over view about bioanalytical method development and validation

1. A OVERVIEW ON BIOANALYTICAL
METHOD DEVELOPMENT AND
VALIDATION
presented by
prashik sudhir shimpi
M.Pharm
R.C. Patel institute of
pharmaceutical Education and
Research,shirpur
1

2. Contents
• Introduction
• Bioanalytical methods
• Chromatographic methods
• Method development
• Bioanalytical method validation
• Conclusion
• References
2

3. introduction
• In this methods determine the drugs in biological fluid are becoming
increasingly important for the study of bioavailability, bioequivalence (BE)
Pharmacokinetics (PK) studies, quantitative evaluation of drugs,
concentration and their metabolites, new drug development, research in
basic biomedical and pharmaceutical sciences and therapeutic drug
monitoring etc.High pressure liquid chromatography (HPLC) most widely
applied analytical techniques because of its highly selective,and high
reliability.especially in pharmaceutical,environmental,food
department,forensic and clinical department.
• The importance of integrating pharmacokinetics, pharmacodynamics and
toxicokinetics in drug development has been emphasized in recent publi-
cations .
• The official test methods that result from these processes are used by
quality control laboratories to ensure the identity, purity, potency and
performance of drug products 1 and includes all the procedures
demonstrating particular method used for quantitative measurement of
analytes in a given biological matrix, such as blood, plasma, serum, or
urine, reliable and reproducible for the intended use
3

4. Bioanalytical methods
• some of the following bioanalytical method:
• Extraction method
• Protein precipitation
• Chromatographic method
• Ligand binding assay(LBA)
4

5. Extraction method
• LIQUID –LIQUID EXTRACTION
• It is based on the principles of difference solubility and partitioning
equilibrium of analyte molecules between aqueous (the sample) and
the organic phases. Liquid-liquid extraction generally involves the
extraction of a substance from one liquid phase to additional liquid
phase.Nowadays liquid extraction replaced with advanced and
improved methods like liquid phase micro extraction and supported
membrane extraction ,single drop liquid phase microextraction.
• Although more and more replaced by solid phase extraction,
liquid–liquid extraction (LLE) is still a widely usedmethod for sample
cleanup in bioanalysis .
• The first consideration to be made is the choice of the extraction
solvent, either a pure solvent or a mixture of solvents.
5

6. (A) Homogenous liquid – liquid extraction. (B) dispersive liquid –liquid
microextraction,and general liquid –liquid extraction. 6

