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Validation of confirmatory methods for

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VALIDATION OF METHODS Validation of confirmatory methods for MRPL and MRL substances in matrices of veterinary origin according to 2002/657/EC: Case Study – PowerPoint PPT presentation

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Title: Validation of confirmatory methods for


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 Departme
nt of Pharmaco-Bromatology Drugs and Hormones
2
Example corticosteroids in liver
  • Allowed substances maximum residue limits (MRL)
  • Forbidden components Minimum Required
    Performance Levels (MRPL)

Dxm (EMEA) MRLb,p,e 2 µg/kg
Btm (EMEA) MRLb,p 2 µg/kg
Prolon (EMEA) MRLb 10 µg/kg
Mprolon (EMEA) MRLb 10 µg/kg
Flm (B) MRPL 2 µg/kg
TrmAt (B) MRPL 2 µg/kg
b bovine p porcine e equidae
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)

4
2002/657/EC Performance Characteristics
  • Allowed substances maximum residue limits (MRL)
  • ? QUANTITATIVE PARAMETERS
  • Forbidden components Minimum Required
    Performance Levels (MRPL)
  • ? QUALITATIVE PARAMETERS

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
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
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
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
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

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
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
Criteria for Decision Limit (CCa)
  • 2002/657/EC
  • MRL Substances (permitted limit a 5)
  • 1. Calibration curve analysis of blank samples
    and blanks fortified around the MRL
  • ? CCa MRL 1,64 sRW
  • OR
  • 2. 20 blank samples fortified at MRL
  • ? CC a MRL1.64 sRW

13
Criteria for Decision Limit (CCa)
  • 2002/657/EC
  • Banned Substances (no permitted limit a 1)
  • 1. Calibration curve analysis of blank samples
    and blanks fortified at and above the MRPL
  • ? CCa Yintercept 2.33 sRW
  • OR
  • 2. 20 blank samples
  • ? CCa 3 x S/NRT

14
Criteria for Detection Capability (CCß)
  • 2002/657/EC
  • MRL Substances (permitted limit)
  • 1. Calibration curve analysis of blank samples
    and blanks fortified around the MRL
  • ? CCß CCa 1,64 sRW
  • OR
  • 2. 20 blank samples fortified at CCa
  • ? CCß CCa 1,64 sRW

15
Detection CapabilityCase of permitted limit
MRL
CCa
CCb
1.64sMRL
1.64ssample
Signal orConcentration
a b 5
16
Criteria for Detection Capability (CCß)
  • 2002/657/EC
  • Banned Substances (no permitted limit)
  • 1. Calibration curve analysis of blank samples
    fortified below and at the MRPL
  • ? CCß CC a 1,64 sRW
  • OR
  • 2. Analysis of 20 blank materials fortified at
    CCa
  • ? CCß CC a 1,64 sRW
  • OR
  • 3. Analysis of 20 blank materials fortified at
    and above CCa
  • ? Less than 5 of false compliant

17
Detection CapabilityCase of no established
permitted limit or banned substance
Xblank
CCa
CCb
2.33sblank
1.64ssample
Signal orConcentration
a 1 ? b 5
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
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)
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
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 (CCb repeatability trueness -
    recovery)
  • ?? completion of validation routine analyses

22
Validation of corticosteroids in liver
3. Mini validation Analysis of other species
(horse, porcine, ovine)
0 MRL/MRPL 0,5 MRL/MRPL 1 MRL/MRPL 1.5 MRL/MRPL 2 MRL/MRPL
pork 1 3 3
horse 1 3 3
Sheep day1 3 3 3 3 3
Sheep day2 3 3 3 3 3
? ovine 2 series of analyses because of
additional extraction step (hexane)
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
RRT - IPs
Identification Points - Precursor Ion ? 1
IP - Transition Product Ion ? 1.5 IP

1 1.5 1.5 4 IPs
25
Linearity
Mandels Fitting Test linear (y ax b) or
quadratic (y ax2 bx c) regression? ?
Calibration curves linear
Dexamethasone 0 4 µg/ kg
26
Matrix effect
Matrix effect Calibration curves slope
comparison mobile phase matrix (T-test) ?
matrix effect
prednisolone
flumethasone
27
Quantitative validation parameters
CCa - CCß ? ISO 11843 is used to calculate
CCa and CCß
Cost Normes (µg/ kg) CCa (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
28
Quantitative validation parameters
CCa - CCß ? ISO 11843 is used to calculate
CCa and CCß 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
29
Quantitative validation parameters
CCa - CCß ? ISO 11843 is used to calculate
CCa and CCß 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
30
Quantitative validation parameters
CCa - CCß ? ISO 11843 is used to calculate
CCa and CCß Repeatability ? recovery
(70-120) and CVr (max. 2/3 CVrw
Horwitz) Reproducibility ? CVrw
Selectivity and specificity ? LC-MSn
selective and specific
31
LC-MSn selectivity - specificity
32
Quantitative validation parameters
CCa - CCß ? ISO 11843 is used to calculate
CCa and CCß Repeatability ? recovery
(70-120) and CVs (max. 2/3 CV Horwitz) Reprod
ucibility ? CV rw Selectivity and
specificity ? LC-MSn selective and specific
Stability ? under current development
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

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

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
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.
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