Analytical Method Validation: Application of the Accuracy profile to a method “Determination of total alkaloids in tobacco” Béatrice Teillet 1 , Thomas Verron 1 , Xavier Cahours 1 and Stéphane Colard 2 1 SEITA, Imperial Tobacco Group – 48 rue Danton, 45404 Fleury-les-Aubrais, France 2 Imperial Tobacco Limited - PO Box 525, Winterstoke Road, Bristol BS99 1LQ, U.K. 2012_SS07_Teillet.pdf Congress2012 - Document not peer-reviewed by CORESTA
Transcript
Analytical Method Validation: Application of the Accuracy profile to a method “Determination of total alkaloids in tobacco”
Béatrice Teillet1, Thomas Verron1, Xavier Cahours1 and Stéphane Colard2
1SEITA, Imperial Tobacco Group – 48 rue Danton, 45404 Fleury-les-Aubrais, France 2Imperial Tobacco Limited - PO Box 525, Winterstoke Road, Bristol BS99 1LQ, U.K.
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Background Analytical method validation1 is the confirmation by examination and provision of objective evidence that particular requirements for a specific intended use are fulfilled.
Classical approach: method is characterized across several independent criteria
interpretation by the lab manager.
THE ACCURACY PROFILE
Decision on validity is not determined by any set of rules
Alternative approach focuses on fitness for purpose2,3,4
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Method validation by the accuracy profile defines whether the method will provide results with the required accuracy, a combination of precision and trueness, for each future sample.
Introduction
the tolerance interval is checked to ensure that if it fits into the acceptability limits
The “fitness for purpose” objective is translated by 2 criteria:
Acceptability limit – gives the expected maximum difference between the result and the target (true) value
+ +
target (true) value Tolerance interval that contains future results with a given confidence (probability β).
To Illustrate Application of this approach to a method “Determination of Total Alkaloids in tobacco by CFA”
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Accuracy profile Validation illustration Set-Up
Definition of the needs
Definition of the quantity to measure
End-User Laboratory Definition
of the target 1
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Definition of the needs
Definition of the quantity to measure
End-User Laboratory Definition
of the target 1
• Sample type • Range of contents • Intended use
Quality control Product comparison Regulatory compliance
process to establish the result in routine conditions (intermediate precision)
including calculation of final results
Routine conditions: 2 replicates
Identified source of variability: days (instruments life), preparation of reagents, calibration standards (as well as stocks solutions), 3 operators
Calculation on a dry weight basis results
Total Alkaloids in tobacco Samples: tobacco (BY, FC, OR) or blends
Select or create reference samples with assigned values
Reference samples
Definition of the target 1
2
Accuracy profile Validation illustration Set-Up
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Select or create reference samples with assigned values
Reference samples 2
Alternative: create dedicated reference materials using a reference method or spiking matrix or using spike calibration
Selection of 5 samples 4 raw tobaccos: 2 BY, 1FC, 1OR 1 blend (3R4F) covering the range 0.2 to 4%
Spike amount in the sample
Response
1 2 3 4 Initial amount in the sample
Spike calibration
Value assignment Spike calibration: 4 levels for each sample (5 replicates)
No reference materials available
Should represent all the matrix interferences covering the range of contents !
Ideally: use of Certified materials At least materials with consensual values
Assigned values must be generated independently of the method for validation !
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Accuracy profile Validation illustration Set-Up
Definition of the needs
Definition of the quantity to measure
End-User Laboratory
Calibration plan (standards) Validation plan (samples)
Select or create reference samples with assigned values
Experimental
Reference samples
Definition of the target 1
2
3
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Calibration plan (standards) Validation plan (samples) Experimental 3
Should represent routine conditions under intermediate precision
5 Days 1 2 3 4 5
2 stock solutions A B 6 Calibration standards CalA CalB
5 Reference samples x 2 replicates T1 T2 T3 T4 T5
3 Operators 2 2 1 1 3
Calibration plan
* Only one set of calibration used for the validation plan
Validation Plan*
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Accuracy profile Validation illustration Set-Up
Definition of the needs
Definition of the quantity to measure
End-User Laboratory
Calibration plan (standards) Validation plan (samples)
graphical representations Accuracy profiles
Data treatment
Select or create reference samples with assigned values
Experimental
Reference samples
Definition of the target 1
2
3
4
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graphical representations Accuracy profiles
Data treatment 4
For each plan, 1 Compute calibration models
(linear or not, with weighting or not)
2 For each level, compute
3 Make a graphical representation for each reference level including recovery, β-expectation tolerance and acceptability intervals
Compare the two intervals for decision on fitness for purpose and estimate the limits of the method
4
The acceptability interval (reference value ± 15%)
• The variance intra and inter days, repeatability and standard deviation of intermediate precision The corresponding tolerance interval (β probability)
• The inverse-predicted concentration and bias (express as recovery)
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70
80
90
100
110
120
130
115%
85%
Upper limit of acceptability interval
Lower limit of acceptability interval LOQ: 0.35%
Rec
over
y %
0.2 0.4 1.0 2.0 3.0 4.0
Standard reference Value (%)
Tolerance interval
Calibration plan
graphical representations Accuracy profiles
Data treatment 4
Calibration model: linear regression
Calibration valid from
0.35 to 4.0%
95% confidence level (β)
++
Acceptability limit criteria of ±15%
95% confidence level (β)
++++
Acceptability limit criteria of ±15%
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Validation plan
graphical representations Accuracy profiles
Data treatment 4
60
80
100
120
0.27 0.89 1.93 3.25 4.10
110
90
70
Rec
over
y %
115%
85%
Tolerance interval
Sample reference Value (% d.w.b*)
Upper limit of acceptability interval
Lower limit of acceptability interval
LOQ: 0.73%
*d.w.b: dry weight basis corrected
Method valid from
0.73 to 4.1%
95% confidence level (β)
++
Acceptability limit criteria of ±15%
95% confidence level (β)
++++
Acceptability limit criteria of ±15%
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Accuracy profile Validation illustration Set-Up
Definition of the needs
Definition of the quantity to measure
End-User Laboratory
Calibration plan (standards) Validation plan (samples)
graphical representations Accuracy profiles
Data treatment
Select or create reference samples with assigned values
Experimental
Reference samples
Definition of the target 1
2
3
4 Decision on the fitness for purpose
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Validation of the method “Determination of Total Alkaloids
in tobacco by CFA”
Decision on the fitness for purpose
Objective evidence is provided to prove that the requirement of the end-user is fully achievable from 0.73 to 4.1% but not for the lowest values from 0.2 to 0.73%.
For lowest values, the end-user can decide:
- to refuse these lower accuracy the method should be adjusted (development phase) and additionally validate to fully fit with this requirement.
- to accept higher variability on the lowest values he will not evaluate the data with his required accuracy
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Accuracy profile approach Conclusion
• This approach is global and pragmatic
Decision on the fitness for purpose
• No conflict between trueness and precision avoiding questionable conclusions produced by independent treatment of criteria
• No limit in the choice of the model of calibration (linear or not, weighting…)
• Focus on concentration levels (possible with non homogeneous variances)
• Methods with very low variability can be validated (not rejected by a H0)
• Risks and guarantees are managed for both end-users and laboratories.
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Accuracy profile Validation Conclusion
Within the expanded regulatory environment, the approach provides to the regulator a pragmatic tool for checking if the method performance fits for the regulatory intended use(s).
Allows a clear and easy comparison between method performance and intended use
