The document discusses analytical method validation. It emphasizes that validation is important to ensure consistent, reliable and accurate data from analytical measurements, especially for regulated environments. The document outlines key aspects of validation including specificity, linearity, range, accuracy, and precision according to ICH and USP guidelines. It also discusses validation requirements for different types of analytical procedures and provides examples of forced degradation studies to demonstrate specificity.
The document discusses analytical method validation. It emphasizes that validation is important to ensure consistent, reliable and accurate data from analytical measurements, especially for regulated environments. The document outlines key aspects of validation including specificity, linearity, range, accuracy, and precision according to ICH and USP guidelines. It also discusses validation requirements for different types of analytical procedures and provides examples of forced degradation studies to demonstrate specificity.
The document discusses analytical method validation. It emphasizes that validation is important to ensure consistent, reliable and accurate data from analytical measurements, especially for regulated environments. The document outlines key aspects of validation including specificity, linearity, range, accuracy, and precision according to ICH and USP guidelines. It also discusses validation requirements for different types of analytical procedures and provides examples of forced degradation studies to demonstrate specificity.
The document discusses analytical method validation. It emphasizes that validation is important to ensure consistent, reliable and accurate data from analytical measurements, especially for regulated environments. The document outlines key aspects of validation including specificity, linearity, range, accuracy, and precision according to ICH and USP guidelines. It also discusses validation requirements for different types of analytical procedures and provides examples of forced degradation studies to demonstrate specificity.
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Analytical Method Validation
Analytical Method Validation
Why Method Validation is Important?
1. The purpose of analytical measurement is to get consistent,
reliable and accurate data. Incorrect measurement results can lead to tremendous costs. 2. Equal importance for those working in a regulated and in an accredited environment. U.S. FDA, EMEA, EPA, AOAC, ISO
Analytical Method Validation
Background NDA and ANDA must include the analytical procedures necessary to ensure: Identity, Strength, Quality, Purity, and Potency of the Drug Substances and Drug product [21CFR 314.50(d)(l) and 314.94(a)(9)(i)] Data to establish and reliability [21CFR 211.169(e) and 211.194(a) (2)]
Analytical Method Validation
Validation is an Old Concept
But There are Many Problems Lack of documented procedures and documented validation results Sampling or Sample preparation step contribute to overall error. Accessories and materials used for equipment qualification are not qualified. Limits for Operational Qualification Lack of software validation and computer system validation Qualification and validation are done at just one particular point in time. Adaptation of acceptance criteria for qualification of new system Analytical Method Validation
Validation Activity Including
the Complete Analytical Procedure Sampling
Sample Preparation
Analysis
Data Evaluation Reporting
Analytical Method Validation
Optimization of Validation
Additional value and Cost VS. Completeness of validation
Analytical Method Validation
Considerations Prior to Method Validation 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 analytical procedure and proper approved protocol with pre-established acceptance criteria Analytical Method Validation
Validation Step
Define the application, purpose and scope of the method.
Analytes? Concentration? Sample matrices? Develop a analytical method. Develop a validation protocol. Qualification of instrument. Qualify/train operator
Analytical Method Validation
Qualification of material. Perform pre-validation experiments. Adjust method parameters and/or acceptance criteria if necessary. Perform full validation experiments. Develop SOP for executing the method in routine analysis. Document validation experiments and results in the validation report. Analytical Method Validation
System Suitability Validation Calibration Pump
Injector
Detector
Data System
Analyst
Method Sample Analytical Method Validation
10
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
Analytical Method Validation
11
Regulations and Guidelines of
Validation US FDA 21 CFR (Code of Federal Regulations) Part 210 and 211 Part 210: cGMP in Manufacturing, Processing, Packaging, or Holding of Drugs; General Part 211: cGMP for Manufacturing Practice for Finished Pharmaceuticals
ICH Guidelines Q2A, Text on Validation of Analytical procedures (March 1995) Q2B, Validation of Analytical Procedures: Methodology (May 1997)
USP Chapter <1225>
Validation of Compendial Methods Analytical Method Validation
12
The accuracy, sensitivity, specificity, and reproducibility of test methods
employed by the firm shall be established and documented. Such validation and documentation may be accomplished in accordance with 211.194(a) (2).
