“DEVELOPMENT AND VALIDATION OF A NEW RP-HPLC …



“DEVELOPMENT AND VALIDATION OF A NEW RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ATORVASTATIN CALCIUM, CLOPIDOGREL BISULPHATE AND ASPIRIN IN BULK DRUGS FORMULATION”

M.PHARM

DISSERTATION PROTOCOL

Submitted to

Rajiv Gandhi University of Health Sciences

Bangalore, Karnataka.

By

Mr. JIVAN RAJARAM PATIL

B. Pharm

Under the Guidance of

Dr. KISHORESINGH. CHATRAPATI

M.Pharm., Ph. D

DEPARTMENT OF PHARMACEUTICAL CHEMISTRY

R.M.E.S’S COLLEGE OF PHARMACY

GULBARGA-585102

2011-2012

Rajiv Gandhi University of Health Sciences, kARNATAKA Bangalore

Curriculum Development Cell

Conformation for Registration of

Subject for Dissertation

Registration No. :

Name of the Candidate : Mr. Jivan Rajaram Patil

Address : R.M.E.S’s College of Pharmacy

Gulbarga, Karnataka.

Name of the Institution : R.M.E.S’s College of Pharmacy

Gulbarga, Karnataka.

Course of Study and Subject : M.Pharm in Pharmaceutical chemistry

Date of Admission to the Course : 25/05/2011

Title of the Topic : Development and Validation of a New RP-

HPLC Method For the Simultaneous

Estimation of Atorvastatin Calcium,

Clopidogrel Bisulphate and Aspirin In Bulk

Drugs Formulation

Brief resume of the intended work : Enclosed

Signature of the student :

Guide Name : Dr. Kishoresingh Chatrapati

M.Pharm Ph.D

Remarks of the Guide : The above information and literature has been

extensively investigated verified and was to be

coreect. The present study will be carried out

under my supervision and guidance

Signature of the Guide :

Co-Guide Name : Mr. H.J Kallur

M. Pharm (Ph.D)

Signature of the Co-Guide :

HOD Name : Dr. Kishoresingh Chatrapati

M.Pharm Ph.D

Signature of the HOD :

Director/Principal Name : Prof. HARIPRASANNA R.C

M.Pharm (Ph.D)

Mobile No : +919845300610

Director/Principal E-mail ID : rmepharmacy@

Remarks of Director/ principal : Recommended for approval

Director/Principal Signature :

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

|1 |Name and Address of the candidate and address |Jivan Rajaram Patil |

| | |A/P Male Masud, Tal –Panhala, Dist- Kolhapur. 416 114. |

|2 |Name of the Institution |R.M.E.S’s College of Pharmacy, Gulbarga, |

| | |Karnataka – 585102 |

| | | |

|3 |Course of study and subject | Master of Pharmacy in |

| | |Pharmaceutical Chemistry |

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|4 |Date of admission to course |25th May 2011 |

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|5 |Title of the topic |“Development and Validation Of a New RP-HPLC Method For the |

| | |Simultaneous Estimation of Atorvastatin Calcium, Clopidogrel |

| | |Bisulphate And Aspirin In Bulk Drugs Formulation.” |

|6 |Brief resume of this intended work | |

| |6.1 Need for the study | |

| | |Enclosure -1 |

| |6.2 Review of Literature | |

| | |Enclosure –II |

| |6.3 Objectives of the study | |

| | |Enclosure -III |

|7 |Material And Methods | |

| |7.1 Source of data | |

| | |Enclosure –IV |

| |7.2 Method of Collection of data | |

| | |Enclosure -V |

| | | |

| |7.3 does the study require any investigation | |

| |or interventions to be conducted on patients | |

| |or other human beings or animal? If so please | |

| |described briefly | |

| |7.4 Has ethical clearance been obtained from | |

| |your institution in case of 7.3 | |

| | |NO |

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| | |NO |

|8 |List of References |Enclosure-VI |

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|9 |Signature of theCandidate | |

|10 |Remarks of the guide |The above information and literature has been extensively |

