Transcript Forced degradation studies

Forced degradation
studies
and
Analytical method
validation
Bujji Kanchi
Main Objective works for stability
• Before performing stability studies, a stability
indicating method is necessary so that any
possible degradants generated during storage
conditions (such as 5°C, 25°C/60%RH and
40°C/75%RH) can be separated, detected, and
quantitated.
Forced degradation
• But This needs to wait months and years
together for storing the stability samples,
analyse as per frequency of its testing.
• So do we need to wait for such long time ?
• What next ?
Got an Idea ?
• To over come this, we need to degrade the sample
forcibly by applying conditions beyond accelerated .
• This is the perfect thought
in the beginning to develop
An accurate and precise
Method.
Stability indicative method(SIM) .
• If the method does the same, Then the method is
called vey good method.
• The so called very good method is stability
indicative method. Hence the term stability
indicative .
• The mehod wakes up to to find the
Anlalytes that forms at any point of
Time during stability studies.
SIMStability indicative method has to
detect the degradation products.
It has to hunt all the kind of
degradants.
Out come
• The main objective of a stability indicating method
is to monitor results during stability studies
• in order to guarantee safety, efficacy and quality. It
represents also a powerful tool when investigating
out-of-trend (OOT) (Swartz et al., 2004) or out-ofspecification (OOS) results.
(CDER, 2006) in quality control processes.
stability indicative ?
• a Stability Indicating Method (SIM) is defined as
a validated analytical procedure that accurate
and precisely measure active ingredients (drug
substance or drug product) free from process
impurities, excipients and degradation products
A car is tested for its highest possible speed
and controllable ability to know its
efficiency
Selectivity
• In the same way we forcibly degrade the sample
to form degradants. These must be detected and
not mix up with other peaks in HPLC
• This is nothing but selectivity of analytical
method
ICH
• ICH terms this forced degradation study as
STRESS TESTING
What ICH Speaks
• Stress testing of the drug substance can help
identify the likely degradation products, which
can in turn help establish the degradation
pathways and the intrinsic stability of the
molecule and validate the stability indicating
power of the analytical procedures used.
Nature of stress testing
• The nature of the stress testing will depend on
the individual drug substance and the type of
drug product involved.
ICH-Stress testing
• Stress testing is likely to be carried out on a
single batch of the drug substance. It should
include the effect of temperatures (in 10°C
increments (e.g., 50°C, 60°C, etc.) above that for
accelerated testing), humidity (e.g., 75% RH or
greater) where appropriate, oxidation, and
photolysis on the drug substance.
Evaluation
• The testing should also evaluate the
susceptibility of the drug substance to hydrolysis
across a wide range of pH values when in
solution or suspension. Photo stability testing
should be an integral part of stress testing.
Sensibility
• The method should be sensitive to the
reportable impurity level. LOQ (limit of
quantitation), which is typically 0.05% of Label
Claim, should be established in the method, and
the method should be linear from LOQ to
typically up to 150% of the nominal standard
(std) concentration.
Ultimately forced degradation
• To know the intrinsic properties of a substance.
1)TO know its degradation products and Path way.
2)To verify stability indicative nature of analytical
method.
Contract R&D labs may helps in this
Stability indicating method
• During storage over time any material will be
degraded. During storage means, Stability studies
samples.
• Degradation may occur in future stability sample.
• The degradants should be identified by our
analytical method, If not identified and quantified
accurately, Its not a stability indicating method.
• If the degradation should be identified and
quantified by the analytical procedures.
• If not identified, the analytical procedures are blind
and not fit for the intended use.
SIM
• In-order to monitor the possible changes to a
product over time, The applied analytical
method must be stability indicating.
• Changes in drug stability can risk patient safety
by formation of degradants(Impurities)
• Therefore needs purity profile under various
experimental conditions.
