The document discusses analytical method validation. It provides an overview of the International Conference on Harmonization (ICH) which aims to harmonize regulatory requirements between Europe, Japan and the United States. It then discusses the key parameters of method validation including specificity, accuracy, precision, linearity, range, robustness, limit of detection, limit of quantification and system suitability. The document provides guidelines on testing and evaluating each validation parameter according to regulatory standards. It emphasizes that analytical methods must be validated before use and whenever the method is changed to ensure reliable and reproducible results.

1. 1

2.  Regulations and quality standards that have an impact
on analytical laboratories require analytical methods to
be validated.
 ICH is a joint regulatory/industry chair, Major initiative in
the process of attempting to harmonize several aspect
like
 Efficacy,
 Safety,
 Quality
2
Introduction

3. 3
ICH
(International Conference on
Harmonization)
EUROPE
JAPAN
UNITED STATES

4. 1. Stability – Q1(Q1A,Q1B,Q1C,Q1D,Q1E,Q1F,Q1G,Q1H0)
2. Analytical Method Validation – Q2 (Q2A,Q2B)
3. Impurity Testing – Q3(Q3A,Q3B.,Q3C)
4. Pharmacopoeial Harmonization-Q4(Q4A)
5. Quality of Biotechnological and Biological products – Q5(Q5A,
Q5B, Q5C, Q5D)
6. Specifications – Q6(Q6A,Q6B)
7. Good Manufacturing practices – Q7(Q7A)
4

5.  Process validation is documented program which
provides a high degree of assurance that a specific
process will consistently produce a desired result /
product meeting its predetermined specification
and quality characteristics
 Method validation is the process of demonstrating
that analytical procedures are suitable for their
intended use
5

6.  Transfer of method from one laboratory to other
 Analyst within the laboratory
 Different days within the laboratory
 In-day variation
 Between instrument variation within the laboratory

7.  The process of method development and validation has a
direct impact on the quality of these Data.
 (The purpose of analytical measurement is to get
consistent, reliable and accurate data)
 To avoid difference between the analytical development
and quality control laboratory
 Regulatory requirement – as per cGMP, GLP 21CFR part
58.113, USFDA 211.165(a), 211.194(b), Pharmaceutical
Inspection Cooperation Scheme PIC/S and Europe, ICH
chapter12 , USP chapter 1225-validation of compendial
drugs, chapter1226-verfication of compendial drugs,
chapter 621 chromatography, ISO/IEC 17025
7

8.  New method Developed
 Before initial use in routine testing
 When transferred to another laboratory
 Whenever the conditions or method parameter are changed (for
example instrument with different characteristics or sample with
different matrix)
 When we make any changes in analytical procedure or Fomulae
change in case of drug product.
(The degree of the revalidation depends on the nature of the change)
8

9.  Identification Tests
 Quantitative Tests for Impurities
 Limit Tests for Control of Impurities
 Quantitative tests for active moiety in samples of drug
substance & drug products and other selected
components in drug product
9
Analytical validation to be validated for

10.  A well designed experimental matrix (Written protocol)
 Step by Step Methodology (STP)
 Standards
 Samples
 Equipments & Analytical instruments should be in calibrated
state.
10

11. 1. Linearity
2. Accuracy
3. Precision
4. Specificity
5. LOD&LOQ (If Applicable )
6. Range
7. Robustness
8. Stability
9. System Suitability
10. Assay
11

12.  Ability of an assay to elicit a
direct and proportional
response to changes in
analyte concentration.
 Use standard drug solution
12

13. Linearity Evaluation :
By Appropriate statistical methods
› Linear Regression (y = mx + b)
› Correlation Coefficient, y-intercept (b), slope of the regression
line(m), residual sum of squares
(5-6 injections of conc. range of 80-120 percent of the expected
conc. Range)
13

14. Linearity Evaluation :
Calibration curve -
Slope - indicates sensitivity of the method
Intercept - indicates response for no analyte (interference)
Residual sum of squares - indicates uncertainty of intercept(in
blank response)
Correlation Coefficient - indicates the relationship chosen is correct
(Line of best fit )
14

15. Definition
The accuracy of an analytical
procedure expresses the closeness
of agreement between the value,
which is accepted either as a
conventional true value or an
accepted reference value and the
value found
15

16. Accuracy is usually determined By:
1. Apply known concentration of reference sample and compare the
measured value to the true value (defined by the organization, from
which the sample received, min.3 conc. of three replicates)
2. Compare the test results obtained by the analytical procedure which is
proved to be accurate with the results obtained from an existing
alternate method that is known to be accurate
3. Recovery Study - By spiking either analyte/impurities into sample matrix
Minimum 9 determination, 3 conc. (80%, 100% , 120%) of 3 injection
4. Accuracy should be reported as % recovery assay by known added
amount and amount found . Find %RSD
5. Accuracy for Drug substance, 99-101%, Drug product, 98-102%
16

17.  Definition
Closeness of agreement (degree of scatter)
between a series of measurements obtained
from multiple sampling of the same
homogenous sample under the prescribed
conditions.
Precision may be considered at 3 levels.
› Repeatability
› Intermediate precision
› Reproducibility
17

