Optimized and Validated RP-HPLC Method for the Determination of Clopidogrel in Bulk and Pharmaceutical Formulation

This study was undertaken to develop a novel, simple, rapid, accurate and precise sensitive reverse phase HPLC method for estimating Clopidogrel in bulk and pharmaceutical dosage form. The current method in this study was achieved by Thermo Hypersil RP C-18 column (100 mm x 4.6 mm, 3.5 μm) using a mobile phase of Phosphate Buffer: Acetonitrile (pH 3.0) is 70: 30 at a column temperature of 25°C. The effluent was monitored by UV detector at 238 nm. The retention time of Clopidogrel was 4.75 min with a flow rate 1.0 mL/min. Calibration curve was linear in the range of 10-60 μg/mL. The method was validated for linearity, precision, robustness and accuracy as per ICH guidelines. The results of all the validation parameters were well within their acceptance values (%RSD <2.0 specified by the USP, ICH and FDA), which prove applicability of the proposed method for routine analyses and quality-control assay of Clopidogrel in pharmaceutical preparations.


Introduction
Clopidogrel is a thienopyridine class of antiplatelet agent, mainly used to prevent blood clots in coronary artery disease, peripheral vascular disease, and cerebrovascular disease.It acts by inhibiting adenosine diphosphate (ADP) induced platelet aggregation and direct inhibition of ADP binding to its receptor and of subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex [1].Clopidogrel was officially listed in the United States of Pharmacopeia (USP) in 2007.Chemically, it is designated as (+)-α-(2chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetic acid methyl ester sulfate with the molecular formula of C 16 H 16 ClNO 2 S (Fig. 1) [2].It is available in the market as tablets dosage form.A detailed literature survey revealed that there is no official method available for the determination of Clopidogrel in pharmaceutical dosage forms but various assays for determining Clopidogrel in pharmaceutical dosage forms, including chemometry [3], spectrophotometry [4], thin-layer chromatography (TLC) [5], high-performance thin-layer chromatography (HPTLC) [6], high-performance liquid chromatography (HPLC) [7][8][9][10], mass spectrometry [11], and voltammetry [12].However, in this study the aim has been designed to develop a new more accurate, simple, precise, reproducible, sensitive reverse phase HPLC method for the determination of Clopidogrel in bulk and tablet dosage forms.This method has been optimized and validated as per as the ICH guidelines.

Materials
Clopidogrel was obtained as a generous sample from Eskayef Bangladesh Limited.HPLC grade acetonitrile was procured from the Active Fine Chemicals Ltd., Bangladesh.Water for Injection (WFI) was obtained as a gift from Globe Pharmaceutical Ltd., Bangladesh.All chemicals and reagents used were of analytical grade in the present study.Anclog (Brand name), commercially available Clopidogrel tablet manufactured by Square Pharmaceutical Ltd. was purchased from our local market.

Instrumentations
The analysis of drugs was carried out on HPLC system (Shimadzu, Japan) on a C18 column with a UV-visible detector.A 20µL syringe was used for injecting the samples.A double-beam Shimadzu UV-1800 visible spectrophotometer was used for spectral studies.Degassing of the mobile phase was done by using an ultrasonic bath sonicator.An Axis balance was used for weighing the materials.

Chromatographic conditions
The mobile phase consisting of sodium dihydrogen phosphate buffer (pH 3.0) and acetonitrile in the ratio 70:30 v/v was filtered through 0.45 µm membrane filter before use, degassed and pumped from the solvent reservoir into the column at a flow rate of 1.0 mL/min.The detection was monitored at 238 nm, and the run time was 4.75 min.The volume of the injection loop was 10 µL and prior to the injection of the drug solution.The column and the HPLC system were kept at 25C.

Preparation of standard solution
About 25 mg of Clopidogrel was accurately weighed and transferred into a 50 mL volumetric flask.Then the reagent methanol was added to make up the volume up to the mark and the mixture was sonicated for 10 min.After filtration 2 mL of this solution was taken into another clean and dry 50 mL volumetric flask and then diluted up to the mark using methanol.

