"Analytical Method Development for Pharmaceutical Formulations:"
“DEVELOPMENT OF NEW ANALYTICAL METHODS AND THEIR VALIDATION FOR THE DETERMINATION OF LAMIVUDINE AND FLUOXETINE HYDROCHLORIDE IN BULK AND MARKETED FORMULATIONS” MASTER OF PHARMACY DISSERTATION PROTOCOL SUBMITTED TO THE RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE BY MAKADIA BINIBEN PRAVINBHAI Under The Guidance of Dr. E.V.S. SUBRAHMANYAM. M.PHARM.Ph.D P.G. DEPARTMENT OF QUALITY ASSURANCE, SRINIVAS COLLEGE OF PHARMACY, MANGALORE – 574143 2011-2013 RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE, KARNATAKA ANNEXURE – II REGISTRATION OF SUBJECT FOR DISSERTATION MAKADIA BINIBEN PRAVINBHAI NAME AND ADDRESS OF DEPARTMENT OF Q.A., 1.0 THE CANDIDATE SRINIVAS COLLEGE OF PHARMACY, VALACHIL,POST PARENGIPETE, MANGALORE TQ-574143 2.0 NAME OF THE SRINIVAS COLLEGE OF PHARMACY, INSTITUTION VALACHIL, MANGALORE. 3.0 COURSE OF STUDY & MASTER OF PHARMACY SUBJECT (QUALITY ASSURANCE) 4.0 DATE OF ADMISSION 31st OCTOBER, 2011 5.0 TITLE OF THE TOPIC: “DEVELOPMENT OF NEW ANALYTICAL METHODS AND THEIR VALIDATION FOR THE DETERMINATION OF LAMIVUDINE AND FLUOXETINE HYDROCHLORIDE IN BULK AND MARKETED FORMULATIONS” 6.0 BRIEF RESUME OF THE INTENDED WORK: 6.1 Need for study: Analytical Method Development for Pharmaceutical Formulations: Analytical methods are essential to characterize drug substances and drug products composition during all stages of pharmaceutical development. For routine analytical purpose it is always necessary to establish methods capable of analyzing large number of samples in a short time period with high accuracy and precision. The number of drugs, which may be either new entities or partial structural modification of the existing ones, introduced into the market is increasing every year. Very often there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. Hence, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. It becomes necessary, therefore to develop new analytical methods for such drugs. These products can present challenges to the analytical chemist responsible for the development and validation of analytical methods. 6.2 Basic criteria for new method development of drug analysis: The drug or drug combination may not be official in any pharmacopoeias. A proper analytical procedure for the drug may not be available in the literature due to patent regulations. Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients. Analytical methods for a drug in combination with other drugs may not be available. The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable. Analytical method development provides the support to track the quality of the product from batch to batch. Estimation can be performed by the following two methods: Titrimetric methods and Instrumental methods. Spectrophotometric Methods Chromatographic Methods Methods for analyzing drugs in dosage forms can be developed, provided one has knowledge about the nature of the sample, its molecular weight, polarity, ionic character and the solubility parameter. Method development involves considerable trial and error procedures. The most difficult problem usually is where to start, what type of column is worth trying with what kind of mobile phase. Method development scheme for a typical HPLC-UV related substance describe below, 1. To define the goals for method development (e.g., what is the intended use of the method?), and to understand the chemistry of the analytes and the drug product. 2. To develop preliminary HPLC conditions to achieve minimally acceptable separations. These HPLC conditions will be used for all subsequent method development experiments. 3. To develop a suitable sample preparation scheme for the drug product. 4. To determine an appropriate standardization method and the use of relative response factors in calculations. 5. To identify the “weaknesses” of the method and optimize the method through experimental design. Understand the method performance with different conditions, different instrument set ups and different samples. 