Validation of Analytical and Bioanalytical methods

• Dr. (Mrs.) Snehalatha Boddu
• Asst. Professor
GUIDANCE BY:
• Sarika K. Kadam
• M. Pharm (Q.A.) Sem I
• Oriental College of Pharmacy, Navi Mumbai
PRESENTED BY:
Sarika Kadam

WHAT IS VALIDATION?
Validation is establishing documented
evidence which provides a high degree
of assurance that a specific process will
consistently produce a product
meeting its predetermined
specifications and
quality attributes.
Sarika Kadam


 To obtain consistent, reliable and accurate data.
 For identification of source and quantization of
potential errors.
 Validation of analytical methods is also required by
most regulations.
 To ensure the products safety and efficacy throughout
all phases of its shelf life.
 Establish a proof that method
can be used for decision making.
Sarika Kadam

 TYPES OF VALIDATION:
Analytical
Method
Validation
Equipment
Validation
Process
Validation
Cleaning
Validation
VALIDATION
Sarika Kadam

 Analytical methods: Set of techniques that allow
us to know qualitatively and/or quantitatively the
analyte in sample.
 Bioanalytical methods: Analytical methods
performed for determination of drug/ drug
substance in biological matrices.
 Analytical Method validation: Process of
documenting/ providing evidence that an
analytical method provides analytical data
acceptable for intended use.
Sarika Kadam


 Identification tests
 Quantitative tests for impurities content
 Limit tests for the control of impurities
 Quantitative tests for active moiety
Sarika Kadam

 Analytical methods need to be
validated:
Before their introduction into routine use.
Whenever conditions change for which
method is validated.
Whenever method is changed and change is
outside the original scope of method.
Sarika Kadam

 Scope and validation criteria should be
defined early in the process which includes:
 What analyte should be detected?
 What are expected concentration levels?
 What are sample matrices?
 Are there interfering substances expected, and if so,
should they be detected and quantified?
 Are there any specific requirements?
 Should information be qualitative or quantitative?
 What are required detection and quantitation limits?
 What precision and accuracy is required?
 How robust the method should be?
Sarika Kadam

Workflow for evaluation and validation of
standard methods:
Define scope of users method
Define validation parameters and limits
Standard method fits scope, parameters,
limits?
Perform part or full validation
Define and perform system suitability testing
NO
YES
Sarika Kadam

ANALYTICAL METHOD AND VALIDATION
 A pharmaceutical drug product must meet all its
specifications throughout its entire shelf life.
 The performance of product characteristic must be
tested by analytical method for the product’s
chemical, physicochemical, microbiological and
biological characteristics.
 The method of analysis used must be validated.
This is required to ensure the product’s safety and
efficacy throughout all phases of its shelf life.
Sarika Kadam

Contents of ICH Guidelines Q2(R1)
 Introduction
 Document History
 Part I:
(Text on Validation of Analytical procedures)
1. Introduction
2. Types of Analytical Procedures to be validated
 Part II:
( Validation of Analytical Procedures: Methodology)
1. Specificity
2. Linearity
3. Range
4. Accuracy
5. Precision
6. Detection limit
7. Quantitation limit
8. Robustness
9. System suitability testing
Sarika Kadam

Identification tests-
 Intended to ensure the identity of an analyte in a sample.
 This is normally achieved by comparison of a property of the sample
 to that of a reference standard (e.g., spectrum, chromatographic
behavior, chemical reactivity, etc)
Quantitative tests for impurities content-
 Intended to accurately reflect the purity characteristics of the sample.
Limit tests for control of impurities-
 Quantitative or semi quantitative test designed to identify and
control small quantities of impurity which is likely to be present in
the substance.
Sarika Kadam

 The objective of the analytical procedure should be clearly understood
since this will govern the validation characteristics which need to be
evaluated.
 Typical validation characteristics to be considered:
 Accuracy
 Precision -
Repeatability
Intermediate Precision
 Specificity
 Detection Limit
 Quantitation Limit
 Linearity
 Range
Sarika Kadam

Furthermore revalidation may be necessary in the following
circumstances:
 Changes in the synthesis of the drug substance;
 Changes in the composition of the finished product;
 Changes in the analytical procedure.
Sarika Kadam

Specificity
Accuracy
Precision
Detection
limitQuantitatio
n limit
Linearit
y
Range
Robust-
ness
Validation
characteristics
Sarika Kadam

