Stability Indicating RP-HPLC method for the determination of Prasugrel in bulk as well as in Pharmaceutical formulation

S. Ashutosh Kumar¹*, Manidipa Debnath¹, J.V.L.N. Seshagiri Rao²

¹A.K.R.G College of Pharmacy, Nallajerla, West Godavari, 534112, A.P

²Prof. Pharmaceutical Analysis, Yalamarty College of Pharmacy, Tarluwada Visakhapatnam, 530052, A.P

*Corresponding Author E-mail:- ashu.mpharm2007@gmail.com

ABSTRACT:

This study was designed to develop and validate a simple, sensitive, precise, specific and stability indicating reverse phase high-performance liquid chromatographic (HPLC) method for the determination of Prasugrel in bulk and its tablet dosage form. The HPLC separation was carried out by reverse phase chromatography on XTerra column C18 (4.6 x 150mm, 5 mm) with a mobile phase composed of Potassium Dihydrogen Phosphate [ pH was adjusted to 3.0 by using Orthophosporic Acid] and Acetonitrile in the ratio of 40:60 v/v in isocratic mode at a flow rate of 1.0 ml/min. The run time was maintained 5mins. The detection was monitored at 210 nm. The Accuracy was calculated and the % Recovery was found 99.0%-101.8% and reproducibility was found to be satisfactory .The calibration curve for Prasugrel was linear from 20 to60 µg/ml. The inter-day and intra-day precision was found to be within limits. The proposed method has adequate sensitivity, reproducibility, and specificity for the determination of Prasugrel in bulk and its tablet dosage forms. The limit of detection and limit of quantification for Prasugrel were found to be 0.07 µg/ml and 0.2 µg /ml respectively. Prasugrel was exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. Peak homogeneity data of prasugrel was obtained using photodiode array detector in the stressed sample chromatograms, which demonstrated the specificity of the method for the estimation in presence of degradants. The present work was undertaken with the aim to develop and validate a rapid and consistent stability indicating RP- HPLC in which the peaks will be appear with a short period of time as per ICH guideline. The proposed method is simple, fast, accurate, and precise for the quantification of Prasugrel in the dosage form, bulk drugs as well as for routine analysis in quality control. Overall, the proposed method was found to be suitable and accurate for quantitative determination and stability study of prasugrel in pharmaceutical dosage form.

KEYWORDS: High Performance Liquid Chromatography; Potassium Dihydrogen Phosphate; Acetonitrile; Prasugrel; Accuracy; Quantification.

INTRODUCTION:

Prasugrel (Fig. 1) is chemically (RS)-5-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4, 5, 6, 7-tetrahydrothieno [3, 2-c] pyridin-2-yl acetate. Prasugrel is a novel third-generation oral thienopyridine that reduces the tendency of platelets, the blood particles responsible for clotting, from sticking or clumping together [1]. Prasugrel blocks a specific receptor on the platelet surface, P2Y12 adenosine diphosphate (ADP), preventing platelets from clumping, which can result in clogged arteries and may lead to a heart attack [2].

Prasugrel is more effective by preventing ischemic events in patients with acute coronary syndrome undergoing percutaneous coronary intervention, increase in bleeding and improved net clinical outcome [3]. Clopidogrel, unlike Prasugrel, was issued a black box warning from the FDA on March 12, 2010, as the estimated 2-14% of the US population that have low levels of the CYP2C19 liver enzyme needed to activate clopidogrel may not get the full effect [4]. Compared to clopidogrel, Prasugrel inhibits adenosine diphosphate–induced platelet aggregation more rapidly, more consistently and to a greater extent than do standard and higher doses of clopidogrel in healthy volunteers and in patients with coronary artery disease [6]. Tests are available to predict if a patient would be susceptible to this problem or not [6-8]. Unlike clopidogrel, Prasugrel is effective in most individuals, although there have been several case reports of decreased responsiveness to Prasugrel [9]. Prasugrel is a member of the thienopyridine class of ADP receptor inhibitors, like Ticlopidine (trade name Ticlid) and Clopidogrel (trade name Plavix). These agents reduce the aggregation ("clumping") of platelets by irreversibly binding to P2Y₁₂ receptors [10-14]. Forced degradation or stress testing is undertaken to demonstrate specificity for developing stability-indicating methods, particularly when little information is available about potential degradation products. The ICH guideline entitled “Stability Testing of New Drug Substances and Products” requires the stress testing to be carried out to elucidate the inherent stability characteristics of the active substances. The aim of stability testing is to prove how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light. It enables recommendation of storage conditions, retest period and shelf lives to be established. Regulatory agencies recommend the use of stability-indicating methods for the assay analysis of stability samples. Thus, stress studies are required in order to generate the stressed samples, method development and its validation [15-17]. Forced degradation of prasugrel was performed under stress conditions (acid, alkaline, photolytic, thermal and oxidative). To establish the stability-indicating nature of the method, stressed samples were analyzed by the proposed method. The proposed RP-HPLC method was validated by assessing its specificity, linearity, accuracy, precision, limits of detection and quantification, system suitability parameters, ruggedness and robustness.