Preparation of sample solution
Ten tablets were weight accurately and powdered.A quantity of tablet powder equivalent to 25 mg of Clopidogrel was accurately weighed and transferred to a clean and dry 50 mL volumetric flask.After this the methanol was added to make up the volume up to the mark and then the mixture was sonicated for 10 min.The solution was filtered through Whatman No. 42 filter paper.After filtration 2 mL of this solution was taken into another clean and dry 50 mL volumetric flask and diluted up to the mark with methanol.

Method validation
The proposed method was validated for linearity, limit of detection, limit of quantification, precision, and accuracy as per International Conference on Harmonization (ICH) guidelines [13,14].

Linearity
The linearity of an analytical procedure is its ability of producing results that are directly proportional to the concentrations of an analyte in the samples.The determination was repeated three times at each concentration (10-60 µg/mL) level.The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method.

Limit of detection (LOD)
Limit of detection (LOD) is defined as the lowest concentration of analyte that gives a detectable response.LOD was determined by the analysis of samples with known concentration of analyte and by establishing the minimum level at which the analyte can be reliably detected.LOD was calculated using the following equation [15].LOD = 3.3 × S o /b, where S o and b are the standard deviation of the response and the slope of the calibration curve.

Limit of quantification (LOQ)
Limit of quantification (LOQ) is defined as the lowest concentration that can be quantified reliably with a specified level of accuracy and precision.LOQ was determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte could be quantified with acceptable accuracy and precision.LOQ was calculated using the following equations [15].LOQ = 10 × S o /b, where S o and b are the standard deviation of the response and the slope of the calibration curve.

Precision
Precision was done by repeatability or intra-assay precision and intermediate precision.Repeatability studies were performed by analysis of concentration of 20 μg/mL six times on the same day.Intermediate precision was determined by repeating the same procedure by another analyst working in the laboratory.

Accuracy
The accuracy of the developed method was evaluated by determination of recovery at three different concentrations, equivalent to 80%, 100%, and 120% of the amount in the pre-analysed dosage form as ICH guidelines and average recoveries were calculated.Triplicated injections were made for each concentration.

Robustness
To determine the robustness of the developed method, experimental conditions were purposely altered.The flow rate of the mobile was 1 mL/min.To study the effect of flow rate on the resolution, it was changed by 0.8 mL/min.The effect of the column temperature on resolution was studied at 30ºC instead of 25ºC.

Results and Discussion
To develop a suitable and robust HPLC method for the determination of Clopidogrel, different mobile phases were employed in this study to achieve the best separation and resolution.Finally, the final mobile phase was selected phosphate buffer with pH 3.0 and acetonitrile in the ratio of 70:30 (v/v).The peak shape was good at wave-length of 238 nm with flow rate of 1 mL/min.Clopidogrel also shows significant UV absorbance at wavelength of 238 nm.Hence, this wavelength has been chosen for detection in analysis of Clopidogrel.The retention of Clopidogrel was found in 4.752 min.(Fig. 2).A linear relationship was obtained between the concentrations of Clopidogrel in the range of 10-60 μg/mL and the respective ratio of peak areas with a correlation coefficient (r 2 ) of 0.997, indicating good linearity with representative linear equation of y = 348.07x+248610 (Fig. 3).The limit of detection was found 1.33 μg/mL while the limit of quantification was 3.86 μg/mL (Table 1).The precision of the RP-HPLC method was validated by studying repeatability and intermediate precision.The obtained precision data and recovery studies data are presented in Tables 2-3, respectively.In both cases, %RSD shows are not more than 2.0% which indicated good repeatability and intermediate precision as well as the result of recovery study reveal that any small change in the drug concentration in the solution could be accurately determined by the proposed methods.Few parameters of the proposed method were deliberately changed to check the robustness of the method.The parameters included variation of flow rate and temperature.The changed flow rate and temperature was 0.8 mL/min.and 30C in lieu of 1.0 mL/min.and 25C, respectively.The method was found to be robust enough by basis of %RSD value (Table 4).

Table 1 .
Linearity and range test of the developed RP-HPLC method for the determination of Clopidogrel in the pharmaceutical formulation.
SEM: Standard error of mean; RSD: Relative standard deviation

Table 3 .
Result of recovery data study of Clopidogrel (Accuracy).