validation

ANALYTICAL & BIO-
ANALYTICAL METHOD
VALIDATION
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VIGNAN PHARMACY COLLEGE,VADLAMUDI.

 Validation is a documented evidence which high degree of
assurance that specific process will produce a product meeting it’s
pre determined specifications and quality attributes..
 Drug substances and drug product manufacturers must perform
validations, it is very important that this understanding be shared
throughout the organization.
 The term validation generally to cover the entire spectrum of
cGMP.
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ANALYTICAL METHOD VALIDATION
 Validation of an analytical method is the process by which it is
established, by laboratory studies, that the performance
characteristics of the method meet the requirements for the
intended analytical applications.
 The process to Confirm that the analytical procedure employed
for a specific test is suitable for intended use and that they
support the identity, quality, purity and potency of the drug
substances and drug products. 4

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CONCEPT OF REVALIDATION
When we make any changes inWhen we make any changes in
 Analytical procedureAnalytical procedure
 Drug substance (e.g. synthetic route)Drug substance (e.g. synthetic route)
 Drug product (e.g. composition)Drug product (e.g. composition)
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PARAMETERSPARAMETERS
Specificity
LOD & LOQ (if applicable)
Linearity
Precision
i. System Suitability
ii. Method Repeatability
iii. Intermediate Precision (or) Ruggedness
iv. Method Reproducibility
Accuracy (or) Recovery
Robustness
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DEFINITION
 SpecificitySpecificity of analytical method as its ability to measure
accurately an analyte in the presence of interference,
such as synthetic precursors, excipients, enantiomers
and known (or likely) degradation product that may be
expected to be present in the sample matrix.
1. SPECIFICITY
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2. LIMIT OF DETECTION AND LIMIT OF
QUANTIFICATION (LOD & LOQ)
2. LIMIT OF DETECTION AND LIMIT OF
QUANTIFICATION (LOD & LOQ)
DEFINITIONDEFINITION
LOD:LOD: Lowest amount of analyte in a sample which can be detectedLowest amount of analyte in a sample which can be detected
but not necessarily quantitated, under the stated experimentalbut not necessarily quantitated, under the stated experimental
conditions (LOD)conditions (LOD)
LOQ:LOQ: Lowest amount of analyte in a sample which can beLowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracyquantitatively determined with suitable precision and accuracy
(LOQ)(LOQ)
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10VIGNAN PHARMACY COLLEGE,VADLAMUDI.

3. LINEARITY
TheThe LinearityLinearity of an analytical procedure is its ability (within a given range) toof an analytical procedure is its ability (within a given range) to
obtain test results that are directly proportional to the concentration (amount) ofobtain test results that are directly proportional to the concentration (amount) of
analyte in the sampleanalyte in the sample
Range: The interval between the upper and lower level( Including these level)Range: The interval between the upper and lower level( Including these level)
that have been demonstrated to be determined with precision, accuracy andthat have been demonstrated to be determined with precision, accuracy and
linearity.linearity.
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4. PRECISION
Closeness of agreement (degree of scatter) between a series ofCloseness of agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the samemeasurements obtained from multiple sampling of the same
homogenous sample under the prescribed conditions.homogenous sample under the prescribed conditions.
Precision may be considered at ….Precision may be considered at ….
1.1. System precision (System suitability)System precision (System suitability)
2.2. Method RepeatabilityMethod Repeatability
3.3. Intermediate precisionIntermediate precision
4.4. ReproducibilityReproducibility 12

