INTRODUCTION:

Clopidogrel bisulfate (CLO), is chemically Methyl 2-(2- Chlorophenyl) -2-(6, 7-dihydro thieno [3, 2-C] Pyridine-5(4H)-yl) Acetate sulphate (Fig. 1). It is an Anti-platelet agent as an ADP receptor blocker mainly to treat patients with the acute coronary syndrome, myocardial infarction (MI), peripheral vascular disease, and some stroke (Ischemic type) patients^1-2.

An extensive literature survey revealed that several HPLC methods were reported for the estimation of clopidogrel bisulfate in bulk and tablet formulation^3-14.

The International Conference on Harmonization (ICH) guideline entitled “Stability testing of new drug substances and products” requires that stress testing be administeredto elucidate the inherent stability characteristics of the active substance¹⁵. An ideal stability-indicating technique is one that resolves the drug and its degradation products efficiently. Consequently, the implementation of an analytical methodology to determine CLO, in the presence of its degradation products is sort ofa challenge for pharmaceutical analysts. Therefore, it was thought necessary to study the stability of CLO under acidic, alkaline, hydrolytic, oxidative, light, and thermal conditions. This paper reports validated stability-indicating HPLC method for the determination of CLO in the presence of their degradation products. The proposed method is simple, accurate, reproducible, stability-indicating, and suitable for routine determination of CLO in bulk and tablet formulations. The method was validated in compliance with ICH guidelines.

Fig. 1: Chemical structure of Clopidogrel

MATERIAL AND METHODS:

Chemicals and reagents:

Pharmaceutical grade Clopidogrel bisulfate was procured as a gift sample from Cadila Pharmaceuticals Ltd., Ahmedabad (India), Clopivas-75a tablet formulation, obtained commercially.

Methanol, orthophosphoric acid, hydrochloric acid, sodium hydroxide, and hydrogen peroxide 30% of analytical grade were used throughout the work.

Instrumentation:

The analysis was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) Shimadzu LC-6AD system equipped with a PDA detector with Lab Solution software were used.

Selection of Solvents:

Based on the solubility study, methanol was selected as the solvent for dissolving CLO.

Chromatographic Conditions:

Chromatographic separation was achieved on a reversed-phase Princeton (C18) column with dimensions (250×4.6 mm, 5μ) at ambient temperature using a mobile phase consisting of a mixture of buffer (pH 3.0, adjusted with orthophosphoric acid) and methanol in the ratio of (20:80) v/v at a flow rate of 1.0ml/min. Detection was carried out at 240nm. The pH of the mobile phase was set at 3.0, Injection volume was 10μl. The optimized chromatographic condition is shown in Table 1.

Table 1: Optimized chromatographic condition

Chromatographic condition

Mobile phase

Flow rate

Water (pH adjusted to 3.0 with orthophosphoric acid): Methanol

(20:80) v/v

1.0 ml/min.

Princeton C18 (250×4.6mm, 5μ)

240nm

30^oC

10 µl

20 minutes

Methanol

4.388minutes

Column

Detector wavelength

Column temperature

Injection volume

Runtime

Diluent

Retention time

Preparation of standard solution of CLO:

For CLO, an accurately weighed 7.5mg of CLO was transferred to a 10.0ml volumetric flask and dissolved in 5.0ml of methanol. The volume was completed to 10.0 ml with methanol. One milliliter ofthe resulting solution was pipetted in 10.0ml volumetric flask and the volume was made up to 10.0ml with methanol to furnish a solution of concentration 75μg/ml of CLO.

Preparation of sample solution of CLO:

Twenty tablets were weighed and finely powdered. An accurately weighed amount of powder equivalent to 7.5 mg of CLO was transferred into a 10.0ml volumetric flask. Then 5.0ml of methanol was added in it. The flask contents were sonicated for 10 min to make the contents homogeneous. This solution was then diluted up to the mark with methanol. The resultant solution was filtered through Whatman Grade I filter paper. One milliliter of the filtrate was transferred to a 10ml volumetric flask and then the volume was made up to the mark with methanol to furnish a sample solution containing 75μg/ mlof CLO. Six replicate of tablet powder equivalent to 7.5mg of CLO were transferred into six 10.0ml volumetric flask and homogenous sample solutions were prepared similarly.

