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LECTURE 13 QUALITY ASSURANCE METHOD VALIDATION

LECTURE 13 QUALITY ASSURANCE METHOD VALIDATION. QUALITY ASSURANCE. A system of activities whose purpose is to provide to the producer or the user of a product or service the assurance that it meets the needs of the user. Quality Assurance vs. Quality Control. Quality Assurance.

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LECTURE 13 QUALITY ASSURANCE METHOD VALIDATION

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  1. LECTURE 13 QUALITY ASSURANCEMETHOD VALIDATION

  2. QUALITY ASSURANCE A system of activities whose purpose is to provide to the producer or the user of a product or service the assurance that it meets the needs of the user.

  3. Quality Assurance vs. Quality Control QualityAssurance Quality Control A series of analytical measurements used to assess the quality of the analytical data (The “tools”) An overall management plan to guarantee the integrity of data (The “system”)

  4. QUALITY CONTROL CHARTS - Visual representation of actual manufacturing data with respect to the target levels.

  5. QUALITY CONTROL MEASURES • Standards and Calibration • Blanks • Recovery Studies • Precision and Accuracy Studies • Method Detection Limits

  6. STANDARDS AND CALIBRATION • Prepared vs. Purchased Standard • Signals: Peak Area, Beer’s Law • Calibration Curves • Continuing Calibration Checks • Internal Standards • Performance Testing.

  7. The concentration of the analyte and CALIBRATION CURVES Graphical representation of the relationship between: • The analytical signal

  8. CONTINUING CALIBRATION VERIFICATION • Many methods don’t require that daily calibration curves are prepared • A “calibration verification” is • analyzed with each batch of samples

  9. SAMPLE BATCH • 10 - 20 samples (method defined) or less • Same matrix • Same sample prep and analysis • Contains a full set of • QC samples

  10. INTERNAL STANDARDS • A compoundchemically similar to the analyte • Not expected to be present in the sample • Cannot interfere in the analysis • Added to the calibration standards and to the samples in identical amounts.

  11. INTERNAL STANDARDS • Refines the calibration process • Analytical signals for calibration standards are compared to those for internal standards • Eliminates differences in random and systematic errors between samples and standards

  12. PERFORMANCE TESTING ? ? Blind samples submitted to laboratories ? Labs must periodically analyze with acceptable results in order to maintain accreditation

  13. BLANKS, BLANKS, BLANKS • Laboratory Reagent Blanks • Instrument Blanks • Field Reagent Blanks • Trip Blanks

  14. LABORATORY REAGENT BLANKS • Contains every reagent used in the analysis • Is subjected to all analytical procedures • Must give signal below detection limit • Most methods require one with every batch

  15. INSTRUMENT BLANK • A clean sample (e.g., distilled water) processed through the instrumental steps of the measurement process; used to determine instrument contamination.

  16. Field Reagent Blanks • Prepared in the lab, taken to the field • Opened at the sampling site, exposed to sampling equipment, returned to the lab.

  17. RECOVERY STUDIES • Matrix Spikes • Laboratory Control Samples • Surrogates .

  18. MATRIX SPIKES • Sample spiked with a known amount of analyte • Subjected to all sample prep and analytical procedures • Determines the effect of the matrix on analyte recovery • Normally one per batch

  19. LABORATORY CONTROL SAMPLE • Analyte spiked into reagent water • Subjected to all sample prep and • analytical procedures

  20. LABORATORY CONTROL SAMPLE Also known as: • Laboratory Fortified Blank (LFB) • Quality Control Sample (QCS)

  21. SURROGATES • Similar to an internal standard • Added to all analytical samples, and to all QC samples to monitor method performance, usually during sample prep • Methods often have specific surrogate recovery criteria • Most common in Organic methods

  22. QUALITY CONTROL MEASURES • Standards and Calibration • Blanks • Recovery Studies • Precision and Accuracy Studies • Method Detection Limits

  23. PRECISION AND ACCURACY • Required for initial certification and annually thereafter • A series of four laboratory control samples • Must meet accuracy (recovery) and precision (standard deviation) requirements, often in method

  24. PRECISION AND ACCURACY • Required with a change in instrumentation or personnel • Specific to the analyst

  25. METHOD DETECTION LIMIT “The minimum concentration of a substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero”

  26. METHOD DETECTION LIMIT • MDLs are determined according to ules from regulating body • Seven replicate laboratory control samples, analyzed for precision • Multiply standard deviation by 3.14 (Student’s t- value)

  27. METHOD DETECTION LIMIT • Must be performed initially for certification • Must meet criteria specified in method • Must be performed with change in instrumentation or test method

