Spectrophotometric Simultaneous Determination of Clopidogrel and Aspirin in Combined Tablet Dosage Form by Ratio Derivative and Area Under Curve Method

 

Arun B. Kale, Prachyasuman Mohanty, Vishnu P. Choudhari*, Abhijit S. Sutar, Swati C. Jagdale and Bhanudas S. Kuchekar

MAEER'S Maharashtra Institute of Pharmacy, S.No. 124, MIT Campus, Paud Road, Kothrud, Pune-411038, MS

*Corresponding Author E-mail: viraj1404@rediffmail.com

ABSTRACT:

A simple, economical, precise and accurate method for simultaneous determination of Clopidogrel (CLOP) and Aspirin (ASP) in combined tablet dosage form has been developed.  The first method is based on Ratio Spectra Derivative (Method A) and second method is based on Area under Curve (AUC) Spectrophotometry (Method B). The amplitudes 218.11 nm and 247.002 nm in the second derivative of the ratio spectra were selected for aspirin and clopidogrel respectively. In the second method (AUC) areas were integrated in the wavelength ranges between 215.66-218.78 nm and 235.15-238.27 with reference to the absorbance curves plotted between the wavelengths 200-350 nm to determine CLOP and ASP.  The drugs obey Beer’s law in the concentration range of 5-25 μg mL^-1 by both the methods. The methods were validated with respect to linearity, precision and accuracy. The results of analysis have been validated statistically and recovery studies confirmed the accuracy and reproducibility of the proposed methods which were carried out by following ICH guidelines. The proposed methods were applied successfully for Simultaneous Determination of Clopidogrel and Aspirin in Combined Tablet formulations.

 

 

 

INTRODUCTION:

Aspirin is also known as acetylsalicylic acid is a salicylate drug, often used as an analgesic, antipyretic, anti-inflammatory and also has an antiplatelet effect by inhibiting the production of thromboxane, which under normal circumstances binds platelet molecule together to create a patch over damage of the walls within blood vessels. Aspirin is also used for long-term, at low doses, to help prevent heart attacks, strokes, and blood clot formation in people at high risk for developing blood clots. Chemically it is 2-acetoxybenzoic acid and shows inhibition of the enzyme cyclooxygenase and it is official in Indian Pharmacopoeia, The United States Pharmacopoeia and British Pharmacopoeia^1-4. Clopidogrel is an oral antiplatelet agent (thienopyridine class) chemically it is a synthetic (+)-(S)-methyl 2-(2-chlorophenyl)-2-(6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)acetate and is official in USP. It inhibits blood clots in coronary artery disease, peripheral vascular disease, and cerebrovascular disease. It is also used, along with aspirin, for the prevention of thrombosis after placement of intracoronary stent or as an alternative antiplatelet drug for patients who are intolerant to aspirin^{2, 4}.

 

Review of the literature revealed that there are Spectrophotometric simultaneous equation, Iso absoprtive, HPLC, RP-HPLC, HPTLC, and Semi-micro column HPLC-UV methods have been reported for simultaneous determination of this combination^5-9. Also for CLOP the HPLC determination of inactive carboxylic acid metabolite in human serum which is an application to a bioequivalence study have been reported¹⁰. Literature survey^11-14 also reveals that there is no Ratio Derivative and Area under Curve spectrophotometric method available for the determination of these analytes in combination. Therefore the aim of the study was to develop simple, rapid, accurate, reproducible and economical Ratio Derivative and Area under Curve methods for both the titled drugs in combined dosage forms. The proposed methods were optimized and validated as per the International Conference on Harmonization (ICH) analytical method validation guidelines¹⁵.

Aspirin                                  Clopidogrel

Fig.1. The chemical structures of analytes

INSTRUMENTATION:

An UV-Visible double beam spectrophotometer (Varian Cary 100) with 10 mm matched quartz cells and Ultrasonicator (Model 5.5 150H) were used. All weighing were done on electronic balance (Model Shimadzu AUW-220D).

 

MATERIALS AND METHODS:

Reagents and chemicals:

Spectroscopic grade Methanol was purchased from LOBA Chemie Pvt. Ltd., Mumbai. Tablet used for analysis were Clavix-As-75 (Batch No. DL 0154) manufactured by Intas Pharmaceuticals, Dehradhun, India containing CLOP 75mg and ASP 75 mg per tablet. Pharmaceutical grade of Clopidogrel (% purity, 99.78) was kindly supplied as a gift sample by Intas Pharmaceutical Ltd. Dehradhun and pure drug sample of Aspirin (% purity, 99.92) was gifted by Vapi Pharmaceutical Ltd, Gujarat. These samples were used without further purification.

