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Published on July 31, 2014

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A PRESENTATION ON : Development and validation of an RP-HPLC method for simultaneous determination of Ramipril and Amlodipine in tablets : A PRESENTATION ON : Development and validation of an RP-HPLC method for simultaneous determination of Ramipril and Amlodipine in tablets 1 PRESENTED BY : AMPATI RAHUL {M PHARMACY:PHARMACEUTICAL ANALYSIS} INTRODUCTION:: INTRODUCTION : RAMIPRIL : ACE inhibitor, anti hypertensive 2 ANGIOTENSINOGEN ANGIOTENSIN-1 ANGIOTENSIN-2 AT 1 RECEPTOR ALDOSTERONE SALT AND WATER RETENTION INCREASES BP RENIN ACE RAMIPRIL ----- PowerPoint Presentation: AMLODIPINE : It is the drug that blocks L-type of voltage gated calcium channel present in blood vessels and heart 3 Why to give drug in combination…..?: Why to give drug in combination…..? 4 PowerPoint Presentation: Large majority of hypertensives ultimately require drug combination to decrease the damage of heart, brain, kidney, etc., Fixed dose combinations of drugs with complementary properties offer the advantages of simplicity, tolerability, convenience and cost effectiveness, as well as the compliance. The combination therapy of ACEI and CCB has proved to be effective. So the combination of RP and AL would also be good therapeutic option. 5 DRUG PROFILE: DRUG PROFILE 6 RAMIPRIL AMLODIPINE STUCTURE CHEMICAL NAME [(2S, 3aS, 6aS)-1-[(S)-2- [[(S)-1-(ethoxycarbonyl)-3-phenylpropyl] amino] propanoyl] octahydro cyclopenta [b] pyrrole-2-carboxylic acid [3-Ethyl5-methyl(4RS)-2-[(2-aminoethoxy) methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate SIDE EFFECTS Hypotension, cough Peripheral edema PowerPoint Presentation: 7 Chemical structure of impurities: Ramipril impurities A (II), B (III), C (IV), D (V) Amlodipine impurity D OBJECTIVE OF WORK:: OBJECTIVE OF WORK : To develop HPLC method for determination of Ramipril and Amlodipine in the same tablet dosage form. To validate the developed methods as per the ICH guidelines. To study stability indicating method of drug. 8 PLAN OF WORK:: PLAN OF WORK : 9 Literature survey Selection and procurement of drug study of physical properties of drug Optimization of analytical method validation of developmental method Forced degradation studies Result and discussion Summary and conclusion REAGENTS AND CHEMICALS: REAGENTS AND CHEMICALS SL NO NAME OF THE CHEMICALS MAKE 1 RP API Green syn co., ltd 2 AL besylate API weihai disu pharmc.Co.,ltd 3 Tablet exepients Chengdu haisco pharmaceutical co.,ltd 4 RS of AL beyslate National institute for control of pharmaceutical products 5 RS of RP European directorate 6 Impurity standards of RP and AL European directorate 7 HPLC-grade acetonitrile Honey well 8 HPLC-grade triethylamine Kermel chemical reagents company 10 HPLC INSTRUMENTS AND ANALYTICAL CONDITION:: HPLC INSTRUMENTS AND ANALYTICAL CONDITION : Analysis was performed on a chromatographic system of shimadzu prominence consisting of AT liquid pump and PDA detector with manual 20 µl sample injection loop. Chromatographic seperation was achieved on Inertsil ODS -3 (250mm * 4.0mm , 3µm) column. The column temperature was maintained at 55°C and flow rate was 1.0mL/min. The detection wavelength was set at 210nm. 11 PowerPoint Presentation: Cont’d…. Data acquisition was made with shimadzu LC solution software. Mobile phase A consisted of 60mM sodium perchlorate buffer (containing 7.2mM triethylamine)- acetonitrile(60:40,v/v) Mobile phase B was 60mM sodium perchlorate buffer (containing 7.2mM triethylamine)- acetonitrile (20:80,v/v). The apparent pH of the mobile phase was adjusted to 2.6 with phosphoric acid. 12 PowerPoint Presentation: Sample preparation: 13 PowerPoint Presentation: Samples were subjected to stressed conditions of light, heat, acid, base and oxidation. In the stress condition, all the solutions were prepared by weighing 4-tablets equivalent mass of sample powders containing