UNIVERSITY SIDI MOHAMED BEN ABDELLAH
FACULTY OF SCIENCES AND TECHNIQUES
FEZ
Uncertainty profile; a global strategy for validation and
estimation of uncertainty: Application to a high-performance
thin-layer chromatographic method
Y. H. Benchekroun1, T. Saffaj, B. Ihssane, S. S. Alaoui, F. Jhilal and H. Bouchafra,
E-mail: yassine.benchekroun@usmba.ac.ma
1 University
SIDI MOHAMED BEN ABDALLAH, Faculty of Techniques and Sciences of
Fez, laboratory of organic applied chemistry, Fez, Morocco.
6th International Conference and Exhibition on
Analytical & Bioanalytical Techniques
Valencia, Spain September 01-03, 2015
2
OBJECTIVES
• Present and suggest a new Strategy of validation of analytical
methods and estimation of measurement uncertainty,
• Expose the efficiency of uncertainty profile as a graphical decisionmaking tool allowing to gather two concepts: analytical validation
and estimation of measurement uncertainty.
• Showing the applicability of the uncertainty profile to evaluate the
performance of an analytical method. “In our case the thin layer
chromatography”
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
3
PLAN
• INTRODUCTION
• VALIDATION OF ANALYTICAL METHODS
• MEASUREMENT UNCERTAINTY
▫ APPROACH OF ISO
▫ APPROACH OF GUM
▫ NEW APPROACH OF UNCERTAINTY
• UNCERTAINTY PROFILE
• EXAMPLE
• CONCLUSION
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
4
INTRODUCTION
• laboratories shall use appropriate methods and procedures for all
tests, hence the validation of analytical methods and estimation of
uncertainty is among techniques to confirm .....,
• Specially nowadays the application of ISO17025 requirement,
request two important concept which are measurement uncertainty
and validation of analytical methods
• Many challenges are in front of the new methods for analytical
validation, especially when old approaches present some failures.
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
5
PROBLEMATIC
UIPAC
FDA
USP
ICH Q2(A4)
Q2(B5)
EURACHEM…
Problematic
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
6
WHAT IS VALIDATION OF ANALYTICAL METHODS ?
• As described in ISO 17025 requirement : Validation is the
confirmation by examination and the provision of objective evidence
that the particular requirements for a specific intended use are
fulfilled.
• Each laboratory shall validate non-standard methods, laboratorydesigned/developed methods, standard methods used outside their
intended scope, and amplifications and modifications of standard
methods to confirm that the methods are fit for their intended use.
• Although these studies have contributed largely to the
implementation and advance the analytical validations, nonetheless,
they present weaknesses regarding the conclusions of the realized
tests and regarding the decisions to be made.
7
VALIDATION OF ANALYTICAL METHODS
• Classic form of validation request a verification of
certain criterion:









Sensitivity & Specificity
Linearity
Robustness & Ruggedness
Accuracy
Quantification & Determination limit
Precision
Trueness
Stability
Range
• Some time even if we verify all those criterion, we
cannot guarantee if this method is valid or not
8
VALIDATION OF ANALYTICAL METHODS
Many strategies of validation suggest a statistical
test to check with all this criterion, also it
recommends a separate assessing of trueness
and precision.
Under-estimate the accuracy
Bad conclusion
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
9
DISADVANTAGE OF DIFFERENT VALIDATION
APPROACH





Enormous guideline of Validation
Different definition of the same criterion
No specific Recommandation for analytical method
Adaptation for use and requirement
The use of statistics
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
10
MEASUREMENT OF UNCERTAINTY
• Uncertainty of measurement” means doubt about the validity of the result
of a measurement.
• Uncertainty is a parameter, associated with the result of a measurement,
that characterizes the dispersion of the values that could reasonably be
attributed to the measurand.
u(Y) = ( RESULT ± DISPEERTION )
(1)
• Although the concept of uncertainty is clearly established for some time by
EURACHEM and GUM approaches, there nevertheless exists a gap
between the normative requirements and daily laboratory practice.
• Indeed, these approaches are complex, expensive and often boring within
the framework of analyses involving multiple sources of errors.
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
11
APPROACH OF ISO
• Total statistical model equation :
Y=µ + δ + B + ∑cix’i + e
(2)
were
µ: True value
δ: Inherent bias
B: Laboratory bias
∑cix’i : combination effect
e: Random error
• Uncertainty u(Z) associated to an observation can be estimated as follow :
u2(y) = u2(δ) + SL2 + ∑ci2u2(x’i) + Sr2
(3)
• We know that:
S2R = SL2 + Sr2
(4)
• The uncertainty expression became :
u2(y) = SR2 + u2(δ) + ∑ci2u2(x’i)
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
(5)
12
APPROACH OF GUM
• Two Type of estimation measurement uncertainty according to GUM :
▫ Evaluation type A : this first type is based on statistical analysis of
a series of observation.
