method development by RP -HPLC ppt.

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method development by RP -HPLC ppt.

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Industrial Guide: Mr. Pankaj Kherde “ Development, Validation and Stability Study for Simultaneous Estimation of Hydrochlorothiazide, Amlodipine Besilate and Losartan Potassium in Combined Dosage Form by HPLC ” Thesis Submitted By: Komal D. Somkuwar Guide: Dr. Manoj S. Charde GOVERNMENT COLLEGE OF PHARMACY, KATHORA NAKA, AMRAVATI. 2011-2012

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7/4/2012 2 Sr. no. Chapter 1 Introduction. 2.1 Aim and objective. 2.2 Plan of work. 2.3 Literature review. 2.4 Drug profile. 2.5 Instrumentation. 3 Experimental and results. 4 Summary and discussion. 5 Conclusion. 6 Future prospective. 7 References. INDEX

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1. INTRODUCTION The Analytical chemistry may be defined as the science and art of developing accurate, precise and sensitive methods for determining the composition of materials in terms of elements or compounds contained. METHOD DEVELOPMENT Method development is a challenging and very much a trial-and-error approach. CONSIDERATIONS BEFORE METHOD DEVELOPMENT Before starting the arduous process, a thorough literature search should be conducted for existing methodologies. It often provides a starting point for method development. New analytical methods are needed for the following reasons: Existing methods are not available. Existing methods are not sufficiently reliable, sensitive, or cost effective. New instrumentation or technique has better performance. An alternate method is required for regulatory compliance.

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7/4/2012 4 Principle of Chromatography It is the method used primarily for the separation of the components of a sample, in which the components are distributed between two phases, one of which is stationary while the other is mobile. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Same principle of separation as classical column chromatography but here mobile phase is pumped through the packed column under high pressure. METHOD DEVELOPMENT IN HPLC Before proceeding with development of method for a particular sample it is absolutely essential to have detailed information about the sample and separation goal should be clearly defined. Information about sample: Number of components present in the sample UV spectra of each analyte Concentration range of each component Solubility behavior Nature of sample (solid, liquid, semisolid) Formula 7/4/2012 4

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7/4/2012 5 2.1 AIM AND OBJECTIVE: The present work is undertaken with an objective to develop economical, simple, precise, accurate and reproducible stability indicating assay method for estimation of Hydrochlorothiazide (HCTZ), Amlodipine Besylate (AMLO) and Losartane Potassium (LOSA) antihypertensive drugs in their combined dosage form by HPLC. 7/4/2012 5

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7/4/2012 6 2.2 PLAN OF WORK Literature Survey 2. Procurement of selected drug for Study: Reference standards were procureted from pharmaceutical company. 3. Selection of multicomponent formulation: By market survey and literature. 4. Selection of analytical technique: HPLC A) Estimation of drugs in combination by RP- HPLC methods: 1) Selection of column. 2) Selection and optimization of Mobile phase. 3) Selection of different chromatographic conditions. 4) System suitability parameters study. 5) Analysis of standard laboratory mixture to see feasibility of Proposed method. 6) To adopt selected method on marketed formulation. 7/4/2012 6

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7/4/2012 7 7) Validation of Proposed Methods. Accuracy Precision Specificity Linearity LOD/LOQ Ruggedness Robustness B) Degradation Profile under different stress condition: Acid Degradation (HCl) . Alkali Degradation ( NaOH). Oxidation Degradation (H 2 O 2 ). Thermal Degradation. 5. Photo Degradation (natural light/ UV radiation ). 7/4/2012 7 CONTD…

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7/4/2012 8 7/4/2012 8 2.3 LITERATURE REVIEW : There are some analytical methods has been reported for simultaneous estimation of HCTZ, AMLO and LOSA by UV, HPLC and HPTLC but no HPLC method has been reported to estimate the simultaneous degradation profile of these drugs. The methods reported for simultaneous estimation of these drugs in combination are: Bagyalakshmi* et la reported Development and optimization of RP-HPLC method for the estimation of s (-) amlodipine in tablet dosage form[34]. Mohammed Mustafa Ali Siddiqui * et la reported Isocratic RP-HPLC method validation and verification of losartan potassium in pharmaceutical formulations with stress test stability for drug substance[35]. Ana Maria Bergold * et la reported Validation of an isocratic HPLC assay of Losartan Potassium in pharmaceutical formulations and stress test for stability evaluation of drug substance[36]. K. Kathiresan * et la reported Analytical method development and validation of Losartan Potassium Tablet by RP-HPLC[37].

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7/4/2012 9 CONTD… 7/4/2012 9 5. B. Wankhede* et la reported Spectrophotometric and HPLC methods for simultaneous estimation of Amlodipine Besylate, Losartan Potassium and Hydrochlorothiazide in tablets[38]. S. Jayaseelan* et la reported RP-HPLC method development and validation for simultaneous estimation of Losartan Potassium, Amlodipine Besylate and Hydrochlorthiazide in tablet dosage form[39]. Anbarasi B.* et la reported Analytical method development and validation of Amlodipine and Hydrochlorothiazide in combined dosage form by RP-HPLC[40]. Priyanka R. Patil* et la reported RP- HPLC method for simultaneous estimation of Losartan potassium and Amlodipine besylate in tablet formulation[41]. D. N. Vora* et la reported Development and validation of a simultaneous HPLC method for estimation of Bisoprolol Fumarate and Amlodipine Besylate from tablets[42]. Pradeep D. Lokhande et la reported Development and validation of a RP-HPLC- PDA method for simultaneous estimation of Hydrochlorothiazide and Irbesartan[43]. D. Boopathy* et la reporte Analytical method development and validation of Losartan Potassium and Atenolol in combined dosage form by RP-HPLC[44].

