about USFDA guidance to bio analytical method validation

1. Bioanalytical Method
Development and Validation
USFDA 2018 Guidance
Presented by;-
M.PAVANKUMAR
219509
Dept of pharmaceutical analysis

2. Bio analytical Method
 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, bioavailablity (BA) and
bioequivalence (BE) studies requiring
pharmacokinetic evaluation
 Bioanalytical method is also used for non human
pharmacology/ toxicology studies (preclinical
studies)

3. Validation
What is Validation and why should it be done ?
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

4. Definition of Bioanalytical Method
Validation
 Bioanalytical method validation include all
the procedure that demonstrate that a particular
method used for quantitative measurement of
analyte in given biological matrix are reliable
and reproducible for intended use
Types of Method validation
 Full validation
Partial validation
Cross validation

5. Validation parameters
 Specificity/selectivity
 Sensitivity
 Accuracy
 Precision
 Linearity
 Recovery
 Matrix Effect
 Stability

6. Selectivity
Ability of an analytical method to differentiate
and quantify the analyte in the presence of
other components in the sample
• Selectivity is evaluated by injecting extracted
blank plasma and comparing with the response
of extracted LLOQ samples processed with
internal standard.

7. Accuracy
Closeness of determined value to the true value.
Accuracy (%) = 100 x Found value -
Theoretical value Found value / Theoretical
value Found value
The mean value should be within ± 15% of the
theoretical value, except at LLOQ, where it
should not deviate by more than ± 20%.

8. Precision
The closeness of replicate determinations of a
sample by an assay. The acceptance criteria is ≤
15% CV.
At LOQ, 20% deviation is acceptable.
% CV (precision) =100 x Standard deviation/Mean It
Includes
 Repeatability
 Intermediate Precision
 Reproducibility

9. 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

10. Matrix effect
The effect on an bio-analytical method caused by
all other components of the sample except the
specific compound to be quantified.
The difference in response between a neat
sample solution and the post-extraction spiked
sample is called the absolute matrix effect

11. Matrix effects can be resulted from
• 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.
• 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 inje

12. Stability
 Chemical stability of an analyte in a given matrix under
specific conditions for given time intervals.
 Analyte change in any respect affect the chromatographic
behavior which may complicate the method development
the following activities should be considered:
 Analyte and IS stock stability in solvent
 Short Term Stability in matrix
 Bench top stability in matrix
 Freeze-thaw stability in matrix
 In-injector stability in matrix
 Long-term stability in matrix