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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Validation of confirmatory methods forMRPL 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
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
• 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
Example: corticosteroids in liver
b = bovinep = porcinee = equidae
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
• 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
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
2002/657/EC: Performance Characteristics
Detection Capability
CCß
Decision Limit CC
Trueness/ Recovery
Precision Selectivity/ Specificity
Applicability/ Ruggedness/
Stability
S + - - - + + Qualitative methods C + + - - + +
S + - - + + + Quantitative methods C + + + + + +
• Allowed substances: maximum residue limits (MRL)
QUANTITATIVE PARAMETERS
• Forbidden components: Minimum Required Performance Levels (MRPL)
QUALITATIVE PARAMETERS
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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
Dark 10 aliquots 10 aliquots 10 aliquots
Light 10 aliquots
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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
OROR
2. 20 blank samples fortified at MRL
CC α = MRL+1.64 σRW
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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
OROR
2. 20 blank samples
CCα = 3 x S/NRT
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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
OROR
2. 20 blank samples fortified at CCα
CCβ = CCα +1,64 σRW
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
MRL CC
Signal orConcentration
CC
=
+1.64MRL +1.64sample
Detection CapabilityCase of permitted limit
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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
OROR
2. Analysis of 20 blank materials fortified at CCα
CCβ = CC α +1,64 σRW
OROR
3. Analysis of 20 blank materials fortified at and above CCα:
Less than 5% of false compliant
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Detection CapabilityCase of no established permitted limit or banned substance
XblankCC
Signal orConcentration
CC
≠
+2.33blank +1.64sample
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Validation of corticosteroids in liver
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
1. Analysis of mix of liver (pork, beef, horse, sheep)
MATRIX EFFECTS: DILUTION EFFECT
Alternative validation strategy
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Validation of corticosteroids in 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
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)
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Validation of corticosteroids in liver
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
3. Mini validation: Analysis of other species (horse, porcine, ovine)
ovine: 2 series of analyses because of additional extraction step (hexane)
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Qualitative validation parametersRelative 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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
RRT - IPs
monsternr. precursor ion 379(%) ion 409(%) RRT20070330refstd03 469 100,00 57,39 0,80 0.25MRPL-afwijking 80,00 45,91 0,78+afwijking 120,00 68,87 0,82I.P. 1 1,5 1,5
monsternr. precursor ion 379(%) besluit ion 409(%) besluit RRT besluit IP20070330spike01 469 100,00 ok 62,88 ok 0,79 ok 4,0 0,5MRPL
Identification Points: - Precursor Ion → 1 IP- Transition Product Ion → 1.5 IP
1 + 1.5 + 1.5 = 4 IPs
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Linearity
Mandel’s Fitting Test :
linear (y = ax + b) or quadratic (y = ax2 + bx + c) regression?
Calibration curves linear
Linear regression
y = 0,2488x + 0,0288R2 = 0,9928
-0,4
-0,2
0
0,20,4
0,6
0,8
1
1,2
-2 -1 0 1 2 3 4 5
X
Y
Dexamethasone0 – 4 µg/ kg
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Matrix effect
-0,5
0
0,5
1
1,5
2
2,5
3
0 5 10 15 20 25
addition
standard
Matrix effect
Calibration curve’s slope: comparison mobile phase – matrix (T-test)
matrix effect
prednisolone
-0,5
0
0,5
1
1,5
2
2,5
0 1 2 3 4 5
addition
standard
flumethasone
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Quantitative validation parametersCCα - CCβ
ISO 11843 is used to calculate CCα and CCβ
Cost Normes (µg/ kg)
CC (µg/ kg)
CC (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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Quantitative validation parametersCCα - 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
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Quantitative validation parametersCCα - 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
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Quantitative validation parametersCCα - 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
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
LC-MSn: selectivity - specificityC:\Xcalibur\data\20070314spike11 03/20/2007 08:18:30 AMsp1: 22-2006-03544+ 1m rpl
RT: 0.00 - 20.06 SM : 5G
0 2 4 6 8 10 12 14 16 18 20Time (min)
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TrmAt
5.10 NL: 4.13E6m/z= 374.50-375.50+412.50-413.50 F : - c ESI Full ms2 [email protected] [ 300.00-496.00] M S 20070314spike11
20070314spike11 #281 RT: 5.10 AV: 1 SB: 43 2.00-3.54, 7.52-9.73 NL: 3.57E6F: - c ESI Full ms2 [email protected] [ 300.00-496.00]
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
Quantitative validation parametersCCα - 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
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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
Dark 10 aliquots 10 aliquots 10 aliquots
Light 10 aliquots
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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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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FEDERAL PUBLIC SERVICE (FPS) HEALTH, FOOD CHAIN SECURITY AND ENVIRONMENT
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.