 

Preparation of Standard Stock Solutions and calibration Curve:

Standard stock solution of pure drug containing 1000 μg mL^-1 of CLOP and 1000 μg mL^-1 of ASP were prepared separately in the methanol. The working standard solutions of these drugs were obtained by dilution of the respective stock solution in double distilled water. The Ratio Derivative amplitudes of spectrum, by using the above mentioned procedures, were used to prepare calibration curves for both the drugs. For verification of Beer’s law a series of dilutions in the concentration range of 5-25 μg mL^-1 of analytes were prepared for both the methods and mixture of both the drugs in same concentration range was prepared.

 

METHODS¹⁶:

Method A: RATIO DERIVATIVE METHOD:

The method involves dividing the spectrum of mixture by the standardized spectra of each of the analyte and deriving the ratio to obtain spectrum that is dependent of concentration of analyte used as a divisor. Using appropriate dilutions of standard stock solution, the two solutions were scanned separately. The ratio spectra of different ASP standards at increasing concentrations were obtained by dividing each with the stored spectrum of the standard solution of CLOP (15μg mL^-1) and the derivative of these spectra traced, illustrated in Fig-2. Wavelength 218.11 nm was selected for the quantification of ASP in ASP + CLOP mixture. The ratio spectra of the solutions of CLOP at increasing concentrations were obtained by dividing each with the stored standard spectrum of the ASP (15 μg mL^-1) and the derivative of these spectra traced, illustrated ins (Fig. 3). Wavelength 247.00 nm was selected for the quantification of CLOP in ASP + CLOP mixture. Measured analytical signals at these wavelengths were proportional to the concentrations of the drugs. Calibration curves were prepared from the measured signals at the selected wavelength and concentration of the standard solutions. The amount of ASP (C_ASP) and CLOP (C_CLOP) in tablets was calculated by using equations 1 and 2, respectively.

C_ASP = [Derivative amplitude at 218.11 – (0.003146)] / (0.06505)  ...   (1)

C_CLOP= [Derivative amplitude at 247.0028 – (0.01119)] / (0.10175) ...   (2)

 

Fig-2. Second derivative of the ratio spectra of ASP solution (5 – 25 μg mL^-1) when 15 μg mL^-1 solution of CLOP is used as divisor.

 

Fig- 3.  Second derivative of the ratio spectra of CLOP solution (5 – 25 μg mL^-1) when 15 μg mL^-1 solution of ASP is used as divisor.

 

Method B: Area Under Curve

For the simultaneous determination using the area under curve (AUC) method, suitable dilutions form the standard stock solutions were prepared separately in distilled water. These solutions were scanned in the range of 200-350 nm and Area Under Curve between 215.66-218.78 nm (λ1-λ2) and 235.15-238.27 nm were integrated  which showed linear response with increasing concentration hence the same wavelength range were used. By using integrated areas two simultaneous equations (eq.3 and 4) were constructed and solved to determine concentrations of analytes.  Concentration of two drugs in mixed standard and the sample solution were calculated using equation (5) and (6).

 

A₁ = 1305 C_CLOP+ 1030 C_ASP….   215.66-218.78 nm     (3)

A₂ = 683.93 C_CLOP + 579.26 C_ASP...   235.15-238.27 nm   (4)

C_CLOP = A₂ × a_X2 - A₁ × a_y2 / a_X2 × a_Y1 - a_X1 × a_Y2 ……… (5)

C_ASP = A₂ - a_X2 × C_CLOP / a_Y2 ………………………… (6)

 

Where,

a_X1 (1305) and a_X2 (683.93) are absorptivities of ASP at (λ1-λ2) and (λ3-λ4), respectively.

a_Y1 (1030.33) and a_y2 (579.26) are absorptivities of CLOP at (λ1-λ2) and (λ3-λ4), respectively.

