International Journal of PharmTech ResearchCODEN (USA): IJPRIF ISSN : 0974-4304Vol.4, No.4, pp 1407-1416, Oct-Dec 2012

Development And Validation Of Reverse PhaseHPLC Method For The Determination OfImpurities In Prasugrel Hydrochloride

Venugopal Sriram1,3*, Kalpana Sriram2, Jagadishbabu Angirekula1,Upendra M. Tripathi and Devanna Nayakanti3

1Analytical Research & Development,Vindhya Pharma (Inida) Pvt. Ltd, Plot No.F-11A&B, Phase-1, IDA Jeedimetla, Hyderabad-500055, Andhra Pradesh, India.

2Startech Labs Pvt. Ltd., Madinaguda, Hyderabad-500055, Andhra Pradesh,India.3Department of Chemistry, Jawaharlal Nehru Technological University,

Anantapur-515002, Andhra Pradesh, India.

*Corres.author: venugopalsriram23@gmail.com

Abstract: The present paper describes the development of reverse phase HPLC method for Prasugrelhydrochloride in the presence of impurities and degradation products generated from the forced degradationstudies. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis andthermal degradation. The degradation of Prasugrel hydrochloride was observed under neutral, acid, base andoxidation environment. The drug was found more sensitive to basic condition. Successful separation of thedrug from the process related impurities and degradation products were achieved on Gemini C18 (250 x 4.6mm) 5 μm particle size column using reverse phase HPLC method. The isocratic method employed with amixture of buffer and (10%v/v water in acetonitrile) mixture of ratio 30:70 respectively. Potassiumdihydrogen orthophosphate (0.05M) is used as buffer. The HPLC method was developed and validated withrespect to linearity, accuracy, precision, specificity and robustness, which is useful for the routinedetermination of Prasugrel hydrochloride.Key words: Prasugrel hydrochloride, RP-HPLC, LC Method development and validation, Forceddegradation studies.

1. INTRODUCTION

Prasugrel Hydrochloride [1-7] is a novel plateletinhibitor used for acute coronary syndromesplanned for percutaneous coronary intervention.Prasugrel hydrochloride is used to reduce the riskof thrombotic cardiovascular events (e.g., stentthrombosis, myocardial infarction [MI]) in patientswith acute coronary syndromes (ACS) undergoingpercutaneous coronary intervention (PCI).Prasugrel (marketing name “Effient” in the US and

India, and “Efient” in the EU) is a plateletinhibitor developed by Daiichi Sankyo Co. andproduced by Ube and currently marketed in theUnited States in cooperation with Eli Lilly andCompany for acute coronary syndromes plannedfor percutaneous coronary intervention (PCI).Prasugrel is a member of the thienopyridine classof ADP receptor inhibitors, like ticlopidine (tradename Ticlid) and clopidogrel (trade name Plavix).These agents reduce the aggregation ("clumping")

Venugopal Sriram et al /Int.J.PharmTech Res.2012,4(4) 1408

of platelets by irreversibly binding to P2Y12

receptors. Compared to clopidogrel, prasugrelinhibits adenosine diphosphate–induced plateletaggregation more rapidly, more consistently, andto a greater extent than do standard and higherdoses of clopidogrel in healthy volunteers and inpatients with coronary artery disease, includingthose undergoing PCI". Clopidogrel, unlikeprasugrel, was issued a black box warning fromthe FDA on March 12, 2010, as the estimated 2-14% of the US population that have low levels ofthe CYP2C19 liver enzyme needed to activateclopidogrel may not get the full effect. Tests areavailable to predict if a patient would besusceptible to this problem or not. Unlikeclopidogrel, Prasugrel is effective in mostindividuals, although there have been several casereports of decreased responsiveness to Prasugrel.

Different analytical techniques reported8-19 so farfor the determination of this drug substance anddrug product. The typical structure of Prasugrelhydrochloride is mentioned in fig.1.HPLC method was developed for thedetermination of Prasugrel and the impuritiesarising during its manufacturing. In the presentstudy, we describe a reverse phase liquidchromatography method for the separation ofprocess and degradation impurities of Prasugrel.The accuracy, linearity, precision, limit ofdetection (LOD), limit of quantification (LOQ)and robustness of the method were determined inaccordance with ICH guidelines 20. This paperreports, a rapid, efficient, simple and validated LCmethod for the separation of impurities anddegradation products.

