Bioanalytical Method Validation

Prof. Dr. Muhammad Rashedul Islam

• The validation of bioanalytical methods and the
analysis of study samples should be performed in
accordance with the principles of Good Laboratory
Practice (GLP)
• As human bioanalytical studies fall outside of the
scope of GLP the sites conducting the human studies
are not required to be monitored as part of a
national GLP compliance programme
• In addition, for clinical trials in humans the
principles of Good Clinical Practice (GCP) should be
followed
• GCP/GLP are required to assure:
– Quality and Credibility
• First, Credibility is critical
– Approval of product based on only 1 or few
studies
• Second, bioanalytical method validation:
– Method development
– Pre-study validation or Method validation
– In-study validation or Analysis of study samples
 Reference standard

• Analysis of drugs and their metabolites in a

biological matrix is carried out using samples
spiked with calibration (reference) standards
and using quality control (QC) samples.
• The purity of the reference standard used to
prepare spiked samples can affect study data.
• For this reason, an authenticated analytical
reference standard of known identity and purity
should be used to prepare solutions of known
concentrations.
• The source and lot number, expiration date,
certificates of analyses when available, and/or
internally or externally generated evidence of
identity and purity should be furnished for each
reference standard.
• If possible, the reference standard should be
identical to the analyte.
• When this is not possible, an established
chemical form (free base or acid, salt or ester)
of known purity can be used.
• Three types of reference standards are usually
used:
– (1) certified reference standards (e.g., USP
compendial standards);
– (2) commercially supplied reference
standards obtained from a reputable
commercial source;
– (3) other materials of documented purity
custom-synthesized by an analytical
laboratory or other noncommercial
establishment
 METHOD DEVELOPMENT
• A specific, detailed description of the bioanalytical method should
be written
• This can be in the form of a protocol, study plan, report, and/or
SOP
• Any method is valid as long as it is validated

Procedures that demonstrate that a method is reliable and

reproducible for the intended use.
Types:
– Full validation: first time, new drug, or addition of
metabolites
– Partial validation: modifications of a validated method
– Cross-validation: comparison between methods
 PRE-STUDY VALIDATION
 Fundamental parameters for validation

 Method used for the determination of drugs and/or

metabolites should be:

Selective Sensitive

Accurate Precise

Stable
 Selectivity

• Selectivity is the ability of an analytical method to

differentiate and quantify the analyte in the
presence of other components in the sample
• For selectivity, 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)
 Selectivity

• Potential interfering substances in a biological

matrix include endogenous matrix components,
metabolites, decomposition products, and in
the actual study, concomitant medication and
other exogenous xenobiotics
• If the method is intended to quantify more
than one analyte, each analyte should be
tested to ensure that there is no interference
 Outliers
• Reported method validation data and the
determination of accuracy and precision
should include all outliers
• However, calculations of accuracy and
precision excluding values that are statistically
determined as outliers can also be reported
 Accuracy
• The accuracy of an analytical method describes
the closeness of mean test results obtained by
the method to the true value (concentration) of
the analyte
• Accuracy is determined by replicate analysis of
samples containing known amounts of the
analyte (QC samples)
• Accuracy should be measured using a minimum
of five determinations per concentration
• A minimum of three concentrations in the
range of expected concentrations is
recommended
• The mean value should be within 15% of the
actual value except at LLOQ, where it should
not deviate by more than 20%
• The deviation of the mean from the true value
serves as the measure of accuracy
 Precision
• The precision of an analytical method describes
the closeness of individual measures of an
analyte when the procedure is applied
repeatedly to multiple aliquots of a single
homogeneous volume of biological matrix
• Precision should be measured using a minimum
of five determinations per concentration
• A minimum of three concentrations in the range
of expected concentrations is recommended
• The precision determined at each concentration
level should not exceed 15% of the coefficient
of variation (CV) except for the LLOQ, where it
should not exceed 20% of the CV.
• Precision is further subdivided into within-run,
intra-batch precision or repeatability, which
assesses precision during a single analytical run,
and between-run, inter-batch precision or
repeatability, which measures precision with
time, and may involve different analysts,
equipment, reagents, and laboratories.
Precision: intra-day and inter-day
 Recovery
• The recovery of an analyte in an assay is the
detector response obtained from an amount of
the analyte added to and extracted from the
biological matrix, compared to the detector
response obtained for the true concentration
of the pure authentic standard
• Recovery pertains to the extraction efficiency
of an analytical method within the limits of
variability
 Recovery

• Recovery of the analyte need not be 100%, but

the extent of recovery of an analyte and of the
internal standard should be consistent, precise,
and reproducible
• Recovery experiments should be performed by
comparing the analytical results for extracted
samples at three concentrations (low, medium,
and high) with unextracted standards that
represent 100% recovery.
 Calibration / Standard Curve
• A calibration (standard) curve is the relationship
between instrument response and known
concentrations of the analyte.
• A calibration curve should be generated for each
analyte in the sample.
• A sufficient number of standards should be used to
adequately define the relationship between
concentration and response. 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.
• 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
internal standard), a zero sample (matrix
sample processed with internal standard), and
six to eight non-zero samples covering the
expected range, including LLOQ.
 Lower Limit of Quantification (LLOQ)

