A New Spectrophotometric Method for the Micro Determination of  Clopin-A with Bromo Cresol Green and its Application to Evaluate the Assay of the Drug

 

I Lakshmi Prasanna, G. T. Naidu, Nuzhath Fathima, G. Abdul Huq

 

ABSTRACT:

A simple, precise, versatile and a new spectrophotometric method is proposed for the estimation of microgram quantities of the drug Clopidogrel in the presence of Aspirin. It is also known as Clopin-A, The drug forms an Ion – pair complex with Bromo Cresol Green (BCG), the stoichiometry of which is established as 1:1 by Job's continuous variation method. The wavelength of the maximum absorbance of the Ion – pair complex is found to be 440nm. The absorbance values of the Ion – pair complex increased linearly with the increase in the amount of the drug Clopidogrel in the presence of Aspirin. This suggests the suitability of the method for the determination of the drug in the range 10μg/ml to 250μg/ml. This also indicates the verification of the Beer-Lambert’s Law in this range. This method is successfully employed to evaluate the assay of commercial tablets in pharmaceutical formulations for Clopidogrel in the presence of Aspirin and the results agreed very well. The molar absorptivity and Sandell Sensitivity of the method are found to be 1.860×10⁴ lit/mole/cm and 0.0225 µg/ml/cm² respectively.

 

 

 

INTRODUCTION:

Clopidogrel was first written about in 1982 and was approved for medical use in 1998. It is on the World Health Organizations List of Essential Medicines, the most effective and safe medicines needed in a health system. It is also used, along with acetylsalicylic acid^[1-6] (ASA, aspirin), for the prevention of thrombosis after placement of a coronary stent or as an alternative antiplatelet drug for people intolerant to aspirin. Clopidogrel^[7-20] is an inhibitor of platelet activation and aggregation through the irreversible binding of platelets. Clopidogrel is a prodrug and is metabolized to a pharmacologically active metabolite and inactive metabolites. It is used in the prevention of ischemic events, myocardial infraction, stroke syndrome, epilepsy, panic disorder. This medication is an anti-platelet agent, that is, a drug that inhibits the ability of platelets to clump together as part of a blood clot.

 

This medication is prescribed either alone or with other medications for prevention or treatment of stroke and heart attack (which are usually caused by blood clots) in persons who are at high risk. Aspirin; 2-acetoxy benzoic acid is cyclo oxygenase inhibitor. It is used as an analgesic, antipyretic, anti-inflammatory and anti thrombic agent. It is used as an anti thrombic agent. A capsule formulation containing 75 mg of aspirin and 75 mg of Clopidogrel bisulphate is available in market as Clopin- A75. A survey of literature revealed that spectrometric method was reported for the determination of aspirin in biological fluids. RP-HPLC methods were reported for the simultaneous estimation of aspirin, paracetamol, caffeine and aspirin with atorvastatin. Spectrofluorimetric method also developed for the estimation of aspirin and dipyridamole. Colorimetry, HPLC and gas chromatographic methods were described in the literature for the estimation of Clopidogrel bisulphate. However less number of UV methods for the simultaneous estimation of aspirin and Clopidogrel bisulphate in combined dosage forms has so far been reported. The present work describes the development of a simple, precise and accurate UV method for the micro determination of Clopidogrel bisulphate in the presence of aspirin^[21-23] in capsules. The tablet formulation is a somewhat new entrant in the Indian market. Several spectrophotometric and HPLC methods are reported for the estimation of aspirin in literature, whereas only a few HPLC methods are available for Clopidogrel bisulphate.

 

MATERIALS AND METHODS:

(A) Instruments used:

(i)  Spectrophotometer: A single beam UV-Spectrophotometer Model SP-UV200 with 1cm matched quartz cuvettes is employed throughout the study for all absorbance measurements.

(ii) pH Meter: A digital ELICO-pH Meter Model LI-120 is used for pH measurements.

 

(B) Preparation of Reagents and Solutions

(i) Clopin solution: 50 mg of pure Clopin is dissolved in methanol and the volume of the resulting solution is adjusted to the mark in the 50 ml standard flask with methanol. This is used as the stock solution of the drug. The working solution with concentration 100 μg/ml of the drug is prepared by suitably diluting the stock solution as and when required.

(ii) Aspirin solution: 50 mg of pure Aspirin is dissolved in Methanol and the volume of the resulting solution is adjusted to the mark in the 50 ml standard flask with methanol. This is used as the stock solution of the drug. The working solution with concentration 100 μg /ml of the drug is prepared by suitably diluting the stock solution as and when required.