Fig. no.1The Chemical structure of Prasugrel

MATERIAL AND METHODS:[18]

Chemical and Reagent Used:

The following chemicals were procured for the process Water [HPLC Grade], Prasugrel [Working Standards], Acetonitrile [HPLC Grade], Ortho phosphoric acid all these chemicals were procured from Standard Solutions, HCl procured from Finar Chemical limited, NaOH procured from S D Fine- Chem Limited and H₂O₂ procured from Alpha Pharma Limited and Tablet 5mg was collected from the Local market and the manufacturing company was Glenmark, Brand Name APLET.

Apparatus and Chromatographic Conditions:

Equipment: High performance liquid chromatography equipped with Auto Sampler and DAD or UV detector.

Column : XTerra column C18 (4.6 x 150mm, 5 mm)

Flow rate : 1.0mL per min

Wavelength : 210 nm

Injection volume : 20 ml

Temperature : Ambient

Run time : 5.0 min

Detector : Photo diode array

Soft ware : Empower 2

MFD by : WATERS

Preparation of Phosphate buffer [19-22]:

2.72 grams of potassium dihydrogen phosphate was weighed and taken in a 1000ml beaker and then it was dissolved and diluted to 1000ml with Water [HPLC Grade]. The pH of the resultant Buffer Solution was adjusted to 3.0 by using Orthophosphoric acid.

Preparation of mobile phase:

The mobile phase was prepared by mixing the above Buffer and Acetonitrile [HPLC grade] in the ratio of 40:60 [400ml and 600ml respectively] and degassed in ultrasonic water bath for 5 minutes. Then the mixture [mobile phase] was filtered through 0.45 µ filter under vacuum filtration.

Diluent Preparation:

The Mobile phase was used as diluent.

Preparation of the Prasugral Standard and Sample Solution:

Standard Solution Preparation:

The Standard Stock Solution was prepared by weighing accurately and transferred 10mg of Prasugrel [Working standard] into a 10 mL volumetric flask. About 7 mL of diluent was added and sonicated to dissolve it completely and the volume was made up to the mark with the same solvent. Further from the above Stock Solution pipette out 0.4 ml into a 10ml volumetric flask and diluted up to the mark with the diluent. The Standard Solution was mixed well and then it was filtered through 0.45µm filter.

Sample Solution Preparation:

The Sample Stock Solution was prepared by weighing 5 Prasugral tablets and calculated the average weight. Tablets were powdered; further 10mg of the powder was weighed accurately and transferred into a 10 mL volumetric flask. About 7 mL of diluent was added and sonicated to dissolve it completely and the volume was made up to the mark with the diluent. The above solution was mixed well and then it was filtered through 0.45µm filter. Further from the above prepared Sample Stock Solution pipette out 0.4 ml into a 10ml volumetric flask and diluted up to the mark with diluent. The resultant solution was mixed well and it was filtered through 0.45µm filter.

Standard and Sample Solutions Injected to the Column [Chromatographic System]:

About 20mL of standard and sample solutions were injecting to the chromatographic system and the area for the drug was measured and the % Assay was calculated by using suitable formulae.

System Suitability [23-26]:

The Tailing factor for the peak due to Prasugral in Standard solution should not be more than 2.0. The theoretical plates for the Prasugral peak in Standard solution should not less than 2000.

Calculation for Assay:

AT WS DT P Avg. Wt.

