INTRODUCTION:

Prasugrel chemically is 5-[2-cyclopropyl-1-(2-fluoro-phenyl)-2-oxoethyl]-4,5,6,7-tetra hydrothieno [3,2-c] pyridin-2-yl acetate¹. Prasugrel^2,3 is a platelet inhibitor belonging to the thieno pyridine class developed by Daiichi Sankyo Co. and produced by Ube and currently marketed in the United States in cooperation with Eli Lilly and Company for acute coronary syndromes planned for percutaneous coronary intervention (PCI). On July 10, 2009, the US Food and Drug Administration approved the use of Prasugrel for the reduction of thrombotic cardiovascular events^4,5

It acts as ADP receptor antagonists, which makes it different, is its safety profile and pharmacokinetic properties. Although clopidogrel is a widely prescribed agent, it has limitations such as: a modest anti-platelet effect, a delayed onset of action and considerable inter-patient variability in drug response.

All these disadvantages motivated the development of more effective and predictable agents, such as the novel Prasugrel. Prasugrel is sparingly soluble in water but completely soluble in methanol. It is available with brand names Effient and Prasita. Literature survey revealed very few methods for the estimation of Prasugrel hydrochloride such as LC-MS⁶, HPTLC⁷, UV-Visible spectrophotometric methods⁸

As the analysis is an important component in the formulation development of any drug molecule. It becomes essential to develop a simple, sensitive, accurate, precise, reproducible method for the estimation of drug samples. Our main concern is development and validation of UV spectrophotometric method as per ICH guideline⁹

MATERIALS AND METHODS:

Apparatus:

After due calibration of the instrument, spectral and absorbance, measurements are made using Labindia UV 3000⁺UV double beam Spectrophotometer with 1cm matched quartz cells. All the chemicals used were of analytical grade. All the solutions were freshly prepared every day.

Reagents Used and Preparations:

(0.1 N) H₂SO₄ Solution:

54 ml of H₂SO₄ ( about 98% purity) was dissolved on distilled water and volume is made upto 1000 ml in a 1000 ml volumetric flask.

0.1M NaOH Solution: (Preparation):

Dissolve about 4gms of NaOH in 1000ml of distilled water.

Bromophenol Blue Preparation:

0.1gm of Bromophenol blue with gentle heating in 1.5 ml of 0.1M NaOH and 20 ml of ethanol, add sufficient water to produce 100ml.

Developed Method 1:

A simple photometric method for the determination of Prasugrel hydrochloride is developed based on the formation of the coloured product with Bromophenol blue exhibiting maximum absorbance at 421nms.

Spectrophotometric parameter was established for the standard of the methods including statistical analysis of the drug. These methods have been successfully extended to the pharmaceutical preparations.

Standard Stock Solution:

Standard stock solution containing 1000 mcg/ml was prepared by dissolving accurately weighed 50 mg of Prasugrel hydrochloride in 50 ml of methanol in a 50 ml volumetric flask. A working standard solution containing 10mcg/ml was prepared with methanol.

Calibration Curves:

Aliquots of standard stock solution containing 0.1mcg/ml - 0.5mcg/ml were transferred into a series of 250 ml of separating funnel. 1 ml of Bromophenol blue and 5 ml of chloroform was added. The absorbance of colored species was measured at 421 nm against reagent blank. The amount of Prasugrel hydrochloride present in sample solution was estimated from its calibration curve.

Sample Solution:

Tablets containing Prasugrel hydrochloride (Prax 5, Torrent Pharmaceuticals) were successfully analyzed by the proposed method. 18 tablets of Prasugrel hydrochloride were accurately weighed and powdered. Tablet powder equivalent to 90 mg of Prasugrel hydrochloride was dissolved in 50 ml of methanol in 50 ml volumetric flask and filtered. The solution was suitably diluted and analysed as given under the procedure for bulk samples.

The results are represented in table (1&2). None of the excipients usually employed on the formulation of tablets interfered in the analysis of Prasugrel hydrochloride by the proposed method.

Developed Method 2:

A simple photometric method for the determination of Prasugrel hydrochloride is developed based on the formation of the product with 0.1 N H₂SO₄ exhibiting maximum absorbance at 239nms.

Standard stock solution:

Standard stock solution containing 1 mg/ml was prepared by dissolving accurately weighed 50 mg of Prasugrel hydrochloride in 20 ml 0.1 N H₂SO₄in a 50 ml volumetric flask and the volume is made up to 50 ml with distilled water.

Calibration Curves:

10 ml of standard stock solution was taken and volume was made up to 100ml with solution containing 2:3 ratios H₂SO₄ (0.1 N) and distilled water. From this solution 0.5 ml to 3 ml of solution was taken and volume is made up to 10 ml with the solution containing 2:3 ratio of H₂SO₄ (0.1 N) and distilled water. The absorbance of coloured species was measured at 223nm against the blank. The amount of Prasugrel hydrochloride present in sample solution was estimated from its calibration curve.

Sample Solution:

Tablets containing Prasugrel hydrochloride (Prax 5, Torrent Pharmaceuticals) were successfully analysed by the proposed method. 18 tablets of Prasugrel hydrochloride were accurately weighed and powdered. Tablet powder equivalent to 90mg of Prasugrel hydrochloride was dissolved in 50 ml 2:3 ratio of 0.1 N H₂SO₄ and distilled water in 50 ml volumetric flask and filtered. The solution was suitably diluted and analysed as given under the procedure for bulk samples.

The results are represented in table (3&4). None of the excipients usually employed on the formulation of tablets interfered in the analysis of Prasugrel hydrochloride by the proposed method.

