1. Dr. Michael Scheutwinkel
Validation by
Dr. Michael Scheutwinkel
International Federation for Consulting GmbH IFC

2. Governmental
regulations
Books ….
Recommendations
of associations

3. References 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)

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

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

7. Definition in clause 5.4.5.1 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.
….fit for purpose

8. 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?

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

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

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

12. When should be validated?
Changes in established methods
New method developed for a particular problem
Established method used in different laboratories, 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.g. a rapid new test against a standard method)

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

14. Description of test method (clause 5.4.4)
Identification / Scope
Parameters in which matrix
Reagents and materials, CRMs
Equipment and environmental
conditions
Calibration
Sampling and
sample preparation
Test performance
including safety instructions
Quality assurance
Documentation , reporting
evaluation criteria
Uncertainty or procedure for
its estimation

15. Please show us the way…

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

17. Determination of method characteristics
Tools
Determination of method characteristics
Perform more
point calibration
Correlation
coefficient:
r² > 0.99 ??
Characteristics
of calibration
Linearity
Sensitivity
Linearity is defined by the
correlation coefficient
Sensitivity is defined by the
slope of the calibration graph

18. Commission Decision 2002/657/EC

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

20. Sensitivity (resolution)
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.

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

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

23. Limit of quantification
Tools
Determination of method characteristics
Characteristics
of the range
Limit of detection
Limit of quantification
Characteristics
of calibration
Characteristics
of accuracy
Robustness /
Ruggedness
Selectivity or
Specifity

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

25. Prerequisites for calibration
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.

26. Characteristics of the range
LOD
Characteristics of the range
Limit of detection by
calculation
LOD
xLOD = 3 . sL / b (Blank value method)
xLOD = 4 . 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

27. Limit of quantification
LOQ
Characteristics of the range by
calculation
Limit of quantification
LOQ
xLOQ = 9 . sL/ b (Blank value method)
xLOQ = 11 . 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

28. Commission Decision 2002/657/EC

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

30. Effect of peak shape on LOD / LOQ

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

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

33. No problem, usually both words are mixed …
The difference between selectivity and specificity
Selectivity
Specifity
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.
Outlines the analytical extent to which an analytical substance or substance group can be determined without interference from sample related components.
No problem, usually both words are mixed …

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

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

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

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

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

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

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

41. Random and systematic deviations

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

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

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

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

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

47. Frame conditions for the determination of the recovery rate
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 samples requires good homogenisation.
The native content shall be below the determination limit.

48. Prove reproducibility
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.

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

50. Classification of the test method
Validation of testing methods
Classification of the test method
Distinguish between
Analysis of contaminants (e.g. pesticides, heavy metals,
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, conductivity)
Qualitative determinations
Microbiological methods

51. Parameters for validation
Validation of testing methods
Parameters for validation

52. Common validation needs of known methods
Validation of testing methods
Common validation needs of known methods

53. The Analysis of Standard Reference Materials
Procedures for method validation
The Analysis of Standard Reference Materials
Generally accepted method for validation.
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.
The analyst must demonstrate that the method provides accuracy and precision.

54. The participation in PT-schemes or in a laboratory Collaborative Study
Procedures for method validation
The participation in PT-schemes or in a laboratory Collaborative Study
The most widely accepted procedure for method validation.
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.

55. Comparison with a currently accepted method
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.
Agreement suggests validation.
Disagreement could also suggest that the currently accepted method is invalid.
In such case, another procedure has to be employed for the method validation.
The more samples are analysed and the wider the range of concentration the higher the credibility of the validation method.

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

57. The Single-Blind Method
Procedures for method validation
The Single-Blind Method
One analyst at the start.
Samples are given to a 2nd analyst to whom levels of analyte are unknown.
Results are compiled and compared by the 1st analyst.
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.

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

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

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

61. Extent of validation in analytical sciences
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

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

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

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

65. Muito
obrigado!

67. Bring them
together ….

71. Trabajo en grupos ”Humedada en Alimentos”
Fuente de incertidumbre
Símbolo
Valor ± [Unidades]
Distribución de probabilidad
Divisor
Incertidum-bre estándar
[unidades]
Calibración de la balance
uEst g
Tipo B / rect
0.7% %
Calibración de la estufa
uPip
0 %
Tiempo
uBurInd
Repetibilidad
uRep
+ 0.1 %
Tipo B
0.1%
ucombinada = 3.43 %
Uexpandida = 7 %

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