Method validation is the process of proving that an analytical method is acceptable for its intended purposes. METHOD VALIDATION = ERROR ASSESSMENT Method validation is the process of demonstrating that analytical procedures are suitable for their intended use and that they support the identity, strength, quality, purity and potency of the drug substances and drug products Validation: Prior ConsiderationsSuitability of Instrument  Status of Qualification and Calibration Suitability of Materials  Status of Reference Standards, Reagents, Placebo Lots Suitability of Analyst  Status of Training and Qualification Records Suitability of Documentation  Written and approved standard test procedure and proper approved protocol with pre-established acceptance criteria  Compendial vs. Non-compendial Methods Compendial methods-Verification Regulatory analytical procedure in USP/NF Non- compendial methods-Validation Alternative analytical procedure proposed by the applicant for use instead of the regulatory analytical procedure Chromatographic Methods Demonstrate Resolution Impurities/Degradants Available Spike with impurities/degradants Show resolution and a lack of interference Impurities/Degradants Not Available Stress SamplesFor assay, Stressed and Unstressed Samples should be compared. Ability of an analytical method to measure the analyte free from interference due to other components. Selectivity describes the ability of an analytical method to differentiate various substances in a sample Original term used in USP Also Preferred by IUPAC and AOAC Also used to characterize chromatographic columns Degree of Bias (Used in USP) The difference in assay results between the two groups the sample containing added impurities, degradation products, related chemical compounds, placebo ingredients Selectivity: For impurity test, impurity profiles should be compared. Temperature (50-60℃) Humidity (70-80%) Acid Hydrolysis (0.1 N HCl) Base Hydrolysis (0.1 N NaOH) Oxidation (3-30%) Light (UV/Vis/Fl) Intent is to create 10 to 30 % Degradation Change in the analytical procedure, drug substance, drug product, the changes, may necessitate revalidation of the analytical procedures. “The degree of revalidation depends on the nature of the change.” “FDA intends to provide guidance in the future on post-approval changes in analytical procedures.” By Visual Inspection of plot of signals vs. analyte concentration By Appropriate statistical methods Linear Regression (y = mx + b) Correlation Coefficient, y-intercept (b), slope (m) Acceptance criteria: Linear regression r2 > 0.999 Requires a minimum of 6 concentration levels Normally derived from Linearity studies. Established by confirming that the method provides acceptable degree of linearity, accuracy, and precision. Specific range dependent upon intended application of the procedure.

1. Validationof AnalyticalValidationof Analytical
Method for DrugMethod for Drug
Substances & DrugProductsSubstances & DrugProducts
Dr. R.Badmanaban., M.Pharm., M.D(A.M).,PhD.,
Associate Professor, Head of Dept - Pharmacognosy
Shri Sarvajanik Pharmacy college - Mehsana-384001
www.sspcmsn.org Email: badu1977@gmail.com

2. Definition:
Method validation is the process of proving that an
analytical method is acceptable for its intended
purposes.
METHOD VALIDATION = ERROR ASSESSMENT
 Method validation is the process of demonstrating that analytical
procedures are suitable for their intended use and that they support the
identity, strength, quality, purity and potency of the
drug substances and drug products

3. Analytical Method
Body Full name Guidance on
Eurachem Focus for Analytical Chemistry in Europe Method validation
CITAC Cooperation of International Traceability in
Analytical Chemistry
Proficiency testing
Quality Assurance
EA European Cooperation for Accreditation Accreditation
CEN European Committee for Normalization Standardization
IUPAC International Union of Pure & Applied Chem. Method validation
ISO International Standardization Organisation Standardisation
AOAC
ILAC
Association of Official Analytical Chemists
International Laboratory Accreditation Cooperat.
Internal qual. Control
Proficiency testing
Accreditation
FDA US Food and Drug Administration Method validation
USP United States Pharmacopoeia Method validation
ICH International Conference on Harmonization Method validation
International regulatory bodies and their
guidelines on different aspects of QA

4. Chromatography todayChromatography today
More than sixty variants of the technique have been developed.
HPLC, GC, SFC, and CE are the most frequently used.
HPLC:
 almost universal
 wide range of equipment and columns is commercially
available
 well-understood separation mechanisms
 sensitive, specific, selective, precise and robust, Rugged,
accurate.
 easy to maintain instrumentation
 flexible in optimizing separations
 More efficient than some of the separation techniques
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Shri Sarvajanik Pharmacy 4

5. Liquid Chromatography (HPLC) instrumentLiquid Chromatography (HPLC) instrument
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6. The ChromatographicThe Chromatographic
Process – TheoreticalProcess – Theoretical
ConsiderationsConsiderations
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7. Elution in Column ChromatographyElution in Column Chromatography
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MobileMobile
phasephase
StationaryStationary
phasephase
tt00 tt11 tt22 tt44tt33