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7/4/2012 10 12. S. D. Kayal * et la reported Method development and validation for the simultaneous determenation of Amlodepine Besilate and Telmisartan in tablet dosage form by RP- HPLC[45]. SS Chitlange* et la reported Stability indicating RP- HPLC Method for Simultaneous Estimation of Valsartan and Amlodipine in Capsule Formulation[46]. Prasad* et la reported simultaneous Determination of Telmisartan, Amlodipine Besilate and Hydrochlorothiazide in a combined poly pill dosage form by Stability indicating HPLC[47]. S. J. Rajput* et la reported development and Validation of a Stability Indicating High Performance Liquid Chromatography method for Atenolol and Hydrochlorothiazide in bulk drug and tablet formulation[48]. Vijaya P. Godse * et la reported ICH guidance in practice: Validated stability-indicating HPLC method for simultaneous determination of Olmesartan medoximil and Hydrochlorothiazide in combination drug products[49]. M. Rama Mohan Reddy * et la reported Stability Indicating High Performance Liquid Chromatography method of low level impurities of Telmisartan and Hydrochlorothiazide in tablet dosage forms[50]. CONTD…

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7/4/2012 11 R. A. Mhaske * et la reported RP- HPLC Method for Simultaneous Estimation of Amlodipine Besilate, Valsartane, Telmisartan, Hydrochlorothiazide and Chlorthalidone: Application to Commercially available Drug Products[51]. A.I. Patel * et la reported RP-HPLC method for the determination of Losartane Potassium and Perindopril Erbumine in combine tablet dosage form[52]. The literature reveals that HCTZ, AMLO and LOSA are official in Indian pharmacopeia, British Pharmacopoeia, United State Pharmacopoeia respectively. Thus from the above literature survey, it reveals that no stability indicating assay method has been so far reported for simultaneous estimation of these drugs in combination. Hence in the present investigation an attempt has been made to develop stability indicating assay method using HPLC method. CONTD… 7/4/2012 11

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7/4/2012 12 2.4 DRUG PROFILE : Hydrochlorthiazid [31]: Structure: Chemical Name : 6-chloro-1,1-dioxo-3,4-dihydro-2 H -1,2,4-benzothiadiazine- 7-sulfonamide. Molecular weight : 297.74 Molecular Formula : C 7 H 8 ClN 3 O 4 S 2 Description : White, Crystalline Powder. Solubility : Slightly Soluble in Water,Alcohol and Soluble in Aceton. 7/4/2012 12

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7/4/2012 13 Pharmacokinetics : Does not undergo significant metabolism, it’s half-life is between 5.6-14.8 hour, primarily excreted unchanged in urine. Mechanisum of Action : Acts by inhibiting Na + reabsorption by kidney. Use : Hypertension, edema, diabetes insipidus and hypercalciuria. Adverse Effect : Hypokalemia, g.i.t and CNS disturbance, Hyperuricemia, Hyperglycemia. CONTD… 7/4/2012 13

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7/4/2012 14 2. Amlodipine Besylate [32]: Structure: Chemical Name : 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4- (2- 6- methyl-1,4 dihydropyridine-3,5- dicarboxylate. Molecular weight : 567.05 Molecular formula : C 20 H 25 ClN 2 O 5 . Description : white to off-white crystalline powder. Solubility : Slightly soluble in water, freely soluble in Methanol, sparingly soluble in ethanol. 7/4/2012 14

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7/4/2012 15 Pharmacokinetics : Almost entirely metabolised to inactive metabolites, the parent substance and the metabolites are excrete in urine. Mechanisum of Action : Calcium channel blocker. Use : Hypertention and Angina Adverse Effect : Peripheral edema, palpitations, gynecomastia, tachycardia. CONTD… 7/4/2012 15

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7/4/2012 16 3. Losartan Potassium [33]: Structure: Chemical Name : (2-butyl-4-chloro-1-{[2'-(1 H -tetrazol-5-yl)biphenyl-4- yl] methyl}-1 H -imidazol-5-yl)methanol. Molecular weight : 461.01 Molecular Formula : C 22 H 23 ClN 6 O Description : white to off-white free flowing crystalline powder . Solubility : It is freely soluble in water, soluble in alcohols, and slightly soluble acetonitrile and methyl ethyl ketone. 7/4/2012 16

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7/4/2012 17 Pharmacokinetics : Undergoes significant first pass metabolism to produce 5-carboxylic acid metabolite, the parent drug and metabolites is excreted in urine and feces. Mechanisum of Action : Selective, competitive angiotensin II receptor antagonist. Use : Hypertension, myocardial infraction and diabetic neuropathy. Adverse effect : Headache, dizziness, weakness and upper g. i. t. side effect are mild. 7/4/2012 17

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7/4/2012 18 2.5 INSTRUMENTATION [14-17]: The various components of HPLC are: Mobile Phase Reservoir and solvent System treatment Pumps (Displacement Pump, Reciprocating Pump, Pneumatic Pump) Sample Injectors Pre columns Liquid chromatographic column (Analytical Column) Detectors UV-Visible detectors Photometric detectors Fluorescence detectors. Refractive index detectors Figure 1.2 : Configuration of HPLC system 7/4/2012 18

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7/4/2012 19 3. EXPERIMENTAL AND RESULTS. I. Materials: A. Standards B. Marketed formulation 7/4/2012 19 STANDARDS USED Sr. No. Name of Working Standards Potency 1 Hydrochlorothiazide 99.50% 2 Amlodipine Besilate 99.58% 3 Losartan Potassium 99.81% Table 3.1: Standard Drugs Used Brand Name Trilopace Manufactured by Sun Pharmaceuticals Ltd. Content Hydrochlorothiazide (12.5 mg) Amlodipine Besylate (5.0 mg) Losartane Potassium (50 mg) Average weight 292.2 mg Table 3.2 : Marketed formulation[53]

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7/4/2012 20 Sr. No. Name of Chemicals Make 1 Acetonitrile HPLC Grade. Rankem 2 Potassium Dihydrophosphate, AR Grade. Qualigens 3 Orthophosphoric Acid, AR Grade. Qualigens 4 Methanol AR Grade. Qualigens Sr. No. Name of Instrument Make Model 1 U V Shimaduz Double beam - 1800 2 HPLC System Shimaduz Pump- LC 2010 CHT Detector- PDA Software- LC Solution (Shimaduz) 3 Analytical Balance Metter AB-204 4 pH Meter pH Tutor Cyber Scan 5 ultrasonicator Servewell Instrument RC-System MU-1700 6 Degasser Tarsons Rockyvac 300 7/4/2012 20 Table 3.3 : Chemicals and Reagents Used Table 3.4 : Instruments Used