SIM
• a Stability Indicating Method (SIM) is defined as a validated
• analytical procedure that accurate and precisely measure
active ingredients (drug substance
• or drug product) free from process impurities, excipients and
degradation products. The
• FDA recommends that all assay procedures for stability
should be stability indicating. The
• main objective of a stability indicating method is to monitor
results during stability studies
• in order to guarantee safety, efficacy and quality. It represents
also a powerful tool when
• investigating out-of-trend (OOT) (Swartz et al., 2004) or outof-specification (OOS) results in quality control processes.
HPLC plays the role
• Liquid chromatography is the most appropriate
technique for developing/validating a SIM.
• The use of diode-array-detector and additionally
mass spectrometers, gives best performances
for people working with SIM development.
HPLC
• The use of HLPC coupled to diode-array
detectors (DAD) in the achievement of peak
purity usually give reasonable results,
• mainly related to reliable determination of the
main active ingredient. It is possible to
guarantee no co-elution with degradation peaks
and other impurities.
When DAD fails to identify similar specta,
MASS spectrum is helpful
• DAD detectors can be limited on occasion the
more similar the spectra,
• and the lower the relative absorbance, the more
difficult it can be to distinguish co-eluted
• compounds. MS detection overcomes many of
these limitations. MS can provide
• unequivocal peak purity information, exact
mass, structural and quantitative information
• depending upon the type of instrument used
Method development
• The goal is to manipulate selectivity by changing
mobile phase composition, wavelength of
detection and pH. Related to mobile phase pH,
Columns mechanically strong, with high
efficiency and that are operate over an extended
pH range, should be preferred.
Method development
• Acidic compounds are more retained at low pH;
while basic
• compounds are more retained at higher pH
(neutral compounds are unaffected). At
• traditionally used pH values (pH 4 - 8), a slight
change in pH would result in a significant
• shift in retention
Experimental design
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Types of degradation
▫ Solid state
▫ Solution state (Liquid state)
Solid state
Conditions
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Room temperature
Elevated temperature (eg: @100°C)
Relative Humidity
Photolysis
Liquid state
Reagents used for degradation
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Water
Acid
Base
Oxidant
Over stressing
• Care should be taken in order to avoid
overstressing or under stressing samples, with
may lead to non representative or nonpurposeful degradation.
Over stressing leads to aberrant results
• So, the use of a properly designed and executed
forced degradation study will generate
representative samples that will help to ensure
that resulting method
reflects adequately
long-term stability
Solid State
• Stress Condition Period of time
• Heat 100° C Up to weak
• Humidity 90% RH Up to 1 week
• Photostability
• 3 mm (powder)
• Exposed and non-exposed
• samples (“control”)
• Follow ICH requirements
• (Q1B)
Solution State
• Stress Condition Period of time
• Acid Hydrolys 0.1 – 1 Mol L-1 HCl Up to 2 hours and 60° C
• Acid Hydrolys 0.1 – 1 Mol L-1 HCl Up to 2 hours and 60° C
• is alkaline 0.1 – 1 Mol L-1 NaOH Up to 2 hours and 60° C
• Oxidation H2O2 3% (v/v) Up to 2 hours and 60° C
Procedure
• Expose the sample to the said conditions of both
solid and liquid state and analyse
• Observe the degradants
• Calculate and Report the impurities.
• Balance the mass by clubbing assay and Total
impurities ,Then compare the mass balance with
respect to Mother sample result
Rate of degradation
• 5-10% degradation is enough,
• Reduce the stress condition , if the degradation
is beyond 10%.
• So avoid over stressing, which is meaning less
for our intended use.
Does the degradants must be identified ?
• degradation products formed in the forced
degradation study are not needed to identify
when these are not formed during Stability
Studies.
• but SIM may assure that these impurities do
not interfere on degradation products
determination.
When sample fails to soluble in reagent
• If the sample is not soluble in the reagent used
for degradation, you may use to dissolve in the
diluent in a little quantity then add the reagent.
Ultimately FD study is useful
• To develop and validate Stability indicating
method
• To understand drug molecule chemistry
• How reactions takes place with different
experiments and forming degradation products.
