Uncertainty profile; a global strategy for validation and estimation of uncertainty: Application to a high-performance thin-layer chromatographic method Y. H. Benchekroun 1, T. Saffaj, B. Ihssane, S. S. Alaoui, F. Jhilal and H. Bouchafra, 1 University SIDI MOHAMED BEN ABDALLAH, Faculty of Techniques and Sciences of Fez, laboratory of organic applied chemistry, Fez, Morocco. UNIVERSITY SIDI MOHAMED BEN ABDELLAH FACULTY OF SCIENCES AND TECHNIQUES FEZ 6 th International Conference and Exhibition on Analytical & Bioanalytical Techniques Valencia, Spain September 01-03, 2015

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

PLAN INTRODUCTION VALIDATION OF ANALYTICAL METHODS MEASUREMENT UNCERTAINTY ▫APPROACH OF ISO ▫APPROACH OF GUM ▫NEW APPROACH OF UNCERTAINTY UNCERTAINTY PROFILE EXAMPLE CONCLUSION 3 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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

Problematic ICH Q2(A4) Q2(B5) EURACHEM… FDA USP 5 PROBLEMATIC UIPAC 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

As described in ISO 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. 6 WHAT IS VALIDATION OF ANALYTICAL METHODS ?

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 7

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 8 VALIDATION OF ANALYTICAL METHODS 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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 9 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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. 10 MEASUREMENT OF UNCERTAINTY 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

APPROACH OF ISO Total statistical model equation : Y=µ + δ + B + ∑c i x’ i + e (2) were µ: True value δ: Inherent bias B: Laboratory bias ∑c i x’ i : combination effect e: Random error Uncertainty u(Z) associated to an observation can be estimated as follow : u 2 (y) = u 2 (δ) + S L 2 + ∑c i 2 u 2 (x’ i ) + S r 2 (3) We know that: S 2 R = S L 2 + S r 2 (4) The uncertainty expression became : u 2 (y) = S R 2 + u 2 (δ) + ∑c i 2 u 2 (x’ i ) (5) 11 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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. 12 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

NEW APPROACH OF UNCERTIANTY Uncertainty Expression of Uncertainty Tolerance intervals Uncertainty Profile Estimation of uncertainty is mainly based on Tolerance intervals u(Z) u 2 (y) = S R 2 + u 2 (δ) + ∑c i 2 u 2 (x’ i ) u 2 (y) = S R 2 + u 2 (δ) β-Content, γ-Confidence β-Expectation Accuracy Profile 13 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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. 14 UNCERTAINTY PROFILE T. Saffaj, B. Ihssane, F. Jhilal, H. Bouchafra, S. Laslami and S. Alaoui Sosse, Analyst, 2013,138, T. Saffaj, B. Ihssane, Talanta 85 (2011) 1535–1542 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

UNCERATINTY PROFILE 15 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 : T. Saffaj, B. Ihssane, F. Jhilal, H. Bouchafra, S. Laslami and S. Alaoui Sosse, Analyst, 2013,138, T. Saffaj, B. Ihssane, Talanta 85 (2011) 1535–1542 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

UNCERTAINTY PROFILE Building Step Decision Step 16 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 T. Saffaj, B. Ihssane, F. Jhilal, H. Bouchafra, S. Laslami and S. Alaoui Sosse, Analyst, 2013,138, T. Saffaj, B. Ihssane, Talanta 85 (2011) 1535–1542 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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 17 LLOQ ULOQ 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain +λ+λ - λ

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

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

20 EXAMPLE 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 S ulfamethoxazole (SMX) Concentration (ng/spot)Uncertainty LimitDecision Relative expanded uncertainty % 120[-2.79, 2.11] Valid [-1.83, 1.00] Valid [-0.10, 1.35] Valid 1.49 T rimethoprim e (TMP) Concentration (ng/spot)Uncertainty Limit Decision Relative expanded uncertainty % 150[-2.68, 3.31] Valid [-3.09, 2.19] Valid [-2.74, 2.58] Valid 6.08

EXAMPLE Figure 1 Uncertainty profile for the determination of Sulfaméthoxazole and Trimethoprim in the tablet combination drug. Acceptance limits are set at 5%. 21 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

22 EXAMPLE Lamivudine (3TC) Concentration (ng/spot)Uncertainty LimitDecision Relative expanded uncertainty % 150[-3.51, 1.79]Valid [-2.88, 1.71]Valid [-3.40, 1.45]Valid3.59 Stavudine (d4T) Concentration (ng/spot)Uncertainty LimitDecision Relative expanded uncertainty % 150[-2.75, 1.33]Valid [-3.32, 1.39]Valid [-3.12, 1.46]Valid3.23 Nevirapine (NVP) Concentration (ng/spot)Uncertainty LimitDecision Relative expanded uncertainty % 150[-3.10, 1.93]Valid [-1.29, 1.41]Valid [-2.87, 1.30]Valid3.18 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

EXAMPLE Figure 2: Uncertainty profile for the determination of : lamivudine, stavudine and Nevirapine in the tablet combination drug. Acceptance limits are set at 5%. 23 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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. 24 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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NEW STRATEGY FOR ANALYTICAL VALIDATION & ESTIMATION OF MEASURMENT UNCERTAINTY Y. H. Benchekroun 1, S. S. Alaoui, F. Jhilal, H. Bouchafra, B.Ihssane, T.Saffaj 1 University SIDI MOHAMED BEN ABDALLAH, Faculty of Techniques and Sciences of Fez, laboratory of organic applied chemistry, Fez, Morocco. UNIVERSITY SIDI MOHAMED BEN ABDELLAH FACULTY OF SCIENCES AND TECHNIQUES FEZ 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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 27

REQUIREMENTS FOR TYPES OF TESTS Validation Characteristics AssayTesting for ImpuritiesIdentification QuantitativeLimit Accuracy++-- Precision - Repeatability++-- Precision - Intermediate Precision ++-- Specificity++++ Detection limit--+- Quantitation limit-+-- Linearity++-- Range++-- Robustness++-- +: YES - : NO 28 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain

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. 29 6th International Conference and Exhibition on Analytical & Bioanalytical Techniques September 01-03, 2015 Valencia, Spain