![PROJECT GUIDE :
DR.ARJUN GOJE
ASSOCIATE PROFESSOR.
PROJECT MEMBERS
R.SWATHI [14AD1R0024]
S.SRAVYA [14AD1R0026]
U.SRAVANTHI [14AD1R0028]
1](https://image.slidesharecdn.com/sravya14ad26-180422062345/85/RP-HPLC-Method-Development-and-Validation-of-Ketoconazole-in-Bulk-and-Pharmaceutical-Dosage-Form-1-320.jpg)
![DRUG PROFILE
• Name :
• Description :
• Structure :
• IUPAC Name :
• Chemical formula
Ketoconazole
Broad spectrum antifungal agent used for long periods at high doses,
especially in immunosuppressed patients.
: C26H28Cl2N4O4
• Molecular mass : 531.431g/mol
• Solubility : Ketoconazole is soluble in organic solvents such as methanol, ethanol.
• Category : Anti-Infective Agents, Antifungal Agents.
1-[4-(4-{[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazin-1-
yl]ethan-1-one
10](https://image.slidesharecdn.com/sravya14ad26-180422062345/85/RP-HPLC-Method-Development-and-Validation-of-Ketoconazole-in-Bulk-and-Pharmaceutical-Dosage-Form-10-320.jpg)
![• Drug profile- www.drug bank.com.
• www. Wikepedia.com.
• Verma Vikrant *, Singh Umesh Kumar, Ketoconazole hplc method
development and validation: a novel approach, international
research journal of pharmacy, 2017, 8 (8), Pages-74-81.
• Rakesh Kumar Jat1*, S Sharma2, R.C. Chhipa1, Rambir Singh1 and
Imran Alam, Development and validation of reverse-phase hplc
method for estimation of ketoconazole in bulk drug, Pharmacophore
2012, Vol. 3 (2), 123-129.
• Olga Popovska*, Zoran Kavrakovski, Vesna Rafajlovska, A RP-
HPLC Method for the Determination of Ketoconazole in
Pharmaceutical Dosage Forms, Current Pharmaceutical Analysis,
Volume 13 , Issue 6 , 2017. Page: [505 - 511].
46](https://image.slidesharecdn.com/sravya14ad26-180422062345/85/RP-HPLC-Method-Development-and-Validation-of-Ketoconazole-in-Bulk-and-Pharmaceutical-Dosage-Form-45-320.jpg)
RP-HPLC Method Development and Validation of Ketoconazole in Bulk and Pharmaceutical Dosage Form
- 1. PROJECT GUIDE : DR.ARJUN GOJE ASSOCIATE PROFESSOR. PROJECT MEMBERS R.SWATHI [14AD1R0024] S.SRAVYA [14AD1R0026] U.SRAVANTHI [14AD1R0028] 1
- 2. INTRODUCTION AIM AND OBJECTIVE PLAN OF WORK LITERATURE SURVEY MATERIALS AND METHODS DRUG PROFILE METHOD DEVELOPMENT METHOD VALIDATION CONCLUSION REFERENCES 2
- 3. High-performance liquid chromatography (HPLC) is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. When a mixture of components are introduced into the column . various chemical and/or physical interactions take place between the sample molecules and the particles of the column packing. 3
- 4. Reverse Phase HPLC: The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is polar(hydrophilic) in nature 4
- 5. The main principle of separation is adsorption. When a mixture of components are introduced in to a HPLC column, They travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the stationary phase, travels slower. The component which has less affinity towards the stationary phase travels faster. 5
- 6. AIM AND OBJECTIVES • Review of literature for Ketoconazole gave information regarding its physical and chemical properties. • Literature survey reveals that several spectrophotometric methods were reported for estimation of Ketoconazole. • In view of the need for a suitable RP-HPLC method for routine analysis of Ketoconazole in formulations, attempts were made to develop simple, precise and accurate analytical method. • Validation is a necessary and important step in both framing and documenting the capabilities of the developed method. 6
- 7. Literature survey Selection of drug Study of physiochemical properties of drugs Result and discussion Validation of developed method Optimization of analytical method Summary and conclusion PLAN OF WORK 7
- 8. S.NO YEAR AUTHOR DESCRIPTION 1. 2017 Verma Vikrant*, et al. •Stationary phase:Inertsil ODS-C18 column (150mm,4.6mm,5μm) . •Mobile phase: Buffer (pH-4) and Methanol in the mixture of 30:70. •Wavelength:274nm. •. Injection volume :20μl. •Flow rates: 1ml/min. 2. 2017 Olga Popovska, et al, •Stationary phase:LiChrospher®100 C-18 column (150 mm length x 4.6 mm i.d., 5 µm particle size) •Mobile phase: Methanol and water (90:10 v/v) •pH :8.9 with phosphate buffer. •Flow rate: 1.0 ml/min. 3. 2012 Rakesh Kumar Jat, et al. •Stationary phase:Promosil C-18, (250 mm, 4.6 mm, 5µm). •Mobile phase: Water : acetonitrile : buffer ph 6.8 (51:45:4 v/v) . •Wavelength:238nm. •Flow rate: 1.0 ml/min. • Retention times: 2.713 min. •Concentration range: 1-50µg/ml. 8