• To generate a degradation impurity that would
reflect in formal stability study Under ICH
Conditions.
Analytical method validation
•.
What is validation
• Validation is term comes from Valid or validity.
• It verifies the validity of a method
It verifies whether an analytical method is valid or
In-valid for the intended use.
What is validation
• Precise and accurate method are successfully
validated methods.
• An out come of a result from a validated method
must be precise and accurate.
Define validation
• Validation
A documented program that provides a high
degree of assurance that a specific process,
method, or system will consistently produce a
result meeting pre-determined acceptance
criteria.
Parameters used for validation
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Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Types of Analytical Procedures to be
Validated
• The discussion of the validation of analytical
procedures is directed to the four most common
types of analytical procedures:
• - Identification tests,
• - Quantitative tests for impurities' content,
• - Limit tests for the control of impurities.
Revalidation
Re-validation
• Changes in synthesis / composition of the drug;
• Changes in the analytical procedure.
• The degree of revalidation required depends on
the nature of the changes. Certain other changes
may require validation as well.
Specificity
• Specificity nothing but selectivity
It is the 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.
How to assess this
• Spike the impurities(about 5% to the principle
peak) in the test samples.
• Compare the retention times of the impurities
individually with respect to spiked samples.
• No co elution should be found.
Specificity
Preparations
• System suitability
• Individual standards
• Spiked sample
• Acceptance criteria :
• 1)Retention times from spiked sample should
match with individual solutions.
• 2)peak purity should pass(DAD detector)
▫ Purity angle should be less than purity threshold
When impurities are not available
• Degrade the sample by applying temperature
about 100°C over a period of 2 days and analyse.
• Check the peak purity of the sample
• The impurities formed during the forced
degradation should not interfere with the major
peak.
Precision
%RSD
• Repeatability
• Six determinations of a test sample at 100%
concentration
• Check the % RSD.
• Usually the %RSD for assay is about 2%,
for impurities tests, it is about 10%
Intermediate precision
• Precision between with in the laboratory by
changing analyst, system and reagents.
• This is also called Ruggedness.
• Precision between laboratories is called
Reproducibility
Linearity
and Range
• The concentration should be linear across the its
range
• The concentration is directly proportional to the
peak response.
• Take 5 concentrations of a standard across the
range calculate the R^2 value(Correlation)
• Correlation should not be less than 0.999
• For assays, the range is 80-120%
• For Impurities test, the range is LOQ -120%
Linearity and Range
Residuals
Residuals
• As per the linear regression line y= mx+c.
• Therotical values will be estimated by taking
area at 100% concentration .
• Practically we get the areas of the same
concentrations by HPLC.
• Substract the areas from HPLC to the areas
obtained from the equation mx+c.
• This value is Residual .
• All residuals should be asymmetric.
Linearity Curve
X-axis : Concentration
Y-axis : Area
y = 50,839,776.00x - 703,602.80
Linearity graph
16000000
14000000
12000000
10000000
8000000
6000000
4000000
2000000
0
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Accuracy
• Testing a reference standard in the laboratory
and compare the result to the reference value.
• Spike the impurities and calculate the %
Recovery
• Verify the accuracy with 9 determinations across
the range is required.
• For assay by HPLC or GC accuracy is
compensated by specificity, precision and
linearity across the range
How to spike standard to get desired
concentration.
• Calculation= standard concentration x 100
Test sample concentration
Example :
Test concentration= 20 mg in 20 ml
Reference concentration =
5 mg / 100 ml(stock), further diluted 10 to 100 ml.
This reference is 0.5% with respect to test sample concentration.
spiking 0.1 % impurity standard into test sample
Spiked preparation:
• Add 2 ml in the test sample after weighing the
sample, then dilute to 20 ml with the diluent.
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Treat this spiked preparation as standard concentration.
Now how this becomes 0.1% spiked.
