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Validation of Analytical and Bioanalytical methods


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Guidelines for Validation of Analytical and Bioanalytical methods as per ICH (Q2R1) and USFDA respectively with an example of Bioanalytical method validation.

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Validation of Analytical and Bioanalytical methods

  1. 1. • Dr. (Mrs.) Snehalatha Boddu • Asst. Professor GUIDANCE BY: • Sarika K. Kadam • M. Pharm (Q.A.) Sem I • Oriental College of Pharmacy, Navi Mumbai PRESENTED BY: Sarika Kadam
  2. 2. WHAT IS VALIDATION? Validation is establishing 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. Sarika Kadam
  3. 3.   To obtain consistent, reliable and accurate data.  For identification of source and quantization of potential errors.  Validation of analytical methods is also required by most regulations.  To ensure the products safety and efficacy throughout all phases of its shelf life.  Establish a proof that method can be used for decision making. Sarika Kadam
  4. 4.  TYPES OF VALIDATION: Analytical Method Validation Equipment Validation Process Validation Cleaning Validation VALIDATION Sarika Kadam
  5. 5.  Analytical methods: Set of techniques that allow us to know qualitatively and/or quantitatively the analyte in sample.  Bioanalytical methods: Analytical methods performed for determination of drug/ drug substance in biological matrices.  Analytical Method validation: Process of documenting/ providing evidence that an analytical method provides analytical data acceptable for intended use. Sarika Kadam
  6. 6.   Identification tests  Quantitative tests for impurities content  Limit tests for the control of impurities  Quantitative tests for active moiety Sarika Kadam
  7. 7. Sarika Kadam
  8. 8.  Analytical methods need to be validated: Before their introduction into routine use. Whenever conditions change for which method is validated. Whenever method is changed and change is outside the original scope of method. Sarika Kadam
  9. 9.  Scope and validation criteria should be defined early in the process which includes:  What analyte should be detected?  What are expected concentration levels?  What are sample matrices?  Are there interfering substances expected, and if so, should they be detected and quantified?  Are there any specific requirements?  Should information be qualitative or quantitative?  What are required detection and quantitation limits?  What precision and accuracy is required?  How robust the method should be? Sarika Kadam
  10. 10. Workflow for evaluation and validation of standard methods: Define scope of users method Define validation parameters and limits Standard method fits scope, parameters, limits? Perform part or full validation Define and perform system suitability testing NO YES Sarika Kadam
  11. 11. ANALYTICAL METHOD AND VALIDATION  A pharmaceutical drug product must meet all its specifications throughout its entire shelf life.  The performance of product characteristic must be tested by analytical method for the product’s chemical, physicochemical, microbiological and biological characteristics.  The method of analysis used must be validated. This is required to ensure the product’s safety and efficacy throughout all phases of its shelf life. Sarika Kadam
  12. 12. Contents of ICH Guidelines Q2(R1)  Introduction  Document History  Part I: (Text on Validation of Analytical procedures) 1. Introduction 2. Types of Analytical Procedures to be validated  Part II: ( Validation of Analytical Procedures: Methodology) 1. Specificity 2. Linearity 3. Range 4. Accuracy 5. Precision 6. Detection limit 7. Quantitation limit 8. Robustness 9. System suitability testing Sarika Kadam
  13. 13. Identification tests-  Intended to ensure the identity of an analyte in a sample.  This is normally achieved by comparison of a property of the sample  to that of a reference standard (e.g., spectrum, chromatographic behavior, chemical reactivity, etc) Quantitative tests for impurities content-  Intended to accurately reflect the purity characteristics of the sample. Limit tests for control of impurities-  Quantitative or semi quantitative test designed to identify and control small quantities of impurity which is likely to be present in the substance. Sarika Kadam