21 CFR PART 211 - CURRENT GOOD MANUFACTURING
PRACTICE FOR FINISHED PHARMACEUTICALS Subpart I-Laboratory Controls 211.165 Testing and release for distribution (e)
Methods validation means establishing, through documented evidence, a
high degree of assurance that an analytical method will consistently yield results that accurately reflect the quality characteristics of the product tested. 21 CFR PART 210 - CURRENT GOOD MANUFACTURING PRACTICE IN MANUFACTURING, PROCESSING, PACKING, OR HOLDING OF DRUGS 210.3 Definitions (b) (25)
Analytical Method Validation
13
The objective of validation of an analytical procedure is to
demonstrate that it is suitable for its intended purpose
ICH Guideline for Industry Q2A
In practice, it is usually possible to design the experimental work such
that the appropriate validation characteristics can be considered simultaneously to provide a sound, overall knowledge of the capabilities of the analytical procedure, for instance: Specificity, Linearity, Range, Accuracy, and Precision.
ICH Guideline for Industry Q2B
Analytical Method Validation
14
ICH/USP Validation Requirements
Precision Specificity Linearity Range Accuracy
Repeatability Intermediate Precision Limit of Detection Limit of Quantitation Robustness
Analytical Method Validation
15
USP Data Elements Required For Assay Validation
Analytical Performance Parameter
Assay Category 2 Assay Category 1
Quantitative
Limit Tests
Assay Category 3
Accuracy
Yes
Yes
Precision
Yes
Yes
No
Yes
Specificity
Yes
Yes
Yes
LOD
No
No
Yes
LOQ
No
Yes
No
Linearity
Yes
Yes
No
Range
Yes
Yes
Ruggedness
Yes
Yes
Yes
Yes
* May be required, depending on the nature of the specific test.
Category 1: Quantitation of major components or active ingredients Category 2: Determination of impurities or degradation products Category 3: Determination of performance characteristics Analytical Method Validation
16
ICH Validation Characteristics vs.
Type of Analytical Procedure
Type of Analytical Procedure
Impurity testing Identification
Assay
Quantitative
Limit Tests
No
Yes
No
Yes
Repeatability
No
Yes
No
Yes
Interm. Prec.
No
Yes
No
Yes
Specificity
Yes
Yes
Yes
Yes
LOD
No
No
Yes
No
LOQ
No
Yes
No
No
Linearity
No
Yes
No
Yes
Range
No
Yes
No
Yes
Accuracy Precision
Analytical Method Validation
17
Specificity Ability of an analytical method to measure the analyte free from interference due to other components.
Selectivity Bias
Analytical Method Validation
18
Specificity: ICH/USP An investigation of specificity should be conducted during the validation of an identification test, an impurities assay, and a potency assay. Procedures used will depend on the intended objective of the analytical procedure. If a method can not completely discriminate, two of more procedures are recommended.
Analytical Method Validation
19
Specificity: Identification Should be able to discriminate between compounds closely related in structure. Confirmed by obtaining negative results for samples with spiked related compounds and positive results for samples with analyte. Choice of potential interfering substances should be based on sensible scientific judgment considering substances that could likely occur. Analytical Method Validation
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.
Analytical Method Validation
21
Pure and Impure HPLC peaks
Peak purity tests can also be evaluated with
The spectra of Photodiode array detectors Mass spectrometry
Analytical Method Validation
22
Specificity: Potential Interference
Placebo Drug Substance Degradants Drug Product Degradants Related Substances Packaging Extractables
Analytical Method Validation
23
Forced Degradation Studies
Heat
High Temperature (50 to 60 oC)
Humidity
Humidity (70 to 80%)
Acid Hydrolysis
Acid Hydrolysis (0.1 N)
Base Hydrolysis
Base Hydrolysis (0.1 N)
Oxidation
Peroxide Oxidation (3 to 30%)
Light
Intense UV/Visible Light
Intent is to create 10 to 30 % Degradation
Analytical Method Validation
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Specificity Study
Condition
DEG #1
DEG #2
DEG #3
DEG #4
Active ingredients
2 N HCl
17.23
4.71
95.17%
0.1 N NaOH
100%
30% H2O2
1.12
1.41
1.65
1.2
99.98%
UV/Vis
4.68
94.67%
Analytical Method Validation
25
Linearity Ability of an assay to elicit a direct and proportional response to changes in analyte concentration. Analytical Method Validation
26
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), residual sum of squares
Requires a minimum of 5 concentration levels
Analytical Method Validation
27
Linearity Example
R square = 0.999 Slope = 0.97 y-intercept = 0.233 Line Eq.: Y = 0.97X + 0.233 Std. Error = 1.319 Std. Deviation of Slopes = 0.0079 Analytical Method Validation
28
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. Analytical Method Validation
29
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.
Analytical Method Validation
30
Minimum Specified Range:
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 Analytical Method Validation
31
Accuracy
Closeness of the test
results obtained by the method to the true value.