 Ability to assess unequivocally the analyte in the presence of
components which may be expected to be present.
Typically these might include : impurities, degradants, matrix, etc.
 Three implications of specification:
SPECIFICITY
Identification
Purity tests
Assay
Impurities are
available
Impurities are not
available
Sarika Kadam

Identification:
 Suitable identification tests should be able to
discriminate between compounds of closely related
structures which are likely to be present.
 The discrimination of a procedure may be confirmed
by obtaining positive results from samples containing
the analyte, coupled with negative results from
samples which do not contain the analyte.
 The identification test may be applied to materials
structurally similar to or closely related to the analyte
to confirm that a positive response is not obtained.
Sarika Kadam

Assay and impurity test (s)
• For chromatographic procedures, representative chromatograms
should be used to demonstrate specificity and individual components
should be appropriately labelled.
• Critical separations in chromatography should be investigated at an
appropriate level. For critical separations, specificity can be
demonstrated by the resolution of the two components which elute
closest to each other.
o Impurities are available: Done by spiking pure substances with
appropriate levels of impurities and/or excipients and demonstrating
that the assay result is unaffected by the presence of these materials
o Impurities are not available: By comparing the test results of
samples containing impurities or degradation products to a second
well-characterized procedure e.g.: pharmacopoeial method or other
validated analytical procedure
Sarika Kadam

ACCURACY( TRUENESS)
 Expresses the closeness of agreement between the
value which is accepted either as a conventional
true value or an accepted reference value and the
value found.
Sarika Kadam

DETERMINATION OF ACCURACY
 Assay:
• Application of an analytical procedure to an analyte of known purity .
• Comparison of the results of the proposed analytical procedure with
those of a second well-characterized procedure.
• Accuracy may be inferred once precision, linearity and specificity have
been established.
Drug substance
• Application of the analytical procedure to synthetic mixtures of the
drug product components to which known quantities of the drug
substance to be analysed have been added.
Drug product
Sarika Kadam

DETERMINATION OF ACCURACY
 Impurities (Quantitation):
Assessed on samples (drug substance/drug product)
spiked with known amounts of impurities.
 Recommended data:
 Minimum 9 determinations = Min. 3 concentration
levels covering specified range.
(3 concentration levels, 3 replicates)
 Reported as percent recovery by the assay of known
added amount of analyte in the sample OR as the
difference between the mean and the accepted true value.
Sarika Kadam

PRECISION
 Expresses the closeness of agreement (degree
of scatter) between a series of measurements
obtained from multiple sampling of the same
homogeneous sample under the prescribed
conditions.
Sarika Kadam

PRECISION
 Investigated using Homogenous authenticated
samples.
 Expressed as:
 Variance
 Standard deviation
 Coefficient of variance
 The value of %CV should be <2
Sarika Kadam

PRECISION
• Precision may be considered at three levels.
• Same operating conditions
• Short interval of time
• Intra-assay precision
Repeatability
• Within-laboratories variations
• Different days
• Different analysts
• Different equipment, etc.
Intermediate Precision
• Between laboratories
Reproducibility
Sarika Kadam

DETERMINATION OF PRECISION
Validation of tests for assay and quantitative
determination of impurities include an investigation
of precision.
Repeatability:
 Min. 9 determinations (3 conc. , 3 replicates) OR
 Min. 6 determinations at 100% test concentration.
Intermediate Precision:
 Extent to which it should be established depends on the
circumstances under which the procedure is intended to
be used.
Sarika Kadam

Reproducibility:
 Assessed by means of an inter-laboratory trial.
 Should be considered in case of the standardization of
an analytical procedure. (For instance, for inclusion of
procedures in pharmacopoeias)
Recommended Data:
 The standard deviation, relative standard deviation
(coefficient of variation).
Sarika Kadam

Concentration Absorption Concentration Absorption
10 g/ml
0.28
10 g/ml
0.21
0.31 0.32
0.29 0.39
0.30 0.53
Mean 0.29 Mean 0.36
HIGH PRECISION LOW PRECISION
Sarika Kadam

ACCURACY AND PRECISION
Sarika Kadam

LIMIT OF DETECTION AND QUANTITATION:
LOD: Lowest amount of analyte in a sample which
can be detected (not necessarily quantitated as an
exact value).
LOQ: Lowest amount of analyte in a sample
which can be quantitatively determined with
suitable accuracy and precision.
Sarika Kadam

