3 - 4 Bioanalytical Method Validation
3 - 4 Bioanalytical Method Validation
3 - 4 Bioanalytical Method Validation
UNIVERSITAS ANDALAS
Bioanalytical
Method
Validation
DR. Harrizul Rivai, M.S.
Lektor Kepala Kimia Farmasi
Fakultas Farmasi
Universitas Andalas
Guidance for Industry : Bioanalytical Method Validation,
U.S.Department of Health and Human Services, Food and Drug
Administration, Center for Drug Evaluation and Research (CDER),
Center for Veterinary Medicine (CVM), September 2013,
Biopharmaceutics, Revision 1
BACKGROUND
Selective, sensitive, and validated analytical
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BACKGROUND
Fundamental parameters for this validation
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BACKGROUND
The following define and characterize the
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BACKGROUND
Full Validation
Full validation of bioanalytical methods is
important:
During development and implementation of
a novel bioanalytical method.
For analysis of a new drug entity.
For revisions to an existing method that add
metabolite quantification
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BACKGROUND
Partial Validation
Partial validations evaluate modifications of
already validated bioanalytical methods.
Partial validation can range from as little as
one intra-assay accuracy and precision
determination to a nearly full validation.
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BACKGROUND
Typical bioanalytical method modifications or changes that fall into this
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BACKGROUND
Cross-Validation
Cross-validation is a comparison of validation
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BACKGROUND
When sample analyses within a single study are
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VALIDATION OF
CHROMATOGRAPHIC METHODS
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A. Reference Standards
Analysis of drugs and their metabolites in a biological matrix
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A. Reference Standards
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B. Bioanalytical Method
Development and Validation
A specific, detailed, written description of the
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B. Bioanalytical Method
Development and Validation
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B. Bioanalytical Method
Development and Validation
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1. Selectivity
Selectivity is the ability of an analytical method to
differentiate and quantify the analyte in the
presence of other components in the sample.
Evidence should be provided that the substance
quantified is the intended analyte.
Analyses of blank samples of the appropriate
biological matrix (plasma, urine, or other matrix)
should be obtained from at least six sources.
Each blank sample should be tested for
interference, and selectivity should be ensured at
the lower limit of quantification (LLOQ).
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1. Selectivity
Potential interfering substances in a biological
matrix include:
endogenous matrix components;
metabolites;
decomposition products; and,
in the actual study, concomitant medication
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3. Calibration Curve
A calibration (standard) curve is the relationship between
instrument response and known concentrations of the
analyte.
The relationship between response and concentration
should be continuous and reproducible.
A calibration curve should be generated for each analyte in
the sample.
The calibration standards can contain more than one
analyte.
A calibration curve should be prepared in the same
biological matrix as the samples in the intended study by
spiking the matrix with known concentrations of the
analyte.
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3. Calibration Curve
In rare cases, matrices may be difficult to obtain (e.g.,
cerebrospinal fluid).
In such cases, calibration curves constructed in surrogate
matrices should be justified.
Concentrations of standards should be chosen on the basis of the
concentration range expected in a particular study.
A calibration curve should consist of
a blank sample (matrix sample processed without analyte or
internal standard),
a zero sample (matrix sample processed without analyte but
with internal standard), and
at least six non-zero samples (matrix samples processed with
analyte and internal standard) covering the expected range,
including LLOQ.
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3. Calibration Curve
Method validation experiments should include
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3. Calibration Curve
a. Lower Limit of Quantification (LLOQ)
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3. Calibration Curve
b. Upper Limit of Quantification (ULOQ)
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3. Calibration Curve
c.Calibration Curve/Standard Curve/Concentration-Response
.The simplest model that adequately describes the
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3. Calibration Curve
d.Quality Control Samples (QCs)
.At least three concentrations of QCs in duplicate should be incorporated into each
run as follows: one within three times the LLOQ (low QC), one in the midrange
(middle QC), and one approaching the high end (high QC) of the range of the
expected study concentrations.
.The QCs provide the basis of accepting or rejecting the run. At least 67% (e.g., at
least four out of six) of the QCs concentration results shouldbe within 15% of their
respective nominal (theoretical) values. At least 50% of QCs at each level should be
within 15% of their nominal concentrations. A confidence interval approach yielding
comparable accuracy and precision in the run is an appropriate alternative.
.The minimum number of QCs should be atleast 5% of the number of unknown
samples or six total QCs, whichever is greater.
.It is recommended that calibration standards and QCs be prepared from separate
stock solutions. However, standards and QCs can be prepared from the same spiking
stock solution, provided the stability and accuracy of the stock solution have been
verified. A single source of blank matrix may also be used, provided absence of
matrix effects on extraction recovery and detection has been verified. At least one
demonstration of precision and accuracy of calibrators and QCs prepared from
separate stock solutions is expected.
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3. Calibration Curve
Acceptance/rejection criteria for spiked,
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4. Sensitivity
Sensitivityis defined as the lowest analyte
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5. Reproducibility
Reproducibility of the method is assessed by
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6. Stability
The chemical stability of an analyte in a
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6. Stability
Drug stability in a biological fluid is a function
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6. Stability
Stability testing should evaluate the stability of the
analytes during sample collection and handling, after longterm (frozen atthe intended storage temperature) and
short-term (bench top, room temperature) storage, and
after freeze and thaw cycles and the analytical process.
Conditions used in stability experiments should reflect
situations likely to be encountered during actual sample
handling and analysis.
If, during sample analysis for a study, storage conditions
changed and/or exceeded the sample storage conditions
evaluated during method validation, stability should be
established under these new conditions.
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6. Stability
The procedure should also include an evaluation of analyte
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6. Stability
a. Freeze and Thaw Stability
During freeze/thaw stability evaluations, the
freezing and thawing of stability samples
should mimic the intended sample handling
conditions to be used during sample analysis.
Stability should be assessed for a minimum
of three freeze-thaw cycles.
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6. Stability
b. Bench-Top Stability
Bench top stability experiments should be
designed and conducted to cover the
laboratory handling conditions that are
expected for study samples.
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6. Stability
c. Long-Term Stability
The storage time in a long-term stability
evaluation should equal or exceed the time
between the date of first sample collection
and the date of last sample analysis.
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6. Stability
d. Stock Solution Stability
The stability of stock solutions of drug and
internal standard should be evaluated.
When the stock solution exists in a different
state (solution vs. solid) or in a different
buffer composition (generally the case for
macromolecules) from the certified reference
standard, the stability data on this stock
solution should be generated to justify the
duration of stock solution storage stability.
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6. Stability
e. Processed Sample Stability
The stability of processed samples, including
the resident time in the autosampler, should
be determined.
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Terima Kasih
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