6. To complete method validation according to ICH guidelines as mentioned in Q2 (R1). 6.3 A BRIEF INTRODUCTION ABOUT LAMIVUDINE1,2 : Chemical structure of Lamivudine: Chemical name: 4-amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2- dihydropyrimidin-2-one Empirical formula: C8H11N3O3S Molecular weight: 229.26 g/mol Solubility: water and alcohol bioavailability: 86% Protein binding: less than 36% Half-life: 5-7 hours Excretion: renal (circa 70%) Melting point: 160-162ºc Boiling point: 475.4ºc Characteristics: white to off white crystalline powder. PHARMACOLOGY1,2: DRUG CATEGORY: Antiretroviral agent Nucleoside and nucleotide reverse transcriptase inhibitors Reverse transcriptase inhibitors MECHANISM OF ACTION: Lamivudine is an analogue of cytidine. It can inhibit both types (1 and 2) of HIV reverse transcriptase and also the reverse transcriptase of hepatitis B. It is phosphorylated to active metabolites that compete for incorporation into viral DNA. They inhibit the HIV reverse transcriptase enzyme competitively and act as a chain terminator of DNA synthesis. The lack of a 3'-OH group in the incorporated nucleoside analogue prevents the formation of the 5' to 3' phosphodiester linkage essential for DNA chain elongation, and therefore, the viral DNA growth is terminated. Lamivudine is administered orally, and it is rapidly absorbed with a bio-availability of over 80%. Some research suggests that Lamivudine can cross the blood brain barrier. Lamivudine is often given in combination with Zidovudine, with which it is highly synergistic. Lamivudine treatment has been shown to restore Zidovudine sensitivity of previously resistant HIV. Lamivudine showed no evidence of carcinogenicity or mutagenicity in in vivo studies in mice and rats at doses from 10 to 58 times those used in humans. SIDE EFFECTS: Lamivudine may cause side effects. Tell your doctor if any of these symptoms are severe or do not go away: diarrhea, headache, fatigue, chills, nausea, vomiting, loss of appetite, dizziness, trouble sleeping, depression, stuffy nose, cough. If you experience any of the following symptoms or those listed in the important warning section, call your doctor immediately: rash, stomach pain, vomiting (in children), nausea (in children), fever, muscle pain, numbness, tingling, or burning in the fingers or toes. 6.4 REVIEW OF LITERATURE: A great deal of work has been done by the scientist about the current application and future possibilities for altering the drug activities and evaluation with new method development by instrumental methods. 1. Srivani Mallepelli, Narasimharao R, Jajow Swapna3 have reported analytical method development and method validation for the simultaneous estimation of Lamivudine and Stavudine in tablet dosage form by RP-HPLC. Chromatography was carried out by isocratic technique on a reversed-phase BDS hypersil C18 column with mobile phase comprising of 0.1M disodium hydrogen phosphate anhydrous buffer: methanol, adjusted to pH 5 with glacial acetic acid in the ratio of 50:50, v/v and the chromatographic condition was set at a flow rate of 1ml/min with the UV detector at 238 nm. The retention time Lamivudine and Stavudine was 4.8 and 5.7 minute, respectively. 2. T.Sudha, V.R.Ravikumar, P.V. Hemalatha4 have reported RP-HPLC method for the simultaneous estimation of Lamivudine and Abacavir sulphate in tablet dosage form. A rapid high performance liquid chromatography methods has been developed and validated for the estimation of Lamivudine and Abacavir simultaneously in combined dosage form, separation was performed on a 5μm C18 column having dimensions (150X4.6mmid) in isocratic mode, with mobile phase containing a mixture of methanol:water (70:30, v/v/) was used. The mobile phase was pumped at a flow rate of 1.4 ml/min and eluents were monitored at 275nm. 3. Venkatesh M, Bhaskar M, Veeranjaneyulu K, Sunitha K, Bhagyasri G, Veliyath S K5 have reported method development and validation of Lamivudine in tablet dosage form by uv-spectrophotometry. Analytical method development and validation play important roles in the