Assay % =--------X--------X-------X--------X-----------------X 100

AS DS WT 100 Label Claim

Where:

AT = Peak Area of Prasugral obtained with test preparation

AS = Peak Area of Prasugral obtained with standard preparation

WS = Weight of working standard taken in mg

WT = Weight of sample taken in mg

DS = Dilution of Standard solution

DT = Dilution of sample solution

P = Percentage purity of working standard

Assay Results:

623234 10 0.4 10 10 99.8 232.4

Assay % =---------X--------X------X--------X-----X-------X------X 100

613972 10 10 233.1 0.4 100 8

= 101.0%

VALIDATION DEVELOPMENT [27]:

Precision: The precision of an analytical procedure expresses the closeness of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. The data was represented in Table no.1

Table no.1The results for Precision were summarized for the drug Prasugrel.

Injection	Area
Injection-1	616550
Injection-2	616002
Injection-3	614198
Injection-4	614670
Injection-5	614644
Average	615213
Standard Deviation	1007.7
%RSD	0.16

Acceptance Criteria: The % RSD for the area of five standard injections results should not be more than 2%.

Intermediate Precision: To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different day by using different make column of same dimensions. The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. The Data were represented in Table no.2.

Table no.2The results for Ruggedness were summarized for the drug Prasugrel.

Injection	Area
Injection-1	618936
Injection-2	618838
Injection-3	620387
Injection-4	620061
Injection-5	619434
Average	619531
Standard Deviation	681.4
%RSD	0.11

Acceptance Criteria: The % RSD for the area of five standard injections results should not be more than 2%.

Accuracy: The accuracy of an analytical procedure expressed the closeness of agreement between the value which was accepted either as a conventional true value or an accepted reference value and the value found. The Standard and Sample were injected to the chromatographic system for Accuracy 50%, 100% and 150%. The amount found was calculated and the amounts added for Prasugrel were calculated. The individual recovery and mean recovery values were also calculated. The Data was represented in Table no.3.

Acceptance Criteria: The % Recovery for each level should be between 98.0 to 102.0%.

Linearity: The linearity of the analytical procedure was its ability to obtain the test results which are directly proportional to the concentration (amount) of analyte in the sample. Different levels were prepared and all Levels were injected individually into the chromatographic system and the peak area was measured. A graph was plotted for peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and the correlation coefficient was calculated. The Data were represented in Table no. 4.

Table no. 4The results for Linearity were summarized for the drug Prasugrel.

Sl. No.	Linearity Level	Concentration	Area
1	I	20µg/ml	295857
2	II	30µg/ml	463117
3	III	40µg/ml	612270
4	IV	50µg/ml	759987
5	V	60µg/ml	924317
Correlation Coefficient			0.999

Acceptance Criteria: The Correlation coefficient should be not less than 0.999.

Limit of Detection: The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantities as an exact value.

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank: 49µV

Signal Obtained from LOD solution (1.5% of target assay concentration): 148 µV

S/N = 148/49= 3.01

Acceptance Criteria: The S/N Ratio value should be 3 for LOD solution.

Limit of Quantification: The Quantification limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank: 49µV

Signal Obtained from LOD solution (5.0% of target assay concentration): 499 µV

S/N = 499/49 = 10.1

Acceptance Criteria: The S/N Ratio value should be 10 for LOQ solution.

Robustness: As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method.

a) The flow rate was varied at 0.9 to1.1 ml/min. The Standard solution 40 µg/ml was prepared and analysed by using the varied flow rates along with developed [actual] flow rate. On evaluation of the above results, it was concluded that the variation in flow rate does not affected the method significantly. Hence it was indicated that the method was robust even by change in the flow rate ±10%. The data are represented in Table no. 5.

Table no. 5The results for Robustness were summarized for the drug Prasugrel with change in flow rate.

Sl. No.	Flow Rate (ml/min)	System Suitability Results
Sl. No.	Flow Rate (ml/min)	USP Plate Count	USP Tailing
1	0.9	2544	1.5
2	1.0	2542	1.5
3	1.1	2234	1.5

b) The Organic composition in the Mobile phase was varied from70% to 50%: The Standard solution 40 µg/ml was prepared and analysed using the varied Mobile phase composition along with the actual mobile phase composition in the method. On evaluation of the above results, it was concluded that the variation in 10% Organic composition in the mobile phase do not affect the method significantly. Hence it indicates that the method was robust even by change in the Mobile phase ±10%. The data was represented in Table no. 6.