8. Intermolecular InteractionsIntermolecular Interactions
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9. ChromatogramChromatogram
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10. Retention RelationshipsRetention Relationships
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11. 11
Validation (4M)
Man
Machine
Material
Method

12. Why Method Validation is
Important
?
Analytical Method

13. Why Method Validation is
Important?
1. Develops confidence in using the method & Proof that
Method is suitable for its intended purpose,
The purpose of analytical measurement is to get
consistent, reliable and accurate data.
Incorrect measurement results can lead to
tremendous costs.
2.2. Regulatory requirementRegulatory requirement, Equal importance for those
working in a regulated and in an accredited environment.

14. When to be validated
?
Analytical Method

15. When to be validated?
Partial validation after development of method.
Complete validation after manufacturing formula is finalized.
Which methods are to be validated ?
Compendia: Pharmacopoeia method
Verification of suitability of method
Non compendia methods: Laboratory developed methods.
Pharmacopoeias methods used outside its scope.

16. Validation: Prior Considerations
Suitability of Instrument
Status of Qualification and Calibration
Suitability of Materials
Status of Reference Standards, Reagents,
Placebo Lots
Suitability of Analyst
Status of Training and Qualification Records
Suitability of Documentation
Written and approved standard test procedure
and proper approved protocol with pre-established
acceptance criteria

17. Validation Activity Including
the Complete Analytical Procedure
Sampling
Sample
Preparation
Analysis
Data Evaluation
Reporting

18. Validation Step
Define the application, purpose and scope of the
method.
Analytes? Concentration?
Develop a analytical method.
Develop a validation protocol.
Qualification of instrument.
Qualify/train operator

19. Qualification of material.
Perform pre-validation experiments.
Adjust method parameters and/or acceptance criteria if
necessary.
Perform full validation experiments.
Develop Procedures for executing the method in routine
analysis.
Document validation experiments and results in the
validation report.
Validation Step

20. Verification vs. Validation
Compendial vs. Non-compendial Methods
Compendial methods-Verification
Regulatory analytical procedure in USP/NF
Non- compendial methods-Validation
Alternative analytical procedure proposed by the
applicant for use instead of the regulatory analytical
procedure

21. Method
Validation
Method
Transfer
Method
Development
Approved
BACKGROUND-LAB METHOD FLOW

22. ICH/USP Validation
Requirements
Precision
Repeatability
Intermediate
Precision
Reproducibility
Limit of Detection
Limit of Quantitation
Robustness
Specificity
System
suitability
Linearity
Range
Accuracy

24. Validation
Parameters
Impurities
Specificity
Linearity and Range
Accuracy
Precision
Robustness
LOD & LOQs
Dissolution
Specificity
Linearity and Range
Accuracy
Precision
Robustness
Assay / CU
Specificity
Linearity and Range
Accuracy
Precision
Robustness

25. Specificity/Selectivity
Ability of an
analytical method
to measure the
analyte free from
interference due
to other
components.
Selectivity
Bias

26. Specificity: ICH/USP
The ability to measure accurately and
specifically the analyte in the presence
of components that may be expected to
be present in the matrix
The degree of interference
Active Ingredients
Excipients
Impurities (synthetic precursors,
enantiomers)
Degradation Products
Placebo Ingredients

27. Analytical Method

28. Continue....
. Combination of 2 or more analytical procedures may be required
to achieve necessary level of discrimination
. Stability indicating analytical methods should always be specific.
. Analysts should ascertain whether the peaks within a sample
chromatogram are pure or consist of more than one
compound. Therefore should know how many compounds are
in the sample or use procedures to detect peak purity
Analytical Method

29. Specificity: Impurities Assay
Chromatographic Methods
Demonstrate Resolution
Impurities/Degradants Available
Spike with impurities/degradants
Show resolution and a lack of interference
Impurities/Degradants Not Available
Stress Samples
For assay, Stressed and Unstressed Samples
should be compared.
29
2009

30. Selectivity
Ability of an analytical method to measure the analyte free
from interference due to other components.
Selectivity describes the ability of an analytical method to
differentiate various substances in a sample
Original term used in USP
Also Preferred by IUPAC and AOAC
Also used to characterize chromatographic columns
Degree of Bias (Used in USP)
The difference in assay results between the two groups
- the sample containing added impurities, degradation products,
related chemical compounds, placebo ingredients
Selectivity: For impurity test, impurity profiles should be
compared. 30
2009