(iii)Bromo Cresol Green solution (0.5% w/v): Bromo Cresol Green is prepared by dissolving 500 mg of Bromo Cresol Green in 100 ml of distilled water.

(iv)Buffer solution pH 3.5 (Potassium acid phthalate - HCl): The potassium acid phthalate – HCl Buffer solution is prepared by diluting a mixture of 50 ml of 0.2M potassium acid phthalate and 8.4 ml of 0.2M HCl to 200 ml with distilled water and the pH is adjusted to 3.5.

 

All other chemical substances and reagents employed in the present investigations are of AR Grade only.

 

RESULTS AND DISCUSSION:

Clopidogrel when treated with Bromo Cresol Green (BCG) forms an Ion Pair complex. This Ion-pair Complex formation reaction is spectrophotometrically monitored to develop a method for the determination of the drug. In the process of carrying detailed investigations, optimization of various parameters such as the wavelength of maximum absorbance (λ_max), the effect of concentration of Buffer solution of pH 3.5 and BCG on the absorbance of the Ion-Pair complex are established and the procedures adopted in each case are described as follows:

Absorption spectrum of Ion-Pair complex:

The complex formed between Clopidogrel in the presence of Aspirin and BCG is obtained in order to fix the wavelength of maximum absorbance in the present study. The experimental procedure adopted is as follows:

 

2ml of Clopidogrel solution (100µg/ml), 2ml of BCG solution (0.5% w/v), 3ml of Buffer solution of pH 3.5, 1 ml of Aspirin solution (100µg/ml) and 1 ml methanol are taken in a 10ml standard flask. The resulting solution is made upto the mark with distilled water. The contents of the flask are shaken well and allowed to stand for a minute for equilibration. Then the absorbance values of the Ion-Pair complex formed are measured in the wavelength range 380nm to 500nm against the reagent blank. The results obtained are used to draw a graph between the wavelength and the absorbance values. The graphical representation is called the Absorption spectrum which is shown in Fig .1 below.

 

It is seen from the above figure 1, of the absorption spectrum, that the maximum absorbance is obtained at 440nm. Hence for all further studies, the wavelength of 440nm is fixed.

 

Effect of volume of Buffer solution pH 3.5:

The effect of Buffer solution pH 3.5 on the absorbance of Ion-pair complex is studied by taking varying volume (x ml) of Buffer solution of pH 3.5 in a series of 10ml standard flasks keeping the volume of Clopidogrel solution fixed at 2ml. To each flask of 2.5ml of BCG solution (0.5% w/v), 1ml of Aspirin and 1ml of methanol are added followed by the addition of distilled water to make up each 10ml flask to mark. The absorbance of each solution is recorded at 440nm against the suitable blank. The results obtained are tabulated in Table.1 below.

 

Table 1: Effect of Buffer solution pH 3.5

S. No	Vol. of Clopidogrel (100µg/ml) in ml	Vol. of Aspirin (100 µg/ml) in ml	Vol. of BCG (0.5% w/v ) in ml	Vol. of Buffer    Solution (pH 3.5) x ml	Vol. of Methanol in ml	Vol. of distilled water in ml (3.5-x)	Total Vol.in each flask in ml	Absorbance
1	2.0	1.0	2.5	0.5	1.0	3.0	10	0.431
2	2.0	1.0	2.5	1.0	1.0	2.5	10	0.496
3	2.0	1.0	2.5	1.5	1.0	2.0	10	0.523
4	2.0	1.0	2.5	2.0	1.0	1.5	10	0.430
5	2.0	1.0	2.5	2.5	1.0	1.0	10	0.445
6	2.0	1.0	2.5	2.5	1.0	0.5	10	0.458

2 ml of Clopidogrel solution (100µg/ml) + 1 ml of Aspirin solution (100µg/ml) + 2.5 ml of BCG solution (0.5% w/v ) + x ml of Buffer solution of  pH 3.5 + 1 ml of methanol +  (3.5-x) ml distilled water = Total volume kept at 10 ml each.  λ_{max =} 440 nm

 

From the data presented in table1 above, it is clear that 1.5 ml of Buffer solution of pH 3.5 is necessary to achieve maximum absorbance.  Hence for all further studies a volume of 1.5 ml of Buffer solution of pH 3.5 is maintained.

