Method Validation for Marine Institute Data | ISO17025 Accreditation | 2002/657/EC Compliance

1. Is My Method Fit For Purpose ? Rebecca Moffat Marine Institute 27th Oct 2016

2. Determining if validation data meets acceptance criteria For Commission Decision 2002/657/EC

3. Contents • Marine Institute • Documents of Reference • Validation under 2002/657/EC • Parameters Commonly Evaluated in Validation • Confirming Positive Sample Criteria • Validation Data For Dyes Analysis by LC-MS/MS • Conclusion

4. Marine Institute

5. Structure CEO Peter Heffernan Marine Environment & Food Safety Irish Maritime Development Office Policy, Innovation and Research Support Corporate Services Fisheries Science Ocean Science Shellfish Safety Chemistry Fish Health Licensing advice & spatial planning Research Vessel Oceanographic Services & modelling Information Services Marine Exploration - mapping Demersal & Pelagic Fisheries Inshore Fisheries Shellfish Fisheries Migratory stocks – salmon & eel Aquaculture Finance Human Resources Communications Facilities Library Policy and Foresight Research Funding International Office Advanced Technology Shipping Industry Liaison Support Marketing & Development

6. Marine Environment and Food Safety • Monitoring • Research • Advice • Sampling • Analysis • Data management • Quality – ISO 17025

7. Marine Environment and Food Safety • Seafood Safety – underpin consumer protection and support market • Science of marine environmental management • Services for management and regulation of aquacluture

8. Quality System • Marine Institute Accredited to ISO17025 since 2002. • 35 Accredited methods – 4 ext to scope in 2017. • Methods Accredited in multidisciplinary areas - Chemistry, (residues, metals, organics), biotoxins, fish diseases, microbiology, phytoplankton, molecular. • MI also holds ISO9001 Certification in the Fish Health Competent Authority Unit.

9. Residues Programme for Aquaculture • MI carries out sampling and analysis in accordance with NRCP • 3 categories of Residues • Banned – Group A compounds • A3 Steroids: Methlytestosterone • Authorized – Group B compounds • B2(c) Carbamates/Pyrethroids: Cypermethrin • Unauthorized – Group B compounds • B3(e) Dyes: Malachite Green

10. Documents of Reference • Commission Decision 2002/657/EC • Eurachem: Fitness for Purpose of Analytical Methods • INAB PS15 – Guide to Method validation for Quantitative Analysis in Chemical Testing Laboratories (ISO17025)

11. Fit for Purpose: Method Validation • Defining an analytical requirement and • Confirming that the method has capabilities consistent with the application • Enables chemists to demonstrate that a method is fit for purpose

12. Parameters commonly evaluated during method validation Performance Characteristics: • Selectivity • Working Range • Analytical Sensitivity • Trueness • Precision • Ruggedness • LOD/LOQ • CCα and CCβ For 2002/657/EC

13. Analysis of Dyes by LCMSMS • Confirmatory method • Based on EU-RL method for Dyes • Dyes analysed include: • BG, CV, LCV, LMG, MG and VB • Awarded INAB accreditation in 2014

14. Validation under 2002/657/EC • Residues Directive Commission Decision 2002/657/EC states: • Confirmatory methods shall provide information in the chemical structure of the analyte and consequently methods based only on chromatographic analysis without the use of spectrometric detection are not suitable on their own for use as a confirmatory method

15. Suitable Confirmatory Methods for Organic Residues or Contaminants

16. Common Performance Criteria and Requirements • Internal Standards: • Where used in the method, a suitable internal standard shall be added to the test portion at the beginning of the extraction procedure. • Depending of availability, either stable isotope-labelled forms of the analyte, or compounds that are structurally related to the analyte shall be used. • Deuterated Dyes

17. Confirming Positive sample acceptance criteria: MS/MS tolerance • Criteria for confirming: MS/MS tolerance ion ratio: • Relative intensities of the detected ions, shall correspond to those of the calibration standard, at comparable concentrations, measured under the same conditions within the following tolerances:

18. Dyes Confirmation Sheet Part 1

19. Confirming Positive Sample Criteria: RRT • Criteria for confirming: Relative Retention Time: • The retention time (or relative retention time) of the analyte in the test portion shall match that of the calibration standard within a specified retention window • The ratio of the chromatographic RT of the analyte to the Internal Standard, shall correspond to that of the calibration solution at a tolerance of ±0.5% for GC and ±2.5% for LC

20. Dyes Confirmation Sheet Part 2

21. Classification of analytical methods by the performance characteristics that have to be determined S= screening methods, C= confirmatory methods, += determination in mandatory