31. Analytical Method

32. Forced Degradation Studies
Temperature (50-60℃)
Humidity (70-80%)
Acid Hydrolysis (0.1 N HCl)
Base Hydrolysis (0.1 N
NaOH)
Oxidation (3-30%)
Light (UV/Vis/Fl)
Intent is to create 10 to 30 % Degradation 32
2009

33. Analytical Method

34. Linearity
Ability of an assay to
elicit a direct and
proportional response to
changes in analyte
concentration.
34
2009

35. Linearity Should be Evaluated
By Visual Inspection of plot of signals vs.
analyte concentration
By Appropriate statistical methods
Linear Regression (y = mx + b)
Correlation Coefficient, y-intercept (b), slope (m)
Acceptance criteria: Linear regression r2
>
0.999
Requires a minimum of 6 concentration 35
2009

36. Range
The interval between the upper and
lower concentrations of analyte in
the sample that have been
demonstrate to have a suitable
level of precision, accuracy, and
linearity.

37. Range
Normally derived from Linearity studies.
Established by confirming that the method
provides acceptable degree of linearity,
accuracy, and precision.
Specific range dependent upon intended
application of the procedure.

38. Range
Acceptable range having linearity, accuracy, precision.
For Drug Substance & Drug product Assay
80 to 120% of test Concentration
For Content Uniformity Assay
70 to 130% of test Concentration
For Dissolution Test Method
+/- 20% over entire Specification Range
38
2009

39. Accuracy
Closeness of the test
results obtained by
the method to the
true value.

40. Accuracy
Should be established across specified
range of analytical procedure.
Should be assessed using a minimum of 3
concentration levels, each in triplicate (total
of 9 determinations)
Should be reported as:
Percent recovery of known amount added or
The difference between the mean assay result and
the accepted value
40
2009

41. Accuracy Data Set (1 of 3)
41
2009

42. Precision
The closeness of agreement
(degree of scatter) between a
series of measurements
obtained from multiple
samplings of the same
homogeneous sample.
Should be investigated using
homogeneous, authentic
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43. Accuracy Vs Precision
Inaccurate &
imprecise
Inaccurate but
precise
Accurate but
imprecise

44. Precision… Considered at 3
Levels
Repeatability
Intermediate
Precision
Reproducibility

45. Repeatability
Express the precision under the same
operating conditions over a short interval of
time.
Also referred to as Intra-assay precision

46. Intermediate
Precision
Express within-laboratory
variations.
Expressed in terms of standard
deviation, relative standard
deviation (coefficient of
variation) and confidence
interval.
Known as part of Ruggedness
in USP (Different Analysts,
Different Laboratories,
Different Instruments, Different
Depends on the
circumstances under
which the procedure is
intended to be used.

47. Repeatability & Intermediate
Precision
Day 1 Day 2
100.6 99.5
100.8 99.9
100.1 98.9
100.3 99.2
100.5 99.7
100.4 99.6
47
Grand
Mean = 100.0
RSD = 0.59%
Mean = 100.5
RSD = 0.24%
Mean = 99.5
RSD = 0.36%
2009

48. Definition: Ability to reproduce data within
the predefined precision
Determination: SD, RSD and confidence
interval
Repeatability test at two different labs.
Reproducibili
ty

49. Reproducibility Study
Lab 1 Lab 2 Lab 3
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
Analys
t1
Analyst
2
Analyst
1
Analyst
2
Analyst
1
Analyst
2
3
Preps
3 Preps 3 Preps 3 Preps 3 Preps 3 Preps

50. Lowest amount of analyte in
a sample that can be
detected but not necessarily
quantitated.
Estimated by Signal to Noise
Ratio of 3:1.
Detection Limit
(DL)
Lowest amount of analyte
in a sample that can be
quantified with suitable
accuracy and precision.
Estimated by Signal to
Noise Ratio of 10:1.
Quantitation Limit
(QL)

51. LOD, LOQ and SNR
Limit of Quantitation (LOQ(
Limit of Detection (LOD(
Signal to Noise Ratio (SNR(
noise
Peak A
LOD
Peak B
LOQ
Baseline

52. S = slope of calibration curve
s = standard deviation of blank readings or
standard deviation of regression line
Validated by assaying samples at DL or QL
52
DL =DL =
3.3s3.3s
QL =QL =
10s10s
SS SS
LOD and LOQ Estimated by
2009

53. 53
Ybl
LOD LOQ
Statistical estimate of LOD & LOQ
LOD = 3.3 Sbl / b LOQ = 10 Sbl / b
Y = b X
+ a
2009

54. Analytical Method

55. Definition: Capacity to remain unaffected
by small & deliberate variations in method
parameters
Determination: Comparison results under
differing conditions with precision under
normal conditions
Variations may include: stability of
analytical solution, variation of pH in a
mobile phase, different column
(lot/supplier), temperature, flow rate.
Robustnes
s