about 10mg RP or 20mg AL pure API. 14 Method development: Method development 15 Gradient HPLC method was adopted To get a shorter run time Higher sensitivity. Stainless steel column(250mm*4.0mm, 3µm) was used (ph. Eur.). Mobile phase A and B all consisted of acetonitrile , Sodium perchlorate buffer and 1mL trimethylamine with different pH values adjusted to 3.6 and 2.6 with phosphoric acid. PowerPoint Presentation: As RP and AL had N-H groups as basic nitrogen centers, the amount of perchlorate and the pH of M.P would affect the peak shape, resolution and symmetry. The results shows that the retention time of RP and AL were lengthened when the amount of perchlorate increased. The RT of RP would be shortened while the RT of AL has no significant change when the pH was decreased. Column diameter of 3µm was chosen Column temperature of 60°C, 55°C and 50°C were studied and 55°C was finally chosen. 16 PowerPoint Presentation: 17 Gradient program proposed for the analysis of AL (Amlodipine), RP (Ramipril) and their related substances PowerPoint Presentation: 18 Chromatograms of sample solution (1mg/mL RP and 2mg/mL AL solution using the final optimized mehtod ).1.Benzene sulfonic acid, 2. Unknown 1, 3.Unknown 2, 4.Amlodipine impurity D, 5.Unknown 3, 6.Ramipril impurity B, 7.Unknown 4, 8.Ramipril impurity C, 9. Unknown 5, 10.Unknown 6, 11.Ramipril impurity D, 12.Unknown 7, and 13.Unknown 8. METHOD VALIDATION:: METHOD VALIDATION : 19 PowerPoint Presentation: System suitability test : The solution containing 0.1mg/mL of RP, 0.2mg/mL of AL besylate and each 0.1 mg/mL of five major impurities was prepared by mobile phase A. System suitability was determined by six replicate injections of the system suitability solution. The resolutions among RP, AL and the closest eluting peaks were bigger than 2 which indicating that this method was reliable for the quantitation of RP and AL 20 PowerPoint Presentation: Acceptance criteria : 21 PowerPoint Presentation: 22 Chromatogram of the system suitability test solution. 1. Benzene sulfonic acid, 2. Ramipril impurity A, 3. Amlodipine impurity D, 4. Ramipril impurity B, 5. Ramipril impurity C, and 6. Ramipril impurity D PowerPoint Presentation: Specificity : The selectivity of the method was confirmed by observing potential interference caused by excipients of tablet formulations and degradation products . The chromatogram of the tablet excipients shows that there were no interference of peaks to the determination of Rp and AL. 23 PowerPoint Presentation: Linearity and range : A standard stock solution containing 0.25mg/mL of RP and 0.5mg/mL of AL besylate was prepared by mobile phase A Dilute with the same solvent to yield solution at different concentrations These solutions were protected from light using aluminium foil Stored at 4ºC in the refrigerator 24 PowerPoint Presentation: The linearity was checked by analyzing six working solution of RP over the concentration range 0.01 – 0.25 mg/mL and 0.014 – 0.36 mg/mL for AL Results : 25 RP AL y=44164x + 303.8 y= 37963x - 597 r 2 = 09998 r 2 = 0.9997 PowerPoint Presentation: Accuracy and precision : Known amount of each standard powder was added to blank sample Mixed , extracted and subsequently dilute to yield three different concentrations of each drug RP: 0.08, 0.10 and 0.12 mg/mL AL: 0.16, 0.20 and 0.24 mg/mL 26 PowerPoint Presentation: 27 Accuracy results [Recovery (%)] for the determination of AL (Amlodipine) and RP (Ramipril ). PowerPoint Presentation: Repeatability or intra – day precision was investigated by injecting six replicate sample solution on the same day. Inter day precision was assessed by analysis newly prepare sample solutions in triplicate over three consecutive days. 28 RP AL RSD% RSD% Intra day 0.46 0.55 Inter day 1.60 1.80 PowerPoint Presentation: LOQ and LOD : The LOQs for RP and AL corresponding to a signal-to-noise ratio of 10 were 0.2µg/mL and 0.07 µg/mL. The LODs corresponding to a signal to noise ratio of 3 were 0.06µg/mL and 0.2 µg/mL. The resultant RSD values for these studies were < 3.5% 29 PowerPoint Presentation: Stability of solutions and robustness : The stability of the standard stock solutions was determined by quantitatively determining each drug in different time comparing to the response obtained for freshly prepared standard solutions. The stability of sample solution was tested for every 2h interval upto 12h. The RSD values of RP and AL were 1.2% and 1.0%, respectively. 