This document provides an overview of analytical method validation. It defines validation as proving a method leads to expected results. Validation is required for analytical tests, equipment, and processes. Once validated, a method is expected to remain in control if unchanged. The document discusses types of analytical procedures that must be validated, including identification, quantitative impurity, limit tests, and assays. It also distinguishes between validation and verification. Key aspects of validation covered include system suitability, specificity, linearity, range, precision, accuracy, recovery, and robustness. The validation characteristics and acceptance criteria are defined.

1. Analytical Method
Validation
Presented By:
Bholakant Raut
Samanjee Kumar Mandal
Keva Pharmaceuticals Pvt. Ltd.

2. WHO validation definition
• The documented act of proving that any procedure, process,
equipment, material, activity, or system actually leads to the expected
results.
• Validation studies are performed for analytical tests, equipment,
facility systems such as air, water, steam, and for processes such as
the manufacturing processes, cleaning, sterilization,
• Once the system or process has been validated, it is expected that it
remains in control, provided no changes are made.

3. Analytical Method Validation
• AMV establishes documented evidence that the analytical method
adopted for a test is fit for the intended purpose in terms of quality,
reliability, reproducibility and consistency of results.
• In other words, if the same method is adopted in any other laboratory
across the world, under the same set of conditions and control
parameters, the results should be in agreement.

4. Analytical Method Validation
• Pharmaceutical Industries governed by rules & regulations. Method
validation is mandatory as per regulatory agencies.
• Analytical methods should be used within GMP and GLP
environments

5. 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.
• Quantitative tests of the active moiety (API) in samples of
drug substance or drug product or other selected component(s)
in the drug product. e.g. ASSAY, DISSOLUTION etc.

6. Validation Vs Verification
• Method Validation
Characterizes performance characteristics
USP 46 - NF 41; General Chapter <1225> Validation of Compendial
Procedures
• Hyperlink the Files〈1225〉 Validation of Compendial Procedures.pdf
• Method Verification
Confirms that the validated method works as intended under conditions of
actual use
USP 46 - NF 41; General Chapter <1226> Verification of Compendial
Procedures
Hyperlink the Files〈1226〉 Verification of Compendial Procedures.pdf

7. Considerations prior to AMV
• Is required throughout the regulatory submission
process/compliance. i.e. for DDA submission, also for Laboratory
accreditation
• Hyperlink the FilesGuideline on Analytical Method Validation-
DDA.pdf
• The laboratory should be following a written SOP that describes the
process of conducting method validation.
• There should be a well-developed and documented test method in
place and an approved AMV protocol should be in place.

8. Considerations prior to AMV cont.
• AMV Plan and Performance Record: Scope, Purpose and
applications of the method. Also describes which method, performance
parameters will be tested, how the parameters will be assessed, and
the acceptance criteria that will be applied. Criteria for revalidation
so on.
• Also Materials and chemicals (Reagents) to be used, Instruments
details and their Status of Qualification and Calibration, AMV Team
members, Status of Training and Qualification Records. i.e. qualified
analyst etc.
• Finally, samples of API or drug product, placebo, and working
standard are needed to perform the validation experiments.

9. Calibration:
• Assessment of proper functioning of instruments
• Instrumental specifications: Liquid chromatography
• Injection reproducibility (injection precision)
• Column performance (Efficiency, Selectivity, Asymmetry, Backpressure)
• Pump performance (Flow rate, Pressure stability)
• Detector performance (Sensitivity, Noise, Drift)
• Temperature calibration of Column Heater,
• Auto Injector Cooler/Heater temperature calibration
• Injection Volume Accuracy of Auto injector
• Electronic Weighing Balance: Must be calibrated by Nepal Bureau of
Standards and Metrology

10. Analytical method validation
characteristics

11. 1. System suitability testing
Sr. No. System Suitability Test (HPLC) Limit
1 Theoretical Plates NLT 2000
2 Tailing factor NMT 2.0
3 RSD of five/six replicate injections NMT 2.0%
4 Resolution between two peaks NLT 2.0
5 RSD (for UV) NMT 3.0%
 System suitability tests should be performed on HPLC systems to determine
the accuracy and precision of the system by injecting five/ six injections of a
solution containing analyte (standard solution) at 100% of test concentration.
Determine relative standard deviation (RSD) of the replicate injections,
theoretical plate and tailing factor.