discovery, development and manufacture of Pharmaceuticals. A simple and reproducible UV- spectrophotometric method for the quantitative determination of Lamivudine in tablet formulation was developed and validated in the present work. The parameters linearity, precision, accuracy was studied according to ICH guidelines. Lamivudine has the maximum wavelength at 275 nm. 4. Bojja Soumya, Thimmaraju Manish Kumar and Nerella Raghunandhan6 have reported simultaneous determination of Tenofovir disoproxil fumarate and Lamivudine by UV spectrophotometric method. A simple and rapid UV spectrophotometric method has been developed for simultaneous estimation of Tenofovir disoproxil fumarate (TDF) and Lamivudine (LAM). The absorption maxima of both drugs were found at 260nm and 280nm and obeyed Beer’s law in the range of 5-45μg/ml and 2-16μg/ml respectively for TDF and LAM in acetonitrile : 0.1N HCl (20:80) solvent system. 5. T. Raja and A. Lakshmana Rao7 have reported development and validation of RP- HPLC method for the estimation of Abacavir, Lamivudine and Zidovudine in pharmaceutical dosage form. A method has been developed and validated for the estimation of Abacavir, Lamivudine and Zidovudine by high performance liquid chromatography (HPLC) on a C18 column with UV detection at 270 nm. The mobile phase composition that provides an optimal resolution of components in an acceptable elution time in water: methanol (70: 30 v/v) with 0.1 % potassium dihydrogen phosphate pH 3.2 (adjusted with ortho phosphoric acid). The powdered tablet were extracted with methanol: water (50:50 v/v) mixture and after addition of Stavudine, an internal standard subjected to HPLC analysis and assayed by comparison of analyte to internal standard peak areas to concentration ratios. 6. B. Raj Kumar, M. Priyanka, B. N. Priyamvada, K. Prasad, K. Mallikarjuna,N. Y. Anuradha, B. Lalithamma8 have reported method development and validation for the determination of Abacavir and Lamivudine by using RP-HPLC in pure and pharmaceutical dosage form. Separation was performed on a 5μm C18 column having dimensions (150X4.6mmid) in isocratic mode, with mobile phase containing a mixture of methanol: water (70:30, v/v/) was used. The mobile phase was pumped at a flow rate of 1.4 ml/min and eluents were monitored at 284nm. 7. Jayaseelan S et al9 have reported the new analytical method development and validation for the simultaneous estimation of Lamivudine and Stavudine in tablet dosage form by RP-HPLC Method by using reverse-phase C18 SYMMETRY column with mobile phase of methanol and water (80:20v/v) with UV detection at 266nm and flow rate is 1.5ml/min. 8. Pawan K S et al10 have reported the development and validation of a HPLC method for the simultaneous analysis of Abacavir sulphate and Lamivudine in combined tablet dosage form using Waters Nova-pak HR silica column 300mm length, mobile phase is acetic acid (0.2%v/v): methanol with UV detection at 254nm and flow rate is 2ml/min. 6.5 A BRIEF INTRODUCTION OF FLUOXETINE HYDROCHLORIDE11,12: Chemical structure of Fluoxetine hydrochloride: Iupac name : (RS)-N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]propan-1-amine .Hydrochloride. Formula : C17H18F3NO.HCl Molecular weight : 309.33 g/mol Solubility : water and alcohol Bioavailability : 72% Protien binding : 94.5% Half life : 1-3 days (acute), 4-6 days (chronic) Excretion : renal (80%), fecal (15%) Melting point :179–182 °C Boiling point:395 °C Characteristics: white to off white crystalline PHARMACOLOGY11,12: MEDICAL USES : Fluoxetine is frequently used to treat major depression, obsessive compulsive disorder, bulimia nervosa, panic disorder, body dysmorphic disorder and premenstrual dysphoric disorder. Caution should be taken when using any SSRI for bipolar disorder as this can increase the likelihood of mania; however, Fluoxetine can be used with an antipsychotic (such as Quetiapine) for bipolar. It has also been used for cataplexy, obesity, and alcohol dependence, as well as binge eating disorder. MECHANISM OF ACTION: Fluoxetine's mechanism of action