  14. 14.  The objective of the analytical procedure should be clearly understood since this will govern the validation characteristics which need to be evaluated.  Typical validation characteristics to be considered:  Accuracy  Precision - Repeatability Intermediate Precision  Specificity  Detection Limit  Quantitation Limit  Linearity  Range Sarika Kadam
  15. 15. Furthermore revalidation may be necessary in the following circumstances:  Changes in the synthesis of the drug substance;  Changes in the composition of the finished product;  Changes in the analytical procedure. Sarika Kadam
  16. 16. Sarika Kadam
  17. 17. Specificity Accuracy Precision Detection limitQuantitatio n limit Linearit y Range Robust- ness Validation characteristics Sarika Kadam
  18. 18.  Ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include : impurities, degradants, matrix, etc.  Three implications of specification: SPECIFICITY Identification Purity tests Assay Impurities are available Impurities are not available Sarika Kadam
  19. 19. Identification:  Suitable identification tests should be able to discriminate between compounds of closely related structures which are likely to be present.  The discrimination of a procedure may be confirmed by obtaining positive results from samples containing the analyte, coupled with negative results from samples which do not contain the analyte.  The identification test may be applied to materials structurally similar to or closely related to the analyte to confirm that a positive response is not obtained. Sarika Kadam
  20. 20. Assay and impurity test (s) • For chromatographic procedures, representative chromatograms should be used to demonstrate specificity and individual components should be appropriately labelled. • Critical separations in chromatography should be investigated at an appropriate level. For critical separations, specificity can be demonstrated by the resolution of the two components which elute closest to each other. o Impurities are available: Done by spiking pure substances with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials o Impurities are not available: By comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure e.g.: pharmacopoeial method or other validated analytical procedure Sarika Kadam
  21. 21. ACCURACY( TRUENESS)  Expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Sarika Kadam
  22. 22. DETERMINATION OF ACCURACY  Assay: • Application of an analytical procedure to an analyte of known purity . • Comparison of the results of the proposed analytical procedure with those of a second well-characterized procedure. • Accuracy may be inferred once precision, linearity and specificity have been established. Drug substance • Application of the analytical procedure to synthetic mixtures of the drug product components to which known quantities of the drug substance to be analysed have been added. Drug product Sarika Kadam
  23. 23. DETERMINATION OF ACCURACY  Impurities (Quantitation): Assessed on samples (drug substance/drug product) spiked with known amounts of impurities.  Recommended data:  Minimum 9 determinations = Min. 3 concentration levels covering specified range. (3 concentration levels, 3 replicates)  Reported as percent recovery by the assay of known added amount of analyte in the sample OR as the difference between the mean and the accepted true value. Sarika Kadam
  24. 24. PRECISION  Expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Sarika Kadam
  25. 25. PRECISION  Investigated using Homogenous authenticated samples.  Expressed as:  Variance  Standard deviation  Coefficient of variance  The value of %CV should be <2 Sarika Kadam
  26. 26. PRECISION • Precision may be considered at three levels. • Same operating conditions • Short interval of time • Intra-assay precision Repeatability • Within-laboratories variations • Different days • Different analysts • Different equipment, etc. Intermediate Precision • Between laboratories Reproducibility Sarika Kadam
  27. 27. DETERMINATION OF PRECISION Validation of tests for assay and quantitative determination of impurities include an investigation of precision. Repeatability:  Min. 9 determinations (3 conc. , 3 replicates) OR  Min. 6 determinations at 100% test concentration. Intermediate Precision:  Extent to which it should be established depends on the circumstances under which the procedure is intended to be used. Sarika Kadam