(Standard concentration / test concentration) x 100
(5/100) x (2/100) x 100
(20/20)
• = 0.1 %
% RECOVERY
• Analyse test sample
and spiked test sample
• Calculate the
 content of impurity from test sample(A)
 content of impurity from spiked test sample(B)
 Amount spiked(C)
• Now % Recovery = (B-A )x 100
C
Precision vs accuracy
Limit of qunatitation(LOQ)
• Least level of analyte should be accurately and
precisely quantified.
• S/N Ratio about 10:1
• Or can be estimated by standard deviation and
slope method.
Limit of detection(LOD)
• Least level of analyte should be visually detected
and not necessarily quantified with accuracy.
• S/N Ratio about 3:1
• Or can be estimated by standard deviation and
slope method.
Signal to noise
S/N Ratio for LOQ
• Inject reference solution and calculate the S/N
Ratio for the standard peak.
• For example
• S/N Ratio for reference solution (0.5%)=200:1
• Now you need the concentration(LOQ) =10:1
• Then LOQ Concentration = (0.5/200) x 10
= 0.025 %
S/N Ratio for LOD
• Inject reference solution and calculate the S/N
Ratio for the standard peak.
• For example
• S/N Ratio for reference solution (0.5%)=200:1
• Now you need the concentration(LOD) =3:1
• Then LOQ Concentration = (0.5/200) x 3
= 0.0075 %
Slope and standard deviation method
LOQ and LOD
• Inject least concentrations up to 30 or 40% level
minimum of 5 levels.
• LOQ = 10 x sigma
S
• LOD = 3.3 x sigma
S
Sigma = standard deviation
In MS-EXCEL the formula is =steyx()
• For slope the formula is
=slope()
Example :
All Concentrations are against test
sample concenration only
Example :
Concentrations and areas for acetone and methanol
Acetone(ppm)
Methanol(ppm)
Robustness
• Deliberately alter the method parameters and
test the sample
• Compare the results with the precision results.
• It the study to know the effectiveness of the
method when small changes are being done as
an error while doing,
• The method is robust, when these results are
unaffected.
Robustness
• Examples of typical variations are:
• - stability of analytical solutions;
• - extraction time.
Robustness
• In the case of liquid chromatography, examples
of typical variations are:
• Influence of variations of pH in a mobile phase;
• Influence of variations in mobile phase
composition;
• Different columns (different lots and/or
suppliers);
• Temperature;
• Flow rate.
Robustness
• In the case of gas-chromatography,
examples of typical variations are:
• Different columns (different lots and/or
suppliers);
• Temperature;
• Flow rate.
Titrations- validation
Specificity
• Titrimetric methods are not exactly specific,
since the similar structural components may
also consume the volumetric solution.
• Blank interference may be verified.
Assay by titration
• Titrimetric methods are linear at beyond the
specified range.
• Because it’s a mole to mole reaction.
• Linearity works here unlike HPLC and GC assays
• Unlimited range
• Accuracy can be done by assaying standards.
• Precision at 100% test concentration
▫ (6 preparations)
selection of validation parameters
Method verification is required for
compendial procedures
• The effectiveness of transferring a validated
method from the original laboratory (or from a
pharmacopoeia) to another laboratory needs to
be verified.
Method verification
• Differences in instrumentation and other
equipment, for example, chromatography
columns of different brands, age etc, capability
of detectors, different filter materials, quality of
reagents used, etc.
Method verification
• It may also be necessary to confirm the precision
of the method or the ability to achieve the
detection levels of the validated method.
• Specificity, precison, LOQ and LOD is
recommonded parameters for verification
Method transfer is required for noncompendial procedures
• Usually verification is performed with USP
procedures and transfer is performed with non
USP procedures.
• Depending the type of data you collected during
the transfer the verification may not be needed.
Alternative compendial analytical
methods
• When An in-house method be better than
compendial method
• You can adopt your own developed method by
justifying
Method equivalency
or
Better results
Ultimately
• Validation is a verification process of analytical
method fit for the intended use.
• A simple comparison for
calibration Vs validation.
 Performance Verification of equipment is calibration
 Performance Verification of method is validation
Questions ?