both pure form and pharmaceutical preparations.

Accuracy and Precision:

The low values of relative standard deviation (RSD) indicate good precision and reproducibility of the method. The average percent recoveries obtained were 97.90–102.99%, indicating a good accuracy of the methods. The results obtained were summarized in Table 4.

Table 4: Direct determinations of CLP in bulk solution using proposed electrodes

C_Taken,(µg/mL)	C_{Found ,}(µg/mL)	R%	SD	RSD%
4198.91	4324.87	102.99	53.19	1.23
419.89	414.01	98.59	7.24	1.75
41.98	41.10	97.90	0.95	2.31
4.19	4.28	102.14	0.12	2.80

Application to the pharmaceutical dosage forms:

The proposed method has been successfully applied to the determination of Clopidogrelbisulphate in pharmaceutical preparations by calibration curve method.

The results obtained were compared with a reference HPLC method [27]. As shown in table 5. Statistical analysis of the result obtained by the proposed and comparison methods using STUDENT’s t-test and variance ratio F-test[28], showed no significant difference between them regarding accuracy and precision, respectively.

Table 5: Determinations of CLP in pharmaceutical preparations using proposed electrode.

Drugs	X ± SD^a	R%	t- value	f- value
Clopiral	75.25±0.56	100.3	0.03	2.71
Platgo	72.62±0.47	96.8	1.51	1.61
Plofexine	73.67±0.61	98.2	2.01	2.21

^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.

DISCUSSION:

New constructed sensor was developed for Clopidogrelbisulphate determination over a wide range of concentration in pH range between 1-3. The electrode showed a very good selectivity to CLP in the presence of various common inorganic cation and different pharmacological related compounds. This electrode can be used as fast, economic, simple and sensitive analytical method for the determination of Clopidogrel in bulk powder and pharmaceutical preparations.

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Received on 29.12.2017 Modified on 25.01.2018

Research J. Pharm. and Tech 2018; 11(5):2017-2021.

DOI: 10.5958/0974-360X.2018.00374.8