Validation of screening methods (2002/657/EC) N. Van Wouwe IPH AFSCA-FAVV.
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Transcript of Validation of screening methods (2002/657/EC) N. Van Wouwe IPH AFSCA-FAVV.
Validation of screening methods (2002/657/EC)
N. Van Wouwe
IPHAFSCA-FAVV
Definition (2002/657/EC)
Screening method : used to detect the presence of a substance
or class of substances at the level of interest. have the capability for a high sample
throughput
=> are used to sift large numbers of samples for potential non-compliant results.
Exemple: ELISA, plate test, biosensor, receptor test,…
Definition (2002/657/EC)
Minimum criteria to use an analytical method as screening method:
must be validated (traceability)
must have a false compliant rate of <5% (β-error) at the level of interest
Performance characteristics for method validation (screening)
Detection Capability
CCß
Decision Limit CC
Trueness/ Recovery
Precision Selectivity/ Specificity
Applicability/ Ruggedness/
Stability
Qualitative methods
S + - - - + +
Quantitative methods
S + - - + + +
+ = determination is mandatory
Qualitative method: identifies a substance on basis of its chemical, biological or physical propriety (binary response: +/-, absence/presence)
Quantitative method: determines the amount or mass fraction of a substance (response: numerical value of appropriate unit)
Validation of screening test
Definition of the scope of the method Analyte of group of analytes Range of concentration List of matrices
Initial validation with the most often used matrice in national monitoring program
Detection capacity (CCβ) Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative method)
If possible: different sources of blank material, different technicians, different days on the same spiked sample
Validation of screening test
Targeted test: for 1 compound validation for this compound
Targeted test: for a family of compounds validation for 1 representative molecule of the family (antibody)
Wide range test: for more than 50 different molecules Validation for at least a list of representative compounds
Common pattern of activity on a specific bacteria? Common way of action (acting target)? Published reference data on validation available?
Proposition of the CRL for antimicrobials (in milk)
Representative Compound Antimicrobial classCefalonium/Cephapirin/Cefquinome CEPHALOSPORINS
Penicillin G/Cloxacillin PENICILLINS
Tetracycline/Doxcycline TETRACYCLINS
Gentamicin/Streptomycin/Spectinomycin AMINOGLYCOSIDES
Enrofloxacin/Flumequine QUINOLONES
Sulfathiazole/sulfaguanidine/Sulfamerazine SULFONAMIDES
Erythromycin/Tylosin MACROLIDES
Lincomycin LINCOSAMIDES
Thiamphenicol PHENICOLATED
Trimethoprim/colistine MISCELLANEOUS
Performance characteristics
Detection capacity Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative
method)
Detection capability (CCβ)
The smallest content of the substance that may be detected, identified and/or quantified in a sample with an error probability of β In case of MRPL, CCβ= lowest concentration at which
the method is able to detect truly contaminated sample with a statistical certainty of 1-β
In case of MRL, CCβ= concentration at which the method is able to detect the MRL concentrations with a statistical certainty of 1-β
Detection capability (CCβ)
No permitted limit Analyse 20 blank materials => CCα = 3x signal/noise
Analyse 20 blank materials fortified at CCα
=> CCβ = CCα + 1.64 x SDRW
Calibration curve procedure (ISO 11843) Analyse of blank material fortified at 0 MRLP, 0.5 MRLP,
1 MRLP, 1.5 MRLP and 2 MRLP Plot analytical results (y-axis) vs concentration(x-axis) CCα = y-intercept (blank) + 2.33 x SDRW
CCβ = CCα + 1.64 x SDRW
Detection capability (CCβ)
No permitted limit If no quantitative results
Analyse fortified blank samples at and above CCα
(n ≥ 20 / concentration level) CCβ = concentration level where only ≤5% false compliant
results remain
Detection capability (CCβ)
CC
Signal orConcentration
CC+2.33xSDblank
+1.64xSDRW
Blank
α=1% β=5%
Detection capability (CCβ)
Permitted limit (MRL) Analyse 20 blank materials fortified at MRL
=> CCα = MRL + 1.64 x SDRW
Analyse 20 blank materials fortified at CCα
=> CCβ = CCα + 1.64 x SDRW
Calibration curve procedure (ISO 11843) Analyse of blank materials fortified at 0.5 MRL, 1 MRL, 1.5
MRL and 2 MRL Plot analytical results (y-axis) vs concentration(x-axis) CCα = MRL + 1.64 x SDRW
CCβ = CCα + 1.64 x SDRW
Detection capability (CCβ)
CC
Signal orConcentration
CCMRL
+1.64xSDMRL +1.64xSDRW
β=5%α=5%
Performance characteristics
Detection capacity Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative
method)
Selectivity/specificity
Specificity: ability of a method to distinguish between analyte being measured and other substances
problem of interference?