| | |investigated, verified and was found to be correct. The present |

| | |study will be carried out under my supervision& guidance. |

|11 |Name & Designation of | |

| | | |

| |11.1 Guide |Dr. Kishoresingh. Chatrapati |

| |11.2 Signature |M.Pharm., Ph.D |

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| |11.3 Co-guide | |

| | |Mr. H.J. Kallur |

| |11.4 Signature |M.Pharm., (Ph.D) |

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| |11.5 Head of the department |Dr. Kishoresingh. Chatrapati |

| | |M.Pharm., Ph.D |

| | |Proffessor and HOD |

| | |Department of Pharmaceutical Chemistry |

| |11.6 Signature | |

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|12 | | |

| |12.1 Remarks of the chairman and the principal |The proposed work can be carried out in our laboratory at R.M.E.S’S |

| |. |college of pharmacy, Gulbarga. Karnataka |

| | | |

| |12.2 Name of the Director/Principal |Prof. HARIPRASANNA R.C |

| | |M.Pharm.,(Ph.D) |

| | |PROFFESSOR & DIRECTOR |

| | |R.M.E.S’S COLLEGE OF  PHARMACY     GULBARGA-585 102 KARNATAKA |

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| | | |

| |Mobile No> |+919845300610 |

| |Director/Principal E-mail ID |rmepharmacy@ |

| | | |

| |Remarks of Director/ principal | |

| |Director/Principal Signature | |

| | | |

| | |Recommended for approval |

| |ENCLOSURE-I |

|6. |6.1 Need of study |

| |Introduction |

| |Most of the drugs in multicomponent dosage form can be analyzed by HPLC method because of the several advantages like rapidity, specificity, |

| |accuracy, precision, and ease of automation in these methods. HPLC method eliminates tedious extraction and isolation procedures. |

| |There are different modes of separation in HPLC. They are Normal Phase Mode, Reversed Phase Mode, Reversed Phase Ion Phase Chromatography, |

| |Affinity Chromatography and Size Exclusion Chromatography. |

| |Normal Phase Mode: In these the stationary phase is polar and the mobile phase is non-polar in nature. In these techniques, non-polar compounds |

| |travel faster and are eluted first. This is because of the lower affinity between the non-polar compounds and the stationary phase. |

| |Reversed Phase Mode: The stationary phase is non-polar hydrophobic packing with Octyl or Octa decyl functional group bounded to silica gel and |

| |the mobile phase is polar solvent. An aqueous mobile phase allows the use of secondary solute chemical equilibrium to control retention and |

| |selectivity. The polar compounds gets eluted first in this mode and non-polar compounds are retained for longer times. As most of the drugs and |

| |Pharmaceuticals are polar in nature, they are not retained for longer times and hence elute faster. The different columns used are C18 Octa |

| |Decyl Silane, C 8 Octasilane, C4 Tetrasilane etc. |

| |Ion Exchange Chromatography: The stationary phase contains ionic groups like NR3+ or SO3- which interact with the ionic groups of the sample |

| |molecules. This is suitable for the separation of charged molecules only. |

| |Affinity Chromatography: In these techniques highly specific biochemical interactions are used for separation. The stationary phase contains |

| |specific group of molecules which can adsorb the sample if certain steric and charge related conditions are satisfied. |

| |Size Exclusion Chromatography: It separates molecules accordingly to their molecular mass. Largest molecules are eluted first and the smallest |

| |molecules last1, 2. |

| |Atorvastatin calcium is chemically [R-(R*, R*)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino) |

| |carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt (2:1) trihydrate3. It is a lipid lowering agent. It is inhibitor of HMGCoA reductase |

| |enzyme4. A very few reports were found for the analysis in the estimation of Atorvastatin calcium. |

| |[pic] |

| |Structure of Atoravastatin calcium |

| |Clopidogrel bisulphate is chemically Methyl (+)-(S) - (o-chlorophenyl)-6, 7-dihydrothieno [3, 2-c] pyridine-5(4H)-acetate, sulfate. It is used |

| |as an anti thrombic agent4, 5. |

| |[pic] |

| |Structure of Clopidogrel bisulphate |

| |Aspirin is chemically 2-acetyloxybenzoic acid a salicylate drug, often used as an analgesic to relieve minor aches and pains, as an antipyretic |

