1. Forced degradation studies and Analytical method validation
Bujji Kanchi

2. 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.

3. 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 ?

4. 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.

5. 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.

6. SIM Stability indicative method has to detect the degradation products. It has to hunt all the kind of degradants.
shoot the degradant

7. 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.

8. 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

9. A car is tested for its highest possible speed and controllable ability to know its efficiency

10. 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

11. ICH
ICH terms this forced degradation study as
STRESS TESTING

12. 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.

13. Nature of stress testing
The nature of the stress testing will depend on the individual drug substance and the type of drug product involved.

14. 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.

15. 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.

16. 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.

17. 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.

18. Contract R&D labs may helps in this

19. 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.

20. 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.

21. 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.

22. 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.

23. 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.

24. 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

25. 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.

26. 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

27. Experimental design Types of degradation Solid state
Solution state (Liquid state)

28. Solid state Conditions
Room temperature
Elevated temperature
Relative Humidity
Photolysis

29. Liquid state Reagents used for degradation
Water
Acid
Base
Oxidant

30. 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.

31. 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

32. 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)

33. 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

34. 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

35. 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.

36. 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.

37. 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.

38. 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.

39. Analytical method validation.

40. 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.

41. 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.

42. 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.

43. Parameters used for validation
Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range

44. 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.

45. Revalidation
Why ?

46. 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.

47. 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.

48. 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.

49. 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

50. 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.

51. 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%

52. 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

53. 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 %
For Impurities test, the range is LOQ -120%

54. Linearity and Range

55. Residuals

56. 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.

57. Linearity Curve X-axis : Concentration Y-axis : Area

58. 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

59. 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.

60. 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 %

61. % 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

62. Precision vs accuracy

63. 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.

64. 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.

65. Signal to noise

66. 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
= %

67. 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
= %

68. 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()

69. Example : All Concentrations are against test sample concenration only

70. Example : Concentrations and areas for acetone and methanol

71. Acetone(ppm)

72. Methanol(ppm)

73. 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.

74. Robustness Examples of typical variations are:
- stability of analytical solutions;
- extraction time.

75. 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.

76. Robustness In the case of gas-chromatography,
examples of typical variations are:
Different columns (different lots and/or suppliers);
Temperature;
Flow rate.

77. 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.

78. 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)

79. 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

80. 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.

81. 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.

82. 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

83. 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.

84. 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

85. 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

86. Questions ?