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7/4/2012 21 A) Development, validation and stablity study for simultaneous estimation of HCTZ, AMLO and LOSA in combined dosage form by RP- HPLC method. Method development strategy : 1. Selection of solvent (Diluent) : Methanol of AR grade was selected as common solvent for preparation of stock solution. 2. Determination of  max of HCTZ, AMLO and LOSA : HCTZ std. stock solution: 50.0 mg of HCTZ was transferred to the 100 ml volumetric flask and the volume was made up to the mark with methanol (500  g/ml). AMLO std. stock solution: 50.0 mg of AMLO was transferred to the 100 ml volumetric flask and the volume was made up to the mark with methanol (500  g/ml). LOSA std. stock solution: 50.0 mg of LOSA was transferred to the 100 ml volumetric flask and the volume was made up to the mark with methanol (500  g/ml). The aliquot portions of std stock solutions of HCTZ, AMLO and LOSA were diluted appropriately with Methanol to obtain conc. 10  g/ml of each drug. The solutions were scanned in the range of 200 – 400 nm. The absorbance spectrum of HCTZ, AMLO and LOSA is shown in Figure 3.1,3.2,3.3. 7/4/2012 21

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7/4/2012 22 Figure 3.1 : Absorbance Spectrum of HCTZ . 7/4/2012 22 Figure 3.2 : Absorbance Spectrum of AMLO.

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7/4/2012 23 Figure 3.2 : Absorbance Spectrum of LOSA. Figure 3.4 : Overlain Spectra of HCTZ, AMLO and LOSA. 7/4/2012 23

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7/4/2012 24 From the overlain spectrum the wavelength selected for estimation of drugs were 232 nm as isoabsorptive point and 271 nm, 236 nm and 254 nm as λ max of HCTZ, AMLO and LOSA respectively. 3. Selection of chromatographic condition for estimation of drugs : Procedure: The mobile phase was allowed to equilibrate with stationary phase until steady baseline was obtained. The std. solution containing mixture of HCTZ, AMLO and LOSA was run and different individual solvents as well as combinations of solvents have been tried to get a good separation and stable peak. Each mobile phase was filtered through 0.45 µm filter membrane. The mobile phases tried are as follows: 7/4/2012 24

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7/4/2012 25 Trial 1: Chromatographic parameters: Column : Princeton C18 (4.6mm × 250mm × 5µm) Flow rate : 1 ml/min Wavelength : 232 nm Injection volume : 20µl Column oven temp. : 30 ˚c Run time : 12.5 minutes Mobile phase : Acetonitrile: Buffer [20:80 ] pH of buffer : 4.0 Observation: By using above method resolution between AMLO and LOSA is very less, hence this method is not suitable. 7/4/2012 25 Figure 3.5 : HCTZ, AMLO and LOSA Std. Chromatogram of Trial 1.

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7/4/2012 26 Trial 2: Chromatographic parameters: Column : Princeton C18 (4.6mm × 250mm × 5µm) Flow rate : 1 ml/min Wavelength : 232 nm Injection volume : 20µl Column oven temp. : 30 ˚c Run time : 12 minutes Mobile phase : Acetonitrile: Buffer [30:70 ] pH of buffer : 3.7 7/4/2012 26 Observation: By using above method tailing of AMLO and LOSA is more than the limit, hence this method is not suitable. Figure 3.6: HCTZ, AMLO and LOSA Std. Chromatogram of Trial 2.

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7/4/2012 27 The following chromatographic conditions were established by trial and error and were kept constant throughout the method. Chromatographic parameters: Column : Princeton C18 (4.6mm × 250mm × 5µm) Flow rate : 1 ml/min Wavelength : 232 nm Injection volume : 20µl Column oven temp. : 30 ˚c Run time : 10.5 minutes Mobile phase : Acetonitrile: Buffer [40:60 ] pH of buffer : 3.7 7/4/2012 27 Figure 3.7 : Separation of the drugs in selected mobile phase for HCTZ, AMLO and LOSA.

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7/4/2012 28 5. System suitability test: Preparation of std. drug solution : Weigh accurately 31.25 mg of HCTZ, 17.36 mg of AMLO and 125 mg of LOSA working std in a 100ml volumetric flask and dissolve by sonication in sufficient mobile phase then make up the volume by mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with mobile phase. 7/4/2012 28 Sr. No. Area Reproducibility Retention Time Tailing Factor Theoretical Plates Resolution 1 2189472 3.602 1.801 2995.53 - 2 2174882 3.578 1.805 2972.77 - 3 2162726 3.558 1.807 2963.6 - 4 2155432 3.546 1.806 2963.11 - 5 2160294 3.554 1.797 3013.3 - Mean 2168561 3.567 1.803 2981.66 - % RSD 0.6322 0.642 0.232 0.739 - Limit NMT 2.0 % NMT 1 % NMT 2 NMT 2000 NLT 1.5 Table 3.6 : System suitability for HCTZ

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7/4/2012 29 Sr. No. Area Reproducibility Retention Time Tailing Factor Theoretical Plates Resolution 1 597906 7.905 1.078 2166.91 13.585 2 596468 7.886 1.069 2181.69 13.607 3 593140 7.842 1.064 2164.15 13.631 4 592081 7.828 1.055 2211.02 13.7 5 591552 7.821 1.069 2207.67 13.7 Mean 594229 7.856 1.067 2186.29 13.645 % RSD 0.4722 0.469 0.787 1.011 0.389 Limit NMT 2.0 % NMT 1 % NMT 2 NMT 2000 NLT 1.5 7/4/2012 29 Table 3.7 : System suitability for AMLO

Table 3.8 : System suitability for LOSA:

