by Dr. Michael Scheutwinkel

International Federation for Consulting GmbH IFC

• Governmental


• Books …. • Recommendations

of associations


EURACHEM Guide The Fitness for Purpose of Analytical Methods A Laboratory Guide to Method Validation and Related Topics (12/1998)

CITAC / EURACHEM Guide Guide to Quality in Analytical Chemistry An Aid to Accreditation (2002)

4 .

Which chapter of ISO/IEC 17025 is dedicated to validation? 5 .

Do not ask me. I am only a simple guy from Alemania! 6 .

4. ….Definition in clause for purpose 7 .1 of ISO/IEC 17025 Validation (ISO/IEC 17025) Validation is the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled.

Validation of an analytical test method Validation is the process of establishing the • Performance characteristics • Limitations of a method • Identification of the influences which may change these characteristics and to what extent. Which analytes can be determined in which matrix in the presence of which interferences? Within these conditions what levels of precision and accuracy can be achieved? 8 .

Equipment and method valdiation Equipment validation DQ IQ Soft and hardware OQ PQ Method validation … different items 9 .

Validation in order to analytical method Standardized-methods Verification Modified standardized and in-house methods Validation 10 .

accuracy).Verification of an analytical test method Verification is the process to demonstrate the competence in test performance of an already validated standardized test method.g. the laboratory needs only to verify that the documented performance characteristics can be met (e. 11 . The tools are the same as for validation.

a rapid new test against a standard method) 12 .g. different equipment or different staff • Out-of-control situations within internal quality assurance • Non-successful participation in PTs • Demonstration of equivalence between two methods (e.When should be validated? • Changes in established methods • New method developed for a particular problem • Established method used in different laboratories.

Validation Strategy A Specific test requirements defined by customer or market B C Comparison of method charcacteristics with requirements D Verification to give proof that the requirements are fulfilled Characterization of the test method 13 .

4. CRMs Equipment and environmental conditions Sampling and sample preparation Quality assurance Uncertainty or procedure for its estimation 14 Calibration Test performance including safety instructions Documentation .4) Identification / Scope Parameters in which matrix Reagents and materials.Description of test method (clause 5. reporting evaluation criteria .

Please show us the way… 15 .

Tools Determination of method characteristics Characteristics of the range Characteristics of calibration Characteristics of accuracy Robustness / Ruggedness Selectivity or Specifity 16 .

99 ?? Linearity is defined by the correlation coefficient Sensitivity is defined by the slope of the calibration graph 17 .Tools Determination of method characteristics Perform more point calibration Characteristics of calibration  Linearity  Sensitivity Correlation coefficient: r² > 0.

Commission Decision 2002/657/EC 18 .

• Lowest point should be the limit of quantification. • In addition. • For calibration certified standard solutions shall be used.Validation of testing methods Calibration • Calibration values should be applied within the working range. • For linear calibration functions at least five concentrations are necessary. also standard solutions have to be added to blank materials. • Test calibration functions for linearity and highlight failing linearity. analysed over all process steps. 19 .

Validation of testing methods Sensitivity (resolution) What is sensitivity?  It is the difference in an analytical concentration that corresponds to the smallest difference of a signal in a method which is still detectable.  Sensitivity can be extracted from the calibration curve or defined by using samples with different concentrations. 20 .

Validation of testing methods Linearity  Definition based on samples with varying concentrations and the calculation of the regression of results. Five standards suffice to produce a calibration curve if  linearity is o. 21 . More standards are necessary once the linearity is  unsatisfactory.  Signal and concentration do not have to be fully related.  Examine repeat samples and standards over a particular working range to experience whether a reliable line can be drawn between proof and detection limit.k.

22 .Homogeneity of variances  Characteristic values for the comparison of standard variations for differing concentration. the working range needs to be split. In case of great differences between standard deviations (in-homogeneity of variances).


Determination of method characteristics
Characteristics of the range Limit of detection Limit of quantification Characteristics of accuracy Robustness / Ruggedness

Characteristics of calibration

Selectivity or Specifity


Validation of testing methods Working range
Finding out the appropriate working range using • • different matrices varying concentrations.

Concentration range within the achievement of acceptable

accuracy and
precision is possible.

In general the working range is broader than the linear range.

Validation of testing methods

Prerequisites for calibration

 Standards must be faultless.

 Precision has to be similar throughout the entire working range.  The model function is applicable: either linear or curved.  Errors only may occur randomly within signals.  Errors have to follow the normal distribution.


sxo (Calibration function method) xLd = Limit of detection sL = Standard deviation of the blanks Sxo = Standard deviation of the calibration function b = Slope of calibration function Various conventions XLOD = Mean of blanks + 3 x SL 26 .LOD Characteristics of the range Limit of detection LOD by calculation xLOD = 3 . sL / b (Blank value method) xLOD = 4 .

LOQ Characteristics of the range Limit of quantification LOQ by calculation xLOQ = 9 . sxo (Calibration function method) xLOQ = Limit of quantification sL = Standard deviation of the blanks Sxo = Standard deviation of the calibration function b = Slope of calibration function Various conventions XLOQ = 5 or 6 or 10 x SL 27 . sL/ b (Blank value method) xLOQ = 11 .