7. • SOLID PHASE EXTRACTION
• In solid phase extraction the analyte is retained on solid phase sorbent while
sample passes through it, followed by elution of analyte with appropriate solvent. A
typical SPE sorbent consist of 40-60µm silica particles to which has been bonded a
hydrocarbon phase. This bonding is achieved by reaction of chlorosilane with the
hydroxyl group of silica gel to form silicon-oxygen-silicon link.Solid phase extraction
is particularly suitable for polar compounds that would otherwise tend to remain in
the aqueous phase. The method is also useful for amphoteric compounds that
cannot be extracted easily from water. Factors governing the adsorption and elution
of drug from the resin column include solvent polarity, flow rate of the solvent
through the column, and the degree of contact the solvent has with the resin beds.
Biological samples can be prepared for clean up by passing the sample through the
resin bed where drug components are adsorbed and finally eluted with an
appropriate solvent.
• Universal sorbent for acidic, basic and neutral compounds Waters OASIS HLB
(hydrophilic lipophilic balance) is water wettable reverse phase sorbent. It is made
from balanced ratio of two monomer the hydrophilic N-vinyl pyrrolidine and
lipophilic divinyl benzene. These sorbents are resistant to pH extremities, show
extraordinary retention of polar compounds, three times relative retention capacity
as compared to traditional silica based SPE sorbent like C-18. In the adsorption
process the hydrophobic portion of the solute . adsorbed on the resin while the
hydrophilic portion of the solute remain in the aqueous phase. The sample is passed
through the resin bed where drug adsorbed and finally eluted with a suitable
solvent.
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8. Fig.2 Solid Phase Extraction
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9. • Advantages of solid phase extraction:
Effective in selective removal of interferences enabling
selective sensitive and robust LCMS/MS. SPE enables the
enrichment of selected analytes without concentrating
interference. SPE technology combined with robotic
automation makes not only cost effective but also a time
efficient sample preparation technique which improves
analytical system performance by introducing analyte in
a MS compatible solvent. Extending the analytical
column life, reduced system maintenance, minimizing ion
suppression while improving signal response
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10. Protein precipitation
• Protein precipitation is widely used in routinely analysis to remove
proteins. Precipitation can be induced by the addition of an organic
modernizer, a salt or by changing the ph which influence the solubility
of the proteins. The samples are centrifuged and the supernatant can
be inserted into the HPLC system or be evaporated to dryness and
dissolved in a suitable solvent. A concentration of the sample is then
achieved. There are some aids with precipitation method as clean-up
technique compared to SPE . It is less time-consuming, little amounts
of organic modifier or other solvents are used. But there are also
disadvantages; the samples often contain protein particles and it is a
no-selective sample cleanup method, there is a risk that endogenous
compounds or other drugs may restrict in the reversed phase-HPLC -
system. However, the protein precipitation technique is often
combined with SPE to produce clean extract. Methanol is generally
favored solvent among the organic solvents as it can produce clear
supernatant which is appropriate for direct addition into HPLC. Salts
are other alternative to acid organic solvent precipitation. This
technique is called as salt induced precipitation. As the salt
concentration of a solution is increased, proteins aggregate and
precipitate from the solution .
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11. Chromatographic method
Reference standards
• Analysis of drugs and their metabolites in biological fluids is performed
using calibration Standards and quality control samples (QCs) spiked with
reference standards. The purity of the reference standard used to prepare
spiked samples can affect study data. For this reason, Authenticated
analytical reference standards of known identity and purity must be used
to prepare solutions of known concentrations. If possible, the reference
standard should be identical to the analyte. When this is not possible, a
predictable chemical form (free base or acid, salt or ester) of known purity
can be used.
• Three types of reference standards are usually used:
• • Certified reference standards (e.g., USP compendial standards).
• • Commercially-supplied reference standards obtained from a reputable
commercial source.
• • Other materials of documented purity custom-synthesized by an
analytical laboratory or other noncommercial establishment.
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12. Method development
• Bioanalytical method development is the process of
making a procedure to unknown compound or novel
compound be identified and measured in a matrix. A
compound can often be measured by several methods
and the choice of analytical method involves, that is,
chemical properties of the analyte, concentrations,
sample matrix, cost of the analysis method and
instruments, speed and time of the analysis,
quantitative or qualitative measurement, precision and
necessary equipment. Method development includes
sample preparation sampling, separation, detection
and evaluation of the results and finally conclusion
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13. Continue..
Sample collection and preparation
• The living media that contain the analyte are usually blood, plasma, urine,
serum, etc. Blood is usually collected from human volunteers/ subjects by
vein puncture with a hypodermic syringe up to 5-7 ml. The venous blood is
withdrawn into tubes with an anticoagulant, generally
ethylenediaminetetraacetic acid, heparin is used. Plasma is obtained by
centrifugation at 4000 rpm for 15 minutes. Around 30-50% of the volume
is collected. The aim of sample preparation is to clean up the sample
before analysis. Material in biological samples that can affect with
analysis, the chromatographic column or the detector includes
endogenous macromolecules, proteins, salts, small molecules, and
metabolic by products. The sample preparation is also to conversation the
analyte from the biological matrix into a solvent suitable for instillation
into the chromatographic system. General methods for sample
preparation such as liquid/liquid extraction, solid-phase extraction (SPE)
and protein precipitation, chromatography, and ligand binding assay (LBA)
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14. Plan for the Method development
• Stock solution preparation and tuning of analyte.
• Selection and optimization of Chromatographic
conditions (i.e. column, mobile phase)
• Selection and tuning of Internal standard
• Checking reproducibility of the method
(trial Precision and Accuracy batches)
• Selection and optimization of extaction method.
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15. Bioanalytical method validation
• Method validation can be defined (as per ICH)
“Establishing documented evidences, which provides a
high degree of assurance that a specific method or
activity will consistently produce a desired result or
product meeting its predetermined specifications and
quality characteristics”. (ICH, 1994) Method validation
is an integral part of method development; it is the
process of demonstrating that analytical procedures
are suitable for their intended use and that they
support the identity, quality, purity and potency of the
drug substances and drug products. Simply method
validation is the process of proving that an analytical
method is acceptable for its intended purpose.
15

