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analytical method validation and validation of hplc


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analytical methods of validation
validation of HPLC.....

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analytical method validation and validation of hplc

  1. 1. SEMINAR ONAnalytical Method Validation& Validation of HPLC• GUIDE: • Presented by: MR. Ishaq Ahmed T.VENKATESH (Asst. Proff) M. Pharmacy (pharmaceutics) Sri Kakatiya Institute of Pharmaceutical Science.
  3. 3. INTRODUCTION Validation Establish a documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes Analytical Validation The principle purpose of analytical validation is to ensure that the selected analytical procedure will give reproducible and reliable results that are adequate for the intended purpose.
  4. 4. Why validation is necessary? It is an important element of quality control. Validation helps provide assurance that a measurement will be reliable. In some fields, validation of methods is a regulatory requirement.
  5. 5. When is validation needed? Before introduction of a new method in to routine use. Whenever condition change for which method has been validation e.g. instrument with different characteristics. Whenever the method is changed and the change is outside the scope of the original method
  6. 6. When Revalidation To Be Done? • Equipment changes • Formula changed • Changed suppliers of critical reagents
  7. 7. Parameters For Method ValidationComparison BP/USP/ICH As per USP and BP As per ICH
  8. 8. AccuracyAccuracy The accuracy is the closeness of the test results obtained by themethod to he true value. Accuracy should be established across its range. Accuracy assessed using a minimum of 9 determinations over a minimum of 3 concentration levels
  9. 9. True value Accurate but imprecise
  10. 10. Precision  Precision : The precision of an analytical method is the degree of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. Repeatability : Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision . a minimum of 9 determinations covering the specified range for the procedure ( e.g., 3 concentrations/3 replicates each); or a minimum of 6 determinations at 100% of the test concentration.
  11. 11. PrecisionIntermediate Precision: Intermediate precision expresses within-laboratories variations, different days, different analysts, different equipment, etc.Reproducibility: Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology).
  12. 12. Relationship between Accuracy and Precision Inaccurate & impreciseInaccurate but Accurate but Accurate AND Precise precise imprecise
  13. 13. Linearity  The linearity of an analytical procedure is its ability to obtain test results that are directly proportional to the concentration of the analyte in the sample.  Linearity is usually demonstrated by the analysis of various concentrations of the analyte (s) across the indented range and represented graphically. A statistical analysis of the data is usually required, such as the calculation of a regression line using the method of least square .  A minimum of 5 concentration is recommended.
  14. 14. Range Range of the analytical procedure is the interval between the upper and the lower concentration of the analyte for which it has been demonstrated that the analytical procedure has a suitable precision, accuracy and linearity. For assay the range is usually not less than 80 to 120% of the test concentration. For determination of content uniformity the range is usually not less than 70 to 130% or the test concentration. For determination of impurities the range is usually not less than the reporting limit of the impurity to 120% or the specification. For dissolution testing the range is usually ±20% over the expected concentration.
  15. 15. Specificity/Selectivity The ability to assess unequivocally the analyte in the presence of components that may be expected to be present. – Impurities – degradants – excipients Specificity must be demonstrated for: – Identification – Impurities Test – Assay Test
  16. 16. Detection limit (limit of detection) Definition : Limit of Detection is the smallest quantity of an analyte that can be detected, but not necessarily quantified. Approaches to calculation :• visual evaluation• signal to noise ratio• standard deviation of the response and the slope of the calibration curve
  17. 17. Calculation 1. visual evaluation- DL is determined by the analysis of a series of samples with known concentrations and establishing the minimum level at which the analyte can be reliably detected. 2. signal to noise ratio- For instrumental procedures that exhibit background noise, it is common to compare measured signals from samples with known low concentrations of analyte with those of the blank samples. The minimum concentration at which the analyte can reliably be detected is established using an acceptable signal - to - noise ratio of 2 : 1 or 3 : 1. 3. standard deviation of the response and the slope of the calibration curve DL=3σ/S where σ is the standard deviation of the response and S is the slope of the calibration curve
