Prequalification programme:
Priority essential medicines
Training Workshop for evaluators from National
Medicines Regulatory Authorities in the East African
Community:
Evaluation of quality and interchangeability
of medicinal products.
Dar Es Salaam
United Republic of Tanzania
10 – 14 September 2007
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Training Workshop on
Evaluation of quality and interchangeability of medicinal products.
ANALYTICAL VALIDATION
Presenter: Drs. J. Welink
Senior pharmacokineticist
Medicines Evaluation Board, NL
WHO adviser
E-mail: j.welink@cbg-meb.nl
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History
Development pharmacokinetics:
computers
separation technics
analytical methods
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History
pg/ml
‘90
‘70
‘60
‘50
'30
mass spectrometry
ng/ml
liquid chromatography
gas chromatrography
μg/ml
chromatography
spectrometry
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mg/ml
Methods
ANALYTICAL METHODS
immunological
methods
LC-MS/MS
GC-MS
GLC
HPLC
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Principle
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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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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 complinace
with the above mentioned principles, the assessor
should rely on the quality of the submitted report.
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Choices of methods
 LC-MS-MS
 GC-MS
 HPLC
 GLC
 Immunological methods
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Choices of methods
 Method used for the determination of drugs
and/or metabolites should be:
Sensitive
Accurate
Discriminative
Precise
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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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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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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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Precision
The analytical method should be presice 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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Validation
To measure is to know!
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Validation
 Specificity
 Range
 Detection limit (LOD)
 Accuracy
 Quantification limit (LOQ)
 Precision
 Linearity
 Robustness
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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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Validation-specificity
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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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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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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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Validation-LOD
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Validation
LOQ, LOD and SNR
 Limit of Quantitation
 Limit of Detection
Peak B
LOQ
 Signal to Noise Ratio
Peak A
LOD
Baseline
noise
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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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Validation-linearity
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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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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 and with confidence
intervals
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Validation-accuracy
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Validation-precision

Repeatability
– concentrations covering the specified range

Intermediate precision
– Like days, analysts, equipment

Reproducibility
– Determined if analyses take place in separate periods

Recommended data
– SD, Coefficient of variations, and confidence intervals
should be reported on each type of precision
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Validation-accuracy/precision
Accuracy/precision:
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Validation-accuracy/precision
Between-day:
Intra-day:
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Validation-accuracy/precision:
Accuracy/precision calibrators:
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Validation-accuracy/precision
FDA
Accuracy
within-run
between-run
Precision
within-run
between-run
normally: <15%
normally: <15%
LLOQ: <20%
LLOQ: <20%
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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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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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Validation-robustness
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Validation-recovery
Recovery:
 Extraction efficiency analytical method
– consistent
– precise
– reproducible
Recovery:
80%
75%
91%
97%
65%
73%
mean: 81.1%
CV: 14.7%
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Recovery:
15%
16%
13%
15%
16%
14%
mean: 14.8%
CV: 7.9%
Validation-stability
Stability assessed prior sample analysis!
 Required data
–
–
–
–
–
Freeze and thaw stability
Short term temperature stability
Long term stability
Stock solution stability
Post preparation stability
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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
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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
QC
QC
QC
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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
- mailfunction
- concentration > HLOQC
- unexpected value
- PK reason
QC
QC
QC
QC
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QC
QC
Analysis clinical samples
- unexpected value
- PK reason
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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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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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Report
The following data are required on the report:
1) Author(s) and their affiliation
2) Name of the institute or company where the
investigations have been performed.
3) Date of publication analytical study
4) Identification number of the report.
N.B. The report should preferably be printed on original
marked paper of the applicant or of the institute where the
analysis has been performed.
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Report
* For which compounds are the samples analysed (active
substance, active and/or quantitatively important metabolite)
* Sample pre-treatment and extraction.
* Analytical method is used.
* Source of the analytical method
- references from literature
- modifications in the procedure.
* Validation of the analytical method
- minimal detectable concentration, stability, reproducibility
- linearity, precision, accuracy, selectivity, sensitivity
- inter- and intraday variability
All individual measurements have to be presented in the report!
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Report
Analysis of subject samples in a separate report
* Reference to validation report
* Handling samples
* Set up analytical run
* Within study validation results
* Re-analysis
* Chromatograms
* Identification results
All individual measurements have to be presented in the report!
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Example
Accuracy/precision:
normally: <15%
LLOQ: <20%
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End
Be organised!
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