Validation of System

1. Dr. Muhammad Shahid
Associate Professor
Department of Biochemistry, UAF.

2. METHOD VALIDATION
It is set of parameters/process used to confirm that
the analytical procedure employed for a specific test
is suitable for its intended use.
Generation of data
Well defined & fully validated
Method does- intended to do
Quantitative measurement
Reliable and reproducible

3. Analytical method validation
Reliable and reproducible for the intended use.
Fundamental parameters
• Accuracy
• Precision
• Selectivity
• Sensitivity
• Linearity
• Stability

4. Why Validation?
 Documenting
 Performance characteristics
 Acceptability of data – criteria
 Methods are Modified
 Modifications should be validated
 Suitable performance

5. Validation
• Full Validation
• Partial Validation
• Cross-Validation
• Pre-study Validation
• In-study Validation

6. Full Validation
• Developing and implementing
a bioanalytical method for the first time.
• A new compound/drug entity.
• Revised assay is important if metabolites
are added to an existing assay for
quantification

7. Partial Validation
one intra-assay accuracy and precision determination
to a nearly full validation.
Typical bioanalytical method changes :
• Method transfers between laboratories or analysts
• Analytical methodology (detection systems)
• Anticoagulant in harvesting biological fluid
• Matrix within species (human plasma to urine)

8. • Sample processing procedures
• Species within matrix (e.g. rat plasma to mouse )
• Relevant concentration range
• Instruments and/or software platforms
• Limited sample volume (e.g. pediatric study)
• Rare matrices
• Selectivity demonstration of an analyte in the
presence of concomitant medications
• Selectivity demonstration of an analyte in the
presence of specific metabolites

9. Cross-Validation
Comparison of validation parameters two or more
analytical methods within the same study or across
different studies.
An original method (reference)
Revised analytical method (comparator).
Interlaboratory reliability
within a single study more than one site or lab,
spiked matrix standards and subject samples
• Different analytical techniques
(LC-MS vs. ELISA) in different studies.

10. In Study Validation
Application of validated method for routine analysis
Accuracy & precision should be monitored
Method works satisfactorily
QC sample in duplicate at 3 concentration
Low , Medium & High QCs
Should be incorporated in each assay run

11. Pre-study Validation
Analytical method development and documentation
Each Biological Matrix and Chemical species
• Selectivity
• Calibration curve & Linearity
• Accuracy, Precision, Recovery
• Stability of analyte
• Acceptance criteria
• Documentation

12. Analytical laboratory conducting
• Good Laboratory Practices (GLPs)
• Sound principles of quality assurance
• Standard Operating Procedures (SOPs)
QC & Assurance
All aspects of analysis
Time of sample collection, reaches, results report

13. SOPs
• Record keeping, security and chain of sample
custody
• Sample preparation
• Analytical tools
Methods
Reagents
Equipment
Instrumentation
Procedures for quality control
Verification of results

14. Process of Analytical method
Developed, validated and used
divided into
• Reference standard preparation
• Method development - assay procedure
• Routine analysis and acceptance criteria for
analytical run and/or batch ( In Study
validation)

15. REFERENCE STANDARD
Highly purified compound, well characterized
To provide accurate data
Quality and purity
Types of reference standards
Chemical
Nuclidic, radiolabel purity & chemical purity

16. Chemical reference standards
1. Certified reference standards (USP compendial
(standards; No need characterization)
2. Commercial
3.Other materials of documented purity
(identity, strength, quality and purity)

17. REFERENCE STANDARD
Source
Lot number
Expiration date
Certificates of analyses when available
Internally or externally generated evidence
of identity and purity

18. For quantitation.
• External standards
• internal standards
External standards
• Analyzed on a separate chromatogram from
the sample
• comparison of the peak area/height
(HPLC or GC) or spot intensity (TLC) of the sample
to that of a reference standard of the analyte
of interest.

19. Internal standard
• Known purity
• No interference in the analysis
• Added to the sample mixture.
Response ratio of
Compound of interest to IS vs
reference standard (HPLC or GC).
1. Complex sample preparation procedures,
(multiple extractions)
2. Low concentration sample (sensitivity)
3. Wide range of concentrations expected

20. Method development & establishment phase
• Selectivity
• Calibration curve & Linearity
• Accuracy, Precision & Recovery
• Stability of analyte
• Acceptance criteria
• Documentation

21. Specificity/ Selectivity
Ability to assess analyte in the presence
of endogenous compounds
Ability to separate analyte from degradation
products, metabolites and co-administerded drugs

22. Selectivity
Differentiate and quantify the analyte in the
presence of other components.
Blank samples - 6 sources.
No interference- LLOQ
If more than one analyte, no interference
Reject Blank with significant interference
> 10 % blank show interference
additional blanks, > 10 % still show interference
Modify method to eliminate interference