▫ Evaluation type B : the second type is based on the analysis of
information and data of uncertainty-type of standard and reference
materials.
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
13
NEW APPROACH OF UNCERTIANTY
Estimation of uncertainty is mainly based on
Tolerance intervals
Uncertainty
Expression
of Uncertainty
Tolerance intervals
u(Z)
u2(y) = SR2 + u2(δ)
β-Expectation
Accuracy Profile
u2(y) = SR2 + u2(δ) + ∑ci2u2(x’i)
β-Content, γ-Confidence
Uncertainty Profile
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
14
UNCERTAINTY PROFILE
• The idea was then to find an universal approach to be applicable to all
kinds of measurements and all type of data.
• Uncertainty profile is a new analytical validation strategy recently
introduced by our Laboratory.
• It’s a graphical criterion that have been developed for assessing the validity
of the analytical methods and measurement uncertainty
• It’s based on the notion of uncertainty and built from the estimates of the
β-content tolerance interval of measures at each level of concentration.
• This graphical tool has the power to assess together the precision and
trueness which allow a better estimate of accuracy of analytical Method.
T. Saffaj, B. Ihssane, F. Jhilal, H. Bouchafra, S. Laslami and S. Alaoui Sosse, Analyst, 2013,138, 4677-4691
T. Saffaj, B. Ihssane, Talanta 85 (2011) 1535–1542
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
15
UNCERATINTY PROFILE
Uncertainty limit are computing with the following formula :
|Ῡ ± k u(Z)| < λ
(6)
|Bias ± k u(Z)| < λ
(7)
For the tolerance interval we use the β content tolerance interval as
following :
Pˆ
M ,ˆ M
P  ˆ
X
M

 kˆ M  X  ˆ M  kˆ M ˆ M ,ˆ M     
T. Saffaj, B. Ihssane, F. Jhilal, H. Bouchafra, S. Laslami and S. Alaoui Sosse, Analyst, 2013,138, 4677-4691
T. Saffaj, B. Ihssane, Talanta 85 (2011) 1535–1542
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
16
UNCERTAINTY PROFILE
• Uncertainty profile Steps:
Building Step
Choice of the appropriate acceptance limit,
It depend
on the field
in which we
work (for example: 1
Generate
all possible
calibration
models,
to
2 %be
onLinear
pharmaceutical
specialties
and 15%
It can
and
non linear
it mean
the relationship
Calculation
ofbulk,
the 5%
inverse
predicted
concentrations,
on
biological
sample) and the concentration in sample
between
the response
Compute the two-sided β-content tolerance intervals,
Determination of the uncertainty for each level,
GPQ
Procedure
Decision Step
Compare the interval of uncertainty (L, U) to
the acceptance limits (-λ, λ)
MLS
Procedure
β-content
tolerance
interval
MEE
Approach
T. Saffaj, B. Ihssane, F. Jhilal, H. Bouchafra, S. Laslami and S. Alaoui Sosse, Analyst, 2013,138, 4677-4691
T. Saffaj, B. Ihssane, Talanta 85 (2011) 1535–1542
Bayesian
Approach
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
17
GRAPHICAL CRITERION
• Uncertainty Profile is graphical criterion based on uncertainty as
principal function.
Bias
% Biais
LLOQ
ULOQ
Interval concentration defined
+λ
Acceptance Limit
0
Uncertainty limit
-λ
Non Defined Interval of Concentration
Conc. level
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
18
EXAMPLE
• Identification of different constituent combined in fixed-dose tablet
with thin layer chromatography :
▫ First the method was assaying sulfamethoxazole and trimethoprim,
▫ Secondly the method assay lamivudine, stavudine and nevirapine.
D.H. Shewiyo, E. Kaale, P.G. Risha, B. Dejaegher, J. De Beer, J. Smeyers-Verbeke, Y.
Vander Heyden, Journal of Chromatography A, 1293 (2013) 159-169
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
19
EXAMPLE
•
The acceptance limits was set at ±5%.
•
The structure plan was chosen for calibration and validation:
assays realized at 3 levels of concentration, on 6 days with 3
replicates.
•
As decision rule, we have selected 4-6-20 rule (i.e. β = 66.7% and γ
= 90%)
• A straight-line response function was estimated for each compound
based on the results of the calibration standards.
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
20
EXAMPLE
Sulfamethoxazole (SMX)
Concentration (ng/spot)
Relative expanded
Uncertainty Limit
Decision
120
[-2.79 , 2.11]
Valid
3.11
150
[-1.83 , 1.00]
Valid
2.42
180
[-0.10 , 1.35]
Valid
1.49
Concentration (ng/spot)
Uncertainty Limit
Decision
150
[-2.68 , 3.31]
Valid
5.08
180
[-3.09 , 2.19]
Valid
5.16
216
[-2.74 , 2.58]
Valid
6.08
uncertainty %
Trimethoprime (TMP)
Relative expanded
uncertainty %
The uncertainty limits (%) obtained did not exceed the acceptance
limits of 5%.
