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					1
                             VALIDATION OF METHODS

             Validation of confirmatory methods for
                    MRPL and MRL substances
                 in matrices of veterinary origin
                   according to 2002/657/EC:
                            Case Study

                   Dr. Apr. S. Impens and Ir. S. Carbonnelle
                   Department of Pharmaco-Bromatology
                   Drugs and Hormones

FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
                                            Example:
2
                                     corticosteroids in liver
    • Allowed substances: maximum residue limits (MRL)

    • Forbidden components: Minimum Required Performance Levels (MRPL)


             Dxm (EMEA)                     MRLb,p,e              2 µg/kg      b = bovine
                                                                               p = porcine
             Btm (EMEA)                      MRLb,p               2 µg/kg
                                                                               e = equidae
             Prolon (EMEA)                    MRLb               10 µg/kg

             Mprolon (EMEA)                   MRLb               10 µg/kg

             Flm (B)                          MRPL                2 µg/kg

             TrmAt (B)                        MRPL                2 µg/kg




    FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
3                                    Corticosteroids in liver



    • 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)




    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

                                   Detection    Decision   Trueness/   Precision   Selectivity/   Applicability/
                                   Capability    Limit     Recovery                Specificity    Ruggedness/
                                     CCß         CC                                                Stability


               Qualitative     S      +            -          -           -            +               +
               methods
                               C      +           +           -           -            +               +

               Quantitative    S      +            -          -           +            +               +
               methods
                               C      +           +           +           +            +               +



    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



    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




     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
                  Criteria for recovery, repeatability and
7
                               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



    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




    FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
9                                   Criteria for stability
                                     2002/657/EC:
         - Analyte in solution at each concentration level

                                         - 20 °C                   + 4 °C       + 20 °C

          Dark                        10 aliquots              10 aliquots     10 aliquots
          Light                                                                10 aliquots


                                     Practical work:
         - Preparation of working solutions
         - Conditions of conservation: tabel
         - Analysis at time 0, after 1 week, 2 weeks, … until instability




    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)




     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




     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 σRW

                                                           OR

     2. 20 blank samples fortified at MRL

                                              CC α = MRL+1.64 σRW




     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 = Yintercept+ 2.33 σRW

                                                           OR

     2. 20 blank samples

                                              CCα = 3 x S/NRT




     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 σRW

                                                           OR

     2. 20 blank samples fortified at CCα

                                              CCβ = CCα +1,64 σRW




     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
15
                                        Detection Capability
                                                 Case of permitted limit




                                  MRL                  CC                      CCb
                                         +1.64sMRL +1.64ssample




                                                                                        Signal or
                                                   = b = 5%
                                                                                      Concentration


     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 σRW

                                                            OR

     2. Analysis of 20 blank materials fortified at CCα

                                               CCβ = CC α +1,64 σRW

                                                            OR

     3. Analysis of 20 blank materials fortified at and above CCα:

                                               Less than 5% of false compliant




      FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
17                                      Detection Capability
                          Case of no established permitted limit or banned substance


                                 Xblank                CC                      CCb
                                        +2.33sblank +1.64ssampl
                                                             e




                                                                                   Signal or
                                  = 1% ≠ b = 5%                                 Concentration
     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
18                   Validation of corticosteroids in liver

              1. Analysis of mix of liver (pork, beef, horse, sheep)
                                  Alternative validation strategy
                          0 MRL          0,5 MRL            1 MRL          1.5 MRL   2 MRL

       Day 1                  5                3               3                3      3

       Day 2                  5                3               3                3      3

       Day 3                  5                3               3                3      3

       Day 4                  5                3               3                3      3

                              MATRIX EFFECTS: DILUTION EFFECT



     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
19                   Validation of corticosteroids in liver

                                        2. Analysis of bovine liver

                               0 MRL          0,5 MRL           1 MRL           1.5 MRL   2 MRL
            Day 1                 4                3                3             3         3

            Day 2                 4                3                3             3         3
            Day 3                 4                3                3             3         3

            Day 4                 4                3                3             3         3


            + 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)




     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




     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
                                            Secondary validation:
             - urine, bile water            - Qual methods:
                                              3 blank samples, 3 fortified samples at or below
             - plasma, blood, milk
                                            MRL/MRPL
             -Faeces
                                            - Quant methods:
             -Feed
                                              3 blank samples, 6 fortified samples at or below
                                            MRL/MRPL (CCb – repeatability – trueness - recovery)
             -Preparations
                                             completion of validation: routine analyses
             -Hair

             -Others (retina, honey, ...)