Table no.6The results for Robustness were summarized for the drug Prasugrel with change in the composition of the Organic Phase.

Sl. No.	Change in Organic Composition in the Mobile Phase	System Suitability Results
Sl. No.	Change in Organic Composition in the Mobile Phase	USP Plate Count	USP Tailing
1	10% less	2488	1.7
2	Actual	2542	1.5
3	10% more	2276	1.5

Degradation studies [28]: The International Conference on Harmonization (ICH) guideline entitled stability testing of new drug substances and products requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance. The aim of this work was to perform the stress degradation studies on the Prasugrel using the proposed method.

Preparation Standard Solution: 10 mg Prasugrel [Working Standard] was weighed accurately and transferred into a 100ml dry volumetric flask, about 70mL of the diluent was added and sonicated to dissolve it completely and the volume was made upto the mark with the same solvent. Further from the above prepared standard solution pipette out 3ml solution and transferred into a 10ml dry volumetric flask, further the volume was made upto the mark with the diluent.

a. Hydrolytic degradation under acidic condition: From the above prepared Stock Solution pipette out 3 ml solution and transferred into a 10ml dry volumetric flask and 3 ml of 0.1N HCl was added. Then the volumetric flask was kept at normal condition for 90 minutes and then it was neutralized with 0.1 N NaOH and the volume was made upto the mark [10ml] with the diluent. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

b. Hydrolytic degradation under alkaline condition: From the above prepared Stock Solution pipette out 3 ml solution and transferred into a 10m dry volumetric flask and 3ml of 0.1N NaOH was added. Then the volumetric flask was kept at normal condition for 90 minutes and then it was neutralized with 0.1 N HCL and the volume was made upto the mark [10ml] with the diluent. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials.

c. Thermal induced degradation: From the above prepared stock solution pipette out 3.0 ml of the solution and transferred into a 10 ml dry volumetric flask and 3 ml of diluent was added. Then the volumetric flask was kept at reflex condition for 60 minutes and the volume was made upto mark [10ml] with the diluent. The resultant solution was filtered with 0.45 microns syringe filters and placed in vials.

d. Oxidative degradation: From the above prepared stock solution pipette out 3.0 ml of the solution and transferred into a 10 ml dry volumetric flask and added 1 ml of 3 % w/v of hydrogen peroxide solution and the volume was made up to the mark [10ml] with the diluent . The volumetric flask was then kept at room temperature for 15 min. The resultant solution was filtered with 0.45 microns syringe filters and placed in the vials. The following results were obtained for the drug Prasugrel under Force degradation studies which were summarized in Table no. 7.

RESULT AND DISCUSSION:

The present study was carried out to develop a sensitive, precise, accurate stability indicating RP-HPLC method for the analysis of the drug Prasugrel in bulk as well as in pharmaceutical dosage forms. In order to develop the method under isocratic conditions, a mixtures of Phosphate Buffer and Acetonitrile[ HPLC grade] in different combinations were tested as mobile phase on a Symmetry C18 (4.6 x 150mm, 5 mm, Make: XTerra) column. A binary mixture of Phosphate Buffer [ whose pH was adjusted to 3 with Orthophosphoric acid] and Acetonitrile [HPLC Grade] in 30:70 v/v proportion was proved to be the most suitable of all the combinations since the chromatographic peaks were better defined and resolved and almost free from tailing. The retention times obtained for the drug Prasugral was around 2.216 min. A model chromatogram was represented in Fig. no.2.

Fig. no.2A model Chromatograph for the drug Prasugrel.

The Precision data was represented in Table no. 1. When Prasugrel was analyzed by the proposed method in the intra and inter-day (Ruggedness) variation results, a low coefficient of variation was observed and the data was represented in Table no. 2. This shows that the present HPLC method was highly precise and it was represented by Fig no. 3. The Accuracy data was summarized in Table no. 3. In order to test the linearity of the method, five dilutions of the [working standard] solutions for the drug in the range of 20 to 60µg per mL were prepared. The data was in Table no. 4. Each of the dilutions was injected into the column [Chromatographic System] and the graph for the Linearity Curve was represented in Fig no. 4.