7/4/2012 30 Table 3.8 : System suitability for LOSA Sr. No. Area Reproducibility Retention Time Tailing Factor Theoretical Plates Resolution 1 8834084 10.504 1.613 7959.58 7.217 2 8812217 10.478 1.613 7980.38 7.247 3 8773530 10.432 1.612 7976.08 7.266 4 8750822 10.405 1.619 7966.6 7.265 5 8751663 10.406 1.618 8004.28 7.294 Mean 8784463 10.445 1.615 8778.33 7.258 % RSD 0.4244 0.424 0.202 0.496 0.392 Limit NMT 2.0 % NMT 1 % NMT 2 NMT 2000 NLT 1.5 7/4/2012 30

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7/4/2012 31 7) Application of proposed method for estimation of HCTZ, AMLO and LOSA on marketed tablet formulation: Preparation of Sample Solution : Weighed accurately 20 tablets and determine the average weight then crushed the tablets into fine powder. Transfer powder equivalent to 125 mg LOSA in 100 ml volumetric flask and dissolve by sonication and make up the volume with mobile phase stir the solution for 30 minutes. Centrifuge, filter and dilute 5.0 ml of this solution to 50.0 ml with mobile phase. Amount of drug in tablet was calculated using following formula : Asp Dst A % Label claim = ----- x ----- x ----- x P Ast Dsp Lc Where, Asp = Area for sample solution. Ast = Area for standard solution. Dst = Dilution factor for standard. Dsp = Dilution factor for sample. Lc = Label claim. A = Average weight. P = Potancy. 7/4/2012 31

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7/4/2012 32 7/4/2012 32 Figure 3.11 : Std. chromatogram of HCTZ, AMLO and LOSA Figure 3.12 : Chromatogram obtained by marketed preparation of HCTZ, AMLO and LOSA

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7/4/2012 33 7/4/2012 33 Wt. of std.(mg) Wt. of Sample(mg) Peak area of std. Peak area of sample % Label claim HCTZ AMLO LOSA HCTZ AMLO LOSA HCTZ AMLO LOSA HCTZ AMLO LOSA 32 17.5 125.9 738.7 2171510 596426 8778331 2118090 590519 8660513 98.27 98.69 98.56 736.0 2111385 588360 8648859 98.19 99.39 98.07 730.8 2125548 584203 8617588 98.62 98.69 98.41 Table 3.9 : Results for estimation of HCTZ, AMLO and LOSA in marketed formulation (I) Sr. No. HCTZ AMLO LOSA Assay (mg) Assay (%) Assay (mg) Assay (%) Assay (mg) Assay (%) 1 12.2837 98.2701 4.9345 98.69 49.2800 98.56 2 12.2737 98.1900 4.9680 99.39 49.0350 98.07 3 12.3362 98.6200 4.9345 98.69 49.2050 98.41 Mean 12.2969 98.3600 4.9457 98.9233 49.1733 98.3467 ±S.D. 0.0336 0.2287 0.0193 0.4041 0.1255 0.2510 % RS.D. 0.2730 0.2325 0.3910 0.4085 0.2552 0.2552 Table 3.10 : Results and for estimation of HCTZ, AMLO and LOSA in marketed formulation (II).

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7/4/2012 34 ACCURACY Procedure: Recovery study was performed by spiking in HCTZ, AMLO and LOSA working std. in the placebo at level 80, 100 and 120 % of label claim in triplicate. Weight taken for Accuracy solutions of HCTZ, AMLO and LOSA: 80 % : 25 mg HCTZ, 13.9 mg AMLO and 100 mg LOSA working std. + placebo. 100 % : 31.3 mg HCTZ, 17.4 mg AMLO and 125 mg LOSA working std. + placebo. 120 % : 37.6 mg HCTZ, 20.9 mg AMLO and 150 mg LOSA working std. + placebo. The % Recovery was then calculated by using formula: Asp Dst P Mg Recovered = ----- X ------ X ----- Ast Dsp 100 Mg Recovered % Recovery = ------------------ X 100 Mg Added B) VALIDATION PARAMETERS: 7/4/2012 34

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7/4/2012 35 7/4/2012 35 % Level of Std. Wt. of working std. (mg) added its dilution (mL) Area at 232 nm Std recovered (mg) % recover Mean recover % R.S.D. 80 25.0/100/5/50 1713040 25.1175 100.47 99.75 0.6227 1703150 1723146 100 31.3/100/5/50 2141300 31.1028 99.37 2101299 2121301 120 37.6/100/5/50 2569560 37.3819 99.42 2529438 2549558 % Level of Std. Actual Conc. (µg/mL) Conc. Calculated (µg/mL) % Accuracy % Deviation 80 25.0 25.1175 100.47 +0.47 100 31.3 31.1028 99.37 -0.63 120 37.6 37.3819 99.42 -0.58 Table 3.11 : Recovery result for HCTZ Table 3.12 : Deviation from recovery for HCTZ

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7/4/2012 36 % Level of Std Wt Of Working Std (mg) added and Its dilution (ml) Area at 232 nm Standard Recovered % Recover Mean Recover % RSD (mg) 472415 80 13.9/100/5/50 475321 13.9105 100.07 477252 590519 100 17.4/100/5/50 591810 17.3091 99.47 99.88 0.3589 590807 708622 120 20.9/100/5/50 717360 20.923 100.11 717358 7/4/2012 36 % Level of Std. Actual Conc. (µg/mL) Conc. Calculated (µg/mL) (%) Accuracy % Deviation 80 13.9 13.9105 100.07 +0.07 100 17.4 17.3091 99.47 -0.53 120 20.9 20.9230 100.11 +0.11 Table 3.13 : Recovery result for AMLO Table 3.14 : Deviation from recovery for AMLO

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7/4/2012 37 7/4/2012 37 % Level of Std. Wt. of working std. (mg) added and its dilution (ml) Area at 232 nm Std. recovered (mg) % Recover Mean recover % RS.D. 80 100/100/5/50 6928410 98.9399 98.93 98.93 0.0058 6917269 6937271 100 125/100/5/50 8660513 123.6754 98.94 8654189 8664088 120 150/100/5/50 10392615 148.4099 98.93 10389897 10391908 % Level of Std. Actual Conc. ( µg /mL) Conc. Calculated (µg /mL) (%) Accuracy % Deviation 80 100 98.9399 98.93 -1.07 100 125 123.6754 98.94 -1.06 120 150 148.4099 98.93 -1.07 Table 3.15 : Recovery result for LOSA Table 3.16 : Deviation from recovery for LOSA

