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.

SIM-

Stability indicative method has to detect the degradation products.

It has to hunt all the kind of degradants.

shoot the degradant

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-of-specification (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 out-

of-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 ofdetection 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

• Types of degradation▫Solid state

▫Solution state (Liquid state)

Solid stateConditions

•Room temperature•Elevated temperature (eg: @100°C) •Relative Humidity•Photolysis

Liquid stateReagents used for degradation

•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 non-purposeful 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

• 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

Why ?

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.

SpecificityPreparations

•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

0.18 0.2 0.22 0.24 0.26 0.28 0.3 0.320

2000000

4000000

6000000

8000000

10000000

12000000

14000000

16000000

f(x) = 50839776.0000002 x − 703602.800000032

Linearity graph

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

Accuracy

How to spike standard to get desired concentration.•Calculation= standard concentration x

100 Test sample

concentration

Example :Test concentration= 20 mg in 20 mlReference 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.

• 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 methodLOQ 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 SSigma = standard deviationIn 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 non-compendial 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 equivalencyorBetter 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 ?