Fig. no.3The Linearity Curve for Drug Prasugrel

The method was duly validated by evaluation of the required parameters. Robustness of the method was found out by testing the effect of small deliberate changes in the chromatographic conditions and the corresponding peak areas. The factors selected for this purpose were flow rate and percentage composition variation in Phosphate buffer and Acetonitrile in the mobile phase. The method was found to be robust enough that the peak area was not apparently affected by small variation in the chromatographic conditions. The Fig.no.4, 5, 6 and 7 were represented the Robust nature of the chromatograph.

Fig. no.4 The Robustness chromatograph with decrease in the flow rate for the drug Prasugrel

Fig. no. 5 The Robustness Chromatograph with increase in the flow rate for the drug Prasugrel

Fig. no. 6The Robustness chromatograph with less change in the composition of Mobile Phase for the drug Prasugrel

Fig. no. 7The Robustness chromatograph with more change in the composition of Mobile Phase for the drug Prasugrel

The system suitability parameters were within the limits as shown in Table 5 and 6 for the drug. Limit of detection and limit of quantification of the method were calculated based on standard deviation of the response and the slope (s) of the calibration curve at approximate levels of the limit of detection and limit of quantification. The LOD for the drug was found to be 0.07µg/ml and LOQ for the Drug was found to be 0.2μg/mL. In order to evaluate the stability of Prasugrel and ability of the method to separate Prasugrel from its degradation products, the drug Prasugrel was subjected to various stress conditions such as Hydrolytic degradation under acidic condition (using 0.1N HCl and 0.1 N NaOH), Hydrolytic degradation under alkaline condition (using0.1N NaOH and 0.1N HCL), Thermal induced degradation (Reflex Condition for 60 mins), Oxidative degradation (by using 3 % w/v of hydrogen peroxide). The following chromatograph represents the degradation studies for the drug [Prasugrel] which were represented in fig. no.8, 9, 10, 11 and 12.

Fig. no.8.The chromatograph represents the Hydrolytic degradation under acidic condition.

Fig. no. 9.The chromatograph represents the Hydrolytic degradation under alkaline condition

Fig. no. 10.The chromatograph represents the Thermal induced degradation.

Fig. no. 11.The chromatograph represents the Oxidative degradation.

Fig. no. 12.The chromatograph represents for the pure drug.

The drug content formulations were quantified by using the proposed analytical method. The low coefficient of variation in the recovery data indicates the reproducibility of the method in dosage forms. It can be concluded that the proposed HPLC method is sufficiently sensitive and reproducible for the analysis of Prasugral in the Tablet formulation dosage forms within a short analysis time.

CONCLUSION:

It was concluded that the proposed stability indicating RP-HPLC method developed for the quantitative determination of Prasugrel in bulk as well as in its formulations was simple, selective, sensitive, accurate, precise and rapid. The method was proved to be superior to most of the reported methods. The mobile phases were simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence the method can be easily adopted as an alternative method to report routine determination of Prasugrel depending upon the availability of chemicals and nature of other ingredients present in the sample. The method also finds use in clinical, biological and pharmacokinetic studies for the drug Prasugrel. The method was validated as per ICH guidelines, and validation acceptance criteria were met in all cases. Application of this method for estimation of Prasugrel from tablet dosage form and stressed samples showed that neither the degradation products nor the excipients interfered in the estimation of drug. Hence, this method was specific, stability-indicating and can be successfully used for the estimation of Prasugrel in bulk and pharmaceutical dosage forms.

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Received on 19.06.2013 Modified on 25.06.2013

Research J. Pharm. and Tech 6(7): July 2013; Page 809-816

%Concentration (At specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	612287	5.0	4.9	99%	99.6%
100%	1220663	10.0	9.9	99.4%
150%	1811336	15.0	14.9	101.8%

Sl. No.	Degradation Studies	Retention Time	Area	Height	Purity Angle	Purity Threshold	% Degradation
1	Hydrolytic degradation under acidic condition	2.219	584725	85986	0.21	0.45	5.1
2	Hydrolytic degradation under alkaline condition	2.216	598658	86748	0.22	0.45	2.84
3	Thermal induced degradation	2.213	597458	85698	0.23	0.42	2.53
4	Oxidative degradation	2.218	600574	86234	0.23	0.46	3.03
5	Standard Drug	2.216	616164	87141	--------	--------	------