is primarily that of an SSRI although it may produce some of its effects through 5-HT2C antagonism in a manner similar to the novel antidepressant agomelatine. Other psychopharmacological substances that exhibit 5-HT2C antagonism are mirtazapine, agomelatine, trazodone, some tricyclic antidepressants and various (mainly atypical) antipsychotics. Fluoxetine acts as an agonist at the σ1-receptor, with a potency greater than that of Citalopram, but less than that of Fluvoxamine. SIDE EFFECTS: skin rash or hives; difficulty breathing; swelling of your face, lips, tongue, or throat, mood or behavior changes, anxiety, panic attacks, trouble sleeping, or if you feel impulsive, irritable, agitated, hostile, aggressive, restless, hyperactive (mentally or physically), more depressed, or have thoughts about suicide or hurting yourself. 6.6 REVIEW OF LITERATURE: 1. Shubhanjali Shukla et.al.13 have reported RP-HPLC method development and its validation for simultaneous estimation of Alprazolam and Fluoxetine hydrochloride in pharmaceutical dosage form. Reverse phase high pressure liquid chromatographic method has been developed for the simultaneous determination of Alprazolam(Alp) and Fluoxetine hydrochloride in tablet dosage form by reverse phase C18 column, phenomenex (250mm x 4.60mm), particle Size 5 µ. The samples were analyzed by using acetonitrile: water in the ratio of 75:25 (pH adjusted to 2.75 with 0.1% orthophosphoric acid) as a mobile phase at the flow rate of 1.1 ml/min in isocratic mode and detection wavelength 224 nm. 2. Rubesh kumar S, P Gayathri, Duganath N, Kiran CH, Sridhar C, Jayaveera K N14 have reported simultaneous estimation of Fluoxetine hcl and Olanzapine in bulk drug and pharmaceutical formulation by using uv-visible spectroscopy method. The developed method is based upon simultaneous equations (Vierodt’s) method by using UV/Visible spectroscopy. Both drugs come in the categories of anti- depressant and antipsychotic agent. The developed method can be used for the simultaneous estimation of FLU and OLZ in pharmaceutical dosage form without separating from each other or from the excipients. Primarily the λ max of Fluoxetine HCl (FLU) and Olanzapine (OLZ) was determined as 226 and 258 nm respectively. The suggested method is validated by using ICH validation parameters like accuracy, precession, linearity and LOD and LOQ respectively. 3. Ibrahim A. Darwish15 have reported development and validation of spectrophotometric methods for determination of Fluoxetine, Sertraline, and Paroxetine in pharmaceutical dosage forms. Three simple and sensitive spectrophotometric methods were developed and validated for determination of the hydrochloride salts of Fluoxetine, Sertraline, and Paroxetine in their pharmaceutical dosage forms. these methods were based on the reaction of the n-alkylvinylamine formed from the interaction of the free secondary amino group in the investigated drugs and acetaldehyde with each of 3 haloquinones, i.e., chloranil, bromanil, and 2,3-dichloro naphtha -quinone, to give colored vinylamino-substituted quinones. The colored products obtained with chloranil, bromanil, and 2,3-dichloronaphthoquinone exhibit absorption maxima at 665, 655, and 580 nm, respectively. 4. M. S. Bhatia, and S. T. Kumbhar16 have reported development and validation of HPTLC method for estimation of Fluoxetine in its capsule formulation. It employs aluminium backed silica gel 60 F254 TLC plates, (20 cm × 10 cm, layer thickness 0.2 mm) prewashed with methanol and mobile phase comprising of toluene: 2-propanol: ammonia 2:2:0.4 (v/v/v). The developing solvent was run upto 80 mm in Camag chamber previously saturated with 10.0 mL of solvent mixture for 30 min. Densitometric scanning was then performed with Camag TLC scanner-3 equipped with winCATS software Version 1.3.0 at λmax 227 nm. The Rf value was found to be 0.74. The recovery of Fluoxetine was found to be 99.90% ± 1.68. 