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7/4/2012 38 2. PRECISION System Precision: Procedure: System Precision was determined by taking five replicate injections of Std. solution into HPLC system. Injection No. Area at 232 nm for Limit HCTZ AMLO LOSA 1 2191200 597904 8834082 2 2189471 600107 8815351 3 2174004 596944 8758132 4 2151882 592328 873672 5 2150994 594847 8748020 Mean 2171510 596426 8778331 ± SD 19511 2970 43564 NMT % RSD 0.898 0.498 0.496 2.0 % 7/4/2012 38 Table 3.17 : System precision data

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7/4/2012 39 2 Method Precision: Procedure: Six sample solution of HCTZ, AMLO and LOSA tablets were prepared and analysed on the same day by HPLC method. Sr. No. Sample % Deviation Form Mean Assay Value Weight (g) Area at 232 % Assay HCTZ AMLO LOSA HCTZ AMLO LOSA HCTZ AMLO LOSA 1 0.7315 2102187 593360 8698271 98.5 100.14 98.27 -0.021 -0.43 1.69 2 0.7313 2099371 592104 8684515 98.39 99.96 99.11 -0.1 -0.25 0.85 3 0.7311 2124008 600844 8808122 99.57 99.65 100.55 -1.28 0.06 -0.59 4 0.7317 2101872 593338 8706062 98.45 100.11 99.3 -0.16 -0.4 0.66 5 0.7319 2060974 583857 8547020 96.51 98.47 98.46 1.78 1.24 1.5 6 0.7322 2100738 592951 8692276 98.33 99.98 99.08 -0.04 -0.27 0.88 Mean 98.29 99.71 99.96 ± SD 0.9887 0.6358 1.04 % RSD 1.0059 0.6376 1.0402 NMT 2.0 % 7/4/2012 39 Table 3.18 : Method precision data

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7/4/2012 40 3. Intermediate precision (Ruggudness) : Procedure: Six sample solution of HCTZ, AMLO and LOSA tablets was prepared as per described method and analysed by different analyst using same make of different HPLC column. 7/4/2012 40 Table 3.19 : Working reference standard data for intermediate precision. Injection no. Area at 232 nm Limit HCTZ AMLO LOSA 1 2281300 597604 8835072 NMT 2.0 % 2 2287900 600102 8938052 3 2271200 597922 8840382 4 2251235 592329 8942282 5 2256323 601087 8850180 Mean 2269591 598009 8881194 ± S.D. 15715 3408 54127 % RS.D. 0.6924 0.5700 0.6095

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7/4/2012 41 7/4/2012 41 Sr. No. Sample % Deviation form mean Assay Value weight (g) Area at 232 % Assay HCTZ AMLO LOSA HCTZ AMLO LOSA HCTZ AMLO LOSA 1 0.731 2116832 589218 8630050 99.25 99.51 98.53 0.27 0.29 0.43 2 0.7308 2099349 591821 8660513 98.46 99.98 98.91 1.06 -0.18 0.05 3 0.7315 2108090 590519 8690976 98.77 99.66 99.16 0.75 0.14 -0.2 4 0.732 2131877 592201 8698271 99.82 99.88 99.17 -0.3 -0.08 -0.21 5 0.7318 2127310 591321 8650361 99.63 99.76 98.65 -0.11 0.04 0.31 6 0.7321 2161800 593361 8715188 101.21 100.06 99.35 -1.69 -0.26 -0.39 Mean 99.52 99.8 98.96 ± SD 0.9713 0.2056 0.3224 % RSD 0.9759 0.2059 0.3258 NMT 2.0 % Drug Analysist A Day 1st Analysist B Day 2 nd Mean assay % % RS.D. (Limit-NMT: 2.0 %) HCTZ 98.29% 99.52 % 98.90% 0.8794 AMLO 99.71% 99.80 % 99.76% 0.0638 LOSA 99.96% 98.96 % 99.46% 0.7109 Table 3.21: Intermediate precision for mean % assay and % RS.D. Table 3.20 :Intermediate precision data for Sample analysis.

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7/4/2012 42 3) SPECIFICITY Placebo Interference study: To identify the interference by excipients, Std. solution, Marketed formulation and placebo sample were analyzed by the developed method. Placebo Preparation: Weigh accurately 571.75 mg of placebo in a 100ml volumetric flask and dissolve by sonication in sufficient mobile phase then make up the volume. Dilute 5.0 ml of this solution to 50.0 ml with mobile phase and mix. Filter the solution and inject the clear filtrate. 7/4/2012 42 Figure 3.13 : Chromatogram of placebo.

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7/4/2012 43 Figure 3.14 : Chromatogram of HCTZ, AMLO and LOSA working standard. 7/4/2012 43 Figure 3.15 : Chromatogram of HCTZ, AMLO and LOSA sample solution.

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7/4/2012 44 4) LINEARITY AND RANGE Procedure: The conc. of HCTZ, AMLO and LOSA were 31.25 µ g/ml, 17.36 µg /ml and 125 µg /ml respectively so the working range of analyte was set between 18 - 43 µg /ml for HCTZ, 10 - 24 µg /ml for AMLO and 75 - 175 µg /ml for LOSA. To show the Linearity and range of sample solutions approximately 60 % to 140 % of the test conc. were prepared. 7/4/2012 44 Sr. No. Wt. of working std (mg) added and its dilution (mL) Conc. (µg/mL) Mean area count At 232 nm Limit 1 31.30/100/3/50 18.78 1312999 NMT : 0.995 2 31.30/100/4/50 25.04 1737298 3 31.30/100/5/50 31.30 2181399 4 31.30/100/6/50 37.56 2604798 5 31.30/100/7/50 43.82 3141098 Y 72264X +(-66331) (r 2 ) 0.9979 Table 3.22 : Linearity and Range study data for HCTZ