- 9. • All experiments will be carried out in the Analytical R & D of Syncorp Clincare Technologies Pvt. Ltd. Dilsuknagar, Hyderabad. • Pure samples of Ketoconazole 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 Analytical R & D of Syncorp Clincare Technologies Pvt. Ltd. Dilsuknagar, Hyderabad. • The methods will be first developed, then Validated as per ICH guidelines, then the method will be applied to the formulations. 9
- 10. DRUG PROFILE • Name : • Description : • Structure : • IUPAC Name : • Chemical formula Ketoconazole Broad spectrum antifungal agent used for long periods at high doses, especially in immunosuppressed patients. : C26H28Cl2N4O4 • Molecular mass : 531.431g/mol • Solubility : Ketoconazole is soluble in organic solvents such as methanol, ethanol. • Category : Anti-Infective Agents, Antifungal Agents. 1-[4-(4-{[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1- ylmethyl)-1,3-dioxolan-4-yl]methoxy}phenyl)piperazin-1- yl]ethan-1-one 10
- 12. • A suitable HPLC having isocratic system equipped with manual injector with UV detector. • LABINDIA T-60 UV – Vis spectrophotometer. • Analytical Balance, capable of measuring the 0.01mg. • Ultra Sonicator • Vaccum filtration kit • Usual laboratory glass ware of class-A 12
- 13. Separations Separation is based upon differential migration between the stationary and mobile phases. Stationary Phase - the phase which remains fixed in the column. e.g. C18, C8. Mobile Phase - carries the sample through the stationary phase as it moves through the column. e.g. methanol, ACN,OPA. Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Waste 13
- 14. Separations Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time High Performance Liquid Chromatograph 14
- 15. Separations Injector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time 15
- 16. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 16
- 17. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 17
- 18. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 18
- 19. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 19
- 20. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 20
- 21. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 21
- 22. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 22
- 23. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 23
- 24. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 24
- 25. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 25
- 26. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 26
- 27. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 27
- 28. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 28
- 29. Separations Injector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time 29
- 30. 30
- 31. METHODOLOGY TRIAL-1 • Stationary phase:Phenomenex Luna C18, 100A, 5m, 250mmx4.6mm i.d. • Mobile phase: Methanol : Water = 50 :50. • Flow rate:0.8 ml/min • Retention time:3.252. • Wavelength:242nm. • Observation: Peak broken at the end. • Result: Method rejected. TRAIL-2 • Stationary phase:Phenomenex Luna C18, 100A, 5m, 250mmx4.6mm i.d. • Mobile phase: Acetonitrile : Water = 60 :40. • Flow rate:1.0 ml/min. • Retention time:2.690. • Wavelength:242nm. • Observation: Splitting of peak • Result: Method rejected. 31
- 32. TRAIL-3 • Stationary phase:Phenomenex Luna C18, 100A, 5m, 250mmx4.6mm i.d. • Mobile phase: Acetonitrile : Phosphate buffer= 60 :40. • Flow rate:1.0 ml/min. • Retention time:4.753. • Wavelength:242nm. • Observation: Splitting of peak • Result: Method rejected. TRAIL-4 • Stationary phase:Phenomenex Luna C18, 100A, 5m, 250mmx4.6mm i.d. • Mobile phase: Acetonitrile : Phosphate buffer= 60 :40. • Flow rate:1.0 ml/min. • Retention time:3.254. • Wavelength:242nm. • Observation: Splitting of peak • Result: Method rejected. 32