30 PowerPoint Presentation: Robustness : The robustness of the method was investigated by a small variety of conditions including changes of pH of the eluent, flow rate and column temperature . The assay results for RP and AL by three different analysts in the same laboratory were also investigated. The RSD values did not exceed 2.5%. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters and by changing analytical operators demonstrated that the method was robust. 31 PowerPoint Presentation: 32 Chromatographic parameter setting applied in the robustness investigation FORCED DEGRADATION STUDIES: FORCED DEGRADATION STUDIES Degradation profile under different stress condition: Acid Degradation (HCL) Alkali Degradation (NaOH) Oxidation Degradation (H 2 O 2 ) Thermal Degradation Photo Degradation 33 PowerPoint Presentation: 34 Acid Degradation : It was conducted using 2mL of 0.1M hydrochloric acid PowerPoint Presentation: Alkali Degradation : It was carried out in 2mL of 0.1M sodium hydroxide. 35 PowerPoint Presentation: Oxidation Degradation : It was performed by adding 2mL of 3% H2O2 and kept in water bath for 5min, and then diluted with mobile phase A. 36 PowerPoint Presentation: Thermal Degradation : For the temperature stress study, compound RPAL tablets, RP and AL besylate API were exposed to dry heat of 60°C in a convention oven for 10 days. 37 PowerPoint Presentation: Photo Degradation : For photo stability studies composed RPAL tablets , RP and AL besylate API were exposed t 4500 lx light in a light cabinet for 10days. 38 PowerPoint Presentation: All stressed samples were compare with an un-stressed sample solution. The peak purity indices for RP and AL in stressed solutions were found to be better ( purity angle < purity threshold) And they also evidence the ability of the method to access unequivocally the analytes of interest in the presence of potential interference. 39 PowerPoint Presentation: Origination of related substance : The origination of the related substances in tablets was investigated. The peaks numbered 6, 8, 11 as defined in the chromatograms of sample solution( RL impurity B, C, D respectively, according to the RRT) would same from RP API. In addition to the known impurities, the unknown impurities peaks numbered 2, 9, 10, 12, 13 originated in RP API And the peaks numbered 1 ( benzoic acid), 3, 4(Amlodipine impurity D), 5 and 7 would originate in AL besylate API. 40 PowerPoint Presentation: 41 Chromatograms of RP (Ramipril) API and AL (Amlodipine) besylate API . PowerPoint Presentation: Assay of AL and RP in tablets: Three batches of compound tablets were analysed using the developed method. 42 PowerPoint Presentation: Conclusion : A gradient LC method has been developed and validated for the analysis of RP and AL in tablet dosage forms. The results of the stress testing revealed that the method was specific and selective. The proposed method has the ability to separate the two main components from their degradation products, related substances found in tablet dosage forms and the tablet excipients . 43 PowerPoint Presentation: Therefore, the chromatographic method can be used to analyze samples obtained during accelerated stability experiments and routine assay of RP and AL in combined tablet dosage forms. In addition, the procedure can be further applied for the detection and determination of the related substances in tablets 44 PowerPoint Presentation: 45

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