  28. 28. DETERMINATION OF PRECISION Reproducibility:  Assessed by means of an inter-laboratory trial.  Should be considered in case of the standardization of an analytical procedure. (For instance, for inclusion of procedures in pharmacopoeias) Recommended Data:  The standard deviation, relative standard deviation (coefficient of variation). Sarika Kadam
  29. 29. DETERMINATION OF PRECISION Concentration Absorption Concentration Absorption 10 g/ml 0.28 10 g/ml 0.21 0.31 0.32 0.29 0.39 0.30 0.53 Mean 0.29 Mean 0.36 HIGH PRECISION LOW PRECISION Sarika Kadam
  30. 30. ACCURACY AND PRECISION Sarika Kadam
  31. 31. LIMIT OF DETECTION AND QUANTITATION: LOD: Lowest amount of analyte in a sample which can be detected (not necessarily quantitated as an exact value). LOQ: Lowest amount of analyte in a sample which can be quantitatively determined with suitable accuracy and precision. Sarika Kadam
  32. 32.  Based on Visual Evaluation:  Establishing the minimum level at which the analyte can be reliably detected.  Based on Signal-to-Noise:  Only be applied to analytical procedures which exhibit baseline noise.  S:N ratio of 3:1 is acceptable  Based on the Standard Deviation of the Response and the Slope:  DL= 3.3 /S (= SD and S= slope of calibration curve) Sarika Kadam
  33. 33. Recommended data:  The detection limit and the method used for determining the detection limit should be presented.  If DL is determined based on visual evaluation or based on signal to noise ratio, the presentation of the relevant chromatograms is considered acceptable for justification. Sarika Kadam
  34. 34. LOQ DETERMINATION:  Based on Visual Evaluation:  Establishing the minimum level at which the analyte can be quantified with reliable accuracy and precision  Based on Signal-to-Noise:  Only be applied to analytical procedures which exhibit baseline noise.  S:N ratio of 10:1 is typical.  Based on the Standard Deviation of the Response and the Slope:  QL= 10 /S (= SD and S= slope of calibration curve) Sarika Kadam
  35. 35. LOQ DETERMINATION: Recommended data:  The quantitation limit and the method used for determining the quantitation limit should be presented.  The limit should be subsequently validated by the analysis of a suitable number of samples known to be near or prepared at the quantitation limit. Sarika Kadam
  36. 36. LOD AND LOQ: Sarika Kadam
  37. 37. LINEARITY: Ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. Sarika Kadam
  38. 38. DETERMINATION OF LINEARITY:  Linear relationship is evaluated across the range of the analytical procedure.  Evaluated by visual inspection of a plot of signals as a function of analyte concentration or content.  If there is a linear relationship, test results should be evaluated by appropriate statistical methods. eg. Regression line by method of least squares.  Some analytical procedures, such as immunoassays, do not demonstrate linearity after any transformation. In this case, the analytical response should be described by an appropriate function of the concentration (amount) of an analyte in a sample.  For the establishment of linearity, a minimum of 5 concentrations is recommended Sarika Kadam
  39. 39. LINEARITY: (Know that it is a straight line) •Mathematical transformations may be needed for some cases to establish linear relationship. Sarika Kadam
  40. 40. RANGE:  Interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. Sarika Kadam
  41. 41. RANGE: These are some min. specified ranges to be considered:  For assay of drug/ drug substance: 80 - 120% of the test concentration.  For content uniformity: min. of 70 to 130 % of the test concentration.  Dissolution testing: +/-20 % over the specified range  For the determination of an impurity: : from the reporting level of an impurity to 120% of the specification. Sarika Kadam
  42. 42. RANGE: ( For what concentration is it a straight line?) Sarika Kadam
  43. 43. ROBUSTNESS:  Measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.  Should be considered during the development phase.  Depends on the type of procedure under study.  Analytical conditions should be suitably controlled. Sarika Kadam
