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RESEARCH ARTICLE

A Novel Stability Indicating HPLC Assay Method for Simultaneous Estimation of Clopidogrel and Aspirin in Tablet dosage form by Using Core shell Technology column

Mahesh HRK*, Dr K Sudhakar Babu

Department of Chemistry, Sri Krishnadevaraya University, Anantapur -515055, Andhra Pradesh. India

*Corresponding Author E-mail: hrkmahesh@gmail.com

ABSTRACT:

A novel, rapid and a stability-indicating reverse phase LC method has been developed and validated for the simultaneous estimation of Clopidogrel and Aspirin in tablet dosage form. The chromatographic separation was achieved on a novel core shell technology phenyl hexyl stationary phase of particle size 2.6µ. The method employed a linear gradient elution and the detection wavelength was set at 220nm. The proposed method was extensively validated as per ICH guidelines. The specificity of the method was proved by performing forced degradation studies. Regression analysis shows r value (correlation coefficient) of greater than 0.999 for individual active drug substances. Accuracy was proved in the range of 50% to 150% of working range.

KEYWORDS: Clopidogrel, Aspirin, Core shell, Simultaneous estimation and Stability indicating.

INTRODUCTION:

Clopidogrel bisulfate (CLP) is chemically designated as thieno [3, 2-c] pyridine-5(4H)-acetic acid, alpha-(2 chlorophenyl)-6, 7-dihydro-methyl ester(S)-sulphate (1:1) [Figure 1] an ADP antagonist. It is used as an anti thrombic agent¹.

Aspirin (ASP) is chemically designated as 2-acetyloxybenzoic acid [Figure 2] is often used as an analgesic, antipyretic, anti-inflammatory and antiplatelet. It suppresses the production of prostaglandins and thromboanes due to inactivation of cyclooxygenas enzyme ².

Different dosage forms of CLP and ASP are available as single or combination dosage forms. The literature survey revealed several analytical methods such as spectrometry, HPTLC, HPLC and LC-MS have been reported for the determination of CLP and ASP in pharmaceutical dosage forms, individually or in combination and in biological samples ^1-17. To our present knowledge, there is no method reported for the simultaneous estimation of CLP and ASP in formulation by using a stability indicating HPLC method with core shell technology column.

Received on 17.12.2014 Modified on 05.01.2015

Research J. Pharm. and Tech. 8(2): Feb. 2015; Page 172-176

DOI: 10.5958/0974-360X.2015.00031.1

Hence the aim was to develop a sensitive, simple, accurate, stability indicating assay method by using a new breakthrough in column technology i.e. core shell technology.

Columns packed with sub-2 micron, totally porous particles are very wide spread nowadays to conduct fast and efficient separation. However sub-2 micron has few limitations with respect to instrumentation. To overcome this limitation, but by not compromising on fast and efficient separation core shell technology columns with sub-3 microns are the better solution. These columns can be effectively used in regular HPLC instruments also.

In the present work forced degradation study with peak purity concept and confirming the separation of known impurities from the analytes was used to prove the stability indicating power of the assay method.


Figure : 1Chemical structure of Clopidogrel	Figure: 2 Chemical structure of Aspirin

MATERIALS AND METHODS:

Chemicals and reagents:

All the reagents were of AR or HPLC grade unless stated otherwise. HPLC grade water was utilized throughout the experiment. Potassium dihydrogen phosphate, Diammonium hydrogen phosphate and octane -1 sulfonic acid sodium salt all these chemicals from Merck India, Methanol and Acetonitrile were taken from Rankem India were used. CLP Standard, ASP standard, and tablet dosage form were obtained from Hetero Labs Ltd (Hyderabad, India).

Equipment:

High performance Liquid chromatography system (from Waters) with, auto sampler and with photo diode array (PDA) detector was used for the study. Data was collected and processed by using Waters Empower software.