5. Zahid Zaheer, Obaid Shaikh, Sucheta Thorat, Rana Ahmed17 have reported development and validation of HPTLC method of Fluoxetine hydrochloride in bulk and pharmaceutical formulation. A simple, accurate, low cost and specific HPTLC method for estimation of Fluoxetine hydrochloride in capsule has been developed. It was performed on Silica gel G60 F254 aluminium foil using acetone : methanol in the ratio of 5:4 as mobile phase. The mobile phase containing chamber was saturated for 10 minutes at room temperature. The Rf value of Fluoxetine was found to be 0.12. The plate was scanned and quantified at 226 nm. 6. Carmen Purdel, Dan Balalau, Mihaela Ilie and Florica Nicolescu18 have reported analytical study of Fluoxetine in biological specimens development and validation of a spectrophotometric method. A simple and accurate method was developed for determination of Fluoxetine in aqueous solution and biological sample. Particular efforts were made to find the best system of solvents for liquid-liquid extraction. n- hexan/izoamyl alcohol (97:3 v/v) proved to be suitable for the extraction of Fluoxetine from biological samples. Good extraction yields were obtained from human urine and bovine serum. The spectrophotometric method was validated through all necessary parameters, according to ICH guidelines. The absorbance intensity vs. concentration plot was linear over the range 40-400 μg/mL Fluoxetine in the range of 250-320 nm in bio spectrophotometer. 7. J.J. Berzas Nevado, A.M. Contento Salcedo, M.J. Villaseñor Llerena, E. Aguas Nuevo19 have reported method development and validation for the simultaneous determination of Fluoxetine and Fluvoxaminein pharmaceutical preparations by capillary electrophoresis. A capillary zone electrophoresis (CZE) method for determining Fluoxetine and Fluvoxamine is proposed. Optimal conditionsfor the quantitative separation were investigated. A background electrolyte solution consisting of 40mMborate buffer adjusted to pH 9.3, hydrodynamic injection and 8 kVof separation voltage were used, obtaining in these conditions analysis times lower than 2.5 min. Main aspects of the validation method are examined and discussed. 6.7 OBJECTIVES OF THE STUDY: In the proposed work, attempt shall be made : To develop a new instrumental method for estimation of Lamivudine and Fluoxetine hydrochloride To develop a validated method according to ICH guidelines. To apply validated method for the estimation of Lamivudine and Fluoxetine hydrochloride in pharmaceutical formulation. 7.0 7.1 MATERIALS AND METHODS: All experiments will be carried out in the Department of Quality Assurance. Srinivas college of Pharmacy, Valachil, Mangalore. Pure sample of Lamivudine and Fluoxetine hydrochloride will be procured from Industries involved in bulk manufacture of this drug. Dosage formulation will be procured from local market. The methods will be developed and validated in Q.A. lab of Srinivas college of Pharmacy. The methods will be first developed, then Validated as per ICH guidelines, then the method will be applied to the formulations. UV spectrophotometer Shimadzu-UV1700 with spectral band width of 2nm and 10nm and matched quartz shall be used for measuring absorbance for Lamivudine and Fluoxetine hydrochloride solutions. Reagents supposed to use for the estimation: 3-methyl,2-benzothiazolinehydrazone(MBTH), 1,10-phenanthroline, 4-amino phenazene, Para dimethyl amino benzaldehyde (PDAB) FolinCiocaltaeau reagent In combinations 3-methyl 2-benzothiazoline hydrazine and cerric ammonium sulphate 3-methyl 2-benzothiazoline hydrazine and and ferric ammonium sulphate etc. can be used as reagents for Spectrophotometric method development. 7.2 SOURCES OF DATA: References from library – Srinivas College of Pharmacy, Valachil, Mangalore. www.pharmainfo.net. www.google.com www.sciencedirect.com www.rxlist.com www.pubmed.com www.medline.com www.wikipedia.com 7.3 Does the study require any investigation to be conducted on patients or animals? No 7.4 Has the ethical clearance been obtained from your institution in case of 7.3 ? Not applicable 8.0 REFERENCES: 1. en.wikipedia.org/wiki/Lamivudine 2. www.drugbank.ca/drug/Lamivudine(DBoo709) 3. Srivani Mallepelli, Narasimharao R, Jajow Swapnahave reported analytical method development and method validation for the simultaneous estimation of Lamivudine and stavudine in tablet dosage form by RP-HPLC. 4. T.Sudha, V.R.Ravikumar, P.V. Hemalatha have reported RP-HPLC method for the simultaneous estimation of Lamivudine and Abacavir sulphate in tablet dosage form. IJPBR 2010;1(4):108-113. 