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7/4/2012 45 7/4/2012 45 Sr. No. Wt. of working std (mg) added and its dilution (ml) Conc. (µg/mL) Mean area count At 232 nm Limit 1 17.4/100/3/50 10.4 367397 NMT: 0.995 2 17.4/100/4/50 13.92 477496 3 17.4/100/5/50 1 7.40 598596 4 17.4/100/6/50 20.88 724695 5 17.4/100/7/50 24.36 843794 Y 34402 X + 4082.2 (r 2 ) 0.9994 Table 3.23 : Linearity and Range study data for AMLO Sr. No. Wt of working std (mg) added and its dilution (ml) Conc. (µg/mL) Mean area count At 232 nm Limit 1 125/ 100/ 3/ 50 75 5366988 NMT: 0.995 2 125/ 100/ 4/ 50 100 7124984 3 125/ 100/ 5/ 50 125 8778981 4 125/ 100/ 6/ 50 150 10434990 5 125/ 100/ 7/ 50 175 12392973 Y 69448 X + 138795 (r 2 ) 0.9991 Table 3.24 : Linearity and Range study data for LOSA

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7/4/2012 46 7/4/2012 46 Figure 3.16 : Linerity plot of HCTZ Figure 3.17 : Linearity plot of AMLO Figure 3.18 : Linerity plot of LOSA

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7/4/2012 47 5) ROBUSTNESS Change in Wavelength (±2 nm) The tablet sample of HCTZ, AMLO and LOSA was analyzed using proposed method after a deliberate change in detection wavelength for estimation by ±2 nm. 7/4/2012 47 Sr. No. Change in wavelength (± 2 nm) % estimation HCTZ AMLO LOSA 1 230 98.06 98.51 98.20 2 232 98.36 98.92 98.35 3 234 98.12 98.23 98.28 Mean 98.18 98.55 98.28  S.D. 0.1587 0.3470 0.0750 % R.S.D. 0.1617 0.3521 0.0763 Table 3.25 : Results of robustness study

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7/4/2012 48 Time (h) Peak area of std. solution Peak area of sample solution % estimation 0 hrs 2172004 2117080 98.2 4 hrs 2170010 2116900 98.29 8 hrs 2168471 2136765 99.28 12 hrs 2155320 2158520 100.6 Mean 99.09 ±S.D. 1.1178 %R.S.D. 1.128 6) STABILITY OF ANALYTICAL SOLUTION Procedure: Prepare std. and sample solutions as per the method and inject one std. and sample preparation initially at 0 hr and after specified time-intervals i.e. after 4hrs, 8 hrs and 12 hrs. Table 3.26 : Result of stability of std. and sample solution for HCTZ. 7/4/2012 48

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7/4/2012 49 Time (h) Peak Area of Std. Solution Peak Area of Sample Solution % estimation 0 hrs 596528 590518 98.68 4 hrs 596489 590764 98.56 8 hrs 596121 589980 99.68 12 hrs 595734 588620 100.88 Mean 99.45 ±S.D. 1.0774 %R.S.D. 1.0834 Time (h) Peak Area of Std. Solution Peak Area of Sample Solution % estimation 0 hrs 8776321 8660412 98.59 4 hrs 8774092 8660269 98.61 8 hrs 8770221 8689649 98.99 12 hrs 8761532 8638718 98.51 Mean 98.68 ±S.D. 0.2143 %R.S.D. 0.2172 Table 3.27 : Result of stability of std. and sample solution for AMLO Table 3.28 : Result of stability of std. and sample solution for LOSA 7/4/2012 49

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7/4/2012 50 C) Forced degradation studies (stress studies) of HCTZ, AMLO and LOSA tablet formulation. In order to established where there the analytical method for assay was stability indicating, the tablet formulation were subjected to various stress conditions. Stress studies were carried out under the condition of acid/alkali hydrolysis, oxidation, thermal and photo degradation in accordance with ICH Q1A(R2). The % degradation was evaluated by the following formula: Area of unstressed – Area of stressed % Degradation = _____________________________________ × 100 Area of unstressed Preparation of sample solution stock: Weighed accurately 20 tablets and determine the average weight then crushed the tablets into fine powder. Transfer powder equivalent to 125 mg LOSA in 100 ml Volumetric flask and dissolve by sonication for 10 mins and make up the volume with mobile phase, stir the solution for 30 mins. Centrifuge and filter the solution. 7/4/2012 50

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7/4/2012 51 1. Acid stress degradation (0.1 N HCl): 5.0 mL from sample stock solution was pipette out in 50 mL volumetric flask and it was subjected to acid stress degradation by treating the sample with 5.0 mL of 0.1 N HCl and the solution was kept at room temp. for 8 hrs. 7/4/2012 51 Figure 6.19 : Chromatogram of acid stressed degradation of drug product.

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7/4/2012 52 7/4/2012 52 Table 3.29 : Acid degradation data Table 3.30 : Result of acid degradation Name of peak Retention time Area % Area Deg I 2.172 14939 0.130 Deg II 2.690 121014 1.049 HCTZ 3.294 1966906 18.350 AMLO 7.784 551485 5.130 LOSA 10.268 8203764 75.341 Stress condition Drugs % Degradation % assay of drugs after degradation Acid degradation: 5.0 mL of Sample solution exposed to 5.0 mL of 0.1N HCl at room temp. for 1hr. HCTZ 7.08 92.92 AMLO 6.61 93.39 LOSA 5.57 94.43

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7/4/2012 53 2. Alkali stress degradation (0.1N NaOH): 5.0 mL from sample stock solution was pipette out in 50 mL volumetric flask and it was subjected to alkali stress degradation by treating the sample with 5.0 mL of 0.1 N NaOH and the solution was kept at room temp. for 8 hrs. 7/4/2012 53 Figure 3.20 : Chromatogram of alkali stressed degradation of drug product.

