Analytical considerations
Drs. Jan Welink
Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Guidance
 FDA 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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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.
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Choice of method
 Method used for the determination of
drugs and/or metabolites should be:
Sensitive
Accurate
Discriminative
Precise
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Discriminative
The method should be able to
discriminate between the selected analyte
and interfering compounds from the
environment or from other compounds
administered simultaneously
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Accuracy
The method must be accurate enough to
measure the true value (concentration) of
the analyte in a relative small sample
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation
To measure is to know!
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation
 Specificity
 Range
 Detection limit (LOD)
 Accuracy
 Quantification limit (LOQ)
 Precision
 Linearity
 Robustness
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-specificity
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation
LOQ, LOD and SNR
 Limit of Quantitation
 Limit of Detection
Peak B
LOQ
 Signal to Noise Ratio
Peak A
LOD
Baseline
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-linearity
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-accuracy
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-precision
 Repeatability
– concentrations covering the specified range
 Intermediate precision
– Like days, analysts, equipment
 Reproducibility
– Determined if analyses take place in separate
periods
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-accuracy/precision
Accuracy/precision:
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-accuracy/precision
Between-day:
Intra-day:
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-accuracy/precision:
Accuracy/precision calibrators:
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-accuracy/precision
FDA
Accuracy
within-run
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between-run
Precision
within-run
between-run
normally: <15%
normally: <15%
LLOQ: <20%
LLOQ: <20%
Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-robustness
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Validation-recovery
Recovery:
 Extraction efficiency analytical method
– consistent
– precise
– reproducible
Recovery:
80%
75%
91%
97%
65%
73%
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mean: 81.1%
CV: 14.7%
Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
Recovery:
15%
16%
13%
15%
16%
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mean: 14.8%
CV: 7.9%
Validation-stability
Stability assessed prior subject sample analysis!
 Required data
–
–
–
–
–
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Freeze and thaw stability
Short term temperature stability
Long term stability
Stock solution stability
Post preparation stability
Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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QC
QC
QC
Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
QC
QC
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
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QC
QC
QC
Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
QC
QC
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
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QC
QC
QC
Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
QC
QC
Analysis clinical samples
- unexpected value
- PK reason
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
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
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
End
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009