uncertainty profile; a global strategy for validation and estimation of uncertainty: application to...

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: [email protected]

1 University SIDI MOHAMED BEN ABDALLAH, Faculty of Techniques and Sciences of Fez, laboratory of organic applied chemistry, Fez, Morocco.

UNIVERSITY SIDI MOHAMED BEN ABDELLAHFACULTY OF SCIENCES AND TECHNIQUES

FEZ

6th International Conference and Exhibition on Analytical & Bioanalytical Techniques Valencia, Spain September 01-03, 2015

mailto:[email protected]

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• 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 decision-making 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”

OBJECTIVES

6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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PLAN

•INTRODUCTION •VALIDATION OF ANALYTICAL METHODS•MEASUREMENT UNCERTAINTY

▫APPROACH OF ISO▫APPROACH OF GUM▫NEW APPROACH OF UNCERTAINTY

•UNCERTAINTY PROFILE•EXAMPLE•CONCLUSION


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• 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.

INTRODUCTION


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Problematic

ICH Q2(A4) Q2(B5)

EURACHEM…

FDAUSP

PROBLEMATIC

UIPAC


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• 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, laboratory-designed/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.

WHAT IS VALIDATION OF ANALYTICAL METHODS ?

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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

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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

VALIDATION OF ANALYTICAL METHODS


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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


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• 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.

MEASUREMENT OF UNCERTAINTY


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APPROACH OF ISO

• Total statistical model equation :

Y=µ + δ + B + ∑cix’i + e (2)

were µ: True value δ: Inherent biasB: 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 + ∑ci

2u2(x’i) + Sr2 (3)

• We know that: S2

R = SL2 + Sr

2 (4)

• The uncertainty expression became :

u2(y) = SR2 + u2(δ) + ∑ci

2u2(x’i) (5)6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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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.


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NEW APPROACH OF UNCERTIANTY

Uncertainty

Expression of Uncertainty

Tolerance intervals

Uncertainty Profile

Estimation of uncertainty is mainly based onTolerance intervals

u(Z)

u2(y) = SR2 + u2(δ) + ∑ci

2u2(x’i) u2(y) = SR2 + u2(δ)

β-Content, γ-Confidence β-Expectation

Accuracy Profile 6th International Conference and Exhibition on Analytical & Bioanalytical

Techniques September 01-03, 2015 Valencia, Spain

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• 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.

UNCERTAINTY PROFILE

T. Saffaj, B. Ihssane, F. Jhilal, H. Bouchafra, S. Laslami and S. Alaoui Sosse, Analyst, 2013,138, 4677-4691T. Saffaj, B. Ihssane, Talanta 85 (2011) 1535–1542


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UNCERATINTY PROFILE

ˆ ˆ,ˆ ˆ ˆ ˆ ˆ ˆ,

M M X M M M M M MP P k X k

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 :



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UNCERTAINTY PROFILE

Building Step

Decision Step

Choice of the appropriate acceptance limit,It depend on the field in which we work (for example: 1 to 2 % on bulk, 5% pharmaceutical specialties and 15% on biological sample)

Generate all possible calibration models,Calculation of the inverse predicted concentrations, Compute the two-sided β-content tolerance intervals,Determination of the uncertainty for each level,

Compare the interval of uncertainty (L, U) to the acceptance limits (-λ, λ)

•Uncertainty profile Steps:

It can be Linear and non linear it mean the relationship between the response and the concentration in sample

β-content tolerance interval

GPQ Procedure

MEE Approach

Bayesian Approach

MLS Procedure



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GRAPHICAL CRITERION• Uncertainty Profile is graphical criterion based on uncertainty as

principal function.

0

Conc. level

%Biais

Uncertainty limit

Acceptance Limit

Bias

Interval concentration defined

Non Defined Interval of Concentration

LLOQ

ULOQ


+λ

- λ

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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


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• 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.

EXAMPLE


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EXAMPLE

The uncertainty limits (%) obtained did not exceed the acceptance limits of 5%.


Sulfamethoxazole (SMX)

Concentration (ng/spot) Uncertainty Limit DecisionRelative expanded

uncertainty %

120 [-2.79 , 2.11] Valid 3.11

150 [-1.83 , 1.00] Valid 2.42

180 [-0.10 , 1.35] Valid 1.49

Trimethoprime (TMP)


uncertainty %

150 [-2.68 , 3.31] Valid 5.08

180 [-3.09 , 2.19] Valid 5.16

216 [-2.74 , 2.58] Valid 6.08

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EXAMPLE

120 150 180

-6

-4

-2

0

2

4

6

Concentration Levels (ng/ml)

Un

cert

ain

ty %

150 180 216

-6

-4

-2

0

2

4

6


Unc

erta

inty

%

Figure 1 Uncertainty profile for the determination of

Sulfaméthoxazole and Trimethoprim in the tablet combination drug.

Acceptance limits are set at 5%.


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EXAMPLELamivudine (3TC)


uncertainty %

150 [-3.51 , 1.79] Valid 2.66

120 [-2.88 , 1.71] Valid 2.93

144 [-3.40 , 1.45] Valid 3.59

Stavudine (d4T)


uncertainty %

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

Nevirapine (NVP)


uncertainty %

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

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

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EXAMPLE

96 120 144

-6

-4

-2

0

2

4

6


Un

cert

ain

ty %

91.2 114.0 136.8

-6

-4

-2

0

2

4

6


Un

cert

ain

ty %

96.00 120.00 144.00

-6

-4

-2

0

2

4

6


Un

cert

ain

ty %

Figure 2: Uncertainty profile for the determination of :

lamivudine, stavudine and Nevirapine in the tablet combination drug.

Acceptance limits are set at 5%.


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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.


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Thank You For Your Attention


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: [email protected]

1 University SIDI MOHAMED BEN ABDALLAH, Faculty of Techniques and Sciences of Fez, laboratory of organic applied chemistry, Fez, Morocco.

UNIVERSITY SIDI MOHAMED BEN ABDELLAHFACULTY OF SCIENCES AND TECHNIQUES

FEZ


mailto:[email protected]

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


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REQUIREMENTS FOR TYPES OF TESTS

Validation Characteristics

Assay Testing for Impurities

Identification

Quantitative

Limit

Accuracy + + - -

Precision - Repeatability

+ + - -

Precision - Intermediate Precision

+ + - -

Specificity + + + +

Detection limit - - + -

Quantitation limit - + - -

Linearity + + - -

Range + + - -

Robustness + + - -+: YES- : NO


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EXAMPLE

• 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.

uncertainty profile; a global strategy for validation and estimation of uncertainty: application to...

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