1. Analytical considerations
Drs. Jan Welink
Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

2. Guidance FDA Guidance for Industry ICH Guidance for industry
Bioanalytical method validation, May 2001
ICH Guidance for industry
Validation of analytical methods: definitions and terminology, June 1995
Validation of analytical procedures: methodology, November 1996

3. GCP/GLP GCP/GLP compliance
Clinical studies have to be performed under conditions complying with the principles of Good Clinical Practice, and for analytical methods and sample data handling conditions complying with the principles of Good Laboratory Practice are required.
For older studies without statement of compliance with the above mentioned principles, the assessor should rely on the quality of the submitted report.

4. Choice of method Sensitive Accurate Discriminative Precise
Method used for the determination of drugs and/or metabolites should be:
Sensitive
Accurate
Discriminative
Precise

5. Sensitivity
Method should be able to quantify the drug in the sampled specimen at least 10 % of the maximum concentration reached after dosing.
Limit of Quantification (LOQ): 1/10 Cmax

6. Discriminative
The method should be able to discriminate between the selected analyte and interfering compounds from the environment or from other compounds administered simultaneously

7. Accuracy
The method must be accurate enough to measure the true value (concentration) of the analyte in a relative small sample

8. Precision
The analytical method should be precise enough to reveal identical results when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of the biological matrix

9. Validation
To measure is to know!

10. Validation Specificity Detection limit (LOD)
Quantification limit (LOQ)
Linearity
Range
Accuracy
Precision
Robustness

11. Validation-specificity
Investigation of specificity should be conducted during the validation phase of the assay
The procedures used to demonstrate specificity should be clearly reported
Must be applied with structurally similar materials
Choices base on scientific judgements

12. Validation-specificity

13. Validation-LOD Various methods possible visual evaluation
minimum level at which the analyte can be detected reliably
signal-to noise
3:1 ratio is acceptable
standard deviation of the slope and response
LOD = 3.3 σ / S
σ = standard deviation of the response
S = slope of the calibration curve

14. Validation-LOQ Based on signal-to noise
Reliable quantification is a 10:1 ratio
Based on SD of the response and the slope
LOQ = 10 σ / S
σ = standard deviation of the response
S = slope of the calibration curve

15. World Health Organization
Validation
World Health Organization
22 April, 2017
LOQ, LOD and SNR
Limit of Quantitation
Limit of Detection
Signal to Noise Ratio
Peak B LOQ
There are no specific criteria set for the Limit of Quantitation (LOQ) and Limit of Detection (LOD) but guidance is available from specifications and pharmacopeias.
The noise is measured by running the instrument at maximum gain with no test being processed. The ripple generated is noise due to the instrument’s electronics, etc. The peak is measured relative to this noise and the ratio is calculated. This is known as the Signal to Noise Ratio (SNR).
Generally:
LOD SNR should be greater than 2:1. Peak A is acceptable for LOD but not for quantitation;
The LOD can be calculated if the standard deviation (SD) of the response (which is standard deviation of the blank) and the slope is determined:
LOD = 3.3 x SD
slope
Similarly LOQ = 10 x SD
The LOQ SNR should generally be above 10:1. Peak B is suitable for quantitation;
Precision as a percentage relative standard deviation should be % at the limit for LOQ.
Peak A LOD
noise
Baseline

16. Validation-LOD/LOQ Recommended data:
The LOD and LOQ and the method used for the LOQ should be presented
The limits should be validated by the analyses of a suitable number of samples prepared at the LOD and LOQ limits

17. Validation-linearity
Should be evaluated across the range of concentrations expected during the study
A minimum of five concentrations used in the range is recommended
The correlation coefficient, y-intercept slope of the regression and residual sum of squares should be submitted
Deviations from the regression line should be analysed for evaluating linearity

18. Validation-linearity

19. Validation-range
The specified range is derived from linearity studies and should cover the extremes of the concentrations probably reached during the study
The range should be justified in the report based on scientific information

20. Validation-accuracy
Accuracy should be assessed on samples spiked with known amounts of the analyte
Accuracy should be assessed using determinations over a minimum of 3 concentration levels (low, medium and high)
Accuracy should be reported as percent recovery from the added amount

21. Validation-accuracy
HQC
MQC
LQC

22. Validation-precision
Repeatability
concentrations covering the specified range
Intermediate precision
Like days, analysts, equipment
Reproducibility
Determined if analyses take place in separate periods

23. Validation-accuracy/precision

24. Validation-accuracy/precision
Between-day:
Intra-day:

25. Validation-accuracy/precision:
Accuracy/precision calibrators:

26. Validation-accuracy/precision
FDA
Accuracy
Precision
within-run
between-run
within-run
between-run
normally: <15%
LLOQ: <20%
normally: <15%
LLOQ: <20%

27. Validation-robustness
Robustness should be considered during development phase
Shows the reliability of the analytical method with respect to variations in the method parameters
In case variations occur they should be suitably controlled and if present adequately tested and documented

28. Validation-robustness
Typical examples:
Stability of the analytical solutions
Influence of variations of pH of the mobile phase
Influence of variations of mobile phase composition
Influence of temperature and flow rate
Extraction conditions
pH and extraction time

29. Validation-robustness

30. Validation-recovery Recovery: Extraction efficiency analytical method
consistent
precise
reproducible
Recovery:
80%
75%
91%
97%
65%
73%
Recovery:
15%
16%
13%
14%
mean: 81.1%
CV: 14.7%
mean: 14.8%
CV: 7.9%

31. Validation-stability
Stability assessed prior subject sample analysis!
Required data
Freeze and thaw stability
Short term temperature stability
Long term stability
Stock solution stability
Post preparation stability

32. Analysis clinical samples
The analytical method should be validated before the start of obtaining clinical samples.
Each analytical run should contain sufficient QC samples at the beginning, middle and end at at least 3 levels (LQC, MQC and HQC). QC QC QC QC QC QC

33. Analysis clinical samples
Acceptation or rejection of a run should be predefined before the actual start of the analysis of the clinical samples.
FDA criteria QC QC QC QC QC QC

34. Analysis clinical samples
All samples of 1 subject in 1 run
Subject sample reanalysis should be predefined before the actual start of the analysis of the clinical samples.
Reasons:
- improper sample injection
- mail function
- concentration > HLOQC
- unexpected value
- PK reason QC QC QC QC QC QC

35. Analysis clinical samples
- unexpected value
- PK reason

36. Report
All methods should be covered by adequate Standard Operating Procedures (SOP’s) for general and analysis specific procedures
Before the start of an analytical procedure an adequate study plan has to be written or be incorporated in the study protocol

37. Report
A specific detailed description of the bioanalytical method should be written
All experiments used to make claims or draw conclusions should be presented in the report
GLP compliance/inspections/audits

38. End