56. Robustness Variations
All Assays
HPLC Assays
GC Assays
-Sample Prep Manipulation
-Extraction Time
-Mobile Phase
Composition
-Different Columns
-Temperature
-Flow Rate
-Different Columns
-Temperature
-Flow Rate

57. Ruggedness
Degree of reproducibility of test
results under a variety of
conditions
Different Laboratories
Different Analysts
Different Instruments
Different Reagents
Different Days
Etc.
Expressed as %RSD
57
2009

58. Solutes may readily decompose prior to chromatographic
investigations e.g. during sample preparation, extraction,
cleanup, phase transfer or storage of prepared vials
(refrigerators or automatic sampler).
Method development should investigate the stability of the
Analytes AND standards.
Stability of analytical solution
Solution stability
• Stability of the samples being analyzed in a sample solution.
e.g. 1 – 48 hours using a single solution.
• should be determined by replicate analysis of the sample
solution.

59. The checking of a system, before or during analysis of
unknowns, to ensure system performance.
“No sample analysis is acceptable unless the
requirements for system suitability have been met.”
(USP Chapter 621)
Plate Count, Tailing, Resolution
Determination of reproducibility (%RSD)
For %RSD < 2.0%, Five replicates
System Suitability "Sample“ - A mixture of main components
and
expected by-products utilized to determine system suitability
“Whenever There is a Significant change in Equipment or
Reagents
SYSTEM SUITABILITY

60. Confuse of Precision Terms
 Repeatabili
ty
 Intermediat
e Precision
 Reproducibilit
y
 Ruggedness
 Robustness

61. Precision Terms
Instrument Precision
Repeatability
Intermediate Precision
Reproducibility
Ruggedness
Robustness
- 6 Standard
Injections
- One Analysis (6
preps)
- Two Analyses
- Two different Lab.
- Many Variables

62. Change in the analytical procedure, drug substance, drug
product, the changes, may necessitate revalidation of the
analytical procedures.
“The degree of revalidation depends on the nature of the
change.”
“FDA intends to provide guidance in the future on post-
approval changes in analytical procedures.”Revalidation should accompany
formulation changes (new samples with new compounds or new matrices)
manufacturing batch changes
new analysts with different skills,
new instruments with different characteristics,
new location with different environmental conditions,
new chemicals and/or reference standards and
modification of analytical parameters.
Revalidation

63. Validation Report
Objective and scope of the method (applicability, type).
Summary of methodology.
Type of compounds and matrix.
All chemicals, reagents, reference standards, QC samples with purity, grade,
their source or detailed instructions on their preparation.
Procedures for quality checks of standards and chemicals used.
Method parameters.
Critical parameters taken from robustness testing.
Listing of equipment and its functional and performance requirements, e.g., cell
dimensions, baseline noise and column temperature range.
Detailed conditions on conduct of experiments, including sample preparation
Statistical procedures and representative calculations.
Procedures for QC in routine analyses, e.g., system suitability tests.
Representative plots, e.g., chromatograms, spectra and calibration curves.
Method acceptance limit performance data and expected uncertainty of
measurement results.
Criteria for revalidation.
The person's) who developed and validated the method.
References (if any).

64. Analytical Method

65. How do we Know the
expectations of the FDA?
FDA Form 483
FDA Warning Letters
Personal Experiences
65
2009

66. 483 Observations
There was inadequate method validation
specificity data to demonstrate that each
method was capable of distinguishing the
active ingredient from its impurities and
degradation products.
Specificity studies did not include the
minimum stress conditions of acid and base
hydrolysis, oxidation, thermal degradation
and photolysis, degradation schematic for the
active ingredient that identifies the major
degradation products was not included for 66
2009

67. FDA Warning Letter
On addition to an example of modifying both
compendia methods and customer supplied
methods, we also observed the use of
invalidated in-house methods
A statement indicating that the method has not
been validated in the particular formulation was
included in the certificate of analysis for…use
of this statement does not absolve…from using
valid, accurate, and
reproducible methods. (June 2009)
67
2009

68. General requirements
Qualified and calibrated instruments
Documented methods
Reliable reference standards
Qualified analysts
Sample integrity
Change control (e.g., synthesis,)
Analytical methods should be used within GMP and GLP
environments, and must be developed using the protocols
and acceptance criteria set out in the ICH guidelines Q2 (R1)

69. Analytical Method

70. Related Site
www.fda.gov
www.fda.gov/cder/
www.waters.com
www.usp.org
www.ich.org
www.aoac.org
www.pharmweb.net
70
2009

71. Thank you