Method Validation:

The developed method was validated following ICH guidelines (ICH Q2R1) for accuracy, precision, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), robustness.

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value, which is accepted either as a standard true value or an accepted reference value, and the value found. It was computed at three different levels, i.e., 80, 100, and 120% of the label claim. Standard addition and recovery experiments were conducted to determine the accuracy of CLO for the quantification of drugs in the sample.

Precision:

The system precision was evaluated by measuring the area of sixqualified working standards for CLO and calculating the percentage of relative standard deviation (RSD). The assay method precision was evaluated by conducting six independent assays of test samples of CLO against qualified working standards and calculating the percentage of relative standard deviation (RSD). The intermediate precision of the method was also verified using different analysts and different days.

Linearity:

Linearity test solutions of CLO were prepared at concentration levels of 45-120µg/ml respectively. Linearity test solutions were prepared by diluting the stock solution to the required concentrations. Linearity was established by the least-squares linear regression analysis of the calibration data. Peak areas were plotted against the respective concentrations and linear regression analysis performed on the resulting curves. The linear curve of clopidogrel bisulfate was shown in Fig. 2.

Specificity:

The specificity of the developed method was established by comparing the chromatograph ofthe standard and sample. It was found that there was no interference due to excipients and impurities at the retention time of the drug.

LOD and LOQ:

The LOD is the lowest analyte concentration that can be detected. LOQ is the lowest analyte concentration that can be quantified with acceptable accuracy and precision. The limits of detection (LOD) and quantification (LOQ) were calculated from the standard deviation of the response and the slope of the calibration plot. LOD and LOQ were established, under ICH definitions, by use of the equations LOD = 3.3σ/S and LOQ = 10σ/S, where σ is the standard deviation of the regression line and S is the slope of the calibration plot.

Robustness:

To evaluate the robustness of the developed method, the chromatographic conditions were deliberately altered and the resolution for CLO was evaluated. To study the effect of wavelength on the estimation, the wavelength was altered by ± 2 nm, i.e., 238 and 242nm from the actual wavelength, 240nm. To study the effect of flow rate on estimation, the flow rate was altered by ± 0.1 ml/min i.e., 0.9 and 1.1ml/min from the actual flow rate, 1.0ml/min.

Stability of the solution:

The stability of both standard and sample solutions was checked up to 24 hr. at room temperature.

Stability-indicating assay:

The drug was subjected to acidic (0.1N HCl), alkaline (0.1N NaOH), oxidative (30% H₂O₂), photo (light), thermal (sand bath at 50°C), and hydrolytic (water) condition and the percentage degradationwere calculated.

RESULTS AND DISCUSSION:

HPLC method development and optimization:

Initially, pure drugs solution was chromatographed usinga mobile phase consisting of a mixture of buffer (pH 3.0, adjusted with orthophosphoric acid) and methanol in the ratio of (20:80) v/v at a flow rate of 1.0 ml/min.gives well-resolved peaks of drugs as well. Detection was carried out at 240nm.The retention time under the optimized condition of clopidogrel bisulfate was found to be 4.388 minutes. The total run time of thechromatogram was about 20 minutes. A typical chromatograph of standard and sample of clopidogrel bisulfate is summarized by Fig. 3 and Fig. 4.

Fig. 2: Linear curve clopidogrel bisulfate (CLO)

Validation of the method:

System Suitability:

The suitability of the system was demonstrated by assessing various parameters. It was established by injecting six replicate injections of the standard solution. Theoretical plates were found to be 3811, tailing factor of 1.54, and %RSD of peak area was 0.8 for CLO respectively (Table 2). All the system suitability parameters were well within the limits, indicating that the system was well suitable for performing the analysis.

Table 2: System suitability results

Parameter

CLO

Theoretical Plate

Retention Time (Rt)

Tailing factor

% RSD

3811

4.388

1.54

0.8

Rt: Retention time, %RSD: Percentage relative standard deviation

Linearity:

Linearity was established bythe least-squares linear regression analysis of the calibration data. Calibration plots were linear over the concentration range 45-120µg/ml for CLO. Peak areas were plotted against the respective concentrations and linear regression analysis performed on the resulting curves. The equation for the calibration plots of CLO was Y=7103.4x+6366with a correlation coefficient of 0.9996. The results of linearity are shown in Table 3.