23. Calibration/Standard Curve
Relationship b/w instrument response & concentrations
 Each analyte- same biological matrix
 A blank sample
(matrix sample processed without IS)
 A zero sample (matrix sample processed with IS)
 5 minimum standards (including LLOQ)
Expected concentration range in the study

24. Concentration-Response
• Simplest model ,Concentration-response relationship
• Selection of weighting and use of a complex
Regression equation should be justified.
Conditions to be met
 20% deviation of the LLOQ
 15% deviation of standards other than LLOQ
 4/ 6 non-zero standards meet the above criteria
 LLOQ and the highest calibration standard
 0.95 or more correlation coefficient

25. Sensitivity/Lower limit of detection ( LOD)
Smallest conc. distinguishable from noise level
Detected only, not quantified
Lower Limit of Quantification (LLOQ)
 Twice the response of LOD
 Lowest standard on the Calibration curve
 5 times the response compared to blank response
 Identifiable, discrete, and reproducible with
 a precision of 20% and accuracy of 80-120%

26. Quality Control Samples
1. Low QC ( < 3X LLOQ)
2. Medium QC ( midway)
3. High QC ( 75-90% highest standard)
Intra- & inter-day precision, accuracy, recovery
& stability studies

27. Accuracy, Precision
Determines the error
Primary criteria for Quality
Accuracy ( Trueness)
Closeness of test results to the true value
 3 QCs concentrations in range of calibration curve
 3 determinations per concentration of QCs
 Deviation within 15% of the actual value
 Should not deviate by > 20% at LLOQ

28. Precision
Closeness of individual measures of an analyte
procedure is applied repeatedly to multiple aliquots
• 3 QCs concentrations in calibration range.
• 3 determinations per QC concentration.
• Should not exceed 15% of CV
• LLOQ should not exceed 20% of CV.

29. Precision
• Within-run
intra-batch precision or repeatability
• Between-run
inter-batch precision or repeatability
time, different analysts, equipment ,
reagents, and laboratories.

30. Recovery
Detector response about analyte added to and
extracted from the biological matrix
Compared
to the true concentration of standard.
 Extraction efficiency need not be 100%
 Extent of recovery of an analyte and IS
Consistent, precise and reproducible
 Compare 3 conc. (low, medium, and high QCs)
with unextracted standards

31. Stability
Storage conditions, Chemical properties of the drug
Matrix & Container system
1. Freeze and Thaw Stability
2. Short-Term Temperature Stability
3. Long-Term Stability
4. Stock Solution Stability
5. Post-Preparative Stability

32. 1. Freeze and Thaw Stability
• 3 freeze and thaw cycles
• 3 LQC and HQC stored at the intended storage
temperature for 24 hours, thawed unassisted at
room temperature.
• Again refrozen for 12 to 24 hours, 2 times more
If unstable,frozen at -70°C during 3 freeze/thaw

33. 2. Short-Term Temperature Stability
Three LQC and HQC
4 to 24 hours ( room temp) & analyzed
3. Long-Term Stability
• Should exceed the time between
the date of first and last sample analysis.
• 3 Low and High QCs compared
with 1st
day long term stability testing

34. 4. Stock Solution Stability
Analyte , Room temperature - 6 hours.
Refrigerated or frozen - relevant period,
Stability should be documented.
Desired storage time
Instrument response - fresh solutions.

35. 5. Post-Preparative Stability
• Stability of processed samples
• Resident time in the auto sampler
• Analyte
• Anticipated run time for the batch size
original calibration standards.

36. Ruggedness/Reproducibility
Studying the eventual effect of different sets of conditions
on the method ( cross validation).
How? Multiple chemists in multiple labs run samples.
Results should be reproducible and can be compared to
method precision.
Result – Samples were run in 3 labs by 3 chemists on 3
different instruments.
IF the results were nearly 100%
Then the method showed Raggedness

37. Robustness
A measure of the analytical procedure’s capability to remain unaffected by
small but deliberate variations
Should be performed during development of the analytical procedure and the
data discussed and / or submitted.
Effect observed, representative instrument output should be submitted.
Conditions are
pH
Buffer Concentration
Temperature
% of Organic part of Mobile phase
Column lot/ type

38. Documentation
Validity- established & verified
Assay Validation Report
SOP & Good record keeping (Essential part)
Data should be documented ( Note book)
Protocols & SOPs : signed & dated
Regularly updated, available for Audit & inspection

39. Documentation
• Analytical method
• Stability studies & supporting data
• Selectivity
• Accuracy, Precision, Recovery
• Linearity & LOQ
(equations and weighting functions)
• Relevant data

40. Summary information
Method development & establishment
Analytical reports of routine sample analysis
Other information applicable

41. Validation though
Tedious but solves most post analytical problems
Quality of data
Consequences of invalid methods
Amount of time & resources exceeds
Wend your way efficiently through the validation maze
& eliminate many of the problems common to
inadequately validated analytical methods