Method developmment, trouble shooting ppt
- 1. R DEEPTHI Assistant Professor Vignan Institute of Pharmaceutical Technology Visakhapatnam
- 2. HPLC/HIGH PERFORMANCE LIQUID CHROMATOGRAPHY HPLC Normal phase chromatography Reverse phase chromatography Normal chromatography Reverse phase chromatography Stationary phase: polar (ex silica) Non-polar (ex: C8, C18, C4) Mobile phase : non-polar (ex n-hexane) Polar (ex: water, methanol)
- 3. Normal phase: non-polar component comes 1st Reverse phase: polar components comes 1st Column Brand name Polarity Column length × diameter Stationary phase particle size Brand names: Thermo hypersil, Iorbax, Ace, xterra, inertsil, phenomenex, agilent Polarity: silica cyano, phenyl, C4, C8, C18 polar Mid polar Non-polar
- 4. Column length: 50 100 150 200 250** 300 Column diameter: 4.6mm (Fixed) Stationary phase particle size: 5µ( generally) 3.5µ 2.9µ Ex: Agilent, c8, 150×4.6mm, 3.5µ
- 5. System suitability: The purpose of the system suitability is to ensure that the Instrument, Method and Analyst is suitable for the intended purpose. Retention time Resolution Tailing Theoretical plates Relative standard deviation Similarity factor Bracketing standard
- 6. Retention time : The time taken by the component to pass through the column is called Rt. Resolution The distance between two peaks Tailing The gap between the midpoint of the peak to tail of the peak is called tailing Theoretical plates Efficiency of method Relative standard deviation(%RSD) It is used to determine “Repeatability of the result” Similarity Factor To check analyst repeatability Bracketing standard To check whether system is suitable NLT2 NMT2 NLT 2500 NMT 2% NMT2%
- 7. Analytical Research & Development Method Development Validation Transfer Method development 1. Mobile phase 2. Column 3. Injection volume 4. Column temperature 5. Flow rate 6. Standard preparation 7. Sample preparation 8. Diluent 9. Wavelength 10. Run time
- 8. Mobile phase: Buffer + solvent(Methanol/Acetonitrile) If component is acidic Basic buffer(pH 7-14) If component is Basic Acidic buffer(pH 0-7) Acidic buffers Orthophosphoric acid Potassium dihydrogen phosphate Sodium dihydrogen phosphate Ammonium acetate Basic Buffers Dipotassium hydrogen phosphate Disodium hydrogen phosphate
- 9. 2. Column: Choosing of the column depends upon the polarity of the components. Component (strong Polar)- choose strong non-polar column (C8, C18) Component (weak polar)-choose weak non-polar column (CN,C4) 3. Injection Volume: 10µL 4. Column Temperature: 250C 5. Flow rate: 1mL/min 6. Diluent: Solubility of the Components 7. Run time: Depends on the Retention time 8. Standard preparation: 1000µg/mL
- 10. 9. Sample Preparation: Wt to be taken = Std wt. × Avg wt Label claim 10. Wavelength: Detector: UV -1st scan in UV and Find λmax Detector : PDA –No need to scan in UV
- 11. Trouble shooting 1. There is no peak? Check the solubility in Diluent Check the Mobile phase Check the Detector wavelength Check the column Polarity 2. High Retention Time? Increase the solvent ratio, reduce buffer ratio Increase Flow rate Decrease column length Increase the column temperature
- 12. 3. Low Retention time with low Theoretical Plates? Increase Buffer ratio Increase column length Decrease Flow rate Decrease column Temperature 4. High Tailing? Injection volume must be reduced Change the diluent Wash the column Change the column (new column)
- 13. 5. Low resolution? Decrease the Particle size Increase the Buffer ratio Increase the column length Gradient Elution 6. Peak splitting? Column washing Take new column
- 14. ICH An agreement between -European union -Japan -United states ICH was created in the year 1990, April in Brussels.
- 15. ICH Safety Efficacy Quality Multidisciplinary Quality has been divided into Q12 Q1: Stability Q2: Analytical Method Validation Q3: Impurities Q4: Pharmacopoeias Q5: Biotechnological quality Q6: Specifications Q7: GMP Q8: Pharmaceutical Development Q9: Quality Risk Management Q10: Pharmaceutical Risk Management Q11: Development and Manufacture of Drug substance Q12: Technical and Regulatory considerations for Pharmaceutical drug product life cycle Management
- 16. Q2 Analytical Method Validation Validation: Parameters: 1. System suitability 2. Accuracy 3. Precision 4. Specificity 5. Robustness 6. Ruggedness 7. LOD 8. LOQ 9. Linearity 10. Range
- 17. 1. System suitability: The purpose of system suitability is to ensure that the instrument, method , analyst are suitable for the intended application. Acceptance criteria: All the system suitability parameters must be within the limits. 2. Accuracy/Recovery: The closeness of agreement between the true value and found value. The method is said to be accurate if individual recovery is between 97-103%. 3. Precision/Repeatability: Closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. The %RSD must be NMT 2%.
- 18. 4. Specificity: is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present Blank Placebo Standard Sample There should be no peak of Blank, Placebo, Impurity chromatograms at Analyte retention time.
- 19. 5. Robustness: The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides its reliability during normal usage. Mobile phase composition Variation pH in mobile phase variation Flow rate variation Column temperature variation Filter validation All the system suitability parameters should be within the limits.
- 20. 6. Ruggedness: System to system variation Column to column variation Analyst to analyst variation Bench top stability of Std. and Sample solution Refrigerator stability of Std. and Sample solution Bench top stability of Mobile phase 7. Limit of Detection (LOD): Minimum concentration of the standard compound in which the peak of the standard get merged with noise. 8. Limit of Quantification (LOQ): Minimum concentration of the standard compound in which the peak of the standard get detected and quantified.
- 21. 9. Linearity: To demonstrate the linearity range of 50%- 150% of target concentration. 6 points: Plot the graph b/n Conc. Vs Peak Area R2 = NLT 0.999 10. Range: Range of concentration in which the method is linear, precise & accurate. Method Transfer QC