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Page 1: International Journal of Research Pharmaceutical and …ijrpns.com/article/METHOD DEVELOPMENT AND VALIDATION OF... · Syeda Bushra Yunus. et al. / International Journal of Research

Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.

Available online: www.uptodateresearchpublication.com May - June 365

Research Article CODEX: IJRPJK ISSN: 2319 – 9563

METHOD DEVELOPMENT AND VALIDATION OF SPECTROPHOTOME TRIC METHOD FOR THE ESTIMATION OF CLOPIDOGREL BISULPHATE IN PURE

AND TABLET DOSAGE FORM

Syeda Bushra Yunus*1, C. Karuppasamy1, Syeda Sheeba Ahamadi1, P. Dilip Kumar 1, G. Madhu Kumar 1, C. Nagaraju1

1*Department of Pharmaceutical Analysis, Creative Educational Society’s College of Pharmacy, Kurnool, Andhra Pradesh, India.

INTRODUCTION Clopidogrel bisulphate is a potent oral antiplatelet gent used in treatment of coronary artery disease. Chemically it is Methyl (2s)-2-(2-chlorophenyl)-2-(6, 7-dihydro-4H-thieno [3,2-c]pyridine-5-yl)acetate. It has a molecular formula of C16H16 ClNO2S and a molecular weight of 419.9g / mol and its structure was given in (Figure No.1). Literature survey revealed, few analytical methods which include A Validated stability indicating HPTLC method for determination of Clopidogrel bisulphate in combined dosage form1, Determination of Clopidogrel bisulphate by Chromatography2,

ABSTRACT A simple, accurate, precise and sensitive Spectrophotometric method was developed for the estimation of Clopidogrel bisulphate in pure and tablet dosage forms. The estimation of Clopidogrel bisulphate was carried out at maximum absorbance of 220 nm. The method was found to be linear and obeys Beer’s law in the concentration range of 1-14 mcg / ml with a correlation coefficient 0.998. The developed method was validated as per ICH guidelines and was found to be accurate and precise. Thus the proposed method can be successfully applied for the estimation of Clopidogrel bisulphate in pure and tablet dosage forms. KEYWORDS Spectrophotometric, Clopidogrel bisulphate, Validation and ICH guidelines.

Author for Correspondence:

Syeda Bushra Yunus,

Department of Pharmaceutical Analysis,

Creative Educational Society’s College of Pharmacy,

Kurnool, Andhra Pradesh, India.

Email: [email protected].

International Journal of Research

in Pharmaceutical and Nano Sciences

Journal homepage: www.ijrpns.com

Page 2: International Journal of Research Pharmaceutical and …ijrpns.com/article/METHOD DEVELOPMENT AND VALIDATION OF... · Syeda Bushra Yunus. et al. / International Journal of Research

Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.

Available online: www.uptodateresearchpublication.com May - June 366

Stability indicating HPTLC Determination of Clopidogrel bisulphate in formulation3, Determination of Clopidogrel bisulphate and its impurity by TLC Method4, Validated HPLC Method for the Estimation of Clopidogrel bisulphate and Aspirin in combined tablet Formulation5, Simultaneous Determination on of Clopidogrel bisulphate and Aspirin in tablet formulation by HPLC and HPTLC6. The present work deals with estimation of Clopidogrel bisulphate in pure API and tablets by UV-Spectrophotometry. MATERIALS AND METHOD Authentic drug sample of Clopidogrel bisulphate was given as a gift sample by Aurobindo Pharma Ltd, Hyderabad. Tablets of Clopidogrel bisulphate were procured from local pharmacy. Distilled water was used for the preparation of solutions. Instrument Labindia - 3000+ UV / Vis double beam Spectrophotometer with a fixed slit width (2 nm) and 10 millimeter quartz cell was used to obtain spectrum and absorbance measurement. Preparation of stock solution 100 mg of standard Clopidogrel bisulphate drug was weighed, transferred to a 100 ml volumetric flask and dissolved in distilled water. The flask was shaken and volume was made up to the mark with distilled water to give a solution containing 1000 μg / ml. Determination of Absorbance Maxima From above stock solution 10ml is taken in 100ml volumetric flask and made upto 100ml to give 100µg/ml concentration and further dilution done and was subjected for scanning between 200-400nm. Effect of dilution on the absorbance maxima was studied by diluting above solution to 1-14µg/ml and scanned from 200-400 nm, shown in Figure No.2. For the standard solution analytical concentration range was found to be 1-14μg / ml and those values were reported in Table No.1. Appropriate volume of aliquots from standard Clopidogrel bisulphate stock solutions were transferred to different volumetric flasks of 10 ml capacity. The volume was adjusted to the mark with