- 33. OPTIMIZED PEAK CONDITIONS • Column: Phenomenex Luna C18, 100A, 5m, 250mmx4.6mm i.d. • Mobile phase:Acetonitrile : Phosphate buffer = 68:32 • Wavelength:242 nm • Flow rate:1.0 ml/ min. • Retention time:4.768. • Sampling System: Automatic • Injection Volume:20µl • Temperature of Column: Ambient • Run Time/ Stop Time:8.0minutes • Concentration of Sample:10ppm 33
- 34. Chromatogram for blank preparation Optimized chromatogram for ketoconazole 35
- 35. 1. Accuracy 2. Precision a) Repeatability b) Intermediate precision • Intra-day(or)intra-assay • Inter-day(or)inter-assay c)Reproducibility 3.Linearity 4.Range 5.Robustness 6.Ruggedness 7.Limit of detection(LOD) 8.Limit of quantification(LOQ) 9.System suitability parameter 10.Assay of marketed formulation METHOD VALIDATION PARAMETERS 36
- 36. To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (80%, 100%, and 120%) of pure drug of Ketoconazole were taken and added to the pre-analyzed formulation of concentration 10g/ml. From that percentage recovery values were calculated Sample ID Concentration (g/ml) Peak Area % Recovery of Pure drug Statistical AnalysisAmount Added Amount Found S1 : 80 % 8 8.157 595625 101.962 Mean= 101.387% S.D. = 0.516599 % R.S.D.= 0.509532 S2 : 80 % 8 8.099 591457 101.237 S3 : 80 % 8 8.077 589875 100.962 Chromatogram for accuracy-1 replicate-1 Chromatogram for accuracy-1 replicate-2 Chromatogram for accuracy-1 replicate-3 Accuracy 37
- 37. Sample ID Concentration (g/ml) Peak Area % Recovery of Pure drug Statistical AnalysisAmount Added Amount Found S4 : 100 % 10 10.077 734587 100.77 Mean= 100.43% S.D. = 0.833727 % R.S.D.= 0.830157 S5 : 100 % 10 9.948 725268 99.48 S6 : 100 % 10 10.104 736524 101.04 Chromatogram for accuracy-2 replicate-1 Chromatogram for accuracy-2 replicate-2 Chromatogram for accuracy-2 replicate-3 38
- 38. Sample ID Concentration (g/ml) Peak Area % Recovery of Pure drug Statistical AnalysisAmount Added Amount Found S7 : 120 % 12 11.989 872949 99.908 Mean= 100.6997% S.D. = 0.841254 % R.S.D.= 0.835409 S8 : 120 % 12 12.190 887456 101.583 S9 : 120 % 12 12.073 878975 100.608 Chromatogram for accuracy-3 replicate-1 Chromatogram for accuracy-3 replicate-2 Chromatogram for accuracy-3 replicate-3 39
- 39. Precision: Repeatability Repeatability Results of Precision HPLC Injections Retention Time (Minutes) Peak Area (AUC) Replicates of Ketoconazole Replicate – 1 4.765 725542 Replicate – 2 4.768 726334 Replicate – 3 4.773 727283 Replicate – 4 4.768 724365 Replicate – 5 4.768 728387 Replicate – 6 4.767 725342 Average 4.768167 726208.8 Standard Deviation 0.002639 1449.807 % RSD 0.055356 0.19964 Chromatogram for Repeatability- 1 Chromatogram for Repeatability-2 40
- 40. Chromatogram for Repeatability-3 Chromatogram for Repeatability-4 Chromatogram for Repeatability-5 Chromatogram for Repeatability-6 41
- 41. Linearity Linearity Results of Ketoconazole S. No. CONC. AUC (n=5) 1 0 0 2 6 425874 3 8 565872 4 10 714542 5 12 865632 6 14 1013121 42
- 42. Chromatogram for linearity-1 Chromatogram for linearity-2 Chromatogram for linearity-3 Chromatogram for linearity-4 Chromatogram for linearity-5 43
- 43. Robustness Change in parameter % RSD Flow (1.1 ml/min) 0.08 Flow (0.9 ml/min) 0.52 Temperature (270C) 0.12 Temperature (230C) 0.17 Wavelength of Detection (244 nm) 0.68 Wavelength of detection (240 nm) 0.73 44
- 44. • HPLC methods play a critical role in analysis of pharmaceutical product • Validation of HPLC should demonstrate that the method is suitable for its intended us • Data for acceptance,release,stability will only be trustworthy if the methods used are reliable 45
- 45. • Drug profile- www.drug bank.com. • www. Wikepedia.com. • Verma Vikrant *, Singh Umesh Kumar, Ketoconazole hplc method development and validation: a novel approach, international research journal of pharmacy, 2017, 8 (8), Pages-74-81. • Rakesh Kumar Jat1*, S Sharma2, R.C. Chhipa1, Rambir Singh1 and Imran Alam, Development and validation of reverse-phase hplc method for estimation of ketoconazole in bulk drug, Pharmacophore 2012, Vol. 3 (2), 123-129. • Olga Popovska*, Zoran Kavrakovski, Vesna Rafajlovska, A RP- HPLC Method for the Determination of Ketoconazole in Pharmaceutical Dosage Forms, Current Pharmaceutical Analysis, Volume 13 , Issue 6 , 2017. Page: [505 - 511]. 46
- 46. 47