Commission Decision 2002/657/EC 28 .

LOD Characteristics of the range Limit of detection From 2 : 1 to 5 : 1 by signal to noise ratio Limit of quantification From 5 : 1 to 10 : 1 29 .

Effect of peak shape on LOD / LOQ 30 .

Tools Determination of method characteristics Characteristics of the range Characteristics of calibration Characteristics of accuracy Robustness or Ruggedness Selectivity or Specifity 31 .

A method which is selective for an analyte is said to be specific. Selective detectors in instrumental analysis Use of antibodies Cross-reactions ? Selective media in microbiology 32 .Selectivity / Specifity Selectivity Specifity Extent to which particular analytes can be determined in complex mixtures.

No problem. Specifity • Outlines the analytical extent to which an analytical substance or substance group can be determined without interference from sample related components. usually both words are mixed … 33 .The difference between selectivity and specificity Selectivity • A selective method gives correct results for all interesting analytes whereas a specific test method gives correct results for the interesting analyte whereas other analytes might interfere each other.

Validation of testing methods Selectivity • Outlines the extent to which an analytical substance can be determined without interference from other components. • Assess selectivity while application to pure solution up to complex matrices. • Method selective to one analytical substance specific. • Document disturbances and restrictions of the method. 34 .

Tools Determination of method characteristics Characteristics of the range Characteristics of calibration Characteristics of accuracy Robustness / Ruggedness Selectivity or Specifity 35 .

• Part of the routine.Validation of testing methods Robustness  Degree of proneness of a method to conscious and unconscious alterations of a working instruction. 36 . as the method-establishing laboratory will have proved robustness before publishing a new method. • Learn from other analytical institutes.

pH. Examples: • Times within process steps • Environmental conditions (e. temperatures) • Minor process changes (e.g.g.Robustness Sensitivity degree of the test method against small deviations in experimental conditions. flow rates in HPLC) 37 .

different equipment. according to USP Ruggedness is the degree of reproducibility obtained under a variety of conditions.Ruggedness.g. etc. expressed as relative SD.: • • • • different analysts. different trade marks of reagents. e. Internal reproducibility 38 .

Tools Determination of method characteristics Characteristics of the range Characteristics of calibration Characteristics of accuracy: Correctness Uncertainty Precision (Repeatability/ Recovery) Reproducibility Selectivity or Specifity 39 Robustness or Ruggedness .

Accuracy = Precision and correctness I Exactness of an analytical method Accuracy Precision Repeatability Degree of repeatability of an analytical method Reproducibility Uncertainty Correctness / Trueness Systematic error / Correct value Recovery / Bias 40 .

Random and systematic deviations precise and correct not precise but correct precise but wrong not precise and wrong 41 .

comparison to a well characterized method) Precision Measure of the degree of repeatability.Accuracy = Precision and correctness II Characteristics of accuracy Correctness Measure of correctness. covering systematic mistakes Repeatability: one laboratory. Better: internal reproducibility Reproducibility: more than one laboratory 42 . covering systematic and non-systematic mistakes (use of CRM.

  Comparison to a reference procedure.  Fortification of a blank sample with the analytical substance.Validation of testing methods Repeatability Use at least one of the following procedures for determination:  Measurement of reference materials. Participation in a co-operative (interlaboratory) test in which the “true content” is known. 43 .

– same method. – same laboratory and – short time-span in-between the analyses. 44 . Repeatability is part of the precision with respect to repeated measurements: – same material. Precision depends on the concentration of the analyte. – same analyst.Validation of testing methods Precision  Information on accuracy of two coinciding and independent analytical results.

• Participation in interlaboratory comparisons. Make an approximation as a first approach to correctness. Purchase reference material.Validation of testing methods Correctness / Trueness Use the one of the following methods to assure correctness: • Analysis of certified reference material. 45 . If not available: • • • • Document any data that prove correctness. Use a similar method in parallel. • Comparison to a known procedure.

• List the recovery rate (in %) and the standard deviation when recovery rates are constant. • A matrix shall be free of the analytical substance. • If not the sample will be enriched with a weak concentration of the analytical sample. • Otherwise issue recovery rate as a function.Validation of testing methods Recovery • Performed by addition of the analytical substance to the matrix. • Or a simulated matrix will be used. 46 . • Recovery can be determined only when the analytical sample is available in pure form.

Validation of testing methods Frame conditions for the determination of the recovery rate The analytical substance needs to be added in that form in which it occurs in nature. The native content shall be below the determination limit. 47 . The samples requires good homogenisation.

Validation of testing methods Reproducibility internally Prove reproducibility externally  Statistically   Comparative standard deviation Replicate standard deviation  Through participation in proficiency testing schemes  or participation in interlaboratory comparisons. 48 .

Tools Determination of method characteristics Characteristics of the range  Limit of detection  Limit of quantification Characteristics of accuracy  Correctness  Uncertainty  Precision (Repeatability/ Recovery)  Reproducibility 49 Characteristics of calibration  Linearity  Sensitivity Robustness or Ruggedness Selectivity or Specifity .