O

F

NS

O

O

H Cl2-[2-(Acetyloxy)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanonehydrochloride( Fig. 1: Structure of Prasugrel Hydrochloride )

O

F

1-Cyclopropyl-2-(2-fluoro phenyl) ethanoneImpurity-A

O

FBr

2-Bromo-1-cyclopropyl-2-(2-fluoro-phenyl)-ethanoneImpurity-B

NHSO HCl

5,6,7,7a-Tetrahydrothieno[3,2-c]pyridine-2(4H)-one hydrochlorideImpurity-C

( Fig. 2: Impurities of Prasugrel Hydrochloride )

Venugopal Sriram et al /Int.J.PharmTech Res.2012,4(4) 1409

2. EXPERIMENTAL SECTION

2. Experimental2.1. Standards and ReagentsPrasugrel hydrochloride sample and impuritiesnamely Impurity-A, Impurity-B and Impurity-C{Fig. 2} were developed and prepared in ResearchDivision of Vindhya. All reagents used were ofanalytical reagent grade stated otherwise. Milli Qwater, HPLC-grade acetonitrile, AR-gradePotassium dihydrogen ortho phosphate werepurchased from Merck.

2.2. InstrumentsThe HPLC system used was equipped withquaternary gradient pumps with auto samplerinjector (Shimadzu LC 2010, Japan) andcontrolled with LC solutions software (Shimadzu).Essae analytical balance was used for allweighing.

2.3. Preparation of system suitability solutionAccurately weighed 25mg of Prasugrelhydrochloride into a 50 mL volumetric flask,added about 10 mL of diluent, sonicated todissolve and diluted up to the mark with diluent.

2.4. Preparation of sample solutionAccurately weighed 25mg of Prasugrelhydrochloride sample into a 50 mL volumetricflask, added about 10 mL of diluent, sonicated todissolve and diluted up to the mark with diluent.

2.5. Chromatographic conditionsThe chromatographic separation was achieved onGemini C-18 250 x 4.6 mm, 5 μm particle sizecolumn. The Isocratic LC method employs withthe composition of solution A and B as mobilephase in the ration of 30:70 respectively. SolutionA contains 0.05M Potassium dihydrogen orthophosphate. Solution B contains HPLC gradeacetonitrile and water 90:10. The flow rate of themobile phase was 1.2 mL per min and the peakshape of the Prasugrel hydrochloride peak wasfound to be symmetrical. Typical systemsuitability chromatogram is presented as figure.4.The detection was performed at UV 220 nm. Theinjection volume was 20 μL. A mixture of waterand acetonitrile (50 : 50 v/v) was used as a diluent.

3. VALIDATION OF THE METHOD

3.1. SpecificitySpecificity is the ability of the method to measurethe analyte response in the presence of its potentialimpurities. The specificity of the developed LCmethod for Prasugrel Hydrochloride was carriedout in the presence of its impurities namely,

Impurity-A, Impurity-B and Impurity-C. Stressstudies were performed for PrasugrelHydrochloride drug substance to provide anindication of the stability indicating property andspecificity of the proposed method. Degradationstudy was performed on 0.50 mg per mL solutionof the drug in diluent. Neutral, acid hydrolysis,base hydrolysis and oxidation study was carriedout by using diluent, 5 mL of 1N hydrochloricacid, 5 mL of 1N sodium hydroxide and 2 mL of50 % hydrogen peroxide solution respectively.These solutions were prepared and each washeated in water bath at 60 °C for 0.5 hr, 1 hr, 1.5hr and 2 hr respectively. Photolytic and thermaldegradation was carried out on drug substance byexposing it separately on short wavelength light(254 nm) and heat (60 °C) for 4 days. Sampleswere withdrawn at appropriate time and subjectedto analysis. All the impurities and degradationproducts were separated with appropriateresolution by the developed method.

3.2. PrecisionThe precision of the related substance method waschecked by injecting six individual preparations ofPrasugrel Hydrochloride spiked with 0.5 % ofeach impurity with respect to the PrasugrelHydrochloride analyte concentration. The % RSDof the area of each impurity was calculated. Theintermediate precision of the method was alsoevaluated using different analyst and instrument inthe same laboratory.

3.3. LOD and LOQThe LOD and LOQ were determined by measuringthe standard deviation of the response and slope.The LOD and LOQ for Prasugrel Hydrochloride,Impurity-A, Impurity-B and Impurity-C weredetermined by injecting a series of dilute solutionswith known concentrations. The precision studywas also carried out at the LOQ level by injectingfive injections of Prasugrel Hydrochloride,Impurity-A, Impurity-B and Impurity-C,calculating % RSD for the areas of each impurity.