Repeatability:Repeatability: precision under same operating conditions (with-in a laboratory overprecision under same operating conditions (with-in a laboratory over
a short period of time using the same analyst with the same equipment)a short period of time using the same analyst with the same equipment)
Measurement / Injection repeatability (System Precision)Measurement / Injection repeatability (System Precision)
Method repeatability (Method Precision)Method repeatability (Method Precision)
Intermediate precisionIntermediate precision: precision under different laboratory conditions (within-: precision under different laboratory conditions (within-
laboratory variation, as on different days, or with different analysts, or equipmentslaboratory variation, as on different days, or with different analysts, or equipments
within the same laboratory)within the same laboratory)
ReproducibilityReproducibility: precision between laboratories / intermediate precision can be: precision between laboratories / intermediate precision can be
considered during the standardization of a procedure before it is submitted to theconsidered during the standardization of a procedure before it is submitted to the
pharmacopoeiapharmacopoeia
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5. ACCURACY
The accuracy of an analytical procedure expresses the closeness ofThe accuracy of an analytical procedure expresses the closeness of
agreement between the value, which is accepted either as aagreement between the value, which is accepted either as a
conventional true value or an accepted reference value and the valueconventional true value or an accepted reference value and the value
foundfound
 It is some times termed as truenessIt is some times termed as trueness
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6. ROBUSTNESS
DEFINTIONDEFINTION
Measure of its capacity to remain unaffected by small, but deliberateMeasure of its capacity to remain unaffected by small, but deliberate
variations in method parameters and provides indication of its reliabilityvariations in method parameters and provides indication of its reliability
during its normal usageduring its normal usage
varying method parameters within a realistic range and the quantitativevarying method parameters within a realistic range and the quantitative
influence of the variables is determined, and, if the influence of theinfluence of the variables is determined, and, if the influence of the
parameter is within a previously specified tolerance, then, the parameter isparameter is within a previously specified tolerance, then, the parameter is
said to be within the method’s robustness range.said to be within the method’s robustness range.
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Typical variations include under validationTypical variations include under validation
programmeprogramme
 Flow rateFlow rate
 WavelengthWavelength
 Mobile phase composition, generally, Organic compositionMobile phase composition, generally, Organic composition
 TemperatureTemperature
 pH of the mobile phasepH of the mobile phase
 Stability of analytical solutionsStability of analytical solutions
 Different columns ( Different lots and suppliers )Different columns ( Different lots and suppliers )
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VALIDATION REPORT
Generally method validation report should have
Objective and scope of the method
Molecule details (IUPAC name, CAS No. Molecular Formulae, Molecular
weight and its Molecular Structure etc.,)
Detailed list of chemicals, reagents, reference standards
Listing of equipment and its functional and performance requirements
Methodology followed
Validation data (parameter wise – procedure, results, conclusion etc.,)
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Instrument out puts, which should represent critical method parameters
Specificity and LOD – for Identification Test (Generally, photographs)
Selectivity / Specificity data (discriminating chromatogram, peak purity data,
blank and placebo chromatograms and stressed samples chromatograms)
Linearity graphs
Resolution and related system suitability chromatograms .
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BIO ANALYTICAL METHOD
VALIDATION
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 INTRODUCTION OF BIO ANLAYTICAL METHOD
VALIDATION.
 BASIC STEPS IN BIO ANALYTICAL METHOD
VALIDATION.
 VALIDATION PARAMETERS.
 CONCLUSION.
 REFERENCE.
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INTRODUCTION
 It involves the quantitative determination of drug and its
metabolites in biological fluids .
 It plays a vital role in the evaluation & interpretation of the
bioavailability, bioequivalence, pharmacokinetic and
toxicokinetic study data.
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BASIC STEPS OF BIOANALYTICAL
PROCESS
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1) SELECTIVITY
It is the ability of an analytical method to identify and
quantify the analyte in the presence of other
components in the sample.
LLOQ – It is the lowest amount of analyte in the sample
that can be quantified with accuracy and precision.
2) MATRIX EFFECT
It is the direct or indirect alteration or interference in
response due to the presence of unintended analytes in
the biological sample.
VALIDATION PARAMETERS
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If MF = 1 ( No matrix effect)
If MF < 1 ( Suppression)
If MF > 1 ( Enhancement)
3) SENSITIVITY
 Sensitivity test should be conducted to prove the
reproducibility for samples at LOQ.
ACCEPTANCE CRITERIA FOR LOQ :
For accuracy : ± 20%
For precision : ≤ 20%
For S/N ratio: 5: 1
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4) CALIBRATION AND QC STANDARDS
 Calibration standard is the biological matrix to which a known amount
of analyte is added.
 QC standard is a spiked sample used to monitor the performance of a
bioanalytical method and is used to assess the integrity and validate
the results of unknown samples.
5) RECOVERY (OR) EXTRACTION EFFICIENCY
 Determined by comparing the detector response of the analyte (or) IS
from an extracted sample to the detector response of the analyte from
an unextracted sample representing 100% recovery.
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6) DILUTION INTEGRITY
 Dilution of the study samples is performed when the
obtained concentration is exceeding the ULOQ, or when
there is less sample availability compared to the method
requirement.( can be tested on dilutions from 1:2 to 1:10
& dilution integrity will be evaluated at 1:2to 1:4)
ACCEPTANCE CRITERIA :
QC samples , Accuracy = ±15%
Precision = ≤15%
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7) CARRYOVER
It is the appearance of an analyte signal in
blank sample peaks after the analysis of
samples with a high analyte concentration.
To evaluate carryover a blank sample will
be placed ULQ standard in a sequence.
It is insignificant when blank sample
response is ≤ 20% of LOQ sample.
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 It is evaluated when different anticoagulants in plasma
are used in the preparation of the QC and Calibration
control standards.
 Anticoagulant effect is nullified ,when QC standard
shows,
 Accuracy = ±15% and Precision = ≤15%
9) STABILITY EVALUATION
 The stability of analyte in the matrix during collection,
storage of samples should be assessed
8) ANTICOAGULANT EFFECT
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Short term stability of analyte and IS In solvent
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MATRIX STABILITY
AUTO SAMPLER STABILITY
 It is evaluated to cover the anticipated run time for the
analytical batch and to handle the situations like system
malfunctioning.
ALL THE QC SAMPLE
CONC. ARE BACK
CALCULATED USING
CALIBRATION CURVE 30

 It is performed to evaluate the stability of the samples,
which are kept on bench during the extraction process.
The anticipated time is usually 4 to 24 hrs
BENCH TOP STABILITY
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LONG TERM STABILITY
 It is performed to demonstrate the stability of the
analyte in the matrix for longer duration of time.
ACCEPTENCE CRITERIA :
 The relative means of back calculated
concentration (test/reference) for both levels must
be within 85-115%.
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FREEZE THAW STABILITY
 It is performed , to evaluate the stability of the
analyte in the matrix after multiple cycles of freezing
and thawing.
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CONCLUSION
 Validation is done according to standard protocol and
used it always produce a product which meets its
predetermined specification and quality control.
 Bioanalytical methods must be validated to objectively
demonstrate the fitness for their intended use.
 Bioanalysis and the production of pharmacokinetic, toxicokinetic
and metabolic data plays a fundamental role in pharmaceutical
research and development involved in the drug discovery and
development process.
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REFERENCE
 Bioanalytical Method Validation and Its
Pharmaceutical Application- A Review article
from Pharmaceutica Analytica
 James agalloco,frederick J. Carleton. Validation
of pharmaceutical process, third edition, Page no
5.
 The United State Pharmacopoeia 24; The National
Formulary 19; 2000: [1225] VALIDATION OF
COMPENDIAL METHODS.
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36VIGNAN PHARMACY COLLEGE,VADLAMUDI.

validation

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