18. Discussion
Repeatability: precision under same operating conditions (with-in a
laboratory over a short period of time using the same analyst with the
same equipment)
-Measurement / Injection repeatability (System Precision)
-Method repeatability (Method Precision)
- Minimum 9 determinations (3 concentrations and 3 injections each)
- Minimum 6 determinations at 100% of the test concentration
Intermediate precision: Variations within the laboratory conditions (as
on different days, different analysts, different equipments, standards
and reagents from different supplier, column from different batch and so
on)
Reproducibility: Expresses the precision between laboratories
(collaborative studies usually applied to standardization of
methodology)
18

19.  Intermediate precision : To verify that in the same
laboratory the method will provide the same results once the
development phase is over.
 Reproducibility : To verify that the method will provide the
same results in different laboratories with different analysts
 Evaluation
Mean, SD, %RSD & 95% CI (System Precision / Method Precision)
Overall mean, SD, %RSD & 95% CI, F-test & T-test
But, the extent is depend on, steps involved (weighing, dilution,
extraction, matrix etc.), technique used, other expected variables
(stability etc.) and intended use of the procedure
19

20.  Definition:
Specificity 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
20
(Contd.,)

21. Discussion:
1. It generally refers to a method that produces a response
for a single analyte only
2. It provides response for a number of chemical entities
that may / may not be distinguished from each other.
3. If each response is distinguished from all other
response, then the method is said to be selective.
Contd.,
21

22.  The term Specificity is used in microbiological assay,
radio-immunoassay etc.,
 Use of the term Selectivity is appropriate for the
methods based on techniques such as HPLC, GC
etc.,
Contd.,
22

23. Procedure :
Suitable identification tests should be done to
discriminate between compounds of closely related
structures which are likely to be present.
A. Identification
B. Assay and Impurity test
A. Impurities are available- Perform blank run, placebo
sample (excipient-without API) & un-spiked , spike with
pure substance, spike with impurities, degradants,
excipients etc.,
Contd.,
23

24. B. Impurities are not available: Compare the test result containing
impurity with standard sample.
Stress Study (Forced Degradation)
 It will help to find the degradative pathway like Alkaline, Acidic,
Oxidative and Photolytic during stability study or Shelf life.
 Heat -40ºC
 Humidity-50%
 Acid Hydrolysis-0.1N HCl
 Base Hydrolysis-0.1N NaOH
 Oxidation-3% H202
 Light-200-300 candle light
Evaluation of Stress Study : By Peak Purity and/or by MS
Conti…
24

25.  LOD: Lowest amount of analyte in
a sample which can be detected
but not necessarily quantitated,
under the stated experimental
conditions (LOD)
 LOQ: Lowest amount of analyte in
a sample which can be
quantitatively determined with
suitable precision and accuracy
(LOQ)
25

26. Estimated By:
Based on Visual Evaluations
- Used for non-instrumental /instrumental methods
Based on Signal-to Noise-Ratio
- 3:1 for Detection Limit
- 10:1 for Quantitation Limit
26

27. 1. Based on Standard Deviation of the Response and
the Slope
LOD =
3.3 σ
LOQ =
10 σ
S S
27

28. Definition
The range of an analytical procedure is the interval between the
upper and lower concentrations (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
Procedure
No separate experiments required
Evaluation
The evaluated data for accuracy, and linearity is enough
28

29. Definition
Measure of its capacity to remain unaffected by small, but
deliberate variations in method parameters and provides
indication of its reliability during its normal usage
Procedure
FOR HPLC FOR GC
Flow rate Flow rate
Mobile phase composition, Temperature
(generally, Organic composition
Temperature
pH of the mobile phase
29

30. EVALUATION
System Suitability parameters at all variable conditions
(change in pH, mobile phase composition, flow rate, column
temp, detection wave length etc.)
% Assay of samples at all variable conditions
Monitor the separation at each variable condition
30

31.  It indicates is degree of reproducibility of test results and determine
the function of variables
 Analysis of the same samples under a variety of conditions, such as
 - Different laboratories,
› Different analyst,
› Different instruments,
› Different lots of reagent,
› Different elapsed assay times,
› Different assay temperatures,
› Different days, etc.
 The reproducibility may be compared to the precision of the assay
under normal condition

32.  It is often essential that solutions (standards, test samples) be
stable enough to allow for delays covering instrument break
downs / overnight analyses
 A minimum of 12 h, 18 h, or 24 h is routinely recommended for
chromatographic methods for which vialed solutions may remain
on an auto-samplers at ambient temperatures due to various
delays.
 A simple logic behind this study is to determine the period of time,
a solution can be held before analysis without compromising
accuracy.
32

33. 33
System Suitability
Sample
Validation
Method
Analyst
Calibration
Pump
Detector
Injector
Data System

34.  Suitable concentration of the sample (tablet, capsule ) should be
prepared and find the absorbance
 By using Linear Regression equation (y = mx + b), calculate
the % obtained. It should be within 98%-102%
 Minimum 2-3 Brand should be assayed or 3 different batch of
the same formulation
 In case formulation is not available, synthetic mixtures of the
drug product components to which known quantities of the
drug substance to be analyzed have been added;
 Compare the results of the proposed analytical procedure with
the reported method if any by using suitable statistical methods

35. THANKS
35