3.4. AccuracyThe accuracy of the method for all the relatedsubstances was determined by analyzing PrasugrelHydrochloride sample solutions spiked with all therelated substances at four different concentrationlevels of LOQ, 50, 100 and 150 % of each intriplicate at the specified limit. The percentage ofrecoveries for the impurities was calculated byinjecting the standard solution for each level.

Venugopal Sriram et al /Int.J.PharmTech Res.2012,4(4) 1410

3.5. LinearityThe Linearity of the method for all the relatedsubstances was determined by analyzing dilutesolution of Prasugrel Hydrochloride and its relatedsubstances at four different concentration levels ofLOQ, 50, 100 and 150 % of each in triplicate atthe specified limit. The correlation coefficient wascalculated for each substance.

3.6. RobustnessTo determine the robustness of the developedmethod, experimental conditions were deliberatelyaltered and the resolution between the PrasugrelHydrochloride, Impurity-A, Impurity-B andImpurity-C was recorded. The parameters selectedwere mobile phase composition and flow rate (±0.2 mL/min).

3.7. Solution and mobile phase stabilityTo determine the stability of sample solution,Prasugrel Hydrochloride samples spiked withrelated substances at specified level were preparedand analyzed after 48 hr. The results of thesestudies indicated the stability of sample solution atroom temperature for 48 hr. The mobile phaseprepared and kept at room temperature for 48 hr.After 48 hr Prasugrel Hydrochloride samplespiked with impurities at specified level wereprepared and analyzed. The results are statistically

evaluated and meeting system suitability andprecision requirement which indicates the mobilephase is stable for 48 hr at room temperature.

4. RESULTS AND DISCUSSION

4.1. Optimization of chromatographicconditionsThe main objective of the chromatographicmethod was to separate Prasugrel Hydrochloridefrom Impurity-A, Impurity-B and Impurity-C.Impurities were co-eluted using differentstationary phases such as C-8, Cyano, phenylcolumn with different mobile phases. Duringevaluation of different column chemistries,Phenomenex Gemini C-18 column was observedto give better resolution with the 0.05M phosphatebuffer. A good resolution and peak shape wasoptimized as mentioned under section“Chromatographic conditions”. In optimizedchromatographic conditions PrasugrelHydrochloride, Impurity-A, Impurity-B andImpurity-C were separated with a good resolutiongreater than 2, typical relative retention times forImpurity-A, Impurity-B and Impurity-C wereapproximately 0.17, 0.46 and 0.62 respectively{Fig. 3}. Specificity details are mentioned in theTable I.

Table I. Specificity details

Parameter Prasugrel HCl Impurity-A Impurity-B Impurity-CRT

RRTNTF

11.591.000178471.008

5.360.46

149591.086

7.160.62

176151.063

1.930.1744241.114

RT= Retention time, RRT=Relative retention time, N= Theoretical plates & TF= Tailing factor.

{ Fig.3 : Prasugrel Hydrochloride spiked with impurities }

Venugopal Sriram et al /Int.J.PharmTech Res.2012,4(4) 1411

{ Fig.4 : Prasugrel hydrochloride system suitability chromatogram}

4.2. Validation of the method4.2.1. Forced degradationDegradation was not observed in PrasugrelHydrochloride sample when subjected to neutral,thermal and photolytic stress conditions.Prasugrel Hydrochloride was degraded underneutral, acid, base and oxidation condition{Fig.4}. The summary of the forced degradationstudy is mentioned in Table II. Prasugrel foundmore sensitive to base medium and oxidationcondition. Significant degradation was observed

in base and oxidation condition. Initially the basehydrolysis conducted with 5 ml of 1N Sodiumhydroxide solution at 60°C and the product iscompletely degraded then 0.1N sodium hydroxidewas used for the study of one hour with 15minutes sampling interval. For oxidation initially2 mL of 50% hydrogen peroxide was used andsignificant degradation was observed at 60°C,then the study was performed using 1 mL of 50%hydrogen peroxide.