| |to reduce fever and as an anti-inflammatory medication6. A very few reports were found for the analysis in combination with other drugs in |

| |pharmaceutical formulations. |

| |[pic] |

| |Structure of Aspirin |

| |NEED FOR THE STUDY : |

| |An exhaustive literature survey on analytical methods led to following observations, |

| |There is no analytical methods were published for new drugs. |

| |The reported methods for many drugs made use of complex or costly methodology and solvents. |

| |Some reported method did’t makes use of internal standards while some used internal standards that were not easily available in market. |

| |There is no consensus among authors about the validation criteria for analytical methods. |

| |Thus the above observations justify the need for development and validation of new methods for simultaneous determination of drugs. |

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| |ENCLOSURE-II |

| |6.2 REVIEW OF LITERATURE: |

| |Review should be based on assimilation of data from various sources for the purpose of establishing a new concept. A preliminary survey of |

| |literature for suitable method development for newer drugs has been made. |

| |Review of literature reveals that extensive of work has been carried out for the routine analysis of drugs and pharmaceuticals in recently |

| |marketed as well as existing formulations and bulk drugs. Development of high performance liquid chromatographic method can be successfully |

| |employed for the analysis of formulations. Some literature survey of the present study is under. |

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| |1. Sdka Erturk,et al., reported an HPLC method for the determination of atorvastatin and its impurities in bulk drug and tablet a gradient |

| |RP-HPLC assay was used with UV detection. Solvent system using methanol or acetonitrile and water or buffer system with a different pH values |

| |were tested. Best resolution has been determined using a Luna C18 column with acetonitrile ammonium acetate buffer pH 4-THF as a mobile phase. |

| |Samples were eluted at flow rate 1.0 ml min-1 and detected at 248 nm7. |

| | |

| |2. Syed Shanaz Qutab et al. Reported his work on simultaneous determination of atorvastatin calcium and ezetimibe in pharmaceutical |

| |formulations by RP-HPLC, separation was achieved on a 250 x 4.6 mm, 5µ Hypersil phenyl-2 column. Eluent was monitored by absorbance at 242 nm |

| |using a mixture of 0.1 M ammonium acetate (pH 6.5) and acetonitrile in the ratio of 28:72 (v/v) 8. |

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| |3. DA Shah, et al. Developed a RP-HPLC method for determination of atorvastatin calcium and nicotinic acid in combined tablet dosage form. A |

| |phenomenex Luna C18, 5 mm column having 250 x 4.6mm i.d. in isocratic mode, with mobile phase containing 0.02 M potassium dihydrogen |

| |phosphate:methanol:acetonitrile(20:40:40, pH 4) was used. The flow rate was 1.0 ml/min and effluents were monitored at 240 nm9. |

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| |4. Patel R. B.,et al. was reported a validated HPLC and HPTLC methods for simultaneous estimation of acetylsalicylic acid (ASA) and clopidogrel|

| |bisulfate (CLP) in pure powder and formulations. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length x 4.6 mm id, 5 microm |

| |particle size) using acetonitrile-phosphate buffer, pH 3.0 (55 + 45, v/v) mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The |

| |HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol-toluene-glacial acetic acid (5.0 + |

| |1.0 + 4.0 + 0.1, v/v) mobile phase. Quantitation was achieved with UV detection at 235 nm10. |

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| |5. M Gandhimathi,et al. Were revels that the reverse phase high performance liquid chromatographic method to determine aspirin and clopidogrel |

| |in combined dosage form is reported. The chromatographic resolution of aspirin and clopidogrel was obtained in a mobile phase consisting of 0.1%|

| |v/v triethylamine (pH 4.0):acetonitrile in the ratio 25:75% (v/v) in an isocratic elution. A detection wavelength of 225 nm and flow rate of 1 |

| |ml/min was used in the study. Nimesulide (20 µg/ml) was used as an internal standard11. |