 Based on Visual Evaluation:
 Establishing the minimum level at which the analyte can
be reliably detected.
 Based on Signal-to-Noise:
 Only be applied to analytical procedures which exhibit
baseline noise.
 S:N ratio of 3:1 is acceptable
 Based on the Standard Deviation of the Response
and the Slope:
 DL= 3.3 /S
(= SD and S= slope of calibration curve)
Sarika Kadam

Recommended data:
 The detection limit and the method used for
determining the detection limit should be presented.
 If DL is determined based on visual evaluation or based
on signal to noise ratio, the presentation of the relevant
chromatograms is considered acceptable for
justification.
Sarika Kadam

LOQ DETERMINATION:
 Based on Visual Evaluation:
 Establishing the minimum level at which the analyte can
be quantified with reliable accuracy and precision
 Based on Signal-to-Noise:
 Only be applied to analytical procedures which exhibit
baseline noise.
 S:N ratio of 10:1 is typical.
 Based on the Standard Deviation of the Response
and the Slope:
 QL= 10 /S
(= SD and S= slope of calibration curve)
Sarika Kadam

LOQ DETERMINATION:
Recommended data:
 The quantitation limit and the method used for
determining the quantitation limit should be presented.
 The limit should be subsequently validated by the
analysis of a suitable number of samples known to be
near or prepared at the quantitation limit.
Sarika Kadam

LINEARITY:
Ability (within a given range) to obtain
test results which are directly
proportional to the concentration
(amount) of analyte in the sample.
Sarika Kadam

DETERMINATION OF LINEARITY:
 Linear relationship is evaluated across the range of the analytical
procedure.
 Evaluated by visual inspection of a plot of signals as a function of
analyte concentration or content.
 If there is a linear relationship, test results should be evaluated
by appropriate statistical methods. eg. Regression line by
method of least squares.
 Some analytical procedures, such as immunoassays, do not
demonstrate linearity after any transformation. In this case, the
analytical response should be described by an appropriate
function of the concentration (amount) of an analyte in a
sample.
 For the establishment of linearity, a minimum of 5
concentrations is recommended
Sarika Kadam

LINEARITY:
(Know that it is a straight line)
•Mathematical transformations may be needed for some
cases to establish linear relationship.
Sarika Kadam

RANGE:
 Interval between the upper and lower
concentration of analyte in the sample for which
it has been demonstrated that the analytical
procedure has a suitable level of precision,
accuracy and linearity.
Sarika Kadam

RANGE:
These are some min. specified ranges to be considered:
 For assay of drug/ drug substance: 80 - 120% of the
test concentration.
 For content uniformity: min. of 70 to 130 % of the
test concentration.
 Dissolution testing: +/-20 % over the specified range
 For the determination of an impurity: : from the
reporting level of an impurity to 120% of the
specification.
Sarika Kadam

RANGE:
( For what concentration is it a straight line?)
Sarika Kadam

ROBUSTNESS:
 Measure of its capacity to remain unaffected by
small, but deliberate variations in method
parameters and provides an indication of its
reliability during normal usage.
 Should be considered during the development phase.
 Depends on the type of procedure under study.
 Analytical conditions should be suitably controlled.
Sarika Kadam

ROBUSTNESS:
Examples of typical variations:
 In the case of liquid chromatography:
 Variations of pH in a mobile phase
 Variations in mobile phase composition
 Different columns (different lots and/or suppliers)
 Temperature
 Flow rate.
 In the case of gas-chromatography:
 Different columns (different lots and/or suppliers)
 Temperature
 Flow
Sarika Kadam

 U.S. Department of Health and Human Services Food
and Drug Administration provides guidelines for
bioanalytical method validation.
 Provides assistance to sponsors of INDs, NDAs,
ANDAs.
 Supplements in developing bioanalytical method
validation information used in human clinical
pharmacology, bioavailability (BA), and
bioequivalence (BE) studies requiring
pharmacokinetic (PK) evaluation.
 Also applies to other bioanalytical methods, such as
immunological and microbiological procedures.
Sarika Kadam

BIOANALYTICAL METHOD VALIDATION:
 Fundamental parameters:
 Accuracy
 Precision
 Selectivity
 Sensitivity
 Reproducibility
 Stability
 Different types of validation:
 Full validation
 Partial validation
 Cross validation
Sarika Kadam