5. Venkatesh M, Bhaskar M, Veeranjaneyulu K, Sunitha K, Bhagyasri G, Veliyath S K have reported method development and validation of Lamivudine in tablet dosage form by uv-spectrophotometry. IJPIR 2012;2(1). 6. Bojja Soumya, Thimmaraju Manish Kumar and Nerella Raghunandhan have reported simultaneous determination of Tenofovir disoproxil fumarate and Lamivudine by UV spectrophotometric method.URP Journals 2012;2(1):9-15. 7. T. Raja and A. Lakshmana Rao have reported development and validation of RP- HPLC method for the estimation of Abacavir, Lamivudine and Zidovudine in pharmaceutical dosage form. IJPRIF 2011; 3(2):852-57. 8. B. Raj Kumar, M. Priyanka, B. N. Priyamvada, K. Prasad, K. Mallikarjuna,N. Y. Anuradha, B. Lalithamma have reported method development and validation for the determination of Abacavir and Lamivudine by using RP-HPLC in pure and pharmaceutical dosage form. IJPWR 2012:3(1). 9. Jayaseelan S , Ganesh S , Rajasekar M , Sekar V, Perumal P. A new analytical method development and validation for the simultaneous estimation of Lamivudine and Stavudine in tablet dosage form by RP-HPLC Method. Pharm Tech Res 2010; 2(2):1539-42. 10. Pawan KS, Chhoten LJ, Raman MS, Satish CM, Gyanendra NS, Muhammed N. Development and validation of a HPLC method for a simultaneous analysis of Abacavir sulphate and Lamivudine in combined tablet dosage form. Pharmbit 2009; 20 (2): 97-106. 11. en.wikipedia.org/wiki/Fluoxetine hydrochloride. 12. www.drugbank.ca/drug/ Fluoxetine hydrochloride. (DBoo472) 13. Shubhanjali Shukla et.al. have reported RP-HPLC method development and its validation for simultaneous estimation of Alprazolam and Fluoxetine hydrochloride in pharmaceutical dosage form. EJAC 2010:5(3):239-45. 14. Rubesh kumar S, P Gayathri, Duganath N, Kiran CH, Sridhar C, Jayaveera K N have reported simultaneous estimation of Fluoxetine HCl and Olanzapine in bulk drug and pharmaceutical formulation by using uv-visible spectroscopy method. International Journal Of Pharmaceutical Sciences And Drug Research 2011;3(1):52-55. 15. Ibrahim A. Darwish have reported development and validation of spectrophotometric methods for determination of Fluoxetine, Sertraline, and Paroxetine in pharmaceutical dosage forms. Journal Of AOAC international 2005;88(1) 16. M. S. Bhatia, and S. T. Kumbhar have reported development and validation of HPTLC method for estimation of Fluoxetine in its capsule formulation. IJPSR 2011;2(6)1582-87. 17. Zahid Zaheer, Obaid Shaikh, Sucheta Thorat, Rana Ahmed have reported developmentand validation of HPTLC method of Fluoxetine hydrochloride in bulk and pharmaceutical formulation. IJPQA 2010;2(3):44-48. 18. Carmen Purdel, Dan Balalau, Mihaela Ilie and Florica Nicolescu have reported analytical study of Fluoxetine in biological specimens development and validation of a spectrophotometric method. Ovidius University Annals Of Chemistry 2009;20(1)111-14. 19. J.J. Berzas Nevado, A.M. Contento Salcedo, M.J. Villaseñor Llerena, E. Aguas Nuevo have reported method development and validation for the simultaneous determination of Fluoxetine and Fluvoxaminein pharmaceutical preparations by capillary electrophoresis. Analytica Chemica Acta 2000:111-14. 9.0 SIGNATURE OF THE CANDIDATE Makadia Biniben Pravinbhai 10.0 REMARKS OF THE GUIDE Forwarded for Approval Dr. E.V.S. Subrahmanyam, 10.1 Professor and Head, NAME AND DESIGNATION OF GUIDE Dept of Quality Assurance 10.2 SIGNATURE 11.0 HEAD OF THE DEPARTMENT Dr. E.V.S. Subrahmanyam, Professor and Head, Dept of Quality Assurance 11.1 SIGNATURE 12.0 REMARKS OF THE PRINCIPAL Forwarded for approval Dr.Ramakrishna Shabaraya A. 12.1 SIGNATURE Principal, Srinivas college of pharmacy, Valachil, Mangalore.