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7/4/2012 54 Table 3.31 : Alkali degradation data Name Of Peak Retention time Area % Area Deg I 2.269 6539 0.11 HCTZ 3.301 1985325 19.565 AMLO 6.728 556623 5.060 LOSA 9.177 8201680 75.265 Table 3.32 : Result of alkali degradation Stress Condition Drugs % Degrdation % assay of drugs after degradition Alkali degradation : Sample is exposed to 5.0 ml of 0.1 N NaOH at room temp. for 1 hr . HCTZ 6.21 93.79 AMLO 5.74 94.26 LOSA 5.30 94.70 7/4/2012 54

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7/4/2012 55 3. Oxidation/Peroxide stress degradation (3 % H 2 O 2 ): 5.0 mL of sample stock solution was pipette out in 50 mL volumetric flask and it was subjected to peroxide stress degradation by treating the sample with 5.0 mL of 3% H 2 O 2 and the solution was kept at room temp. in dark area for 8 hrs. Figure 3.21 : Chromatogram of peroxide stressed degradation 7/4/2012 55

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7/4/2012 56 Name Of peaks Retention time Area % Area Deg I 2.300 5110192 31.862 HCTZ 3.297 194249 13.186 AMLO 6.801 544675 3.396 LOSA 9.17 81686795 51.556 7/4/2012 56 Stress Condition Drugs % degradation % assay of drugs after degradation Oxidative degradation: 5.0 mL of sample solution is exposed to 5.0 mL of 3% H 2 O 2 at room temp. in dark for 1 hr . HCTZ 8.24 91.76 AMLO 7.76 92.24 LOSA 5.68 94.32 Table 3.33 : Oxidative stress degradation data. Table 3.34 : Result of oxidative stress degradation

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7/4/2012 57 7/4/2012 57 4. Thermal stress degradation (Heat at 80 º c for 1hr): 5.0 mL of sample stock solution was pipette out in 50 mL volumetric flask, make up the volume with mobile phase and then the solution was subjected to thermal stress degradation by heating the sample on boiling water bath at 80ºc for 8 hrs. Figure 3.22 : Chromatogram of thermal stressed degradation

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7/4/2012 58 7/4/2012 58 Table 3.35 : Thermal stressed degradation data. Table 3.36 : Result of thermal stressed degradation Name of peaks Retention time Area % Area Deg I 2.401 4370 0.085 HCTZ 3.282 1963985 19.437 Deg II 4.646 18002 0.163 AMLO 6.779 550670 4.981 LOSA 9.142 8229227 75.334 Stress Condition Drugs % degradation % assay of drugs after degradation Thermal degradation: 5.0 mL of Sample solution is heated at 80 ◦ c for 1 hr . HCTZ 7.22 92.78 AMLO 5.76 94.24 LOSA 5.00 95.00

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7/4/2012 59 7/4/2012 59 5. Photo Degradation Photo degradation studies were carried out on solid dosage form (fine tablet powder). The sample in a petri plate was spread as a thin layer (1 mm) and exposed to direct UV radiation for 24 hrs in UV chamber. The withdrawn sample was dissolve and then diluted with mobile phase as per sample preparation. Figure 3.23 : Chromatogram of photo stressed degradation

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7/4/2012 60 Name Of peaks Retention time Area % Area Deg I 2.253 3674 0.112 HCTZ 3.299 1961938 19.479 LOSA 9.158 8195180 75.351 Table 3.37 : Photo stressed degradation data Table 3.38 : Result of photo stressed degradation 7/4/2012 60 Stress condition Drugs % degradation % assay of drugs after degradation Photo degradation: Tablet powder exposed to direct UV radiation for 24 hrs in UV chamber. HCTZ 7.31 92.69 AMLO 6.84 93.16 LOSA 5.37 94.63

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7/4/2012 61 7/4/2012 61 Sampling in acid, alkali, oxidative and thermal stress degradation study: In acid, alkali, oxidative and thermal stress degradation studies of tablet formulation the samples (5.0 mL) were withdrawn at one hour time interval to study the extent of degradation. If reasonable degradation (5-20 %) is seen, stress testing can be terminated at that point. (Note: The sample of acid and alkali degradation were neutralized prior to dilution)

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7/4/2012 62 7/4/2012 62 4. SUMMARY AND DISCUSSION : In the present investigation an attempt has been made to develop stability indicating assay method for simultaneous estimation of HCTZ, AMLO, LOSA by RP- HPLC. The present experimental study was divided into three parts: Development of RP-HPLC method for simultaneous estimation of HCTZ, AMLO and LOSA . Validation of developed Method. Forced degradation study (Stress study). DEVELOPMENT OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF HCTZ, AMLO AND LOSA. The separation was done on Princeton C18 column (250mm x 4.6mm × 5µm). The mobile phase ACN:Buffer (40:60) was found to be more satisfactory as it gave good resolution of drugs with reasonably symmetrical sharp peak. A detection wavelength 232 nm was selected. This system gave good resolution and optimum retention time with appropriate tailing factor (< 2). The total run time of chromatogram was about 10.5 min. The observed number of theoretical plates were more than 2000 indicates high column efficiency. The detector response was found to be linear. The optimized HPLC methods were then adopted for the assay.

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7/4/2012 63 7/4/2012 63 VALIDATION OF PROPOSED METHOD. 1. Accuracy: It was ascertained from the recovery studies over the range of 80, 100 and 120 % of labeled claim. Recovery results were well within the range 98-102%.Thus the method was found to be accurate. 2. Precision: Replicate estimation of HCTZ, AMLO and LOSA in tablet by proposed method have yielded quite concurrent results which speaks about repeatability of the method. 3. Specificity: It was carried out to ascertained how accurately and specifically, the analyte of interest are estimated in presence of other components. 4. Linearity & Range: Graphs plotted (Figure 6.19,6.20 & 6.21 ) as detector response as a function of labeled claim shows a linear relationship over 60 - 140 % of labeled claim. This has indicated the capability of method to estimate accurately the drug over a wide range. 5. Robustness: The variation of detection wavelength by 2 nm has not shown any significant change in results. 6. Stability of analytical solution: The std. and sample solutions of drugs have reasonably good stability over a period of about 12 hrs.