Chromatographic Conditions:

The analysis was carried out on Kinetex phenyl hexyl, 100x4.6 mm, 2.6mm particle size. The column Oven temperature was maintained at 35°C. The mobile phase A consists of diammonium hydrogen phosphate salt (1.32gm per Liter), and octane -1 sulphonic sodium salt (2gm per Liter), pH adjusted to 2.3±0.05. A mixture of Acetonitrile and Methanol in the ratio of 50:50 was used as Mobile phase B. Flow rate was set to 1.0 mL/minute in gradient elution mode. Gradient time program was set as T/%B: 0/30, 2/45, 4/55, 10/55, 11/30 and 15/30. Before delivering the mobile phase into the system, it was degassed and filtered through 0.22 µm filters using vacuum. The injection volume was 10µL and the detection was performed at 220nm using PDA detector. The typical retention times of ASP and CLP are 2.6 minutes and 8 minutes respectively in the final optimized conditions [Figure 3]. The criticality of this method are to elute both the active ingredients with optimum separation and symmetric peak shapes with no interference due to placebo or any known and potential impurities arising due to degradation during shelf life.

Figure: 3 Typical Chromatogram of Standard.

Sample Preparation:

Diluent Preparation:

1.36 g of potassium dihydrogen phosphate was dissolved in 1000 mL water and pH adjusted to 2.5±0.05. Prepared a degassed mixture of this buffer and acetonitrile in the ratio of 55:45 %v/v respectively.

Standard Preparation:

Standard Stock Solution was prepared by weighing accurately 49 mg of CLP and 50 mg ASP standard in 100 ml volumetric flask, added about 60 mL diluent ,sonicated to dissolve and diluted to volume with diluent. Working Standard solution was prepared by diluting 5 ml of stock solution to 50 ml with diluent and mixed well.

Test Preparation:

Crushed 10 tablets (Hetero labs Ltd.) into a fine powder, transferred powder equivalent to 50 mg ASP in to a 100ml volumetric flask, added about 60 ml diluent and sonicated for not less than 30 minutes with intermediate shaking. Care was taken during sonication to avoid the heating. Diluted to volume with diluent and mixed well. Filtered a portion of solution through 0.45µm membrane filters.

Further diluted 5.0 mL of this solution into a 50 mL volumetric flask, diluted to volume with diluent and mixed well.

Experimental Design:

Method Validation:

The aim of method validation was to confirm that the present method was suitable for its intended purpose as described in ICH guidelines ¹⁸. The described method has been extensively validated in terms of specificity, precision, linearity, accuracy and robustness. The precision was expressed in terms of coefficient of variation (RSD) calculated from % assay results. The accuracy was expressed in terms of percent recovery of the known amount of analytes added to the placebo.

RESULTS AND DISCUSSIONS:

System suitability:

System suitability tests are an integral part of a liquid chromatographic method. As integral part of chromatographic method system suitability parameters like USP Tailing, Theoretical plates and Relative standard deviation (RSD) for replicate injections were evaluated and found to be satisfactory as per common chromatographic practices. According to the results presented, the proposed method fulfills these requirements within the accepted limits Results are shown in Table No 1.

Table 1: Results of System Suitability Test

Name of Drug substances	Theoretical plates	USP Tailing factor	%RSD for replicate injections
Aspirin	6306	1.1	0.1
Clopidogrel	30881	1.0	0.1

Figure 4: Typical Chromatogram of Spiked sample with Impurities

Table 2: Results of Forced degradation Studies with Peak purity details

Stress Conditions	Aspirin		Clopidogrel
Stress Conditions	PA	PT	PA	PT
Acid Degradation	0.201	0.359	0.191	0.392
Alkali Degradation	0.152	0.334	0.167	0.369
Peroxide Degradation	0.214	0.331	0.181	0.377

PA = Purity Angle, PT= Purity Threshold

Note: Purity Angle should be less than Purity Threshold to meet Peak purity criteria acceptance criteria

Linearity:

The linearity of the method was tested in order to demonstrate proportional relationship of detector response versus analyte concentration over the working range. It is usual practice to perform linearity experiments over a wide range of analyte. This gives confidence that the detector response and concentration are proportional and consequently ensures that calculations can be performed using a single reference standard/working standard, rather than the equation of a calibration line.