 

Effect of Bromo Cresol Green (BCG) concentration:  The effect of BCG on the absorbance of the Ion-Pair complex is studied by taking varying volumes (x ml) of BCG in a series of 10 ml standard flask. After taking x ml (0.5 ml to 2.5 ml) of BCG in each flask, 1.5 ml of Buffer solution of pH 3.5, 2 ml of drug solution of Clopidogrel, 1 ml of Aspirin solution, 2 ml of methanol are added and the resulting solution is made up to 10 ml using distilled water. The absorbance of each solution is recorded at 440 nm against a suitable blank. The results obtained are tabulated in Table .2 below.  

 

Table 2: Effect of BCG on Ion-Pair complex

S. No	Vol. of Clopidogrel (100 µg/ml) in ml	Vol. of Aspirin (100 µg/ml) in ml	Vol. of BCG solution (0.5% w/v) x ml	Vol. of Buffer (pH 3.5) in ml	Vol. of methanol in ml	Vol. of distilled water in ml (3.5-x)	Total Vol.in ml	Absorbance
1	2.0	1.0	0.5	1.5	2.0	3.0	10	0.173
2	2.0	1.0	1.0	1.5	2.0	2.5	10	0.227
3	2.0	1.0	1.5	1.5	2.0	2.0	10	0.323
4	2.0	1.0	2.0	1.5	2.0	1.5	10	0.364
5	2.0	1.0	2.5	1.5	2.0	1.0	10	0.495
6	2.0	1.0	3.0	1.5	2.0	0.5	10	0.488

2 ml of Clopidogrel solution (100µg/ml) + 1 ml of Aspirin solution (100µg/ml) + x ml (0.5 ml to 2.5 ml) of BCG solution (0.5% w/v) + 1.5 ml of Buffer solution (pH 3.5) + 2 ml of methanol + (3.5 – x) ml distilled water = Total volume kept at 10 ml each . λ_max  = 440 nm

 

From the data presented in Table. 2 above, it is clear that 2.5ml of BCG solution is required for maximum absorbance. Hence for all further studies a volume of 2.5 ml of BCG solution is fixed.

 

Effect of concentration of drug Clopidogrel:

This study pertains to the effect of the drug Clopidogrel concentration on the absorbance of the Ion-Pair complex under the established optimal experimental conditions. The recommended procedure for the calibration curve and for the obedience of Beer-Lambert’s law for the quantitative spectrophotometric determination of the drug Clopidogrel is as follows:

 

Calibration Curve: Obedience of Beer – Lambert’s Law: Various aliquots x ml i.e., (0.5ml to 2.5ml) of Clopidogrel solution (100µg/ml) are taken in a series of 10ml standard flask. To each flask, 1ml of Aspirin solution (100µg/ml), 1.5 ml of Buffer solution of pH 3.5, 2.5ml of BCG solution (0.5% w/v), 2ml methanol followed by (3-x) ml of distilled water are added so as to make the total volume in each case at 10ml. The contents of each flask are shaken well and allowed to stand for a minute for equilibration. The absorbance of each solution is measured at 440 nm against a suitable reagent blank which is prepared in a similar manner but devoid of drug solution. The results obtained are mentioned in Table.3 and Figure 2 below.

 

Table.3: Calibration curve – Obedience of Beer-Lambert’s law

S.No	Vol. in ml Clopidogrel (100 μg/ml) x ml	Vol. of Clopidogrel in μg/ml	Vol. of Aspirin (100 μg/ml) in ml	Vol. of Buffer pH3.5 in ml	Vol. of BCG (0.5% w/v) in ml	Vol. of Methanol in ml	Vol. of distilled water in ml (3-x)	Total vol. in ml	Absorbance
1	0.5	50	1.0	1.5	2.5	2.0	2.5	10	0.137
2	1.0	100	1.0	1.5	2.5	2.0	2.0	10	0.251
3	1.5	150	1.0	1.5	2.5	2.0	1.5	10	0.356
4	2.0	200	1.0	1.5	2.5	2.0	1.0	10	0.467
5	2.5	250	1.0	1.5	2.5	2.0	0.5	10	0.578

x ml (0.5 ml to 2.5 ml) of Clopidogrel solution (100 µg/ml) + 1 ml of Aspirin solution (100 µg/ml) + 1.5 ml of Buffer solution of pH 3.5 + 2.5 ml of BCG solution (0.5% w/v) +2 ml methanol + (3-x) ml distilled water = Total volume kept at 10 ml in each.   λ_max = 440nm

 

It is obviously clear from the data presented in above table.3 and from this calibration straight line as shown in above Figure.2 that the absorbance values increased linearly with the increase in the amount of the drug.  This verifies the Beer-Lambert’s Law and suggests that the method can be successfully employed for the spectrophotometric quantitative determination of the drug Clopidogrel in the range 10µg/ml to 250µg/ml. The molar absorptivity and the Sandell sensitivity of the method are found to be 1.860×10⁴ lit/mole/cm and 0.0225µg/ml/cm² respectively.