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7/4/2012 64 . FORCED DEGRADATION STUDY: The acid, alkali and oxidative stress studies were performed by exposing the DP to 0.1N HCl, 0.1N NaOH and 3% H 2 O 2 in dark respectively at room temperature for 8 hrs. Thermal degradation was performed by exposing the DP to 80 ◦ c for 8 hrs. Photo degradation was attempted by exposing the DP to direct UV radiation for 24 hrs in UV chamber. The periodically withdrawn stressed samples were chromatographed under optimized conditions. HPLC method was developed to resolve the degradants from each other and also from the parent drug.

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7/4/2012 65 7/4/2012 65 5) CONCLUSION: The optimized chromatographic parameters of the HPLC method proves to be simple, rapid and sensitive. The total runtime was about 10.5 min. within which three drugs and their degradation products were separated. Linear relationships between conc. and corresponding response have been shown by the proposed methods over a wide conc. range. The result of assay of HCTZ, AMLO and LOSA tablet obtained by proposed method are quit concurrent and reproducible indicating precise and ruggedness of the method. The content estimation for HCTZ, AMLO and LOSA tablet was found to be 12.2969 mg, 4.9457 mg and 49.1733 mg respectively. The recoveries of the drugs were about 100 % indicating accuracy of the method and non- interference of excipient states method specificity, hence the Developed method is stability indicating and can be used for routine assay of HCTZ, AMLO and LOSA free of interferences from their degradation products in tablet formulations. From the studies it can be concluded that RP-HPLC can be successfully used for estimation of HCTZ, AMLO and LOSA in their combined dosage form.

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7/4/2012 66 6. FUTURE PROSPECTIVE HCTZ, AMLO and LOSA is a combination recommended as antihypertention. Comparative estimation of different brands can be conducted. The method can be developed for estimation of the proposed drugs in biofluids. The method can be further validated in terms of : Specificity studies – The degraded portion can be detected and analysed by IR, NMR and Mass spectra. Ruggedness – Different parameters like different laboratory, different source of reagents and solution can be performed.

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7/4/2012 71 Kathiresan K*., S. Gothandaraman, M. Swamivel Manickam, S. Mathan Kumar and R. Manavalan, “Analytical Method Development and Validation of Losartane Potassium Tablet by RP-HPLC” , Rasayan J. Chem , (2008), 1(3 ), 521-525. Wankhede S. B.*, K. C. Raka, S. B. Wadkar, and S. S. Chitlange, “Spectrophotometric and HPLC Methods for Simultaneous Estimation of Amlodipine Besilate, Losartan Potassium and Hydrochlorothiazide in Tablets”, Indian Journal of Pharmaceutical Sciences , (2010 Jan-Feb), 72(1), 136–140. Jayaseelan S.*,M. Rajasekar, S. Ganesh, V. Sekar and P. Perumal, “RP-HPLC method development and validation for simultaneous estimation of Losartan Potassium, Amlodipine Besilate and Hydrochlorthiazide in tablet dosage form”, Scholars Research Library Der Pharma Chemica , 2010, 2(3),31-36. Anbarasi B.*, K. Safeer and N.Senthil Kumar, “Analytical Method Development and Validation of Amlodipine and Hydrochlorothiazide in combined dosage form by RP-HPLC”, International Journal of ChemTech Research, Jan-Mar 2010, 2(1), 21-25. Priyanka R. Patil*, Sachin U. Rakesh, Prof. P. N. Dhabale and Prof. K. B. Burade, “RP- HPLC Method for Simultaneous Estimation of Losartan potassium and Amlodipine besylate in Tablet Formulation”, International Journal of ChemTech Research , (July-Sept 2009),1(3),464-469. Vora D N.* and A. A. Kadav, “Development and Validation of a Simultaneous HPLC Method for Estimation of Bisoprolol Fumarate and Amlodipine Besylate from Tablets”, Indian Journal of Pharmaceutical Sciences, ( 2008 Jul-Aug), 70(4), 542–546.

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7/4/2012 72 Aniruddha R. Chabukswar, C. Swati, Jagdale, S. Bhanudas S. Kuchekar, Pradeep D Lokhande1, Santosh N. Shinde, Kunal D. Ingale, Anuja K. Kolsure, “Development and validation of a RP-HPLC-PDA method for simultaneous estimation of Hydrochlorothiazide and Irbesartan”, Der Pharma Chemica , (2010), 2(4), 148-156. 44)Boopathy D.*, Abdussaleem And K, P. Perrumal, Analytical Method “Development and Validation of Losartan Potassium and Atenolol in combined dosage form by RP-HPLC”, International Journal of PharmTech Research, (Jan-Mar 2010), 2(1), 471-474. 45) Kayal1 S. D.*, F.A.Khan1, A. G. Tated1, R. L. Bakal1, and A. V. Chandewar1et “Method Development and Validation for the simultaneous Determenation of Amlodepine Besilate and Telmisartan in tablet dosage form by RP- HPLC”, International Journal of Pharmaceutical Research and Development,( 2011), 3(5), 144 - 153. 46)Chitlange1 S. S.*, Kiran Bagri1 and D. M. Sakarkar, “Stability Indicating RP- HPLC Method for Simultaneous Estimation of Valsartan and Amlodipine in Capsule Formulation ”, Asian J. Research Chem. ( July-Sept . 2008), 1(1). 47)C. V. N. Prasad1*, Ch. Santhosh Kumari1 and Sree Ramulu J., “Simultaneous Determination of Telmisartan, Amlodipine Besilate and Hydrochlorothiazide in a combined poly pill dosage form by Stability Indicating HPLC”, International Journal Of Research In Pharmacy And Chemistry, ( 2011), 1(3) ,2231-2781. 48)N. Chheta, S. P.Gandhi and S. J. Rajput *, “Development and Validation of a Stability Indicating High Performance Liquid Chromatography method for Atenolol and Hydrochlorothiazide in bulk drug and tablet formulation” International Journal of ChemTech Research, ( July-Sept 2009), 1(3), 654-662.

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7/4/2012 74 Thank You! 7/4/2012 74