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| |6. K Anandakumar,et al. Developed a reverse phase high performance liquid chromatography method has been reported for the simultaneous |

| |estimation of aspirin and Clopidogrel, bisulphate in formulation. The separation was achieved by octadecyl column (C 18) and acetonitrile: |

| |methanol: 20 mM phosphate buffer at pH 3 (50:7:43 v/v) as eluent, at a flow rate of 1 ml/min. Detection was carried out at 240 nm. Quantitation |

| |was done by external standard calibration method. The retention time of aspirin and Clopidogrel, bisulphate was found to be 2.40 and 9.27 min, |

| |respectively12. |

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| |7. P Mishra et al. Was developed a simultaneous equation method and absorbnce ratio method were reported for the determination of aspirin and |

| |clopidogrel in pharmaceutical formulations. First method is based on the additivity of absorbances. Second method is based on the determination |

| |of graphical absorbance ratio at two selected wavelengths, one being the isoabsorptive point for the two drugs (225 nm) and the other being the |

| |absorption maximum of hydrolysed aspirin (235.7 nm). The accuracy and reproducibility of the reported methods was statistically validated by |

| |recovery studies13. |

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| |8. Hassan H.Hammud et al. Developed a stability-indicating Spectrofluorimetric and RP-HPLC methods for aspirin and dipyridamole, |

| |Spectrofluorimetric method used first and second derivatives ratio of the emission with zero crossing technique. RP-HPLC method utilized an |

| |adsorbosil C8, 10µM, 250 mm x 4.6 mm i.d. column,mobile phase consisted of water : acetonitrile:ortho-phosphoric acid (65:35:2 v/v/v) with flow |

| |rate at 1.5 ml/min and UV detection at 250 nm14. |

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| |9. Purushotam K.Sinha et al. reported method involving separation of aspirin and clopidogrel bisulphate by HPTLC on TLC aluminum plates |

| |precoated with silica gel 60 F 254 as a stationary phase with solvent system of carbon tetrachloride:acetone (6:2.4 v/v) and detection was |

| |carried out densitometrically in absorbance mode at 220 nm15. |

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| |10. Suresh Kumar S. et al. Developed a HPLC method for analysis of aspirin by using Hypersil BDC C18 (100 x 4.6 mm 5µ) column. Mobile phase |

| |consistet of sodium perchlorate buffer (pH 2.5) acetonitrile:isopropyl alcohol (85:14:1 % v/v) at flow rate of 1.5 ml min-1 and detected at 275 |

| |nm16. |

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| |11. Rupali Hirave et al. reported her work on RP-HPLC method for simultaneous estimation of atorvastatin calcium and fenofibrate in tablet |

| |dosage form by using a mobile phase consisting of methanol:water pH 3.2 (90:10 v/v) at a flow rate of 1 ml/min, sepration was achieved by HiQ |

| |sil C8 (4.6x250mm) column. Detection was carried out at 260 nm17. |

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| |12. Antaryami Jena et al. reported his work on analytical method development and validation of simultaneous determination of atorvastatin |

| |calcium and amlodipine besilate in tablet dosage form by RP-HPLC by using mobile phase consisting of a mixture of a phosphate buffer (1 ml ortho|

| |phosphoric acid in 1000 ml of water) acetonitrile and methanol (53:43:4 v/v) at a flow rate of 1 ml/min and UV detection at 246 nm, using |

| |photodine array detector, by using Grace Smart RP C18 column(250 x 4.6 , 5µm)18. |

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| |13. Suresh Kumar GV et al. developed and validated a RP-HPLC method for simultaneous estimation of atorvastatin calcium and telmisartan in |

| |tablet dosage form, sepration was achieved on (Waters symmetry C18, 250mm x 4.6mm, 5µ) column, by using mixture of ammonium acetate (0.02M, pH |

| |4.0 adjusted with glacial acetic acid) and acetonitrile in ratio (40:60 v/v) at flow rate of 1.0 ml/min and detected wavelength 254 nm19. |

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| |14. Anandkumar K et al. Developed a reverse phase high performance liquid chromatography method for simultaneous estimation of aspirin and |

| |clopidogrel bisulphate in formulation. The separation was achieved by octadecyl column C18 and acetonitrile:methanol: 20 mM phosphate buffer at |

| |pH 3 (50:7:43 v/v/v) as eluent, at a flow rate of 1 ml/min. Detection was carried out at 240 nm20. |