distilled water to obtain concentrations of 1,2,3,4,5,6,8,10,12,14μg / ml. Absorbance spectra of each solution against distilled water as blank were measured at 220 nm and the graphs of absorbance against concentration was plotted and are shown in Figure No.2. The regression equation and correlation coefficient was determined. ANALYSIS OF MARKETED FORMULATION Accurately weigh 20 tablets of Clopidogrel bisulphate and finely powdered. The powder equivalent to 100mg of Clopidogrel bisulphate was transferred into 100ml volumetric flask and 20 ml of distilled water added and sonicated for 15 min to dissolve the Clopidogrel bisulphate in it and made volume to mark with distilled water. 10 ml of this diluted with distilled water. The concentration of Clopidogrel bisulphate in marketed formulation was determined in Table No.2. To examine the absence of either positive or negative interference of excipients used in formulation, recovery studies were carried out. Accuracy was determined by recovery studies. The recovery studies were carried out by adding the known amount of Standard Clopidogrel bisulphate drug to the sample solution of the tablets. Precision for assay were determined by repeatability, interday, intraday precision for drug (each in three replicate). Limit of Detection (LOD) and limit of Quantitation (LOQ) The LOD and LOQ of Clopidogrel bisulphate are determined by using calibration standards. Value of LOD is determined by using the formula: 3.3ơ/S And the value of LOQ is determined by: 10ơ/S, where ‘ơ ‘ is the standard deviation of the y intercept of the regression equation and ‘S’ is the slope of calibration curve. RESULTS AND DISCUSSION The absorption spectral analysis shows the λmax of Clopidogrel bisulphate at 220 nm (Figure No.3). The calibration curve was obtained for a series of concentration in the range of 1-14mcg/ml. It was found to be linear and hence, suitable for the estimation of the drug. The slope, intercept,

Page 3: International Journal of Research Pharmaceutical and …ijrpns.com/article/METHOD DEVELOPMENT AND VALIDATION OF... · Syeda Bushra Yunus. et al. / International Journal of Research

Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.

Available online: www.uptodateresearchpublication.com May - June 367

correlation coefficient and optical characteristics are summarized in Table No.2. Regression analysis of Beer’s law plot revealed a good correlation. The effects of various excipients generally present in the tablet dosage form of Clopidogrel bisulphate were investigated. The results indicated that they did not interfere in the assay in amounts far in excess of their normal occurrence in it. The proposed method was validated as per the ICH guidelines7,8. The precision was measured in terms of repeatability, which was determined by sufficient number of aliquots of a homogenous sample. The % RSD was found to be within the limits. This showed that the precision of the method is satisfactory. The recovery technique was performed to study the accuracy and

reproducibility of the proposed method. For this, known quantities of the Clopidogrel bisulphate solution was mixed with definite amounts of pre-analyzed formulations and the mixtures were analyzed. The total amount of Clopidogrel bisulphate was determined by using the proposed methods and the amount of added drug was calculated by the difference. The % RSD was less than ± 2.0 (Table No.3). This showed that the recoveries of Clopidogrel bisulphate by the proposed methods are satisfactory and the results are shown in Table No.4 and 5. Limit of detection (LOD) and Limit of quantitation (LOQ) were determined by the proposed method.

Table No.1: Results of Clopidogrel bisulphate calibration curve at 220 nm for by UV Spectroscopy

S.No Concentration

(mcg / ml)

Absorbance at

220 nm

1 1 0.035

2 2 0.068

3 3 0.098

4 4 0.135

5 5 0.165

6 6 0.215

7 8 0.290

8 10 0.376

9 12 0.451

10 14 0.532

Page 4: International Journal of Research Pharmaceutical and …ijrpns.com/article/METHOD DEVELOPMENT AND VALIDATION OF... · Syeda Bushra Yunus. et al. / International Journal of Research

Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.

Available online: www.uptodateresearchpublication.com May - June 368

Table No.2: Shows Optical Characteristics of Clopidogrel bisulphate

*Y=mx+c where X is the concentration of Clopidogrel bisulphate in mcg/ml and Y is the absorbance at the respective λmax. **Average of six determinations.