Analytical Method Validation
32
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 (reference material) or The difference between the mean assay result and the accepted value Analytical Method Validation
33
Accuracy Data Set (1 of 3)
Amount Added (mg)
Amount Found (mg)
Percent Recovery
0.0
0.0
---
50.2
50.4
100.5
79.6
80.1
100.6
99.9
100.7
100.8
% Recovery
% Recovery
99.2
98.9
99.3
99.3
99.4
99.7
99.3
99.3
Std.dev.
0.1
0.4
95%C.I
99.30.25
99.30.99
Mean
Analytical Method Validation
34
Analyte recovery at different
concentration Analyte Ingred. (%)
Analyte ratio
Unit
Mean recovery (%)
100
100 %
98-102
10
10-1
10 %
98-102
10-2
1%
97-103
0.1
10-3
0.1%
95-105
0.01
10-4
100 ppm
90-107
0.001
10-5
10 ppm
80-110
0.0001
10-6
1 ppm
80-110
0.00001
10-7
100 ppb
80-110
0.000001
10-8
10 ppb
60-115
0.0000001
10-9
1 ppb
40-120
AOAC manual for the Peer-Verified Methods program
Analytical Method Validation
35
Precision The closeness of agreement
Ball Ball Ball Ball Ball Ball Ball Ball
Strik Strike Strike e Strike Strike Strike
(degree of scatter) between a
series of measurements obtained from multiple samplings of the same homogeneous sample. Should be investigated using homogeneous, authentic samples.
predefined precision Determination: SD, RSD and confidence interval Repeatability test at two different labs.
Note: Data not required for BLA/NDA
Analytical Method Validation
41
Reproducibility Study Lab 1
Lab 2
Lab 3
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Analyst 1
Analyst 2
Analyst 1
Analyst 2
Analyst 1
Analyst 2
3 Preps
3 Preps
3 Preps
3 Preps
3 Preps
3 Preps
Analytical Method Validation
42
Analyte concentration versus precision
Analyte %
Analyte ratio
Unit
RSD (%)
100
100 %
1.3
10
10-1
10 %
2.7
10-2
1%
2.8
0.1
10-3
0.1%
3.7
0.01
10-4
100 ppm
5.3
0.001
10-5
10 ppm
7.3
0.0001
10-6
1 ppm
11
0.00001
10-7
100 ppb
15
0.000001
10-8
10 ppb
21
0.0000001
10-9
1 ppb
30
AOAC manual for the Peer-Verified Methods program
Analytical Method Validation
43
Detection Limit (DL)
Quantitation Limit (QL)
Lowest amount of analyte in a
Lowest amount of analyte in a
sample that can be detected
sample that can be quantified
but not necessarily quantitated.
with suitable accuracy and
Estimated by Signal to Noise
Ratio of 3:1.
precision. Estimated by Signal to Noise Ratio of 10:1.
Analytical Method Validation
44
Detection Limit (DL) and Quantitation
Limit (QL) Estimated by 1. Based in Visual Evaluations - Used for non-instrumental methods
2. Based on Signal-to Noise-Ratio
- 3:1 for Detection Limit - 10:1 for Quantitation Limit
3. Based on Standard Deviation of the Response
and the Slope Analytical Method Validation
45
Based on Signal-to Noise-Ratio
Analytical Method Validation
46
Detection Limit (DL) and
Quantitation Limit (QL)
DL =
3.3s S
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 Analytical Method Validation
47
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 Variations may include: stability of analytical solution, variation of pH in a mobile phase, different column (lot/supplier), temperature, flow rate.
USP 23 <621> Definition: evaluation of equipment, electronic, analytical operations and samples as a whole Determination: repeatability, tailing factor (T), capacity
System Suitability Requirements
Parameters
Recommendations
In general k 2.0
R > 2, between the peak of
interest and the closest potential interferent (degradant, internal STD, impurity, excipient, etc..)
factor (k), resolution (R), and
theoretical Plates (N)
T2
In general N > 2000
Repeatability
RSD 2.0% (n 5)
Analytical Method Validation
53
Guidance on Re-Validation When sponsors make changes 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. Analytical Method Validation
54
References Analytical Methods Validation for FDA Compliance The Center for Professional Advancement 2003. 3.12-14. Guideline for submitting samples and analytical data for kethods validation (Feb. 1987) ICH Q2A ICH Q2B 21 Code of Federal Registrations Part 210 and 211 Michael E. Swatrz and Ira S. Krull, Analytical method development and validation. Mrcel Dekker, Inc. New York, 1997. USP 23 <1225> http://www.waters.com Ludwig Huber, Validation and Qualification in Analytical Laboratories, Interpharm Press Inc. Buffalo Grove, Illinois, 1999. Analytical Method Validation