22. Specificity • Specificity: • Ability of a method to distinguish between the analyte being measured and other substances • Determined by • Analysing blank representative samples and showing no interfering peaks in mass range window • Analysing each standard individually

23. Specificity • LC-MS/MS offers high degree of selectivity and specificity • Small interfering peaks were observed at RT for some analytes in chromatograms of non-fortified samples • Areas so low to be of little consequence • Quantification of each analyte not influeneced notably by levels detected in negative controls

24. Specificity Blanks - BG

25. Specificity Standards - BG

26. Working Range • Linearity of matrix matched calibration curve • Upper Linear Range • Saturation Point and method range

27. Matrix matched curve • Fortify negative control salmon tissue across range of concentrations and bring through extraction procedure • Matrix can have effect on the signal produced (suppression or enhancement) • MM calibration curve used to ensure these effects are accounted for in quantification of unknowns

28. Calibration Standard Levels

29. Example CV extracted calibration curve

30. Upper Linear Range • Matrix matched Calibration Curves • Calibration standards concentrations range: • 0µg/kg – 15µg/kg • Most suitable range for all analyes: • 0µg/kg – 10µg/kg • Lowest standard with analyte present 0.5µg/kg • 0.25*MRPL

31. Saturation point and method range • Saturation point examined in triplicate at 5 and 7.5 * MRPL for all dyes • Equivalent to 10µg/kg and 15µg/kg • Acceptable recoveries achievable for 4/6 analytes at 15µg/kg • Acceptable recoveries achievable for 6/6 analytes at 10µg/kg • Method range: 0.5µg/kg – 10µg/kg (15µg/kg) CV, MG, LCV, LMG BG, CV, MG, VB, LCV, LMG

32. Recovery/Trueness 2002/657/EC • The inter-laboratory CV for the repeated analysis of a reference or fortified material under reproducibility conditions, shall not exceed the level in table below: * For mass fraction lower than 100g/kg the Horwitz equation gives unacceptable high vales. Therefore the CV’s for concentrations lower than 100µg/kg shall be as low as possible

33. Trueness • When no CRMS are available it is acceptable that the trueness measurements are assessed through the recovery of additions of known amounts of analyte to a blank matrix. • Data corrected with the mean recovery are only acceptable when they fulfil the following:

34. Recovery/Trueness data

35. Precision • Falls under 2 categories • Within Batch Repeatability • Within Lab Reproducability

36. Within Batch Repeatability • Prepare a set f samples of identical matrices, fortified with the analyte to yield concentrations equivalent to 1, 1.5 and 2 times MRPL • Each level analysed with 7 replicates

37. Results of Repeatability 1*MRPL

38. Results of Repeatability 1.5* MRPL

39. Results of Repeatability 2*MRPL

40. Within Lab Reproducability • Samples spiked at approx 1, 1.5 and 2*MRPL were analysed on 3 occasions by 2 analysts • Analyst A – twice using different batches of reagents and solvents • Analyst B

41. Within Lab Reproducibility 1*MRPL Within lab reproducibility results at approximately 100%MRPL (2µg/kg)

42. Within Lab Reproducibility 1.5*MRPL Within lab reproducibility results at approximately 150%MRPL (3µg/kg)

43. Within Lab Reproducibility 2*MRPL Within lab reproducibility results at approximately 200%MRPL (4µg/kg)

44. Ruggedness • Examined using the Youden approach • Choose a subset of 8 combinations that have a balance between capital and small letters • 8 determinations are made which will use a combination of the chosen factors A-G

45. Chosen Factors

46. Experimental Design for Ruggedness Study

47. Ruggedness Results

48. Decision Limit (CCα) • Decision Limit • The limit at and above which it can be concluded with error on probability, α, that a sample is non-compliant • Substances with no permitted limit • CCα established by analysing 20 blanks and calculate the signal to noise at the time window analyte is expected • 3 times S/N can be used as CCα • Weakest qualifier used to determine S/N

49. S/N Ratio Data for Transition 2 Ext 6 is the lowest standards in the calibration curve and its S/N ration is greater than 3* S/N of blanks CCα for confirmatory analysis if 0.5µg/kg for each analyte

50. Detection Capability CCβ • Detection Capability • The lowest concentration of an analyte that may be detected, identified and/or quantified in a sample with an error of probability β • Substances with no permitted limit • CCβ established by analysing 20 blanks fortified at the decision limit. • Decision limit + 1.64 times the std dev of within laboratory reproducibility of the measured content equals detection capability (β = 5%)