F(measuring technique, class of compounds, matrices,…)
Selectivity/specificity
How to test specificity for qualitative screening method? Analyse 20 different blank samples and 20 positive samples
(blind study, same or different days/technicians)
True positive (N+)
True negative (N-)
test result positive
Positive agreement
(PA)
False positive (FP)
test result negative
False negative (FN)
Negative agreement (NA)
Specificity= 100* NA/N-
Other parameters:
Accuracy= 100* (PA+NA)/(N- + N+)
Sensitivity= 100* PA/N+
False positive= 100* FP/(N- + N+)
False negative= 100* FN/(N- + N+)
Selectivity/specificity
How to test specificity for semi-quantitative screening methods? Select potentially interfering substances (metabolites,
derivatives,…) Analyse relevant blank samples (n ≥ 20) Analyse fortified blank samples with interfering substances at a
relevant concentration Estimate the effect of the interferences
False identification? Influence in quantification? Identification of the target analyte is hindered?
Performance characteristics
Detection capacity Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative
method)
Applicability
Scope of the method must be define in term of : Matrix (solid/liquid matrix, type of tissue) Animal species
To introduce a new matrix Analyse at least 10 different blank material fortified at level
of interest for the new matrix (CCβ) + test of interferences If 10 positive results => method applicable for the new matrix If 1 negative result => 10 additional analyses
If 1 negative result=> CCβ must be recalculated for the new matrix
Ruggedness
Ruggedness: the susceptibility of an analytical method to changes in experimental conditions
sample material analytes storage condition environmental condition sample preparation condition
Ruggedness
How to test ruggedness? (during development) Identify possible factor that could influence the results
(the analyst, solvents, pH, T°, rate of heating,…) Vary each factor slightly If one factor is found to influence results of the
representative molecule, conduct further experiments
=> acceptability limits for this factor
(in the method protocol)
Recommendation of CRL: analyses of 10 blank and 10 spiked samples at the same concentration and with minor change of factor to detect influence on results
Stability Test are not necessary if stability data already
exist (from other lab or from publication) To include in the validation report
Stability test: the analyte in solution the analyte in matrix
Aliquots of a fresh solution or sample stored under different conditions (T° and/or storing time)
Performance characteristics
Detection capacity Selectivity/Specificity Applicability/ Ruggedness/Stability Precision (only for semi-quantitative
method)
Precision (for quantitative screening) Precision: the closseness of agreement
between independent test results obtained under predetermined conditions
Expressed in terms of imprecision / standard deviation of test results
How to test precision? Repeatability test within-laboratory reproducibility test(or intermediate
precision) Reproducibility test (between laboratories: interlaboratory
studies)
determination of RSD (%) < Precision criteria
Precision (for quantitative screening)
Precision (for quantitative screening) Repeatability
3 concentrations: 1x; 1,5x; 2x MRPL 0,5; 1x; 1,5x MRL
6 replicates/level 3 times same conditions
Within-laboratory Reproducibility 3 concentrations:
1x; 1,5x; 2x MRPL 0,5; 1x; 1,5x MRL
6 replicates/level 3 times different conditions
(analyst, env. condition,…)
Precision (for quantitative screening) ANOVA treatment of data => RSDr & RSDRW
Comparison with precision criteria: Horwitz equation: RSDR(%) = 2(1-0.5logC)
Criteria for repeatability: RSDr = 1/2 to 2/3 RSDR
Criteria for within-lab reproducibility: RSDRW = 2/3 to 1 RSDR
! For concentration < 100 µg/kg, RSDR becomes too high!
Other recommendations False negative rate <5%: Analyses of 20 negative and 20
positive samples in order to test the screening method (see selectivity).
One QC sample must be added in routine and results must be added to the validation file
Method transfer/Commercial test Bibliographical survey to compil the evaluation of performance of the
test Collection of data from supplier on validation study Experimental plan to test skillness of technician to perform the test Use of QC sample Participation to proficiency test
Exemple: analyse of PCDD/F by CALUX bioassay
PCDD/F: 17 toxic congeners to analyse in various matrices (TCDD=most toxic dioxin)
Results expressed in TEQ (=Sum (CCixTEFi)i=1-17) MRL for each matrix (milk, meat, egg, fish oil,…) MRL expressed in pg TEQ/g fat or ng TEQ/ kg Reference method: GC-HRMS Screening method: immunoassay, bioassay,…
Exemple: analyse of PCDD/F by CALUX bioassay
Gene expression
LIGHT
All substances fixing the Ah receptor
CALUX bioassay= genetically modified cell-based bioassay (luciferase)
Amount of light produced is proportional to the toxicity (TEQ) of extracts
Analyse of PCDD/F by CALUX bioassay Advantage:
RapidCheaper than GC-HRMSTime for analyses
Disadvantage:Various compounds can fix the Ah receptor
(PAH, PCB, PHDD/F,…)
specificity!!!!
Analyse of PCDD/F by CALUX bioassay : protocol
Extraction of fat
Clean-up on silica acid + carbon columns
Fraction with PCBs
Fraction with PCDD/F
Evaporation
Dosing plateReading plate
Fraction with interfering compounds
Analyse of PCDD/F by CALUX bioassay : validation
Selectivity/specificity Ruggedness/Stability Precision Detection capability
Analyse of PCDD/F by CALUX bioassay : selectivity
Possible interfering compounds? PAH : mostly in environmental sample PCB: fractionation during clean-up Other compounds? (PHDD/F): dependant of the matrix? (matrix
effect?)