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
21
Uncertainty %
EXAMPLE
Concentration Levels (ng/ml)
Figure 1 Uncertainty profile for
the determination of
Uncertainty %
Sulfaméthoxazole and
Trimethoprim in the tablet
combination drug.
Concentration Levels (ng/ml)
Acceptance limits are set at 5%.
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
22
EXAMPLE
Lamivudine (3TC)
Relative expanded
Concentration (ng/spot)
Uncertainty Limit
Decision
150
[-3.51 , 1.79]
Valid
2.66
120
[-2.88 , 1.71]
Valid
2.93
144
[-3.40 , 1.45]
Valid
3.59
Concentration (ng/spot)
Uncertainty Limit
Decision
150
[-2.75 , 1.33]
Valid
1.94
114.0
[-3.32 , 1.39]
Valid
2.83
136.8
[-3.12 , 1.46]
Valid
3.23
Concentration (ng/spot)
Uncertainty Limit
Decision
150
[-3.10 , 1.93]
Valid
2.71
120.00
[-1.29 , 1.41]
Valid
1.78
144.00
[-2.87 , 1.30]
Valid
3.18
uncertainty %
Stavudine (d4T)
Relative expanded
uncertainty %
Nevirapine (NVP)
Relative expanded
uncertainty %
The uncertainty limits (%) obtained did not exceed the acceptance limits of
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
5%.
23
Uncertainty %
Uncertainty %
EXAMPLE
Concentration Levels (ng/ml)
Figure 2: Uncertainty profile
for the determination of :
lamivudine, stavudine
Nevirapine in the tablet
combination drug.
and
Concentration Levels (ng/ml)
Uncertainty %
Acceptance limits are set at 5%.
6th International
Conference
and Exhibition on Analytical & Bioanalytical Techniques September
Concentration
Levels
(ng/ml)
01-03, 2015 Valencia, Spain
24
CONCLUSION
• Uncertainty profile approach presented here can easily be applied to
validate an analytical method, and to estimate the uncertainty
measurement.
• This approach prove it efficiency to replace the classical and accuracy
profile for validation, with it’s application on chromatography techniques.
• This approach provide a good estimation of measurement uncertainty using
validation data and without recourse to other experiment .
• Using this graphical tool we can easily make decision concerning the
validity or not of an analytical method, also it’s allow a good estimate of
uncertainty.
• The transfer of analytical method.
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
25
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
UNIVERSITY SIDI MOHAMED BEN ABDELLAH
FACULTY OF SCIENCES AND TECHNIQUES
FEZ
NEW STRATEGY FOR ANALYTICAL
VALIDATION & ESTIMATION OF MEASURMENT
UNCERTAINTY
Y. H. Benchekroun1, S. S. Alaoui, F. Jhilal, H. Bouchafra, B.Ihssane, T.Saffaj
E-mail: yassine.benchekroun@usmba.ac.ma
1 University
SIDI MOHAMED BEN ABDALLAH, Faculty of Techniques and
Sciences of Fez, laboratory of organic applied chemistry, Fez, Morocco.
6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September
01-03, 2015 Valencia, Spain
27
DRUG TEST AND ANALYSIS
• 3 Type of analytical Method are :
▫ Identification test are intended to ensure the identity of
an analyte in a sample
▫ Testing for impurities can be either a quantitative test
or a limit test for the impurity in a sample
▫ Assay procedures are intended to measure the analyte
present in a given sample
28
6th International
Conference and
Exhibition on Analytical
& Bioanalytical
Techniques September
01-03, 2015 Valencia,
Spain
REQUIREMENTS FOR TYPES OF TESTS
Validation
Characteristics
Assay
Testing for Impurities
Quantitative
Limit
Identification
Accuracy
+
+
-
-
Precision - Repeatability
+
+
-
-
Precision - Intermediate
Precision
+
+
-
-
Specificity
+
+
+
+
Detection limit
-
-
+
-
Quantitation limit
-
+
-
-
Linearity
+
+
-
-
Range
+
+
-
-
Robustness
+
+
-
-
+: YES
- : NO
29
EXAMPLE
6th International
Conference and
Exhibition on Analytical
& Bioanalytical
Techniques September
01-03, 2015 Valencia,
Spain
• Thin layer chromatography methods provide faster separations,
simplicity, versatility, reproducibility, and cost effectiveness, in
addition to being eco-friendly.
• The use of HPTLC in quantitative analysis need that each method
be validated in accordance with the appropriate regulatory
guidelines.
• The two assay method have already validated using the total error
approach.