1. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 1 Validation of confirmatory methods for MRPL and MRL substances in matrices of veterinary origin according to 2002/657/EC: Case Study VALIDATION OF METHODS Dr. Apr. S. Impens and Ir. S. Carbonnelle Department of Pharmaco-Bromatology Drugs and Hormones

2. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 2 Allowed substances: maximum residue limits (MRL) Forbidden components: Minimum Required Performance Levels (MRPL) Dxm (EMEA)MRL b,p,e 2 µg/kg Btm (EMEA)MRL b,p 2 µg/kg Prolon (EMEA)MRL b 10 µg/kg Mprolon (EMEA)MRL b 10 µg/kg Flm (B)MRPL2 µg/kg TrmAt (B)MRPL2 µg/kg Example: corticosteroids in liver b = bovine p = porcine e = equidae

3. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 3 Analysis portion: 20  5 g Hydrolysis: H. Pomatia extract (2 h – 60  5 °C) L/L-extraction: methanol – diethylether Purification: Solid Phase Extraction (C18-column) Separation – Detection: LC-MSn (ThermoFisher Deca XP ion trap) Corticosteroids in liver

4. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 4 2002/657/EC: Performance Characteristics Allowed substances: maximum residue limits (MRL)  QUANTITATIVE PARAMETERS Forbidden components: Minimum Required Performance Levels (MRPL)  QUALITATIVE PARAMETERS

5. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 5 Criteria for specificity 2002/657/EC: - Chemically related components (metabolites, isomers,…) - Representative number of blank samples Practical work: - 20 blank samples - 20 blank samples fortified with possibly interfering compounds

6. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 6 Criteria for trueness 2002/657/EC: - 6 replicates of Certified Reference Materials (CRM) if available - Calculation of mean, standard deviation and coefficient of variation - Trueness (%)= mean recovery-corrected concentration detected x 100 certified value - Remark: CRM not available  recovery Practical work: -6 identical blank samples fortified at MRL/MRPL

7. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 7 Criteria for recovery, repeatability and reproducibility 2002/657/EC: - Recovery: 18 samples fortified at : - 6 x 0.5 MRL or 1 MRPL - 6 x 1 MRL or 1.5 MRPL -6 x 1.5 MRL or 2 MRPL - Repeatability: 3 series of recovery analyses -Within-laboratory reproducibility: 3 series of recovery analyses (different operators, different environmental conditions, …) -For between-laboratory reproducibility: Proficiency tests

8. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 8 Criteria for applicability and ruggedness 2002/657/EC: -Determination of factors influencing measurements results (Method of Youden) Practical work: -Development of the method: minimalisation of the factors influencing the results

9. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 9 Criteria for stability 2002/657/EC: - Analyte in solution at each concentration level Practical work: - Preparation of working solutions - Conditions of conservation: tabel - Analysis at time 0, after 1 week, 2 weeks, … until instability - 20 °C+ 4 °C+ 20 °C Dark10 aliquots Light10 aliquots

10. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 10 Criteria for stability 2002/657/EC: - Analyte in matrix at detection capability level Practical work: - Analysis of incurred material - preparation of fortified blank samples - Conservation at min. – 20 °C - Analysis at time 0, after 1 week, 2 weeks, … until instability (max. 20 weeks)

11. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 11 Criteria for calibration curve 2002/657/EC: - At least 5 levels (including 0) - Description of working range - Determination of the mathematical formula, goodness of fit and acceptability ranges Practical work: -5 levels of concentrations: 0 – 0,5 – 1 – 1,5 – 2 MRL/MRPL

12. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 12 Criteria for Decision Limit (CCα) 2002 /657/EC: - MRL Substances (permitted limit – α = 5%): 1. Calibration curve: analysis of blank samples and blanks fortified around the MRL  CCα = MRL+ 1,64 σ RWOR 2. 20 blank samples fortified at MRL  CC α = MRL+1.64 σ RW

13. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 13 Criteria for Decision Limit (CCα) 2002/657/EC: - Banned Substances (no permitted limit – α = 1%): 1. Calibration curve: analysis of blank samples and blanks fortified at and above the MRPL  CCα = Y intercept + 2.33 σ RWOR 2. 20 blank samples  CCα = 3 x S/N RT

14. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 14 Criteria for Detection Capability (CCβ) 2002/657/EC: - MRL Substances (permitted limit): 1. Calibration curve: analysis of blank samples and blanks fortified around the MRL  CCβ = CCα + 1,64 σ RWOR 2. 20 blank samples fortified at CCα  CCβ = CCα +1,64 σ RW

15. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 15 MRL CC  Signal or Concentration CC   =  +1.64  MRL +1.64  sample Detection Capability Case of permitted limit

16. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 16 Criteria for Detection Capability (CCβ) 2002/657/EC: - Banned Substances (no permitted limit): 1. Calibration curve: analysis of blank samples fortified below and at the MRPL  CCβ = CC α +1,64 σ RWOR 2. Analysis of 20 blank materials fortified at CCα  CCβ = CC α +1,64 σ RWOR 3. Analysis of 20 blank materials fortified at and above CCα:  Less than 5% of false compliant

17. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 17 Detection Capability Case of no established permitted limit or banned substance X blank CC  Signal or Concentration CC   ≠  +2.33  blank +1.64  sampl e

18. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 18 Validation of corticosteroids in liver 0 MRL0,5 MRL1 MRL1.5 MRL2 MRL Day 153333 Day 253333 Day 353333 Day 453333 1. Analysis of mix of liver (pork, beef, horse, sheep) MATRIX EFFECTS: DILUTION EFFECT Alternative validation strategy

19. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 19 Validation of corticosteroids in liver 0 MRL0,5 MRL1 MRL1.5 MRL2 MRL Day 143333 Day 243333 Day 343333 Day 443333 2. Analysis of bovine liver + calibration curve in matrix (0 - 0,25 - 0,5 - 1 - 1,5 - 2 MRL/MRPL) + calibration curve in mobile phase (0 - 0,25 - 0,5 - 1 - 1,5 - 2 MRL/MRPL)

20. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 20 Flexible scope Criteria that should be fulfilled by ISO 17025 accredited laboratories working with the principle of flexible scope for analyses concerning EC Decision 96/23/EC – 2002/657/EC BELAC 2 – 105 rev1-2006

21. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 21 Flexible scope - Continuous changes of normes  flexibility - Classes of matrices for flexible scope: - eggs and protein-rich tissues (muscle, kidney, liver, thyroid,fish, aquaculture, eggs) - fat - urine, bile water - plasma, blood, milk -Faeces -Feed -Preparations -Hair -Others (retina, honey,...) Secondary validation: - Qual methods:  3 blank samples, 3 fortified samples at or below MRL/MRPL - Quant methods:  3 blank samples, 6 fortified samples at or below MRL/MRPL (CC  – repeatability – trueness - recovery)  completion of validation: routine analyses

22. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 22 Validation of corticosteroids in liver 0 MRL/MRPL 0,5 MRL/MRPL 1 MRL/MRPL 1.5 MRL/MRPL 2 MRL/MRPL pork133 horse133 Sheep day1 33333 Sheep day2 33333 3. Mini validation: Analysis of other species (horse, porcine, ovine)  ovine: 2 series of analyses because of additional extraction step (hexane)

23. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 23 Qualitative validation parameters Relative retention time (RRT) RRT = absolute retention time target absolute retention time internal std  2002/657/EC: RRT (LC-MSn) = ± 2,5 % RRT reference std or control sample Diagnostic ions - identification points (IPs) -Flm,TrmAt (banned)  2002/657/EC: min. 4 IPs -Dxm, Btm, Prolon, Mprolon  2002/657/EC: min. 3 (MRL bovine) or 4 IPs (horse, sheep)

24. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 24 RRT - IPs Identification Points: - Precursor Ion → 1 IP - Transition Product Ion → 1.5 IP 1 + 1.5 + 1.5 = 4 IPs

25. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 25 Linearity Mandel’s Fitting Test : linear (y = ax + b) or quadratic (y = ax 2 + bx + c) regression?  Calibration curves linear Dexamethasone 0 – 4 µg/ kg

26. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 26 Matrix effect Calibration curve’s slope: comparison mobile phase – matrix (T-test)  matrix effect prednisolone flumethasone

27. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 27 Quantitative validation parameters CCα - CCβ  ISO 11843 is used to calculate CCα and CCβ CostNormes (µg/ kg) CC  (µg/ kg)CC  (µg/ kg) Btm22.53.0 Dxm22.42.7 Flm20.81.3 MProlon1013.717.4 Prolon1013.617.2 TrmAt20.40.8

28. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 28 Quantitative validation parameters CCα - CCβ  ISO 11843 is used to calculate CCα and CCβ Repeatability  recovery (70-110%) and CVr (max. 2/3 CVrw Horwitz) Cost CVr (%)Recovery (%) Btm17.499 Dxm2.696 MProlon11.9104 Prolon9.5105

29. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 29 Quantitative validation parameters CCα - CCβ  ISO 11843 is used to calculate CCα and CCβ Repeatability  recovery (70-120%) and CVr (max. 2/3 CVrw Horwitz) Reproducibility  CV rw Cost CVrw (%) Btm19 Dxm9 MProlon11.9 Prolon25.2

30. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 30 Quantitative validation parameters CCα - CCβ  ISO 11843 is used to calculate CCα and CCβ Repeatability  recovery (70-120%) and CVr (max. 2/3 CVrw Horwitz) Reproducibility  CV rw Selectivity and specificity  LC-MSn: selective and specific

31. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 31 LC-MSn: selectivity - specificity

32. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 32 Quantitative validation parameters CCα - CCβ  ISO 11843 is used to calculate CCα and CCβ Repeatability  recovery (70-120%) and CVs (max. 2/3 CV Horwitz) Reproducibility  CV rw Selectivity and specificity  LC-MSn: selective and specific Stability  under current development

33. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 33 Stability in solution 2002/657/EC: - Analyte in solution at each concentration level Practical work: - Analysis: - Cost in Meoh: 1 mg/ ml, 100 µg/ml, fortification mix - Cost in mobile phase: reference std mix - Analysis at time 0, after 1 week, 2 weeks, … until instability - 20 °C+ 4 °C+ 20 °C Dark10 aliquots Light10 aliquots

34. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 34 Stability in solution concentrationSolventconservation 1 mg/ mlMeoh12 months (- 20 °C) 100 µg/ mlMeoh12 months (- 20 °C) Fort mixMeoh3 months (8 °C) 100 µg/ mlMobile phase1 week (8 °C) Ref std mixMobile phase1 week(8 °C)  reference std in mobile phase with fortification mix in meoh

35. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 35 Stability in matrix 2002/657/EC: - Analyte in matrix at detection capability level Practical work: - Preparation of fortified blank samples, conservation at min. – 20 °C - non-compliant samples: conservation for max. 6 months  under development

36. FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT 36 Conclusions All 2002/657/EC validation criteria for the determination of corticosteroids in liver using LC-MSn are fulfilled CONCLUSION The analytical procedure is suitable for identification and quantification of Betamethasone, Dexamethasone, Flumethasone, Methylprednisolone, Triamcinolone-Acetonide in bovine, porcine, horse and ovine liver.