1. ANALYTICAL METHOD VALIDATION

2. Method Validation
Validation of analytical procedures is the process of determining the suitability of a given methodology for providing useful
analytical data.
Validation is the formal and systematic proof that a method compiles with the requirements for testing a product when
observing a defined procedures.

3. 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
Method validation is primarily concerned with:
identification of the sources of potential errors
quantification of the potential errors in the method
A method validation describes in mathematical and quantifiable terms the performance characteristics of an assay

4. Examples of Methods That Require Validation Documentation
Chromatographic Methods - HPLC, GC, TLC, GC/MS, etc.
Pharmaceutical Analysis - In support of CMC.
Bioanalytical Analysis - In support of PK/PD/Clinical Studies.
Spectrophotometric Methods – UV/VIS, IR, NIR, AA, NMR, XRD,MS
Capillary Electrophoresis Methods - Zone, Isoelectric Focusing
Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC, etc.

5. Purpose of Method Validation
Identification of Sources and Quantitation of Potential errors
Determination if Method is Acceptable for Intended Use
Establish Proof that a Method Can be Used for Decision Making
Satisfy FDA Requirements

6. What is not Analytical Method Validation?
Calibration
The Process of Performing Tests on Individual System Components to Ensure Proper function
For example) HPLC Detector calibration
Wavelength Accuracy/ Linear Range/ Noise Level

7. System Suitability
Test to verify the proper functioning of the operating system, i.e., the electronics, the equipment, the specimens and the analytical operations.
Minimum Resolution of 3.0 between the analyte peak and internal standard peaks
Relative Standard Deviation of replicate standard injections of not more than 2.0%

8. System Suitability Validation Calibration Analyst Method Sample Pump
Injector
Detector
Data System
Analyst
Method
Sample

9. Method Life Cycle
Validation
Development
Optimization

10. for its intended purpose
The objective of validation of an analytical procedure is to demonstrate that it is suitable
for its intended purpose
ICH Guideline for Industry
Q2A, Text on Validation of Analytical Procedures
March 1995

11. Specificity/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
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
the sample without added substances

12. 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.
For impurity test, impurity profiles should be compared.

13. 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

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

15. 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 > 0.95
Requires a minimum of 5 concentration levels

16. 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
For Impurity Assays
From Reporting Level to 120% of Impurity Specification for Impurity Assays
From Reporting Level to 120% of Assay Specification for Impurity/Assay Methods

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

18. 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

19. Accuracy Data Set (1 of 3) Amount Added (mg) Found (mg)
Percent Recovery
0.0
---
50.2
50.4
100.5
79.6
80.1
100.6
99.9
100.7
100.8
120.2
119.8
99.7
150.4
149.7
99.5

20. 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 samples.

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

22. Repeatability
Express the precision under the same operating conditions over a short interval of time.
Also referred to as Intra-assay precision
Should be assessed using minimum of 9 determinations
(3 concentrations/ 3 replicates) or

23. Intermediate Precision
Express within-laboratory variations.
Expressed in terms of standard deviation, relative standard deviation (coefficient of variation) and confidence interval.
Depends on the circumstances under which the procedure is intended to be used.
Studies should include varying days, analysts, equipment, etc.

24. 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
Mean = 100.5
RSD = 0.24%
Mean = 99.5
RSD = 0.36%

25. Reproducibility
Definition: Ability to reproduce data within the predefined precision
Determination: SD, RSD
Repeatability test at two different labs.
Lab 1
Lab 2
Lab 3
Day 1
Day 2
Day 2
Man 1
Man 2
3 Prep

26. Detection Limit (LOD)/ Quantitation Limit (LOQ)
Lowest amount of analyte in a sample that can be detected but not necessarily quantitated.
Estimated by Signal to Noise Ratio of 3:1.
LOQ
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.

27. LOD and LOQ Estimated by
Based in Visual Evaluations
- Used for non-instrumental methods
Based on Signal-to Noise-Ratio
- 3:1 for Detection Limit
- 10:1 for Quantitation Limit
Based on Standard Deviation of the Response and the Slope

28. LOD and LOQ Estimated by
QL =
10s S
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

29. Robustness
Definition: Capacity to remain unaffected by small but deliberate variations in method parameters
Determination: Comparison results under differing conditions with precision under normal conditions
Examples of typical variations in LC
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

30. 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

31. Thank you