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| |15. Patel G.F. et al. Developed simple and reproducible spectrophotometric method for the estimation of aspirin and atorvastatin calcium; |

| |second order derivative spectrpscopy method was adopted to eliminate spectral interference, using 266.78 nm and 237.35 nm as zero crossing |

| |points for aspirin and atorvastatin calcium respectively. Methanol was used as a solvent21. |

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| |The literature survey revealed some HPLC methods and spectrophotometric methods for determination of atorvastatin calcium and aspirin |

| |individually and in combinations with other drugs. The present work describes the development of simple, sensitive, precise and accurate reverse|

| |phase HPLC method for simultaneous estimation of atorvastatin calcium and aspirin in bulk drug and combined capsule dosage form. |

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| |ENCLOSURE-III |

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| |6.3 OBJECTIVES OF THE STUDY: |

| |Objectives of present work are: |

|. |To develop a new RP-HPLC method for simultaneous determination of atorvastatin calcium, Clopidogrel bisulphate and aspirin by using simple |

| |mobile phase. |

| |To establish simple, sensitive, precise, accurate, and cost effective, RP-HPLC method for simultaneous estimation of atorvastatin calcium and |

| |aspirin in bulk drug and dosage form. |

| |To validate developed method as per ICH and USP guidelines. |

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| |6.4 PLAN OF WORK : |

| |1. Literature survey for selection of drug and to gain therotical knowledge on analytical |

| |method development and validation. |

| |2. Procurement and standardization of drugs. |

| |3. Development and optimization of analytical method. |

| |4. Description of final method for drug. |

| |5. Validation of developed method as per ICH guidelines. |

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| |ENCLOSURE-IV |

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| |MATERIALS AND METHODS: |

| |7.1. SOURCE OF DATA: |

| |a) Internet |

| |b) Gulbarga University Library, Gulbarga |

| |c) RGUHS ( Helinet ) |

| |d) International Pharmaceutical Abstracts |

| |e) All the basic facilities required for these are available in our college |

| |Laboratory. |

| |7.2 MATERIAL : |

| |Cyberlab HPLC Model High pressure system-2003 series comprising of LC-P-100 pump and LC-UV-100 as UV-VIS detector, Rheodyne manual Injector |

| |fitted with 100 µl loop and cyberstore chromatography software. |

| |Other instrument to be use UV visible Spectrophotometer of PG labs, analytical balance, sonnicator etc. |

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| |ENCLOSURE-V |

| |7.3 METHODS : |

| |Method development included the following stages; |

| |Procurement and Standerdization of drugs reference standards by measurement of physical constants and spectroscopy techniques like IR |

| |Spectroscopy. Accordingly this analysis and standardization is done. |

| |Procurement of HPLC grade chemical. |

| |Selection and Optimization of chromatographic conditions. |

| |The stationary and mobile phase will be selected and optimized in active best resolution between drug and internal standards. Retention |

| |wavelength will be selected to improve specificity and sensitivity. |

| |Calibration Experiment. |

| |Validation: The method will be validated using the validation criteria provided by U.S.FDA and ICH in it guidance document. Validation |

| |experiment will be perform using Quality Control (QC) sample. |

| |7.3 (a) Does the study require any investigation to be conducted on patients or other humans or animals? If so, please describe briefly. |

| |NOT APPLICABLE |

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| |7.3(b) Has ethical clearance been obtained from your institution in case of 7.3 |

| |NOT APPLICABLE |

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| |ENCLOSURE-VI |

| |8. LIST OF REFERENCES: |

| |Swarbrick James and Boylan James.C, Encyclopedia of pharmaceutical technology, volume I, Marcel Dekker Inc. New York, 1998: 217-224. |

| |Beckett AH, Stenlake JB, Practical Pharmaceutical chemistry, 1997, 4th edition,part 2, CBS publication and distributors:275-337. |

| |Martindale (2007) The complete drug reference 35th edition Pharmaceutical Press, London: 866. |

| |Satoskar RS, Bhandarkar SD, Ainapure SS, editors. Pharmacology and Pharmaco- therapeutics. 15 th ed. Mumbai: Popular Prakashan; 1997, 462. |

| |Budavari, S, eds., In; the Merck Index. 12 th ed. New Jersey: Merck and Co., Inc; 1996, 406. |