RESULTS AND DISCUSSION:

In the present study, the method 1 was based on the drugs formation of a complex with Bromophenol blue. All the conditions required were optimized. Statistical analysis was carried out and results of which were satisfactory. The optical characteristics such as absorption maxima, beers law limits, molar absorbtivity and regression analysis and correlation coefficients are presented in the Table.No.1.

Recovery studies were close to 100% that indicate the accuracy and precision of proposed method and it indicates the non-interference of the formulation excipients. All the validated parameters are summarized in the Table.No.2.

The method 2 was based on the detection of drug in 0.1 N H₂SO_4. All the conditions required were optimized. Statistical analysis was carried out and results of which were satisfactory. The optical characteristics such as absorption maxima, beers law limits, molar absorbtivity and regression analysis and correlation coefficient are presented in the Table.No.3.

CONCLUSION:

A Simple Visible Spectrophotometric method was developed for the determination of Prasugrel hydrochloride in pure and its dosage form using Bromophenol blue. The absorbance of the complex formed by Bromophenol blue and drug was found maximum at wavelength 421 nm against the corresponding reagent blank.

A Simple UV Spectrophotometric method was developed for the determination of Prasugrel hydrochloride in pure and its dosage form using 0.1 N H₂SO_4. The absorbance of the drug in 0.1 N H₂SO₄was found maximum at the wavelength 223 nm against the corresponding blank.

Both the methods were found to be simple, precise, and rapid for detection of Prasugrel hydrochloride in pure and its dosage form. The statistical parameter and recovery study data clearly indicates reproducibility and accuracy of the method. This can be conveniently adopted for routine analysis of Prasugrel hydrochloride in pure and its dosage form.

Table-1 -Optical characteristics and precision data of method 1

PARAMETERS
Lambda max	421nm
Beers law limits (mcg/mL)	0.1-0.5
Molar Absorbtivity (L/mol cm)	6.53×10^-3
Sandells sensitivity (mcg/cm²/0.001absorbance unit)	0.07142
Régression Equation(Y) · Slope (m) · Intercept (c)	0.0501 0.0010
Correlation Coefficient(r)	0.99
Precision (standard deviation %)	0.102

Table-2- Assay of prasugrel hydrochloride in pharmaceutical formulation method 1:

Labeled Amount	Obtained % By Proposed Method	Recovery By Proposed Method
5 mg	100.9	99.5
5 mg	100	98.9

Table-3- Optical characteristics and precision data of method 2

PARAMETERS
λ max	223 nm
Beers law limits (mcg/mL)	0.5-3
Molar Absortivity (L/mol cm)	0.00741
Sandells sensitivity (mcg/cm²/0.001 absorbance unit)	0.0721
Regression Equation(Y) · Slope (m) · Intercept (c)	0.0020 0.0040
Correlation Coefficient(r)	0.95
Precision (standard deviation %)	0.97

Table-4-Assay of prasugrel hydrochloride in pharmaceutical formulation method 2:

Labeled Amount	Obtained % By Proposed Method	Recovery By Proposed Method
5 mg	100.5	99.7
5 mg	100.8	99.6

Figure 2: 0.1N H2SO4

Linearity of Prasugrel HCl:

Figure 3: Bromophenol Blue

Figure 4: 0.1N H₂SO₄

REFERENCES:

1. Raja Kumar Viriyala, Fakir Mohan Jena, B V V Ravi Kumar et al, validated new spectrophotometric methods for the estimation of prasugrel in bulk and pharmaceutical dosage forms, pharmacie globale international journal of comprehensive pharmacy, Vol. 02, Issue 06, page 1-3.

2. Amy B. Riley, Mohammad J. Tafreshi and Stacy L. Haber, Prasugrel: A novel antiplatelet agent, American Journal of Health-System Pharmacy, 65(11), 1019-28.

3. Dovlatova NL, Jakubowski JA, Sugidachi A and Heptinstall S, The reversible P2Y antagonist cangrelor influences the ability of the active metabolites of clopidogrel and Prasugrel to produce irreversible inhibition of platelet function. J Thromb Haemost, Jul 2008, 6(7), 1153-59.

4. Baker WL and White CM, Role of Prasugrel: a Novel P2Y12 Receptor Antagonist in the Management of Acute Coronary Syndromes, American Journal of Cardiovascular Drugs, Aug 2009, 9(4), 213-29.

5. Food and Drug Administration (United States), March 12, 2010.

6. Farid NA, McIntosh M, Garofolo F, Wong E, Shwajch A, Kennedy Z et al. Determination of the active and inactive metabolites of Prasugrel in human plasma by liquid chromatography/tandem mass spectrometry, Rapid Communications in Mass Spectrometry. 2007 Jan 30,, 169-79.

7. TC Borole, R Mehendre, MC Damle and KG Bothara. Development and validation of stability indicating HPTLC method for determination of Prasugrel, J Chem Pharm Res, 2010, 2(4), 907-13.

8. Harshini B, SVR Alekhya, G Manasa and K Vanitha Prakash, Extractive Spectrophotometric Estimation Of Prasugrel In Pharmaceutical Formulation, Research Journal of Pharmaceutical, Biological and Chemical Sciences, 2011, 2(3), 426-30.

9. Sagar Tatane, Development of UV Spectrophotometric Method of Telmisartan in Tablet Formulation, Journal of Advances in Pharmacy and Healthcare Research, 2011, Issue1, p.g 23-26.

Received on 11.02.2013 Modified on 01.03.2013

Research J. Pharm. and Tech 6(6): June 2013; Page 641-644