  18. 18. Limit of Quantitation• The quantitation limit is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.• Used particularly for the determination of impurities and/or degradation products.
  19. 19. Limit of Quantitation Various approaches of determining the Quantitation Limits are- Based on visual evaluation- Based on signal-to-noise- Based On Standard Deviation Of Response And Slope DL= 10 σ/S σ = the standard deviation of the response S = the slope of the calibration curve LOQ vary with detector sensitivity. Lamp aging, different manufacturer of detector.
  20. 20. Ruggedness  Ruggedness: The ruggedness of an analytical method is the degree of reproducibility of the test results obtained by the analysis of the same samples under a variety of conditions, such as  Day-to-day variations  Analyst-to-analyst  Laboratory-to-laboratory  Instrument-to-instrument  Chromatographic column-to-column  Reagent kit-to-kit  Instability of analytical reagents
  21. 21. 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 an indication of its reliability during use.
  22. 22. VALIDATION OF HPLCThe goal of equipment validation is to produce constant result withminimal variation with out compromising the product and performance ofequipment.
  23. 23. Qualification Qualification is a subset of the validation process that verifies module and systemperformance prior to the instrument being placed on-line.
  24. 24. DesignQualification(DQ) •For setting the functional and performance specifications •DQ can be very simple for similar equipment e.g. just another HPLC system
  25. 25. InstallationQualification(IQ)•for performing anddocumenting theinstallation in the selecteduser environment•safety, servicerequirements
  26. 26. OperationalQualification(OQ)•For testing theequipment in theselected userenvironment to ensure itmeets our definedfunctional andperformancespecifications
  27. 27. PerformanceQualification(PQ)•For testing that the systemconsistently performs as intendedfor the selected application•Periodic calibration /maintenance•Must be signed back by user•May use Method SystemSuitability Checks as part of PQ
  28. 28. TYPICAL PARAMETERS USED INHPLC Method validation Accuracy Detection limit and quantitation limit Linearity Precision Repeatability Reproducibility• Recovery• Robustness• Sample solution stability• Specificity
  29. 29. System suitability The simplest form of an HPLC system suitability test involves a comparison of chromatogram trace with a standard trace. This allows a comparison of the peak shape, peak width, baseline resolution. Parameters to be calculated to provide a system suitability test report.
  30. 30. System Suitability Number of theoretical plates (efficiency) Capacity factor Separation (relative retention) Resolution Tailing factor Relative standard deviation These are measured on a peak or peaks of known retention time and peak width.
  31. 31. Retention Factor or capacity factor •The capacity factor is a measure of how long each component is retained on the column. • k is used in preference to retention time. •Generally the value ok K’ is > 2. •Inpractice the k value for the first peak of interest should be >1 to assure that it is separated from solvent. •Hear Tr is retention time of peak of interest & T0 is unretained peaks retention time.
  32. 32. Relative Retention/ Separation•This describes the relative position of two adjacent peaks. Ideally it iscalculated using the capacity factor•Because the peak separation depends on the components interction withthe stationary phase.
  33. 33. Number of theoretical plate •Thisis a measure of sharpness of the peaks and therefore the efficiency of the column that is , how many peaks can be located per unit run time of the chromatogram. •This cam be calculated in various ways. •ex: USP uses the peak width at base and BP at half the height. •The theoretical plate number depends on elution time but in general should be > 2000
  34. 34. Tailing factor T • This is a measure for asymmetry of peak. • Peak asymmetry is measured at 5% of full peak height.
  35. 35. Peak Resolution R• This is not only a measure of separation between twopeaks, but also the efficiency of the column.• It is expressed as ratio of the distance between the twopeak maxima to mean value of peak width.
  36. 36. Precision• Injection repeatability (i.e. 6)• If RSD (relative standard deviation) of ≤ 2% is required then 5 replicate injections should be used.• Data from six injections are used if the RSD is more than 2.0%
  37. 37. CONCLUSION This summarizes the validation parameters that are required according to the requirement of ICH/USP&BP. Summarized the extent, need & necessary of validation. Validation may cost initially but it avoids the risk of breaking down, for this reason even small industries are concentrating more on validation their by fulfilling goal of GMP. Validation when done according to standard protocol and used it always produce a product which meets its predetermined specifications and quality.
  38. 38. References Article on Validation of Analytical Procedures: comparison of ICH vs Pharmacopoeia by katrai Sahil. British Pharmacopoeia, 2007 (4) A523 and A159-163 United states pharmacopoeia 30, 1920-1924 and 2149-2152. CDER guideline Nov 1994 validation of chromatographic methods