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

Dr. Apr. S. Impens and Ir. S. Carbonnelle Departme
nt of Pharmaco-Bromatology Drugs and Hormones
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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
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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)

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2002/657/EC Performance Characteristics

• Allowed substances maximum residue limits (MRL)
  
• ? QUANTITATIVE PARAMETERS
• Forbidden components Minimum Required
  Performance Levels (MRPL)
• ? QUALITATIVE PARAMETERS

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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

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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

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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

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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

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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

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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)

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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

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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

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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

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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

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Detection CapabilityCase of permitted limit
MRL
CCa
CCb
1.64sMRL
1.64ssample
Signal orConcentration
a b 5
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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

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Detection CapabilityCase of no established
permitted limit or banned substance
Xblank
CCa
CCb
2.33sblank
1.64ssample
Signal orConcentration
a 1 ? b 5
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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
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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)
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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

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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

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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)
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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)

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RRT - IPs
Identification Points - Precursor Ion ? 1
IP - Transition Product Ion ? 1.5 IP

1 1.5 1.5 4 IPs
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Linearity
Mandels Fitting Test linear (y ax b) or
quadratic (y ax2 bx c) regression? ?
Calibration curves linear
Dexamethasone 0 4 µg/ kg
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Matrix effect
Matrix effect Calibration curves slope
comparison mobile phase matrix (T-test) ?
matrix effect
prednisolone
flumethasone
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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
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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
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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
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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
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LC-MSn selectivity - specificity
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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
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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

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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

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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

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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.