 

Table 1: Optical characteristics of the proposed methods and results of precision and formulation analysis

Parameter			Clopidogrel		Aspirin
			Method A	Method B	Method A	Method B
λ (nm)			247 nm	235.15 -238.27 nm	218.11nm	215.66  -218.78 nm
Beer’s law range (μg mL-1)			5-25	5-25	5-25	5-25
Regression Equation (y = mx + c)		Slope (m)	0.10175	0.10489	0.06505	0.1242
		Intercept (c)	0.01119	0.02469	0.00314	0.0489
Correlation coefficient			0.999	0.999	0.999	0.999
Precision (%R.S.D.)	Repeatability(n=6)		0.64	0.55	1.01	0.67
	Intra-day(3×5) times)		0.54	0.67	1.19	0.83
	Inter-day(3×5) days)		0.51	0.61	1.03	0.69
	Analyst		0.49	0.58	0.98	0.59
Formulation Analysis (%)Assay,  %RSD) n=6			98.98, 0.86	100.91, 0.98	99.89,1.03	101.07, 1.13

R.S.D. is Relative Standard Deviation.

 

 

Table 2: Results of Recovery studies of CLOP and ASP by the proposed methods

Formulation studies	Recovery Level	Recovery of	% Mean  Recovery,  % RSD by
			Method A	Method B
Formulation  (Tablet) n = 3	50%	CLOP	101.25, 0.57	99.12,   0.42
		ASP	99.50,   0.48	101.52,  0.89
	100%	CLOP	100.98,  0.55	102.12,  1.27
		ASP	100.09,  1.24	101.09,  0.97
	150%	CLOP	101.95,  0.59	102.32,  0.84
		ASP	102.45,  0.76	101.23,  0.48

 

 

Fig.4: Overlay spectrum of ASP (5-25µg mL^-1) and CLOP (5-25 µg mL^-1) in methanol/Distilled water.

 

FORMULATION ANALYSIS:

A quantity of powder from twenty tablets equivalent to 100 mg of CLOP (100 mg of ASP) was weighed and transferred to 100 mL flask containing 75 mL methanol and ultrasonicated for 10 min. The solution was filtered, filter was washed with methanol by adding washings to flask and volume was made up to mark with methanol. The solution was then suitably diluted with distilled water to have 15μg mL^-1 of CLOP. The proposed methods were then followed to determine concentration of analytes in the sample solutions.

 

RECOVERY METHOD:

The accuracy of the proposed methods was checked by recovery study, by addition of standard drug solution to preanalysed sample solution at three different concentration levels (50%, 100% and 150%) within the range of linearity for both the drugs. The basic concentration level of sample solution selected for spiking of the drugs standard solution was 7.5 μg mL^-1 of both analytes for both the methods.

 

PRECISION OF METHOD:

Repeatability of the methods was determined by repeating the procedure six times. To study intraday precision, methods were repeated 5 times in a day and the average % RSD was calculated. Similarly the methods were repeated on five different days and average % RSD was calculated. Method was repeated by another analyst working in the same laboratory to know the precision of analyst. The values confirm the intra and inter day precision.

 

RESULTS AND DISCUSSION:

The proposed methods for simultaneous estimation of CLOP and ASP in combined dosage form were found to be accurate, simple and rapid. The developed methods can be used for routine analysis of two drugs in combined dosage forms. Practically no interference from tablet excipients was observed in these methods. Both the methods are accurate, simple, rapid, precise, reliable, sensitive, reproducible and economical (Table 1). The values of % RSD For second order ratio derivative spectra (Tablet) were found to be < 2 (% RSD 0.49-1.19 and 0.55-0.83). The result of recovery studies for Tablet was found to be 99.50-102.45 for method A and 99.12-102.45 for method B (Table 2), indicates that there is no interference due to excipients present in the formulation. Thus the methods can be easily and conveniently adopted for routine quality control analysis.

 

CONCLUSION:

The proposed methods are simple, precise, accurate, economical and rapid for the determination of CLOP and ASP in combined tablet dosage forms. Analysis of authentic samples containing CLOP and ASP shows no interference from the common additives and excipients. Hence, recommended procedure is well suited for the assay and evaluation of drugs in pharmaceutical preparations. It can be easily and conveniently adopted for routine quality control analysis.

ACKNOWLEDGEMENT:

The authors are thankful Intas Pharmaceutical Ltd. Dehradhun, India and Vapi Pharmaceutical Ltd, Gujrat, India for providing gift samples of Clopidogrel and Aspirin, respectively. The authors are thankful to Management of MAEER’s Maharashtra Institute of Pharmacy, Pune for providing necessary facility for the work.

 

REFERENCES:

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Received on 26.05.2010       Modified on 23.08.2010

Accepted on 30.09.2010      © RJPT All right reserved