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Validation for Confirmatory Methods

Validation for Confirmatory Methods. Dr. Richard Maguire. Plan. Speaker Background MBRS Background Disclaimer Forensic Toxicology Literature Resources Pre-Validation. Plan. Validation Protocol Validation Parameters Acknowledgements. Speaker Background. Analytical Sceince, DCU

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Validation for Confirmatory Methods

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  1. Validation for Confirmatory Methods Dr. Richard Maguire

  2. Plan • Speaker Background • MBRS Background • Disclaimer • Forensic Toxicology • Literature Resources • Pre-Validation

  3. Plan • Validation Protocol • Validation Parameters • Acknowledgements

  4. Speaker Background • Analytical Sceince, DCU • Ph.D. Analytical Science, Trinity College, University of Dublin • 9 years as Principal Analyst Medical Bureau of Road Safety • 6 years in National Drug Treatment Centre, 3 as Principal Analyst • UKAS Assessor

  5. MBRS Background • Based in Health Science Centre in UCD • Responsible for testing intoxicants in drivers arrested under suspicion of driving under the influence of Alcohol and/or drugs • Alcohol in Blood, Urine and Breath • Drugs in Blood, Urine and Oral Fluid • Results must be legally defensible

  6. Disclaimer • MBRS approach to Validation is Industry specific (Forensic Toxicology) • Different Industries have similar approaches but there are different norms in each industry • Some industries have specific regulations and guidelines that must be adhered to • Focus on quantitation, qualitative analysis may require less validation effort • Stating the obvious

  7. Forensic Toxicology • Analytical toxicology is concerned with the detection, identification and measurement of drugs and/or foreign compounds and their metabolites in biological and related specimens • Forensic Toxicology is the application of Analytical Toxicology for the purposes of Law Enforcement

  8. Key Validation Resources • ISO 17025 (1999, 2005), INAB PS15 • Society of Forensic Toxicology Guidelines (2006) • Peters, Drummer and Mushoff FSI 165 (2007), 216-224 • UK and Ireland Association of Forensic Toxicologists (2010) • SWGTOX (2013) • EU Guidelines (Implementing CD 96/23/EC)

  9. Validation Resources • ISO 17025 • States requirements only Section 5.4.5

  10. Validation Resources • INAB PS15 • Fills gaps in ISO 17025 • General and detailed

  11. Key Validation Resources • Society of Forensic Toxicology Guidelines • Overlap with ISO 17025 • Good content on Validation

  12. Key Validation Resources • Peters, Drummer and Mushoff (2007) • Detailed and Instructive • Well referenced

  13. Key Validation Resources • UKIAFT(2011) • Based on SOFT 2006 • Detailed and Instructive • Under review

  14. Key Validation Resources • SWGTOX • American Standard • Scientific Working Group • Detailed and Instructive (Worked Examples) • Very well referenced

  15. Key Validation Resources • EU Guidelines (Implementing CD 96/23/EC) • Concerning performance of analytical methods and interpreting results • Very detailed with good instruction on identification of analytes

  16. Pre-Validation Work

  17. Validation Essentials • Validation Protocol and Plan • Matrix (Blood, Urine or Oral Fluid) • Preparation(Dil./Ext (SLE/SPE) Hydrolysis) • Separation (LC or GC) • Detection (MS or MS/MS) • Reference Standards • Internal Standard • (Deuterated, check for analyte impurities)

  18. Validation Essentials cont’d • Calibration Range • Controls (Internal, External and existing specimens) • Proficiency Testing • Validation Report(include narrative) • Accreditation (flexible scope) • Ongoing QC and Validation

  19. Validation Parameters • Selectivity/Specificity • Calibration (Model) • Accuracy (Bias) • Precision • LOD/LLOQ/ULOQ • Carryover • Recovery • Stability • Robustness • Measurement Uncertainty

  20. Validation Protocol and Plan • Must cover all aspects of validation • Must clearly outline what needs to be done for each parameter • This should be broken down into • What is required • How is this to be done (n=x etc.) • Acceptance criteria • Report Requirements

  21. Selectivity/Specificity • Any chemicals which are not of interest but we are likely to find in our specimens which will cause interference problems • Prep and inject into system to see if there is any interference • If there is a problem the method may need to be adapted • Methods like MS/MS and HRMS are very selective

  22. Selectivity/Specificity

  23. Selectivity/Specificity • Test at levels likely to be found in specimens • May not need to put through extraction initially • If a problem then can subject to extraction, if not extracted may not be a problem • If extracted and interfering you may need to adapt method and repeat selectivity work

  24. Calibration Model • Establish mathematical relationship between signal and concentration (don’t change) • 6 points over relevant range (Matrix Match) • Cal level less than cut-off/decision point • Separated by equal concentration intervals good (more complicated prep?) • Serial dilution

  25. Calibration Model • Don’t need to maintain 6 points, useful for dropping points if a cal level is out of spec • May be linear, may be 1/x, 1/x2 • Check residuals • Calculate error on residuals for the different calibration models and select one with the lowest • Don’t force through 0,0 • R2 >0.99

  26. Calibration Model

  27. Accuracy (Bias) • Closeness to expected value • Use spikes (fortified samples at least 20% and 80% of concentration range (at cut-off, at halfway point)

  28. Accuracy (Bias) • Matrix matched, n=7 at each concentration level (SWGTOX advocate n=3) • Can utilize External Controls and PTs here also • Compare observed and expected values and express as % • SWGTOX allows ±20% for drugs (10% for Ethanol)

  29. Precision • Ability to be accurate/biased consistently • Check at each control level over cal. range • 6x6, 2x11(UKAS), 3x5 (SWGTOX) • 7x6 can drop a point using Grubbs Test and still have 6 data points

  30. Precison • Within and Between Run • Calculate %CV at each level • Can use one way ANOVA to estimate • Grouping is batch to batch • Would expect to get RSD less than 15% for within batch and also between batch

  31. LOD/LLOQ/UPLOQ • LOD (S/NRMS >3) • Lower Limit of Quantitation; • Check at the cut-off (accuracy and precision should be acceptable). • LLOQ (S/NRMS >10) • Policy on out of range results • Report as greater than calibration range • If diluting what to dilute with? Blank Matrix? • Check accuracy and precision on dilutions

  32. Carryover • Monitor with washes between specimens and cals, controls etc. • Eliminate if possible • Manage if not possible • Carryover must be not more than 10% of lowest calibrator • Results can be reported provided specimen after carryover has 10 times signal of carryover wash (SWGTOX)

  33. Recovery • Applies to methods with extraction • Important where sensitivity of instrument is poor at desired LOD/LLOQ • Spike solvent and don’t extract, evaporate and reconstitute, compare to same volume of matrix spiked at same concentration and reconstituted in same volume • Peak Area Solvent and Extract compared as a %

  34. Stability • Need to take into consideration your turnaround time • Can be checked using spikes over time frame and planned storage conditions (Fridge/Freezer) • Post preparation stability should also be checked (instrument outages), sometimes called onboard stability • Freeze/Thaw

  35. Robustness • How resilient is method to expected changes • Change of column • Mobile Phase • Analysts • SPE cartridge batch • Environmental variations • Build into validation plan if conducting validation over several weeks • Treat like casework

  36. Uncertainty of Measurement • GUM • Top down is quick but does not allow you to identify what the different parts of the method contribute to UM • Bottom up allows you to do this, it is more time consuming to calculate, but helps identify main UM contributors • Control data feeds into this

  37. Acknowledgements • Everyone involved in putting standards and guidelines together • MBRS staff who carry out the validation work

  38. THANK YOU!! richard.maguire@ucd.ie

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