REFERENCE STANDARD
 Analysis is carried out by using Samples spiked with reference
standards and using QC samples.
 Three types of reference standards:
 certified reference standards (e.g., USP compendial standards)
 commercially supplied reference standards.
 other materials of documented purity custom-synthesized by an
analytical laboratory.
Sarika Kadam

SELECTIVITY:
Ability of an analytical method to differentiate
and quantify the analyte in the presence of other
components in the sample.
 Blank samples (appropriate biological matrix)from at
least 6 different sources is analyzed.
 Selectivity is ensured at LLOQ.
Sarika Kadam

ACCURACY
 Determined by taking three different concentrations and
five determinations per concentration.
(3 conc.  5 replications)
 Mean value should be within 15% of actual value
 Except for LLOQ: Deviation within 20% is allowed
 deviation of the mean from the true value serves as the
measure of accuracy.
Sarika Kadam

PRECISION:
 3 concentrations and 5determinations per conc.
 Precision determined at each concentration level
should not exceed 15% of the coefficient of variation
(CV).
 Except for the LLOQ, where it should not exceed
20% of the CV.
Sarika Kadam

RECOVERY:
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.
 Performed by comparing analytical results for extracted
samples at three concentrations (low, medium, and high)
with unextracted standards that represent 100% recovery.
Sarika Kadam

STABILITY:
Function of the storage conditions, the chemical
properties of the drug, the matrix, and the
container system
 Determined stability is relevant only to that matrix and
container system and should not be extrapolated to others.
Sarika Kadam

Freeze and Thaw Stability
Short-Term Stability
Long-Term Stability
Stock Solution Stability
Post-Preparative Stability
Evaluationofstability
Sarika Kadam

FREEZE AND THAW STABILITY:
• Stability is determined after 3 freeze and thaw cycles.
• One cycle is as follows:
At least 3 aliquots each at low and high conc.
Store at intended storage temperature for 24 hrs
Thaw unassisted at RT
Sarika Kadam

SHORT-TERM TEMPERATURE STABILITY:
At least 3 aliquots each at low and high conc.
Thaw at RT
Keep at this temp. for 4-24 hrs
Analyze
Sarika Kadam

STABILITY:
Long-Term Stability:
Storage time : time between the date of first sample collection
and the date of last sample analysis.
At least three aliquots of each of the low and high
concentrations.
Stock Solution Stability:
Evaluated at room temperature for at least 6 hours
Tested by comparing the instrument response with that of
freshly prepared solutions.
Post-Preparative Stability:
Stability of processed samples, including the resident time in
the auto sampler.
Compared with freshly prepared samples.
Sarika Kadam

Example: Simultaneous determination of vancomycin and ceftazidime in
cerebrospinal fluid in craniotomy patients by HPLC.
 EXPERIMENTAL CONDITIONS:
 Internal standard: Tinidazole
 Column: Diamonsil C18 column (200mm×4.6mmI.D., 5µm)
 Mobile phase : Acetonitrile and acetate buffer (pH 3.5) (8:92, v/v)
at room temperature (25 ◦C)
 Detection wavelength: 240 nm
 METHOD VALIDATION :
Sarika Kadam

Selectivity
 Six separate lots of drug-free control CSF were analyzed
• Free of interferences with compound of interest
• Retention times: Vancomycin 7 min
Ceftazidime 11 min
Tinidazole 20 min
Control
CSF spiked with drugs
CSF sample 12hrs after receiving drugs .
TypicalChromatogram
Sarika Kadam

SENSITIVITY AND LINEARITY:
 LLOQ were found to be –
 For vancomycin: 0.1 g/ml
 For ceftazidime: 1 g/ml
 Reproducibility of LLOQ was determined by examining five LLOQ
samples
 Calibration curves were constructed by plotting the peak area
ratios (analytes/IS) of plasma standards versus nominal
concentration
Calibration curves showed a linear range of 0.1–10 g/ml for
vancomycin and 1–100 g/ml for ceftazidime.
Unknown sample concentrations exceeding the range were diluted
appropriately with control blank CSF and re-assayed. The difference
between the nominal standard concentration and the back-calculated
concentration from the weighted linear regression line was within
15% for each point on the standard curve indicating that the linear
regression analysis applied provided an adequate fit of the data.
Sarika Kadam