Results of the selectivity test: No interferences for feedstuff, milk, egg, fat Interferences for fish oil
CALUX results = 2 x GC-HRMS results
Analyse of PCDD/F by CALUX bioassay : selectivity Matrix effect for fish oil
y = 1,9161x + 1,999
R2 = 0,9287
0
5
10
15
20
25
0 2 4 6 8 10 12
GC-HRMS TEQ value (pg TEQ/g oil)
CA
LU
X T
EQ
va
lue
(p
g T
EQ
/g o
il)
Analyse of PCDD/F by CALUX bioassay : ruggedness What are the critical point in the protocol?
Carbon column (interferences)Solvent (interferences)Curve (results)Evaporation time (recovery)Age of CALUX cell line (RSD)
Analyse of PCDD/F by CALUX bioassay : ruggedness Carbon column: amount of carbon used
0,7cc XCARB
0
10
20
30
40
50
60
70
80
90
100
PCB81
PCB77
PCB123
PCB118
PCB114
PCB105
PCB126
PCB167
PCB156
PCB157
PCB169
PCB189
% r
eco
very
1cc XCARB
0
10
20
30
40
50
60
70
80
90
100
PCB81
PCB77
PCB123
PCB118
PCB114
PCB105
PCB126
PCB167
PCB156
PCB157
PCB169
PCB189
% r
eco
very
1,4cc XCARB
0
10
20
30
40
50
60
70
80
90
100
PCB81
PCB77
PCB123
PCB118
PCB114
PCB105
PCB126
PCB167
PCB156
PCB157
PCB169
PCB189
% r
eco
very
1,9cc XCARB
0
10
20
30
40
50
60
70
80
90
100
PCB81
PCB77
PCB123
PCB118
PCB114
PCB105
PCB126
PCB167
PCB156
PCB157
PCB169
PCB189
% r
eco
very
DX fraction
PCB fraction
Not collected fraction
(Rdt PCDD/F= 60%) (Rdt PCDD/F= 80%)
(Rdt PCDD/F= 80%)
Analyse of PCDD/F by CALUX bioassay : ruggedness Evaporation time
0
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30
evaporation time with dry extract
CA
LU
X r
esp
on
se
Analyse of PCDD/F by CALUX bioassay : ruggedness
Solvent: tested before use on a TCDD solution (antagonist/agonist effect)
Curve: tested with an independant TCDD solution
Age of CALUX cells: new cell every 2 months
Analyse of PCDD/F by CALUX bioassay : precision
Validation protocol
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Blank solvent 1 1 1 1 1 1
Blank sample 1 1 1 1
Sample at MRL/2 3 3 2
Sample at MRL 6 2 3 3
Sample at 2MRL 3 2 3
Quality sample 1 1 1 1 10 10
Analyse of PCDD/F by CALUX bioassay : precision
ANOVA results for the TEQ determination of PCDD/F in
feedstuff by CALUX bioassay At MRL (0.75ng TEQ/kg) : XMRL= 0.751 ng TEQ/kg
Sr= 0.063 => RSDr= 8.4%
SRW=0.073 =>RSDRW= 9.7%
At MRL/2 (0.376ng TEQ/kg) : XMRL/2= 0.464 ng TEQ/kg
Sr= 0.051 => RSDr= 11%
SRW=0.051 =>RSDRW= 11%
At 2MRL (1.5ng TEQ/kg): X2MRL= 1.571 ng TEQ/kg
Sr= 0.107 => RSDr= 6.8%
SRW=0.115 =>RSDRW= 7.3%
RSD < 30%
(2002/70/EC)
Analyse of PCDD/F by CALUX bioassay : detection capacity CCβ for the TEQ determination of PCDD/F in feedstuff
by CALUX bioassay
CCα = MRL + 1.64 x SRW
CCα = 0.75 + 1.64 x 0.073 = 0.87 ng TEQ/kg
CCβ = CCα + 2.33 x SRW
CCβ = 0.87 + 2.33 x 0.073 = 1.04 ng TEQ/kg2002/70/EC: false negative rate < 1% !
=> At a concentration of 1.04ng TEQ/kg, we are sure that the sample is a positive sample with 99% certainty
Analyse of PCDD/F by CALUX bioassay :confirmatory range
MRL CC
Signal orConcentration
CC
NON COMPLIANTCOMPLIANT SUSPICIOUS
CC*+1.64MRL +2.33sample
-2.33MRL
?
α=5%β=5% * =1%
Analyse of PCDD/F by CALUX bioassay :confirmatory range
Lower limit of the confirmatory range for the TEQ determination of PCDD/F in feedstuff by CALUX bioassay CC*= MRL-2.33 x SDRW
CC*= 0.75- 2.33 x 0.073 = 0.58 ng TEQ/kg
Conclusion Sample lower than 0.58 ng TEQ/kg are negative with 99%
certainty (false negative rate < 1%) Sample above 0.58 ng TEQ/kg must be confirmed by GC-HRMS