  44. 44. ROBUSTNESS: Examples of typical variations:  In the case of liquid chromatography:  Variations of pH in a mobile phase  Variations in mobile phase composition  Different columns (different lots and/or suppliers)  Temperature  Flow rate.  In the case of gas-chromatography:  Different columns (different lots and/or suppliers)  Temperature  Flow Sarika Kadam
  45. 45. Sarika Kadam
  46. 46.  U.S. Department of Health and Human Services Food and Drug Administration provides guidelines for bioanalytical method validation.  Provides assistance to sponsors of INDs, NDAs, ANDAs.  Supplements in developing bioanalytical method validation information used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies requiring pharmacokinetic (PK) evaluation.  Also applies to other bioanalytical methods, such as immunological and microbiological procedures. Sarika Kadam
  47. 47. BIOANALYTICAL METHOD VALIDATION:  Fundamental parameters:  Accuracy  Precision  Selectivity  Sensitivity  Reproducibility  Stability  Different types of validation:  Full validation  Partial validation  Cross validation Sarika Kadam
  48. 48. REFERENCE STANDARD  Analysis is carried out by using Samples spiked with reference standards and using QC samples.  Three types of reference standards:  certified reference standards (e.g., USP compendial standards)  commercially supplied reference standards.  other materials of documented purity custom-synthesized by an analytical laboratory. Sarika Kadam
  49. 49. SELECTIVITY: Ability of an analytical method to differentiate and quantify the analyte in the presence of other components in the sample.  Blank samples (appropriate biological matrix)from at least 6 different sources is analyzed.  Selectivity is ensured at LLOQ. Sarika Kadam
  50. 50. ACCURACY  Determined by taking three different concentrations and five determinations per concentration. (3 conc.  5 replications)  Mean value should be within 15% of actual value  Except for LLOQ: Deviation within 20% is allowed  deviation of the mean from the true value serves as the measure of accuracy. Sarika Kadam
  51. 51. PRECISION:  3 concentrations and 5determinations per conc.  Precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV).  Except for the LLOQ, where it should not exceed 20% of the CV. Sarika Kadam
  52. 52. RECOVERY: Detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard.  Performed by comparing analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100% recovery. Sarika Kadam
  53. 53. STABILITY: Function of the storage conditions, the chemical properties of the drug, the matrix, and the container system  Determined stability is relevant only to that matrix and container system and should not be extrapolated to others. Sarika Kadam
  54. 54. Freeze and Thaw Stability Short-Term Stability Long-Term Stability Stock Solution Stability Post-Preparative Stability Evaluationofstability Sarika Kadam
  55. 55. FREEZE AND THAW STABILITY: • Stability is determined after 3 freeze and thaw cycles. • One cycle is as follows: At least 3 aliquots each at low and high conc. Store at intended storage temperature for 24 hrs Thaw unassisted at RT Sarika Kadam
  56. 56. SHORT-TERM TEMPERATURE STABILITY: At least 3 aliquots each at low and high conc. Thaw at RT Keep at this temp. for 4-24 hrs Analyze Sarika Kadam
  57. 57. STABILITY: Long-Term Stability: Storage time : time between the date of first sample collection and the date of last sample analysis. At least three aliquots of each of the low and high concentrations. Stock Solution Stability: Evaluated at room temperature for at least 6 hours Tested by comparing the instrument response with that of freshly prepared solutions. Post-Preparative Stability: Stability of processed samples, including the resident time in the auto sampler. Compared with freshly prepared samples. Sarika Kadam
  58. 58. Example: Simultaneous determination of vancomycin and ceftazidime in cerebrospinal fluid in craniotomy patients by HPLC.  EXPERIMENTAL CONDITIONS:  Internal standard: Tinidazole  Column: Diamonsil C18 column (200mm×4.6mmI.D., 5µm)  Mobile phase : Acetonitrile and acetate buffer (pH 3.5) (8:92, v/v) at room temperature (25 ◦C)  Detection wavelength: 240 nm  METHOD VALIDATION : Sarika Kadam
  59. 59. Selectivity  Six separate lots of drug-free control CSF were analyzed • Free of interferences with compound of interest • Retention times: Vancomycin 7 min Ceftazidime 11 min Tinidazole 20 min Control CSF spiked with drugs CSF sample 12hrs after receiving drugs . TypicalChromatogram Sarika Kadam