ACCURACY AND PRECISION:
Nominal
conc (g/ml)
Intra day (n=5) Inter day (n=5)
Mean det.
Conc.
(g/ml)
Accuracy
(%)
CV
(%)
Mean det.
Conc. (g/ml)
Accuracy
(%)
CV (%)
Vancomycin
0.2 0.22 110.0 7.3 0.19 95.0 6.8
1.0 0.97 97.0 3.8 1.04 104.0 4.5
5.0 4.93 98.6 2.1 4.94 98.8 1.8
Ceftazidime
2.0 2.13 106.5 6.9 1.92 96.0 7.9
10.0 10.28 102.8 4.2 10.03 101.3 3.2
50.0 49.53 99.1 1.9 5.27 100.5 2.0
Intra- and Inter-day accuracy and precision (%CV) acceptance criteria for each QC was
≤15%
3 Different conc,. 5 replicates
Sarika Kadam

ROBUSTNESS
Some experimental conditions varied are-
 Mobile-phase buffer pH (±0.3 pH units),
 Only small changes in retention time
 Slightly longer times when increased pH
 Slightly shorter times when decreased pH
 Mobile-phase composition (±2% to the acetonitrile percentage)
 General change in run times, but had no great influence on
separation performance
 Flow rate (±0.1ml/min)
 Inverse changes in run times without generating problem with
respect to resolution, efficiency or peak shape.
Sarika Kadam

RECOVERY
 A set of samples (n = 5 at 3 conc.) was prepared by
spiking vancomycin/ceftazidime into CSF at 0.2/2, 1/10,
and 5/50 µg/ml and each was spiked with IS tinidazole 50
µg/ml.
 Mean extraction recoveries of vancomycin/ceftazidime at
concentrations of 0.2/2, 1/10, and 5/50 µg/ml were
97.17/102.04%, 105.18/103.02% and 100.77/100.98%,
respectively.
Sarika Kadam

STABILITY
 Freeze thaw stability:
 QC samples were used.
 Freeze remove from freezer thaw at RT for 4 hrs
refreeze at -20c.
 Showed accepted stability.
 Long-term stability:
 Performed at -20C.
 1 month stability data is given in table indicates drugs are
stable at least for 1 month.
 Short- term stability:
 3 sets of samples left at RT for 12hrs.
 Stable during exposure period.
 Post- preparative stability:
 Kept in autosampler for 24hrs before injection.
 Found to be stable.Sarika Kadam

STABILITY
Nominal
conc.
(g/ml)
Freeze thaw
(n=3)
Long term
(n=3)
Short term (n=3) Post preparative
(n=3)
Found
conc
(g/ml)
CV
(%)
Found
conc
(g/ml)
CV (%) Found
conc
(g/ml)
CV (%) Found
conc
(g/ml)
CV (%)
Vancomycin
0.2 0.19 5.3 0.21 9.5 0.19 5.3 0.20 5.0
1.0 0.99 4.0 0.98 3.1 1.04 5.8 0.98 2.0
5.0 4.88 2.5 5.13 2.9 4.97 2.2 4.95 1.0
Ceftazidime
2.0 2012 7.1 2.04 5.4 1.92 7.3 2.03 3.0
10.0 9.89 3.2 10.07 3.6 10.10 2.4 9.95 1.7
50.0 50.18 2.1 49.89 2.2 50.21 2.0 49.94 1.4
Sarika Kadam

CONCLUSION
 The method reported describes a selective and
accurate HPLC method for simultaneous
determination of vancomycin and ceftazidime in CSF
with rapid and simple sample pretreatment.
Sarika Kadam

REFERENCES:
1. ICH HARMONISED TRIPARTITE GUIDELINE - VALIDATION OF ANALYTICAL
PROCEDURES: TEXT AND METHODOLOGY Q2(R1) Current step 4 version
2. Guidance for industry Bioanalytical Method Validation, U.S.
Department of Health and Human Services, Food and Drug
Administration, May 2001 BP
3. Validation and qualification in analytical laboratories, 2nd edition, by
Ludwig Huber, Page no. 125-154
4. Journal of Pharmaceutical and Biomedical Analysis 48 (2008)
860-865
5. Validation of pharmaceutical processes, 2nd edition, Fredrick J.
Carleton, page nos. 1-16
6. Pharmaceutical master validation plan, The ultimate guide to FDA,
GMP& GLP compliance, syed Imtiaz Haider, Page nos. 1-36
7. Pharmaceutical analysis, modern method; James W. Munson,Page
nos. 1-15
Sarika Kadam

Validation of Analytical and Bioanalytical methods

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