Introduction to Bioanalytical method validation, importance, types and parameters

1. 1
AISSMS COLLEGE OF PHARMACY, PUNE-01
Prepared by:- Guided by:-
SHIVANI K. CHAUDHARI DR. S.V. GANDHI
M. Pharmacy
21 March 2018

2. Validation
 Validation is the process of determining the
suitability of a given methodology for
providing useful analytical data.
 Validation is required for any new method to
ensure that it is capable to give reproducible
and reliable results, when used by different
operators employing the same equipment in
the same or different laboratories
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3. BIO-
ANALYTICAL
MEHOD
• Bioanalytical Method relates specifically to
determine the concentration of drug or its
metabolite or both in biological matrix such as
plasma, serum, urine , etc.
• Bioanalytical information used in human
clinical pharmacology, bioavailability (BA)
and bioequivalence (BE) studies requiring
pharmacokinetic evaluation.
• Bioanalytical method is also used for non
human pharmacology/toxicology studies
(preclinical studies).
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4. Objective
ofStudy
• To develop and validate highly specific,
reliable and cost effective chromatographic
and spectroscopic methods for determination
of drug in human plasma.
• The scope of developing and validating the
bio analytical method is to get a suitable
method which is more accurate and precise
for the analyte of interest under given set of
lab condition by using resources available.
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5. Why Validate
Bioanalytical
Methods?
• Is to demonstrate the performance and
reliability of a method and hence the
confidence can be placed on the results.
• If the results are used to support registration of
a new drug or the reformulation of an existing
one.
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6. Need of
Bioanalytical
Method
Validation
• It is essential to used well-characterized and
fully validated bioanalytical methods to yield
reliable results that can be satisfactorily
interpreted.
• It is also important to emphasize that each
bioanalytical technique has its own
characteristics, which will vary from analyte
to analyte, specific validation criteria may
need to be developed for each analyte.
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7. TYPES OF
VALIDATION
FULL
VALIDATION
PARTIAL
VALIDATION
CROSS
VALIDATION
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8. FULL
VALIDATION
• For developing and implementing
a novel bioanalytical method.
• For analysis of a new drug entity.
• For revisions to an existing method
that add metabolite quantification.
Full validation of bioanalytical methods is
important:
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9. PARTIAL
VALIDATION
• Bioanalytical method transfers between
laboratories or analysts.
• Change in analytical methodology.
• Change in anticoagulant in harvesting
biological fluid.
• Change in matrix within species.
• Change in sample processing procedures.
• Changes in instruments and/or software
platforms.
Partial validations are modifications of already
validated bioanalytical methods-
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10. CROSS
VALIDATION
 Cross-validation is a comparison of validation
parameters when two or more bioanalytical
methods are used to generate data within the
same study or across different studies.
 An example of cross-validation would be a
situation in which an original validated
bioanalytical method serves as the reference, and
the revised bioanalytical method is the
comparator. The comparisons should be done
both ways.
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12. SELECTIVITY
 The ability of the bioanalytical method to measure
and differentiate the analytes in the presence of
components that may be expected to be present.
 Selectivity is the documented demonstration of the
ability of bioanalytical procedure to
discriminate the analyte from interfering
components.
 Selectivity is evaluated by injecting extracted
blank plasma and comparing with the response of
extracted LLOQ samples processed with internal
standard.
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13. LLOQ
 Lower limit of quantification
 LLOQ is the lowest concentration of an
analyte at which the analyte can be quantified
with reliable accuracy and precision.
 The analyte response at the LLOQ should be at
least 5 times the response of a blank sample.
Mean accuracy and precision at the LLOQ
should be within ±20% of the nominal
(theoratical) concentration and not more than
20%, respectively.
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14. ACCURACY
 Closeness of determined value to the true Value.
Accuracy(%) =Measured value - True value x100
True value
 The mean value should be within ± 15% of the
theoretical value, except at LLOQ, where it
should not deviate by more than ± 20%.
 Accuracy should be measured using a minimum
of five determinations per concentration.
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15. PRECISION
 Precision is an analytical method describes
variations between the individual concentrations
determined in repeated measurements.
 It is typically measured as coefficient of
variation (%CV) or relative standard deviation.
% CV = standard deviation ×100
mean
 The acceptance criteria is = 15% CV.
 At LOQ, 20% deviation is acceptable
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17. LINEARITY
 The ability of the Bioanalytical procedure to obtain
test results that are directly proportional to the
concentration of analyte in the sample within the
range of the standard curve(calibration curve).
 For the establishment of linearity, a minimum of
five concentrations is recommended.
 The validation report should include the regression
equation and the correlation coefficient used.
 DETERMINATION- Linearity should be evaluated
by visual inspection of a plot of signals as a
function of analyte concentration or content.
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18. STABILITY
 The aim of a stability test is to detect any
degradation of the analytes of interest during
the entire period of sample collection,
processing, storing, preparing, and analysis.
 The condition under which the stability is
determined is largely dependent on the nature of
the analyte, the biological matrix, and the
anticipated time period of storage.
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19. • Freeze remove from freezer thaw at
RT for 4 hrs refreeze at -200C.
Freeze-thaw
stability in matrix
• 3 sets of samples left at RT for 12hrs.
• Stable during exposure period.
ShortTerm
Stability in matrix
• Performed at -200C.
• Observe the stability for months and record
the time period.
Long-term
stability
• Stability of stock solutions of drug.
• Stock solution can be in different state or in
different buffer concentration.
Stock solution
stability
• Kept in autosampler for 24hrs before
injection.
Post-preparative
stability
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20. CARRY
OVER
 Carry over is an alteration of the measured
concentration due to a left over analyte in a
analytical instrument used
 The carryover should be evaluated by analysing
a blank sample following the highest
concentration calibration standard.
 The response in the blank sample obtained after
measurement of highest concentration standard
should not be greater than 20% of the analyte
response at the LLOQ and 5% of the response
of internal standard.
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21. MATRIX
EFFECT
 The effect on an bio-analytical method caused
by all other components of the sample except
the specific compound to be quantified.
 Due to ion suppression/enhancement by the
others ions present in the biological matrix
which might get ionized during detection and
will give false results.
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22.  Matrix effect studied by comparing the response
of extracted samples spiked before extraction
with response of the blank matrix sample to
which analyte has been added at the same
nominal concentration just before injection.
MF= Analyte response in presence of matrix
Analyte response in absence of matrix
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23. RECOVERY
 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.
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24. REFERENCES
 Food and Drug Administration, FDA, Guidance
for Industry: Bioanalytical Method Validation,
Rockville, MD: US Department of Health and
Human Services, Food and Drug Administration,
Center for Drug Evaluation and Research, 2013.
 http://www.ema.europa.eu/ema/index.jsp?curl=p
ages/includes/document/document_detail.jsp?we
bContentId=WC500109686&mid=WC0b01ac05
8009a3dc
 Analytical method validation and instrument
performance verification by chung chow chan,
Y.C. Lee, Herman Lam, Xue-ming Zhang,
published by CBS publishers,pg. no. 105-138.
 http://www.ich.org/fileadmin/Public_Web_Site/I
CH_Products/Guidelines/Multidisciplinary/M10/
ICH_M10_Concept_paper_final_7Oct2016.pdf
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