     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
22                       Validation of corticosteroids in liver
                   3. Mini validation: Analysis of other species (horse, porcine, ovine)
                              0       0,5       1                                   1.5        2
                          MRL/MRPL MRL/MRPL MRL/MRPL                              MRL/MRPL MRL/MRPL
         pork                    1                   3                  3

         horse                   1                   3                  3
         Sheep                   3                   3                  3            3        3
         day1
         Sheep                   3                   3                  3            3        3
         day2

      ovine: 2 series of analyses because of additional extraction step (hexane)




       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)




     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

     monsternr.         precursor ion 379(%)           ion 409(%)           RRT
     20070330refstd03         469      100,00                57,39                0,80 0.25MRPL
     -afwijking                         80,00                45,91                0,78
     +afwijking                        120,00                68,87                0,82
     I.P.                       1         1,5                  1,5

     monsternr.         precursor ion 379(%) besluit   ion 409(%) besluit   RRT        besluit   IP
     20070330spike01          469      100,00 ok             62,88 ok             0,79 ok             4,0 0,5MRPL

                            1         +         1.5          +       1.5 = 4 IPs




      FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
25                                                    Linearity

         Mandel’s Fitting Test :
                  linear (y = ax + b) or quadratic (y = ax2 + bx + c) regression?
                                             Calibration curves linear
                                    Linear regression

                            1,2
                                                        y = 0,2488x + 0,0288
                              1
                            0,8
                                                             R2 = 0,9928               Dexamethasone
                            0,6                                                        0 – 4 µg/ kg
         Y




                            0,4
                            0,2
                              0
             -2       -1   -0,2 0       1         2             3              4   5
                           -0,4
                                             X




     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
26                                             Matrix effect

                                                     Matrix effect
                Calibration curve’s slope: comparison mobile phase – matrix (T-test)
                                      matrix effect

       3                                                           2,5

     2,5                                                             2

       2
                                                                   1,5
     1,5                                                addition                                         addition
                                                        standard     1                                   standard
       1
                                                                   0,5
     0,5

       0                                                             0
            0       5     10    15      20      25                        0   1      2    3      4   5
     -0,5                                                          -0,5




                        prednisolone                                              flumethasone



      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


                      Cost                Normes (µg/ kg) CC (µg/ kg) CCb (µg/ kg)
                      Btm                             2                         2.5    3.0
                      Dxm                             2                         2.4    2.7
                      Flm                             2                         0.8    1.3
                      MProlon                        10                         13.7   17.4
                      Prolon                         10                         13.6   17.2
                      TrmAt                           2                         0.4    0.8




     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 (%)
                           Btm                           17.4                    99
                           Dxm                            2.6                    96
                           MProlon                       11.9                   104
                           Prolon                         9.5                   105




     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
                          CVrw
                                Cost                                   CVrw (%)
                                Btm                                         19
                                Dxm                                             9
                                MProlon                                    11.9
                                Prolon                                     25.2




     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
                          CVrw
                                                     Selectivity and specificity
                          LC-MSn: selective and specific




     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
31                                                     LC-MSn: selectivity - specificity
                                  C:\Xcalibur\data\20070314s pike11                                              03/20/2007 08:18:30 AM
                                  s p1: 22-2006-03544+ 1m rpl
                                   RT: 0.00 - 20.06 SM : 5G                                                                                         20070314spike11 #281 RT: 5.10 AV: 1 SB: 43 2.00-3.54, 7.52-9.73 NL: 3.57E6
                                                              5.10                                                  NL: 4.13E6
                                      100                                                                                                           F: - c ESI Full ms2 493.00@24.50 [ 300.00-496.00]
                                                                                                                    m/z=
                                                                                                                    374.50-375.50+
                                                                                                                                                                                            375
                                                                                                                                                       100
                                                                                                                    412.50-413.50 F: - c ESI Full
                                       95                                                                           ms2 493.00@24.50 [
                                                                                                                    300.00-496.00] M S
                                                                                                                    2007031 4spike1 1                  95
                                       90
                                                                                                                                                       90
                                       85
                                                                                                                                                       85
                                       80
                                                                                                                                                       80

                                       75
                                                                                                                                                       75

                                       70
                                                                                                                                                       70

                                       65
                                                                                                                                                       65

                                       60
                                                                                                                                                       60




                                                                                                                        Relative Abundance
     Relative Abundance




                                       55
                                                                                                                                                       55

                                       50                                                                                                                                                              413
                                                                                                                                                       50

                                       45
                                                                                                                                                       45

                                       40                                                                                                              40

                                       35                                                                                                              35
                                                                                                                                                                                                                433
                                       30                                                                                                              30


                                       25                                                                                                              25


                                                                                                                                                       20                       357
                                       20
                                                                                                                                                                                                                                       491

                                       15                                                                                                              15


                                       10
                                                                                                                                                       10               337                              415
                                                                                                                                                                                                                                         493
                                                                                                                                                        5
                                        5

                                                                         TrmA t                                                                         0
                                        0
                                            0     2       4          6       8       10      12   14   16   18     20
                                                                                                                                                         300     320     340     360     380     400      420     440     460    480
                                                                                  Time (min)                                                                                                     m/z




                          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



     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
33                                    Stability in solution
                                      2002/657/EC:
          - Analyte in solution at each concentration level

                                          - 20 °C                   + 4 °C       + 20 °C

           Dark                        10 aliquots              10 aliquots     10 aliquots
           Light                                                                10 aliquots
                                                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




     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
34                                   Stability in solution

              concentration                            Solvent                     conservation

                  1 mg/ ml                              Meoh                    12 months (- 20 °C)
                 100 µg/ ml                             Meoh                    12 months (- 20 °C)
                   Fort mix                             Meoh                     3 months (8 °C)
                 100 µg/ ml                       Mobile phase                    1 week (8 °C)
                Ref std mix                       Mobile phase                     1 week(8 °C)



       reference std in mobile phase with fortification mix in meoh




     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




     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.



     FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT

				
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