{ Fig.5: Prasugrel Hydrochloride neutral degradation sample with diluent (2hr at 60°C)- Majordegradation products were observed at RT 2.13, 6.66 and 8.19 min.}

Venugopal Sriram et al /Int.J.PharmTech Res.2012,4(4) 1412

{ Fig.6: Prasugrel Hydrochloride acid hydrolysis study sample with 5 mL of 1N HCl (2 hr at 60 °C)-Major degradation products were observed at RT 2.13, 4.12, 4.63, 6.66, 6.84, 7.57, 8.20 and 14.28min.}

{ Fig.7: Prasugrel Hydrochloride base hydrolysis study sample with 5 mL of 0.1N sodium hydroxide(15 min at 60 °C)- Major degradation products were observed at RT 3.58, 4.60, 6.59, 6.76, 8.10 and14.02 min.}

Venugopal Sriram et al /Int.J.PharmTech Res.2012,4(4) 1413

{ Fig.8: Prasugrel Hydrochloride peroxide degradation study sample with 1 mL of 50% hydrogenperoxide (30 min at 60 °C)- Major degradation products were observed at RT 2.21, 6.55, 6.71, 8.04and 13.89 min.}

Table II. Forced degradation study result of Prasugrel hydrochloride

Degradation condition % Purity RRT of majordegradation peaks

Control sample

Neutral condition (60 °C, 2 hr only diluent)

Acid hydrolysis (60 °C, 2 hr, 5 mL, 1 N HCl)

Base hydrolysis (60 °C, 15 min, 5 mL, 0.1 N

NaOH)

Oxidation (60 °C, 30 min, 1 mL, 50 % Hydrogen

peroxide)

Photolytic degradation (UV 254, 4 days)

Thermal degradation (60 °C, 4 days )

99.63

97.36

96.77

27.27

70.75

99.48

99.54

-

0.15, 0.48, 0.49 & 0.59

0.20, 0.42, 0.58,

0.59,0.66 & 0.71

0.24, 0.28, 0.31, 0.40,

0.43, 0.58, 0.59 & 0.71

0.20, 0.24, 0.43, 0.49,

0.58, 0.60 & 0.71

No major degradationpeak was observed

No major degradationpeak was observed

4.2.2. PrecisionThe Precision was determined at the LOQ to150% concentration for Prasugrel Hydrochloride,Impurity-A, Impurity-B and Impurity-C and the% RSD was found to be below 6.0 for allimpurities.

4.2.3. Limit of detection and limit ofquantificationThe values of LOD and LOQ for PrasugrelHydrochloride, Impurity-A, Impurity-B andImpurity-C are mentioned in the Table. III.

Venugopal Sriram et al /Int.J.PharmTech Res.2012,4(4) 1414

Table III. Validation results summary

Parameter Prasugrel HCl Impurity-A Impurity-B Impurity-C

R2 value

LOD in μg/ml

LOQ in μg/ml

%RSD at LOQ

Precision

Accuracy at LOQ

Accuracy at 50%

Accuracy at 100%

Accuracy at 150%

0.9998

0.0167

0.0493

2.016

0.198

--

--

--

--

0.9988

0.0081

0.0246

4.504

0.984

104.54

97.08

97.20

97.94

0.9987

0.0077

0.0234

5.758

1.933

95.73

92.52

96.38

95.49

0.9990

0.0041

0.0125

1.743

0.870

97.02

106.22

103.79

96.63

4.2.4. LinearityLinearity calibration plot for the relatedsubstances method was obtained over thecalibration ranges tested i.e. LOQ, 50 %, 100 %and 150 % of the specification limit. Thecorrelation co-efficient obtained was greater than0.99. The above result shows that an excellent

correlation existed between the peak area and theconcentration of three impurities. Linearity graphsfor Prasugrel hydrochloride, Impurity-A,Impurity-B and Impurity were presented below.Linearity details were mentioned table III.

Venugopal Sriram et al /Int.J.PharmTech Res.2012,4(4) 1415

4.2.5. AccuracyThe Accuracy of all these related substances wasfound to be in between the predefined acceptancecriteria of 80 to 120 % and the data given in TableIII.

4.2.6. RobustnessWhen the chromatographic conditions like flowrate and mobile phase composition weredeliberately varied and resolution between any twopeaks is found greater than 2.0, which illustratesthe robustness of the method.

4.2.7. Solution stabilityThere were no significant changes in the amountof the impurities during solution stabilityexperiment performed using the related substancesmethod. The results from the studies indicated, thesample solution was stable at room temperature for48 hours.

5. CONCLUSION

A new, accurate and selective isocratic HPLCmethod was developed for the determination ofimpurities in Prasugrel hydrochloride andvalidated as per the ICH guidelines. The methodwas found to be simple, selective, precise, accurateand robust. Therefore, this method can be used forroutine testing as well as stability analysis ofPrasugrel hydrochloride. All statistical resultswere within the acceptance criteria.

ACKNOWLEDGEMENT

We acknowledge the support of the Vindhya groupof companies for their many practicalcontributions in AR&D. We thank Mr.D.N.Reddy,Managing Director for permitting this work to bepublished. The authors acknowledge the helprendered by other colleagues for the technicalsupport and useful discussion.

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