Table 3: Linearity results

Parameter

CLO

Concentration Range (µg/ml)

Slope (m)

Intercept

Coefficient correlation (r²)

45-120

7103.4

6366

0.9996

Accuracy:

The percentage recoveries were 99.72-101.09% for CLO. The %RSD value was found to be <2%. The results of recovery are shown in Table 4.

Table 4: Recovery results

Drug

Level

(%)

Amount taken (µg/ml)

Amount found* (µg/ml)

% Recovery*

CLO

100

120

59.83

75.81

90.75

99.72

101.09

100.83

*Average of three determinations

Precision:

The result of intraday and interday for CLO wasfound to be 0.8 and 0.4 respectively. The percentage RSD of system, method, and intermediate precision study was well within ± 2.0%, indicate that the method was precise.

LOD and LOQ:

The LOD and LOQ of CLO were found to be 6.90 µg/ml. and 20.91µg/ml. respectively.

Robustness:

Table 5: Robustness results

Condition		CLO
Condition		*Amount estimated [%]**	RSD [%]
Change in wavelength (240±2 nm)	238 nm	100.16	0.0900
Change in wavelength (240±2 nm)	242 nm	100.08	0.1417
Change in flow rate (1.0±0.1 ml/min.)	0.9 ml/min.	99.37	0.2068
Change in flow rate (1.0±0.1 ml/min.)	1.1 ml/min.	99.56	0.2009

*Average of three determinations, %RSD: Percentage relative standard deviation

Analysis of clopidogrel bisulfate from marketed tablets:

The percentage assay of tablet formulation was found to be 99.92% for CLO. The stability of the drug solutions was observed for 24 h. %RSD of 0.8 indicates the stability of the method for 24 h. In degradation studies, the drug was exposed to various stress conditions. From the chromatograms of stressed samples, it was found that no interference from degradants was observed at the retention time of clopidogrel bisulfate. Optimum degradation was observed in the presence of acid and alkali. No degradation was observed in the presence of water, peroxide, light, and heat for CLO. The results of the percentageof degradation are presented in Table 6 and Fig. 5-10. Hence, the method was found to be specific.

Table 6: Stability-indicating method data for CLO

Stress condition

CLO (%Degradation)

Acidic (0.1 N HCl for 24 hrs.)

Alkaline (0.1 N NaOH for 24 hrs.)

Hydrolytic (HPLC waters for 24 hrs.)

Oxidative (30% H₂O₂ for 24 hrs.)

Photo (Sun light for 30 days)

Thermal (Sand bath at 50^oC for 24 hrs.)

8.26

6.24

No degradation

HPLC: High Performance Liquid Chromatography.

Table 7: Summary of validation parameter

Parameter

CLO

Calibration range (µg/ml)

Optimized wavelength (nm)

Retention Time

45-120

240

4.388

Regression equation (Y)

Slope

Intercept

Coefficient correlation (r²)

Precision (% RSD)

Intraday

Interday

% Assay*

LOD (µg/ml)

LOQ (µg/ml)

Y = 7103.4x+6366

7103.4

6366

0.9996

0.8

0.4

99.92

6.90

20.91

*Average of five determinations, LOD: Limit of detection, LOQ: Limit of quantification

CONCLUSION:

The method enables simple, rapid, accurate, precise, specific, economical, and sensitive analysis of clopidogrel bisulfate in bulk and tablet dosage form. This method was validated under ICH guidelines. The method can, therefore, be used for routine qualitycontrol analysis clopidogrel bisulfate in bulk and tablet dosage form.

ACKNOWLEDGMENTS:

The authors extend their sincere thanks to Cadila Pharmaceuticals Ltd, Ahmadabad (India), for providing gift sample of pure clopidogrel bisulfate. We also extend our thanks to the Head of Department, Department of Pharmaceutical Sciences; RTM Nagpur University, Nagpur for providing the necessary facilities.

CONFLICT OF INTEREST:

Authors declare that they have no conflict of interest exists in this investigation.

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Received on 27.04.2020 Revised on 30.05.2020

Asian J. Pharm. Res. 2020; 10(4):253-259.

DOI: 10.5958/2231-5691.2020.00044.1