The linearity of detector response to different concentrations of both active ingredients was studied by preparing a series of solutions using CLP and ASP standards. The data were subjected to statistical analysis using a linear-regression model. The results have indicated good linearity. Results are shown in Table No 3.

Table 3: Results of Linearity Studies (Response Vs Concentration)

Name of Drug substances	Correlation coefficient	Slope	Intercept
Aspirin	0.999	21573	438.4
Clopidogrel	0.999	21830	4476.5

Precision:

Six sample solutions were prepared using single sample Lot of CLP and ASP tablets and the precision of the method was tested. The % relative standard deviation (RSD) indicates that proposed method has got an acceptable level of repeatability. Results indicate the proposed method has got a good precision. Results are tabulated in Table No 4.

Table 4: Precision data

Active Name	Mean % Assay	%RSD
Aspirin	101.4	0.2
Clopidogrel	100.8	0.2

N= six sample preparations

Accuracy:

Accuracy of the proposed method was established by recovery experiments. This study was employed by spiking of known amounts of active ingredients into the placebo samples of at 50%, 100% and 150% of targeted concentration, in triplicate and injected into the chromatographic system. The resulting mixtures were analyzed as described in proposed method. Results obtained from recovery studies are given in Table No 5.

Table 5: Results of Recovery Study at Different Levels

Name of Drug substances	50% level	100% level	150% level
Aspirin	100.5	100.5	100.3
Clopidogrel	100.5	100.2	100.5

Note: Number of samples analyzed at each level is in triplicate

Robustness:

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate, variations in method parameters, and provides an indication of its reliability during normal usage. In the present study, an experimental design was planned for robustness testing varying some conditions, e.g. flow rate, column temperature and variation of buffer pH in the mobile phase. It can be seen that, with every employed condition, there were no dramatic changes in the chromatographic behavior. All parameters have been observed within the limits required for system suitability tests. The results are shown in Table No 6.

Solution stability:

Solution Stability studies were performed for Standards and test spiked solution for about 24 hours at 5°C.Results from this study were compared against initial standard area and % Assay for test solution. Data indicates that both standard and test solutions are stable at 5°C for about 24 hours with acceptance criteria of ± 2.0 for both test and standard solution. Results obtained from solution stability studies are given in Table No 7.

CONCLUSION:

The validated stability-indicating HPLC method has proved to be simple, accurate, precise and reliable. The developed method reported herein was validated by evaluation of the validation parameters as described in ICH guidelines. System suitability, specificity, linearity, precision, accuracy and robustness of the proposed technique were obtained during the validation studies. The developed method is also stability-indicating and can be used for the routine analysis of combined tablet dosage form of Clopidogrel and Aspirin also check the purity and stability of the active substance in pharmaceutical dosage forms.

ACKNOWLEDGEMENT:

The authors are thankful to Hetero Labs Ltd, Hyderabad for providing the necessary facilities to complete this research.

Table 6: Results of Robustness Study

Parameter	Deliberate change	Theoretical plates		Tailing factor
Parameter	Deliberate change	Aspirin	Clopidogrel	Aspirin	Clopidogrel
Flow rate (1.0mL/min)	0.8mL/min	7523	45220	1.1	1.02
Flow rate (1.0mL/min)	1.2mL/min	5503	27564	1.05	1.01
Temperature (35°C)	30°C	7502	43205	1.05	1.02
Temperature (35°C)	40°C	5003	28034	1.05	1.01
pH of buffer (2.3)	2.1	6552	40220	1.05	1.02
pH of buffer (2.3)	2.5	4856	28502	1.10	1.01

Table 7: Results of Solution Stability Studies

Name	Initial	After 24 Hours	Difference from the Initial
Area of Standard for Aspirin	1088036	1100018	2.2%
Area of Standard for Clopidogrel	826193	825884	4.8%
% of Aspirin Assay in Test	101.5	101.5	0.0
% of Clopidogrel Assay in Test	100.7	101	0.3

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