  28. QUALITY ASSESSMENTS The overall system of activities whose purpose is to provide assurance that the overall control job is being done effectively. Two types: - Evaluation of the precision and accuracy of methods of measurements - Evaluation of the quality of manufactured goods sold for public consumption. (control charts)

  29. SPECIFICATIONS State how good the numbers should be and what precautions are required in the analytical procedure. Quality Asssurance begins with sampling. False Positive - False Negative -

  30. METHOD VALIDATION • Confimation by examination

  31. WHEN SHOULD METHODS BE VALIDATED? • A new method developed for a particular problem • Established methods revised for a particular problem • Established methods have changed with time • Established method used in different laboratory with different analysts at different times. • Establish the equivalence between two methods.

  32. METHOD VALIDATIONUSE OBJECTIVES • States the pupose for which the results will be used. Example: Analytical data and results will be used to determine whether the new antibacterial ingredient will be effective in removing 99.9% of the gram negative bacteria.

  33. SPECIFICATIONS will detail on how the numbers need to be and what precautions are required in the analytical procedures. Specifications might include: • Sampling requirements calibration checks • Accuracy and precision quality control samples • Rate of false results • Selectivity • Sensitivity • Acceptable blank values • Recovery of fortification

  34. METHOD VALIDATION FOR REGULATORY SUBMISSION, THE FOLLOWING ARE REQUIRED: Method Specificity / Confirmation of identity Linearity Accuracy Precision Range Limit of detection, Limit of quantification Rubostness

  35. CONFIRMATION OF IDENTITY • It is necessary to establish that the signal being produced at the measurement stage, which has been attributed to the analyte, is only due to the analyte and not from the presence of something chemically or physically similar.

  36. SPECIFICITY Specificity is the ability of an analytical method to distinguish the analyte from everything else that might be in the sample.

  37. LINEARITY LINEARITY measure how well a calibration curve follows a straight line. A common measure of linearity is R2, square of the correlation coefficient. Good linear fit when R2 = 0.995 and above Prepare independently at leas 6 samples + blank and measure linearity.

  38. SAMPLE PROBLEM: An AAS method to analyze Pb+2 in water has just been developed and is in the process of being validated. The validation protocol states the following criteria to be met (in addition to proof of identity and setting dynamic range): Determine whether the new method is validated. Linearity: R2 > 0.995 Accuracy: % Recovery = 100.0 +/- 5% Precision: Intermediate Precision, STD < 0.5% Intralab Precision, STD < 2.0% The following validation experiments were done: Linearity Test. Blank samples were spiked with several known amounts of Pb+2. The following data were collected.

  39. Linearity Test:

  40. RECOVERY TEST. Blank samples were spiked with 0.10 and 0.15 ppb of Pb+2 and analyzed using the method. The following data were collected.

  41. Precision. An analyst in the laboratory as well as two ther analysts from different laboratories analyzed the same blank sample spiked with 0.125 ppm Pb+2 with the following results.

  42. ACCURACY ACCURACY is “nearness to the truth”. Analyze blank , sample and a certified reference material (CRM) and compare the means. Do a recovery test. Analyze a blank sample spiked with 0.5, 1.0, 1.5X level of your analyte. And analyze for % Recovery.

  43. PRECISION PRECISION is how well replicate measurements agree with one another, usually express as standard deviation. Instrument precision – reproducibility observed when same quantity of a sample is analyzed using a particular instrument repeatedly (>10 times) Intra-assay precision – analyzing aliquots of a homogeneous material several times by one analyst on one day using same equipment. Inter-lab precision (reproducibility) aliquots of the same sample are analyzed by different analyst in different labs using different (?) instrument

  44. RANGE RANGE is the concentration interval over which linearity, accuracy, and precision are all acceptable. Correlation coefficient of R2 equal or greater than 0.995 Interlab precision of +/-3% Spike recovery of 100 +/-2%

  45. LIMITS OF DETECTION AND QUANTITATION The detection limit is the smallest quantity of analyte that is “significantly different” from the blank. LoD: Measure blank samples at least 7 times and get the mean. Do similar analyses with 7 samples with analyte and calculate the standard deviation. LoD = blank mean + 3S LoQ = 6x to 10x the SD of blank sample LoQ = 2x to 3x of LoD

  46. RUGGEDNESS / ROBUSTNESS Robustness is the ability of an analytical method to be an affected by small deliberate changes in operating parameters.

  47. STANDARD ADDITION METHOD • INTERNAL STANDARD METHOD

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