12. 2. Specificity
• Specificity is the ability of the analytical method to measure only the
analyte of interest without interference from other components in the
sample that are likely to be present, such as impurities, degradants, matrix
components, etc.
• Should be investigated by injecting the blank (solvent)/ placebo (matrix
solution), standard solution, sample solution to demonstrate the absence of
interference with the elution of analytes.
• HPLC-Well separation of all peaks. Drug from Impurities
• Peak purity- No overlapping of peaks / coelution

13. Fig. An example of method specificity

14. 3. Linearity
• The analytical method should be linear, i.e., there should be a direct relationship
between the concentration of the analyte(s) and the signal produced.
• Standard solutions should be prepared at minimum of 5/6 concentrations within
the range of typically 80%, 100 %, 120 %, of target concentration.
• A plot of signals as a function of analyte concentration or content is then created,
usually by means of a regression line. The correlation coefficient, y-intercept,
slope of the regression line, and residual sum of squares should be calculated.
• Correlation coefficient (r2) ≥ 0.99 (For AssaY)
• r2 ≥ 0.98 (For Dissolution)

15. Fig. An example of a regression line
Peak area as a function of Crisaborole concentration
Crisaborole Concentration (mg/ml)

16. 4. Range
The analytical method must be able to detect and quantify low levels of analytes
accurately. The specified range is normally derived from linearity studies.
The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations)
for which it has been demonstrated that the analytical procedure has a suitable
level of precision, accuracy and linearity.
The following minimum specified ranges should be considered:
Assay of drug substances (80 % to 120 % of the test concentration)
Content Uniformity (minimum 70% to 130 % of the test concentration)
Dissolution testing (+/-20 % over the specified range)

17. 4. Range Cont.
• Limit Of Detection (LOD): The detection limit of an individual
analytical procedure is the lowest amount of analyte in a sample which
can be detected but not necessarily quantitated as an exact value.
• LOD may be expressed as:
LOD = 3.3 σ/S
• Where σ = the standard deviation of the response,
• S = the slope of the calibration curve. The slope S may be estimated
from the calibration curve of the analyte.

18. 4. Range Cont.
• Limit Of Quantitation (LOQ):The quantitation limit of an individual
analytical procedure is the lowest amount of analyte in a sample which
can be quantitatively determined with suitable precision and accuracy.
• LOQ may be expressed as:
LOQ = 10σ/S
Where σ = the standard deviation of the response,
S = the slope of the calibration curve. The slope S may be estimated
from the calibration curve of the analyte.

20. 5. Precision
• Precision is the degree of agreement (degree of scatter) between
replicate measurements of the same sample under the same
conditions.
• Precision may be considered at three levels: Repeatability,
Intermediate precision and Reproducibility.

21. 5. Precision Cont.
•Repeatability expresses the precision under the same
operating conditions over a short interval of time.
For instrument precision determinations of five replicate of
reference standard should be made.
For the method precision at least nine determinations covering
specified range of 3 concentration and 3 replicates should be made or a
minimum of 6 determinations at 100% of the test concentration.

22. 5. Precision Cont.
•Intermediate Precision
• Test procedure Intermediate precision (within-laboratory
variation) should be demonstrated by at least two
analysts, using at least two HPLC/UV-vis
spectrophotometer on different days and evaluating
the relative percent purity data across the two systems of
triplicate sample of one concentration.

23. 5. Precision Cont.
•Reproducibility:
• Reproducibility expresses the precision between laboratories
(collaborative studies, usually applied to standardization of
methodology). Reproducibility is assessed by means of an inter-
laboratory trial.
• i.e. Different analysts in different laboratories should be able to get
similar results. This is usually tested in the case of Method transfer.

24. 6. Accuracy and Recovery..Hyperlink the
Files5. Accuracy Test.pdf
• The accuracy is closeness between the experimental value and true
value/theoretical value.
• Spiked samples should be prepared at three concentrations over the
range of 80 %, 100 % and 120 % of the target concentration. Three
individually prepared triplicates at each concentration will be
analyzed.
• Limit:

25. 6. Accuracy Cont.
Accuracy = Trueness + Precision

26. 7. Robustness
a) Deliberate variation
The investigation of robustness can be done by change of flow rate of
the mobile phase, change of temperature of column, change of
composition of the mobile phase, change in the pH of the mobile
phase and use of different column.
Changes should be within the limits that produce acceptable
chromatography & UV spectrum.

27. 7. Robustness Cont.
a) Deliberate variation Cont.

28. 7. Robustness Cont.
b) Stability of the standard and sample solution
Solutions of drug product should be analyzed in comparison to the
fresh prepared solutions stored at room temperature in auto sampler
and stored at 2 - 8 °C, in refrigerator at least 24 hours.
98.0 % to 102.0 % in comparison to the freshly prepared solutions
Hyper link

29. Revalidation
The degree of revalidation depends on the nature of the change.
Change in the analytical procedure, drug substance, drug product may
necessitate revalidation of the analytical procedures.
Every 5 years even if no change in any of the above.
When the method is properly validated – consistent, reliable and
accurate results are obtained.
METHOD VALIDATION =ERROR ASSESSMENT

30. References
1) ICH harmonized tripartite guideline - Validation of Analytical
Procedures : Text and methodology - ICH Q2(R1)
2) United States Pharmacopoeia, Chapter <1225> Validation of
compendial methods.
3) Guideline on Analytical Method Validation on Non-pharmacopoeial
Products for Regulatory Approval. Doc. No.: AMVP/076-77-02,
Supersede No:1 (AMVP/073/01).