16. Continue..
• Selective and sensitive analytical methods for the
quantitative evaluation of drug and their
metabolites (analytes) are critical for the
successful conduct preclinical and/or
biopharmaceuticals and pharmacology studies.
Bioanalytical method validation includes all of the
procedures that demonstrate that a particular
method used for quantitative measurement of
analytes in a given biological matrix, such as
blood, plasma, serum and urine is reliable and
reproducible for the intended use.
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17. Continue..
• Types of method validation
1. Full Validation
• Full validation is important when
developing and implementing a bioanalytical
method for the first time.
• Full validation is important for a new drug
entity. A Full validation of the revised assay is
important if metabolites are added to an
existing assay for quantification.
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18. 2. Partial Validation
Partial validations are 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. Typical bioanalytical method changes that
fall into this category include, but are not limited to
• Bioanalytical method transfers between
laboratories or analysts
• Change in analytical methodology
(e.g., change in detection systems)
• Change in anticoagulant in harvesting biological fluid
• Change in matrix within species
(e.g., human plasma to human urine) 18

19. Continue..
• Change in sample processing procedures
Change in species within matrix (e.g., rat
plasma to mouse plasma)
• Change in relevant concentration range
• Limited sample volume (e.g., pediatric study)
• Selectivity demonstration of an analyte in the
presence of concomitant medications or of
specific metabolites.
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20. Continue..
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 where an original validated bioanalytical method serves as the reference and the revised
bioanalytical method is the comparator. When sample analyses within a single study are conducted at more
than one site or more than one laboratory, cross-validation with spiked matrix standards and subject samples
should be conducted at each site or laboratory to establish interlaboratory reliability. Cross-validation should
also be considered when data generated using different analytical techniques (e.g., LC-MS-MS vs.ELISA4) in
different studies are included in a regulatory submission. The bioanalytical method for human bioavailability,
bioequivalence, pharmacokinetics and drug interaction studies must meet the criteria in 21 CFR 320.29. The
analytical laboratory should have a written set of standard operating procedures (SOPs) to ensure a complete
system of quality control and assurance. The SOPs should cover all aspects of analysis from the time the
sample is collected and reaches the laboratory until the results of the analysis are reported. The SOPs also
should include record keeping, security and chain of sample custody, sample preparation, and analytical tools
such as methods, reagents, equipment, instrumentation, and procedures for quality control and verification
of results. (Shah Vinod P et al. 2006) Each method developed is validated for the following fundamental
parameters:
• Selectivity
• Sensitivity
• Linearity
• Accuracy
• Precision
• Matrix effect,
• anticoagulant effect
• Carryover
• Recovery
• Stability
• Ruggedness
• Dilution integrity
• Partial volume (USFDA, 2001) 20

21. Conclusion
• Bioanalysis and the production of pharmacokinetic,
toxicokinetic and metabolic data play a fundamental
role in pharmaceutical research and development;
therefore the data must be produced to acceptable
scientific standards. For this reason and the need to
satisfy regulatory authority requirements,
• all bioanalytical methods should be properly
validated and documented and explain the
bioanalytical method development and validation
from a quality assurance department point view.
Some of the method and how is validation carried
out were described in different situations
encountered in the study sample analysis
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22. REFERENCES
• 1 .C. Hartmann, D.L. Massart and R.D. McDowall, J.Pharm.
Biomed. Anal., 12 (1994) 1337 1343.
• 2. Evans. G, Handbook of bioanalysis and drug metabolism, 8-69,
(2004)
• 3. 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; 2001 May.
22

23. REFERENCES
• 4. L. R. Synder, J. J. Kirkland, L. J. Glajch, Practical HPLC Method Development, 2nd
edn., John Wiley & sons, Inc, 2 (1997)
• 5. Heftman., ‘Chromatography- Fundamentals and applications of Chromatography
and Related differential migration methods’ 6th edn., Elsevier, Amsterdam 04, Vol.
69A,(2003), pp.72-87.
• 6. Braggio S., Barnaby R. J., Grosi P , Cugola M., ‘A strategy for validation of
bioanalytical methods’, Journal of Pharmaceutical and Biomedical Analysis 14,
(1996), pp.375- 388
• 7. A. Kirthi et al, Shanmugam R., “A Review on Bioanalytical Method Development
and Validation by RP-HPLC”, Journal of Global Trends in Pharmaceutical Sciences, 5(4)
(2014): pp 2265-2271.
• 8. Bolton Sanford, “Pharmaceutical Statistics Practical and Clinical Applications”,
Third Edition, Marcel Dekker Inc, 1984, Page No.- 216-257, 265-315.
• 9. Dean John R., “Extraction Techniques in Analytical Sciences”, 2009, John Wiley &
Sons, Ltd, Page No.-22-29, 39-45, 49-52
• 10. Lingerman H., R. D. McDowall and U. A. Th. Brinkman, “Guidelines for Bioanalysis
Using Column Liquid Chromatography”, Trends in Analytical Chemistry 10:2 (1991)
pp- 48-59
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