Table No.3: Determination of Precision results for Clopidogrel bisulphate at 220 nm

S.No Conc

mcg / ml

Inter-day

Absorbance Mean ± SD** % RSD

Intra-day

Absorbance Mean ± SD** %

RSD

1 LQC

(3mcg/ml) 0.162±0.000516 0.318 0.098±0.000516 0.526

2 MQC

(6mcg/ml) 0.099±0.0010 1.01 0.215±0.00154 0.716

3 HQC

(12mcg/ml) 0.455±0.0060 1.31 0.451±0.00206 0.456

**Average of six determinations.

Table No.4: Assay of Clopidogrel bisulphate formulation

S.No Formulation

Label claim

(mg/tab)

Amount found (mg)

(n=3)

Mean ± SD

Assay %RSD

1 Clopilet 75 75.009±0.075 98.73 % 1.002

S.No Parameter UV method

1 λmax (nm) 220

2 Beer’s law limits (mcg / ml) 1-14

3 Regression equation (Y*) Y = 0.038x+0.015

4 Slope (m) 0.038

5 Intercept (c) 0.015

6 Correlation coefficient(r2) 0.998

7 % RSD** < 2%

8 Limit of detection (mcg / ml) 0.0151

9 Limit of quantitation (mcg / ml) 0.0457

Page 5: International Journal of Research Pharmaceutical and …ijrpns.com/article/METHOD DEVELOPMENT AND VALIDATION OF... · Syeda Bushra Yunus. et al. / International Journal of Research

Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.

Available online: www.uptodateresearchpublication.com May - June 369

Table No.5: Determination of Accuracy results for Clopidogrel bisulphate at 220 nm

Figure No.1: Structure of Clopidogrel Bisulphate

Figure No.2: Calibration curve of Clopidogrel bisulphate

y = 0.038x + 0.015

R² = 0.998

0

0.1

0.2

0.3

0.4

0.5

0.6

0 2 4 6 8 10 12 14 16

Ab

sorb

an

ce

concentration ( mcg)

S.No Brand name Amount of sample

(mcg / ml)

Amount of drug

added (mcg / ml)

Amount

Recovered % Recovery ± SD**

1 Clopilet 10 8 17.91 98.85±0.51

2 Clopilet 10 10 19.92 99.82±0.43

3 Clopilet 10 12 21.83 99.77±0.12

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Syeda Bushra Yunus. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 2(3), 2013, 365 - 370.

Available online: www.uptodateresearchpublication.com May - June 370

Figure No.3: Spectra of Clopidogrel bisulphate

CONCLUSION Thus it can be concluded that the methods developed in the present investigation are simple, sensitive, accurate, rapid and precise. Hence, the above said method can be successfully applied for the estimation of Clopidogrel bisulphate in tablet dosage form. ACKNOWLEDGEMENT The authors are sincerely thanks to Creative Educational Society’s College of Pharmacy, Kurnool, Andhra Pradesh, India for providing the facilities to complete this research work. CONFLICT OF INTEREST We declare that we have no conflict of interest. REFERENCES 1. Shinha P K, Mrinalini C D, Bothra K G. A

Validated stability indicating HPTLC Method for determination of Aspirin and Clopidogrel bisulphate in combined dosage form, Eur J Chem, 4(2), 2009, 152-160.

2. Vocilkova L, Opatrilova R, Sramek V. Determination of Clopidogrel bisulphate by Chromatography, Cur Pharm Anal, 5, 2009, 424-431.

3. Himani A, Neeraj K, Paradkar A R, Mahadik K R. Stability indicating HPTLC Determination of

Clopidogrel bisulphate as Bulk drug and in pharmaceutical dosage form, J Pharm and Biomed Anal, 61(5), 2003, 581-589.

4. Anti D, Filipi S, Agbaba D. A simple and sensitive TLC Method for determination of Clopidogrel bisulphate and its impurity in pharmaceutical product, Acta chromatogr, 18(1), 2007, 199-206.

5. Pankaj K, Kachhadia A S, Doshi H S. Validated Column HPLC Method for Determination of Aspirin and Clopidogrel bisulphate in combined Tablets in the presence of Degradation Products Formed under ICH Recommended Stress Conditions, JAOAC Int, 92(1), 2009, 152-7.

6. Patel R B, Shankar M B, Patel M R, Bhatt K K. Simultaneous Estimation of Acetylsalicylic Acid and Clopidogrel bisulphate in Pure Powder and Tablet Formulations by HPLC and HPTLC, JAOAC International, 91(4), 2008, 750-755.

7. ICH/CGMP guidelines Q2A, Text on Validation of Analytical Procedures, 1994.

8. ICH/CGMP guidelines Q2B, Validation of Analytical Procedures-Methodology, 1996.

Please cite this article in press as: Syeda Bushra Yunus. et al. Method development and validation of spectrophotometric method for the estimation of clopidogrel bisulphate in pure and tablet dosage form, International Journal of Research in Pharmaceutical and Nano Sciences, 2(3), 2013, 365-370.