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

This is nothing but selectivity of analytical method 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 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 Types of degradation Solid state Solution state (Liquid state)

Solid state Conditions Room temperature Elevated temperature (eg: @100°C) Relative Humidity Photolysis

Liquid state Reagents 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 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.

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

Accuracy 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. 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 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 - signifies that this characteristic is not normally evaluated + signifies that this characteristic is normally evaluated (1) in cases where reproducibility (see glossary) has been performed, intermediate precision is not needed (2) lack of specificity of one analytical procedure could be compensated by other supporting analytical procedure(s) (3) may be needed in some cases

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 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 ?