• The lowest standard on the calibration curve

should be accepted as the limit of quantification
if the following conditions are met:
• The analyte response at the LLOQ should be at
least 5 times the response compared to blank
response.
• Analyte peak (response) should be identifiable,
discrete, and reproducible with a precision of
20% and accuracy of 80-120%.
 Dilution of samples

• The ability to dilute samples originally above

the upper limit of the standard curve should
be demonstrated by accuracy and precision
parameters in the validation
 Stability
• Drug stability in a biological fluid is a function of the
storage conditions, the chemical properties of the
drug, the matrix, and the container system.
• The stability of an analyte in a particular matrix and
container system is relevant only to that matrix and
container system and should not be extrapolated to
other matrices and container systems.
• The stability of the analyte should be established
preferably prior to sample analysis.
• Stability procedures should evaluate the
stability of the analytes during sample
collection and handling, after long-term
(frozen at the intended storage temperature)
and short-term (bench top, room
temperature) storage, and after going through
freeze and hot cycles and the analytical
process
• Conditions used in stability experiments
should reflect situations likely to be
encountered during actual sample handling
and analysis.
• The procedure should also include an evaluation
of analyte stability in stock solution.
• All stability determinations should use a set of
samples prepared from a freshly made stock
solution of the analyte in the appropriate
analyte-free, interference-free biological matrix.
• Stock solutions of the analyte for stability
evaluation should be prepared in an appropriate
solvent at known concentrations.
 APPLICATION TO
ROUTINE DRUG ANALYSIS
• Assays of all samples of an analyte in a biological
matrix should be completed within the time period
for which stability data are available.
• Samples can be analyzed with a single
determination without duplicate or replicate
analysis if the assay method has acceptable
variability.
• Where high precision and accuracy may be difficult
to achieve, duplicate or even triplicate analyses can
be performed for a better estimate of analyte.
 Calibration curve
• A calibration curve should be generated for
each analyte to assay samples in each analytical
run and should be used to calculate the
concentration of the analyte in the unknown
samples in the run.
• An analytical run can consist of QC samples,
calibration standards, and either (1) all the
processed samples to be analyzed as one batch
or (2) a batch composed of processed unknown
samples of one or more volunteers in a study.
• A matrix-based standard curve should consist of
a minimum of six standard points, excluding
blanks (either single or replicate), covering the
entire range.
• The same curve fitting, weighting, and goodness
of fit determined during pre-study validation
should be used for the standard curve within
the study.
• Changes in the response function relationship
between pre-study validation and routine run
validation indicate potential problems.
Acceptance range for calibration standards

• Matrix-based standard calibration samples: 75%, or

a minimum of six standards, when back-calculated
(including ULOQ) should fall within ±15%, except for
LLOQ, when it should be ±20% of the nominal value
– Values falling outside these limits can be discarded,
provided they do not change the established model
• Acceptance criteria for accuracy and precision
should be provided for both the intra-day and intra-
run experiment
– Acceptance criteria of pre-study validation (15%)
 Quality Controls to monitor
• It is preferable to analyze all study samples
from a subject in a single run.
• Once the analytical method has been validated
for routine use, its accuracy and precision
should be monitored regularly to ensure that
the method continues to perform satisfactorily.
• To achieve this objective, a number of QC
samples prepared separately should be
analyzed with processed test samples at
intervals based on the total number of samples.
 Acceptance criteria for QCs
• At least 67% (four of every six) QC samples should be
within 15% of their respective nominal value.
• Two of the six (33%) QC samples may be outside the
15% of their respective nominal value, but not both at
the same concentration.
• The minimum number of samples (in multiples of
three) should be at least 5% of the number of unknown
samples or six total QCs, whichever is greater.
• The data from rejected runs need not be documented,
but the fact that a run was rejected and the reason for
failure should be recorded.
Re-analysis of samples or repeat analyses
• The rationale and the reporting should be clearly documented
• Documentation should include
– the initial and repeat analysis results
– the reported result
– assay run identification
– the reason for the repeat analysis
– the requestor of the repeat analysis, and
– the manager authorizing reanalysis.
• Repeat analysis of a clinical or preclinical sample should be
performed only under a predefined SOP.
 Repeat analysis
• SOP or guideline for repeat analysis, reasons for repeating and
acceptance criteria
• Reasons for repeat analyses could include:
– Repeat analysis of clinical or preclinical samples for regulatory purposes,
– inconsistent replicate analysis,
– samples outside of the assay range,
– sample processing errors,
– equipment failure,
– poor chromatography, and
– Inconsistent pharmacokinetic data

• Reassays should be done in triplicate if sample volume allows

 DOCUMENTATION
• Documentation of successful completion of
validation studies should be provided in the assay
validation report.
• General and specific SOPs and good record
keeping are an essential part of a validated
analytical method.
• The data generated for bioanalytical method
establishment and the QCs should be documented
and available for data audit and inspection.
 Documentation to submit
• Documentation for submission to the Agency
should include:
– (1) summary information,
– (2) method development and establishment,
– (3) bioanalytical reports of the application of any
methods to routine sample analysis, and
– (4) other information applicable to method
development and establishment and/or to routine
sample analysis.