  60. 60. SENSITIVITY AND LINEARITY:  LLOQ were found to be –  For vancomycin: 0.1 g/ml  For ceftazidime: 1 g/ml  Reproducibility of LLOQ was determined by examining five LLOQ samples  Calibration curves were constructed by plotting the peak area ratios (analytes/IS) of plasma standards versus nominal concentration Calibration curves showed a linear range of 0.1–10 g/ml for vancomycin and 1–100 g/ml for ceftazidime. Unknown sample concentrations exceeding the range were diluted appropriately with control blank CSF and re-assayed. The difference between the nominal standard concentration and the back-calculated concentration from the weighted linear regression line was within 15% for each point on the standard curve indicating that the linear regression analysis applied provided an adequate fit of the data. Sarika Kadam
  61. 61. ACCURACY AND PRECISION: Nominal conc (g/ml) Intra day (n=5) Inter day (n=5) Mean det. Conc. (g/ml) Accuracy (%) CV (%) Mean det. Conc. (g/ml) Accuracy (%) CV (%) Vancomycin 0.2 0.22 110.0 7.3 0.19 95.0 6.8 1.0 0.97 97.0 3.8 1.04 104.0 4.5 5.0 4.93 98.6 2.1 4.94 98.8 1.8 Ceftazidime 2.0 2.13 106.5 6.9 1.92 96.0 7.9 10.0 10.28 102.8 4.2 10.03 101.3 3.2 50.0 49.53 99.1 1.9 5.27 100.5 2.0 Intra- and Inter-day accuracy and precision (%CV) acceptance criteria for each QC was ≤15% 3 Different conc,. 5 replicates Sarika Kadam
  62. 62. ROBUSTNESS Some experimental conditions varied are-  Mobile-phase buffer pH (±0.3 pH units),  Only small changes in retention time  Slightly longer times when increased pH  Slightly shorter times when decreased pH  Mobile-phase composition (±2% to the acetonitrile percentage)  General change in run times, but had no great influence on separation performance  Flow rate (±0.1ml/min)  Inverse changes in run times without generating problem with respect to resolution, efficiency or peak shape. Sarika Kadam
  63. 63. RECOVERY  A set of samples (n = 5 at 3 conc.) was prepared by spiking vancomycin/ceftazidime into CSF at 0.2/2, 1/10, and 5/50 µg/ml and each was spiked with IS tinidazole 50 µg/ml.  Mean extraction recoveries of vancomycin/ceftazidime at concentrations of 0.2/2, 1/10, and 5/50 µg/ml were 97.17/102.04%, 105.18/103.02% and 100.77/100.98%, respectively. Sarika Kadam
  64. 64. STABILITY  Freeze thaw stability:  QC samples were used.  Freeze remove from freezer thaw at RT for 4 hrs refreeze at -20c.  Showed accepted stability.  Long-term stability:  Performed at -20C.  1 month stability data is given in table indicates drugs are stable at least for 1 month.  Short- term stability:  3 sets of samples left at RT for 12hrs.  Stable during exposure period.  Post- preparative stability:  Kept in autosampler for 24hrs before injection.  Found to be stable.Sarika Kadam
  65. 65. STABILITY Nominal conc. (g/ml) Freeze thaw (n=3) Long term (n=3) Short term (n=3) Post preparative (n=3) Found conc (g/ml) CV (%) Found conc (g/ml) CV (%) Found conc (g/ml) CV (%) Found conc (g/ml) CV (%) Vancomycin 0.2 0.19 5.3 0.21 9.5 0.19 5.3 0.20 5.0 1.0 0.99 4.0 0.98 3.1 1.04 5.8 0.98 2.0 5.0 4.88 2.5 5.13 2.9 4.97 2.2 4.95 1.0 Ceftazidime 2.0 2012 7.1 2.04 5.4 1.92 7.3 2.03 3.0 10.0 9.89 3.2 10.07 3.6 10.10 2.4 9.95 1.7 50.0 50.18 2.1 49.89 2.2 50.21 2.0 49.94 1.4 Sarika Kadam
  66. 66. CONCLUSION  The method reported describes a selective and accurate HPLC method for simultaneous determination of vancomycin and ceftazidime in CSF with rapid and simple sample pretreatment. Sarika Kadam
  67. 67. REFERENCES: 1. ICH HARMONISED TRIPARTITE GUIDELINE - VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY Q2(R1) Current step 4 version 2. Guidance for industry Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, May 2001 BP 3. Validation and qualification in analytical laboratories, 2nd edition, by Ludwig Huber, Page no. 125-154 4. Journal of Pharmaceutical and Biomedical Analysis 48 (2008) 860-865 5. Validation of pharmaceutical processes, 2nd edition, Fredrick J. Carleton, page nos. 1-16 6. Pharmaceutical master validation plan, The ultimate guide to FDA, GMP& GLP compliance, syed Imtiaz Haider, Page nos. 1-36 7. Pharmaceutical analysis, modern method; James W. Munson,Page nos. 1-15 Sarika Kadam
  68. 68. Sarika Kadam