Validation of testing methods Classification of the test method Distinguish between • Analysis of contaminants (e. conductivity) • Qualitative determinations • Microbiological methods 50 . food additives) • Analysis of food constituents (salt and minerals) • Methods which are determining true values • Test methods which have to be calibrated with instruments • Physical measurement (pH value. heavy metals.g. pesticides.

Validation of testing methods Parameters for validation Parameter Analysis of trace contaminants Description of test method Calibration Working range Detection limit Determination limit Recovery Repeatability Correctness Specificity Reproducibility X X X X if possible X X X X if possible if possible X X X X if possible if possible X X X if possible X if possible X X X X X X X X X X X X X X X X X X X X X X Analysis of constituents Conventional methods Methods not necessary to calibrate X X X X Physical methods Qualitative methods Bacteriological methods 51 .

RF Polarography/ Voltametry Titration Water assay Chromatography/ Capillary Electrophoresis X Spectroscopy Release TOC Enzyme Immuno Assays X X X X X X X X X X X X X X X X X X X X X X X X X Accuracy Precision X X X X X X Specificity/ Selectivity (X) Linearity X X X X X Detection/ Determination limits X X Robustness X X X 52 . AES. ICP.Validation of testing methods Common validation needs of known methods Method AAS.

Such standards are provided with guarantee on the market. It may be necessary to contract the preparation of a unique sample in particular matrix in order to utilise this procedure for method validation.Procedures for method validation The Analysis of Standard Reference Materials • • • Generally accepted method for validation. • 53 . The analyst must demonstrate that the method provides accuracy and precision.

    54 . Serious practical draw backs: Costly and time consuming Effort in co-ordination Shipping of samples and data Statistical analysis and interpretation of results. This method is rarely used for the first description of a method in the literature.Procedures for method validation The participation in PT-schemes or in a laboratory Collaborative Study The most widely accepted procedure for method validation.

Procedures for method validation Comparison with a currently accepted method • • • Usually done by one analyst or two using a split-sample. using results from the currently accepted method for verification. 55 . another procedure has to be employed for the method validation. Disagreement could also suggest that the currently accepted method is invalid. • The more samples are analysed and the wider the range of concentration the higher the credibility of the validation method. • In such case. Agreement suggests validation.

samples and cost. simple and useful but it leads to subjective results. a good start for the overall validation process. accuracy and precision. Fast. 56 . Suitable for a first approximation requiring minimal time. Using samples at known levels of analyte to demonstrate recovery. In general. manpower.Procedures for method validation The Zero-Blind Method • • • • • One analyst. If this methods fails there is no reason to proceed with further validation of the method.

57 . Comparison by the first analyst makes objectivity questionable. This method still is biased on behalf of the 1st analyst. Suitable after the zero-blind method has been successful and before additional analysts or the management will be involved. Results are compiled and compared by the 1st analyst. Samples are given to a 2nd analyst to whom levels of analyte are unknown.Procedures for method validation The Single-Blind Method • • • • • • One analyst at the start.

Only the 3rd analyst has access to these data. Most effective approach to objectiveness 58 . 3rd analyst (or administrator) compares both data from the first two analysts.Procedures for method validation The Double-Blind Method • • • • • Three analysts. 1st analyst prepares samples at known levels. 2nd analyst does the actual analysis.

Validation of testing methods Clearance of validated test procedures Quality Manager  Validation results Customer requirements 59 .

wide-spread? Risk potential for client/ company? Political decisions super-ordinated criteria Actual aim of the validation? Consideration of normative and other requirements? 60 .Validation of testing methods Decision criteria for the extent of a validation Technical feasibility and staff disposition? Analytical viewpoints facts Measuring principle. method complexity? Occurrence of the sample.

Validation of testing methods Extent of validation in analytical sciences Purpose • Suitability of the method • „Marketability“ of the method Analytical sensibility Costs Requirements from the outside 61 .

risks and technical possibilities. The validation shall be completed by a statement by the laboratory that the method is fit for the intended use. 62 .ISO/IEC 17025 states: Validation is always a balance between costs.

Validation is always a balance betwee costs. risks and technical possibilities. 63 .

64 .Do not forget: Self developed software or excel sheets have to be validated. Records for that must be available.

Muito obrigado! 65 .

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67 .Bring them together ….

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70 .

003 g Tipo B / rect 3 0.1 % Tipo B 3 0.1% 3 ucombinada = 3.7% 0.Trabajo en grupos ”Humedada en Alimentos” Valor ± [Unidades] Incertidumbre estándar [unidades] Fuente de incertidumbre Símbolo Distribución de probabilidad Divisor Calibración de la balance uEst + 0.43 % Uexpandida = 7 % 71 .4% 3 Calibración de la estufa uPip 0% Tipo B / rect Tiempo uBurInd 0% Repetibilidad uRep + 0.

Background and general aspects Why proficiency testing?  PT schemes are just one important tool in quality control and quality management.IfEP Workshop: Proficiency Testing .  Participating in Proficiency Tests  Use of certified reference material  Validation of methods QM 72 .