 

Stoichiometric composition of Ion-Pair Complex: Job’s continuous variation method: The composition of the Ion – Pair complex between the drug Clopidogrel in the presence of aspirin and the reagent BCG is established by the Job’s continuous variation method. In this, the equimolar concentrations (5 x 10^-4 M) of both the drug and BCG are varied continuously keeping the total volume of mixed solution as constant at 10ml. In each case, the absorbance is measured at 440nm against a suitable blank. The data obtained is presented in the Table.4 and the Figure.3 and is as shown below.

 

Table.4: Job’s method of continuous variation  

S. No.	Vol. of Clopidogrel (5 x 10-4M) V1 in ml	Vol. of Aspirin (5 x10-4M) in ml	Vol. of Buffer Solution of pH 3.5 in ml	Vol. of BCG (5 x 10-4 M) V2 in ml	Vol. of Methanol in ml	Total vol. in ml	Vol. fraction (x) of the drug (V1/V1+V2)	Absorbance
1	0.5	2.0	1.0	4.5	2.0	10	0.1	0.251
2	1.0	2.0	1.0	4.0	2.0	10	0.2	0.438
3	1.5	2.0	1.0	3.5	2.0	10	0.3	0.767
4	2.0	2.0	1.0	3.0	2.0	10	0.4	0.992
5	2.5	2.0	1.0	2.5	2.0	10	0.5	1.229
6	3.0	2.0	1.0	2.0	2.0	10	0.6	0.897
7	3.5	2.0	1.0	1.5	2.0	10	0.7	0.564
8	4.0	2.0	1.0	1.0	2.0	10	0.8	0.287
9	4.5	2.0	1.0	0.5	2.0	10	0.9	0.221

0.5 ml to 4.5 ml of Clopidogrel solution (5 x 10^-4 M) +2 ml of Aspirin solution (5 x 10^-4 M) +1 ml of Buffer solution of pH3.5 + 4.5 ml to 0.5 ml of BCG solution (5 x 10^-4 M) +2 ml of Methanol = Total volume kept at 10 ml in each case.  λ_max = 440 nm

 

The data in the above Table.4 are plotted in the form of a graph between volume fraction of the drug i.e., (V₁/V₁+V₂) on X- axis and the absorbance values on Y-axis. The graph obtained is as shown in figure 3 below.

 

From the graph shown above, it is found that one mole of the drug is reacting with 1 mole of BCG, there by establishing the stoichiometry of the Ion-Pair complex as 1:1 (Drug: BCG)

Assay of Clopidogrel drug in pharmaceutical formulations:

The recommended procedure for the quantitative micro determination of Clopidogrel drug is applied for the assay of the drug in the dosage form of the commercial tablets and also in pharmaceutical formulations. The assay is carried out as follows:

 

20 tablets of Clopidogrel are weighed and finely powdered. An accurately weighed portion of the powdered sample equivalent to 50mg of Clopidogrel is taken in a 50ml volumetric flask containing 25ml of methanol and is sonicated for about 20 minutes. The resultant solution is filtered through Whatman filter paper No.41 into another 50ml volumetric flask. The filter paper is washed several times with methanol and the washings are added to filtrate. The final volume is made upto the mark with methanol. Now, 5ml of filtrate of the sample solution is diluted to 10 ml with methanol and treated as per the recommended procedure of calibration. From this, the amount of the drug present in the sample is computed from the calibration curve. The results obtained are as shown in Table.5 below.

 

Table.5: Assay of Clopin-A in Tablets

Sample	Labelled amount in mg	Amount found by present method ±SD*	Percentage of Label claim	*tcal	% RSD
Tablet I	20	20.07±0.08	100.07	1.9565	0.40
Tablet II	20	20.06±0.14	100.06	0.9582	0.30

* Average of 5 determinations based on label claim.

 

ACKNOWLEDGEMENTS:

The authors (ILP and NF) express their gratitude to the authorities of the Departments of Physics and Chemistry respectively of Rayalaseema University, Kurnool, and the Central Instrumentation Cell of Prof. T. Jayashankar Telangana State Agricultural University, Hyderabad for providing necessary facilities to carry out the proposed work.

 

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Received on 25.07.2019         Modified on 25.10.2019

Accepted on 20.12.2019         © RJPT All right reserved