Bioanalytical Method Validation procedures and guidelines

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Bioanalytical Method Development And
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2.  Introduction
 Objective
 Method Development
 Method Validation
 Validation Parameter
 Plan of Work
 Reference
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3.  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)

4.  LC/MS/MS is a hyphenated technique that combines
physical separation power of liquid chromatography
with detection power of Mass spectrometry.
 LC/MS/MS is a powerful technique used for many
application which have very high sensitivity and
specificity
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5. • To develop and validate highly specific, reliable and cost
effective LC-MS/MS method for determination of drug
in human plasma
• The scope of developing and validating the bioanalytical
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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Steps In Method Development
 Literature search for drugs.
 Identification of analytical techniques and optimization
 Reference standard preparation.
 Selection of internal standard
Sample pre- treatment (Extraction procedure)
 Sample storage

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Literature Search For Drugs

8.  Mode of separation
 Selection of column
 Selection of mobile phase
 Role of pH, Buffer
 Role of Temperature
 Role of flow rate
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9.  Liquid-Liquid Extraction
 Solid phase Extraction
 Reduced organic solvent consumption
 Easier collection of the total analyte fraction
 More efficient separation of interferences
 Protein precipitation
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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

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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

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Validation parameters
Specificity/selectivity
 Sensitivity
Accuracy
Precision
Linearity
Recovery
 Matrix Effect
Stability

13.  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.
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14. Accuracy
Closeness of determined value to the true
value.
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15. 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

16.  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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17.  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.
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18.  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 injection
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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

20.  literature survey
 Selection of analytical technique.
 Method Development
 Method Validation
 Compilation of data
 Result & conclusion
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21.  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.
 Peters F.T., Review: Bioanalytical method validation – How, how much and why, Department
of Experimental and Clinical Toxicology, Institute of Experimental and Clinical
Pharmacology and Toxicology, University of Saarland.
 Pranay W, Bioanalytical Method Development –Determination of Drugs in Biological Fluids
2010.
 Skoog DA, West DM, Holler FJ, Crouch SR. Fundamentals of Analytical chemistry. 18th ed.
Thomson Asia pvt Ltd. Chapter 1. The nature of analytical chemistry. 2004; 2-5 & 973-996.
 Chatwal R. G., Anand K. S. Instrumental method of chemical analysis, Himalaya Publishing
House , Mumbai, 2007, 3rd edition, pp. 2.566 – 2.587, 2.624 – 2.639, 2.272- 2.302.
 ICH, Validation of analytical procedure, International conference on Harmonization, IFPMA,
Q2B Validation of Analytical Procedures: Methodology
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