| |Harvey PS, Goa KL, Drugs, 1999, 58:469-475. |

| |Erturk S, Aktas ES, Ersoy L, Fccoglu S. An HPLC method for the determination of atorvastatin and its impurities in bulk drug and tablets. J. |

| |Pharm. and Biomed.Anal., 33; 2003, 1017-1023. |

| |Qutab SS, Razzaq SN, Khan IU, Ashfaq M, Shuja ZA. Simultaneous Determination of Atorvastatin Calcium and Ezetimibe in Pharmaceutical |

| |Formulations by Liquid Chromatography. J. Food and Drug Anal., 2007; vol. 15(2):139-144. |

| |Shah DA, Bhat KK, Mehta RS, Shankar MB, Baldania SL, RP-HPLC method for the determination of atorvastatin calcium and nicotinic acid in combined|

| |tablet dosage form. Ind. J. Pharm. Sci., 2007; 69 (5): 700-703 |

| |Patel RB, Shankar MB, Patel MR, Bhatt KK, Simultaneous estimation of acetylsalicylic acid and clopidogrel bisulfate in pure powder and tablet |

| |formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography. J AOAC Int. 2008, |

| |Jul-Aug;91(4):750-755. |

| |M Gandhimathi, TK Ravi, High performance liquid chromatographic determination of aspirin and clopidogrel in tablets, 2007, 69(1), 123-125. |

| |K Anandakumar, T Ayyappan, V Raghu Raman, T Vetrichelvan, A.S.K Sankar, D Nagavalli, RP-HPLC analysis of aspirin and clopidogrel bisulphate in |

| |combination, 2007, 69(4), 597-599. |

| |P Mishra, Archana Dolly, Simultaneous determination of clopidogrel and aspirin in pharmaceutical dosage forms, 2006, 68(3), 365-368. |

| |Hassan HH, Fawzy AY, Mohamad EM, Ghassan MS and Nada MS. Stability-Indicating Spectrophotometric Fluorimetric and RP-HPLC methods for the |

| |determination of aspirin and dipyridamole in their combinations. The Open Spect. J., 2008; vol.2: 19-28. |

| |Sinha PK, Damale MC, Bothara KG. A Validated Stability Indicating HPTLC Method for Determination of Aspirin and Clopidogrel Bisulphate in |

| |Combined Dosage Form. Euras. J. Anal. Chem., 2009 ;vol.4(2):152-160. |

| |Kumar SS., Jamadar LD, Bhat K, Musmade P, Vasantharaju SG, Udupa N. Analytical method development and validation for aspirin. Interna. J. |

| |ChemTech Research, 2010; vol.2 (1):389-399. |

| |Hirave R, Bendagude R, Kondawar M. RP-HPLC method for simultaneous estimation of Atorvatatin Calcium and Fenofibrate in tablet dosage forms. |

| |J.Pharmacy Research, 2010;vol.3 (10):2400-2401. |

| |Jena A, Madhu M, Latha S.Analytical Method Development and Validation of Simultaneous Determination of Atorvastatin Calcium and Amlodipine |

| |Besilate in Tablet Dosage form by RP-HPLC. Interna. J.Pharm. Sci. and Research, 2010; vol.1(11):100-106. |

| |SureshKumar GV, Rajendraprasad Y, Chandrashekar SM. Development and Validation of reversed-phase HPLC method for Simultaneous Estimation of |

| |Atorvastatin calcium and Telmisartan in Tablet dosage form. Interna. J. PharmTech Research, 2010; vol 2(1):463-470. |

| |Anadakumar K, Ayyappn T, Raghu R, Vettrichelvan T, Sankar ASK, Nagavalli D. RP-HPLC analysis of aspirin and clopidogrel bisulphate in |

| |combination. Ind. J. Pharm. Sci., 2010; Vol.69 (4):597-599. |

|7 |Patel GF, Vekaria NR, Dholakiaya .B. Estimation of Aspirin and Atorvastatin Calcium in Combined Dosage Form Using Derivative Spectrophotometric |

| |Method. Intern. J. Pharm. Research, 2010; vol.2 (1):62 66. |

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