analytical method validation raaj gprac [compatibility mode]

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Analytical Method Validation 1 Analytical Method Validation Analytical Method Validation Rajashri Rajashri Ojha Ojha Director, Director, Raaj Raaj GPRAC GPRAC

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Page 1: Analytical method validation raaj gprac [compatibility mode]

Analytical Method Validation 1

Analytical Method Validation Analytical Method Validation

RajashriRajashri OjhaOjha

Director, Director, RaajRaaj GPRACGPRAC

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Analytical Method Validation 2

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

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Analytical Method Validation 3

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)]

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Analytical Method Validation 4

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

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Analytical Method Validation 5

Validation Activity Including the Complete Analytical Procedure

Sampling

Sample Preparation

Analysis

Data Evaluation Reporting

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Analytical Method Validation 6

Optimization of Validation

Additional value and Cost VS. Completeness of validation

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Analytical Method Validation 7

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

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Analytical Method Validation 8

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

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Analytical Method Validation 9

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.

Validation Step

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Analytical Method Validation 10

System Suitability

Sample

Validation

MethodAnalyst

Calibration

Pump

Detector

Injector

Data System

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Analytical Method Validation 11

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

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Analytical Method Validation 12

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

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Analytical Method Validation 13

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)

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Analytical Method Validation 14

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

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Analytical Method Validation 15

ICH/USP Validation Requirements

Precision

Repeatability

Intermediate Precision

Limit of Detection

Limit of Quantitation

Robustness

Specificity

Linearity

Range

Accuracy

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Analytical Method Validation 16

USP Data Elements Required For Assay Validation

Analytical

Performance

Parameter

Assay Category 1Assay Category 2

Assay Category 3Quantitative Limit Tests

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

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Analytical Method Validation 17

ICH Validation Characteristics vs.

Type of Analytical Procedure

Type of Analytical

ProcedureIdentification

Impurity testingAssay

Quantitative Limit Tests

Accuracy No Yes No Yes

Precision

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

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Analytical Method Validation 18

Specificity

Ability of an

analytical method

to measure the

analyte free from

interference due to

other components.Selectivity

Bias

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Analytical Method Validation 19

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.

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Analytical Method Validation 20

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.

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Analytical Method Validation 21

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.

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Analytical Method Validation 22

Pure and Impure HPLC peaks

Peak purity tests can also be evaluated with

The spectra of Photodiode array detectors

Mass spectrometry

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Analytical Method Validation 23

Specificity: Potential Interference

Placebo

Drug Substance Degradants

Drug Product Degradants

Related Substances

Packaging Extractables

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Analytical Method Validation 24

Forced Degradation Studies

Heat

Humidity

Acid Hydrolysis

Base Hydrolysis

Oxidation

Light

High Temperature (50 to 60 oC)

Humidity (70 to 80%)

Acid Hydrolysis (0.1 N)

Base Hydrolysis (0.1 N)

Peroxide Oxidation (3 to 30%)

Intense UV/Visible Light

Intent is to create 10 to 30 % Degradation

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Analytical Method Validation 25

Specificity Study

ConditionDEG

#1

DEG

#2

DEG

#3

DEG

#4

Active ingredients

2 N HCl 0 0 17.23 4.71 95.17%

0.1 N

NaOH0 0 0 0 100%

30%

H2O2

1.12 1.41 1.65 1.2 99.98%

UV/Vis 0 0 4.68 0 94.67%

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Analytical Method Validation 26

Linearity

Ability of an assay

to elicit a direct and

proportional

response to

changes in analyte

concentration.

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Analytical Method Validation 27

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

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Analytical Method Validation 28

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

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Analytical Method Validation 29

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.

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Analytical Method Validation 30

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.

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Analytical Method Validation 31

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

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Analytical Method Validation 32

Accuracy

Closeness of the test

results obtained by the

method to the true value.

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Analytical Method Validation 33

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

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Analytical Method Validation 34

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

Mean 99.3 99.3

Std.dev. 0.1 0.4

95%C.I 99.3±0.25 99.3±0.99

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Analytical Method Validation 35

Analyte recovery at different concentration

AOAC manual for the Peer-Verified Methods program

Analyte Ingred. (%) Analyte ratio Unit Mean recovery (%)

100 1 100 % 98-102≥ 10 10-1 10 % 98-102≥ 1 10-2 1% 97-103

≥ 0.1 10-3 0.1% 95-1050.01 10-4 100 ppm 90-107

0.001 10-5 10 ppm 80-1100.0001 10-6 1 ppm 80-110

0.00001 10-7 100 ppb 80-1100.000001 10-8 10 ppb 60-115

0.0000001 10-9 1 ppb 40-120

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Analytical Method Validation 36

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.

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Analytical Method Validation 37

Precision… Considered at 3 Levels

Repeatability

Intermediate Precision

Reproducibility

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Analytical Method Validation 38

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 or

Minimum of 6

determinations at the

100% level.

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Analytical Method Validation 39

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

Depends on the

circumstances under which

the procedure is intended

to be used.

Studies should include

varying days, analysts,

equipment, etc.

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Analytical Method Validation 40

GrandMean = 100.0RSD = 0.59%

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 = 100.5RSD = 0.24%CI = 100.5 ± 0.24

Mean = 99.5RSD = 0.36%CI = 99.5 ± 0.36

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Analytical Method Validation 41

Definition: Ability reproduce data within the

predefined precision

Determination: SD, RSD and confidence interval

Repeatability test at two different labs.

Note: Data not required for BLA/NDA

Reproducibility

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Analytical Method Validation 42

Reproducibility Study

Lab 1 Lab 2 Lab 3

Day 1 Day 2 Day 1 Day 2 Day 1 Day 2

Analyst1

Analyst

2

Analyst

1

Analyst

2

Analyst

1

Analyst

2

3 Preps 3 Preps 3 Preps 3 Preps 3 Preps 3 Preps

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Analytical Method Validation 43

Analyte concentration versus precision

AOAC manual for the Peer-Verified Methods program

Analyte % Analyte ratio Unit RSD (%)

100 1 100 % 1.3≥ 10 10-1 10 % 2.7≥ 1 10-2 1 % 2.8

≥ 0.1 10-3 0.1% 3.70.01 10-4 100 ppm 5.3

0.001 10-5 10 ppm 7.30.0001 10-6 1 ppm 11

0.00001 10-7 100 ppb 150.000001 10-8 10 ppb 21

0.0000001 10-9 1 ppb 30

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Analytical Method Validation 44

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)

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Analytical Method Validation 45

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

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Analytical Method Validation 46

Based on Signal-to Noise-Ratio

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Analytical Method Validation 47

Detection Limit (DL) and

Quantitation Limit (QL)

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

DL =3.3s

QL =10s

S S

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Analytical Method Validation 48

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.

Robustness

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Analytical Method Validation 49

Confuse of Precision Terms

Repeatability

Intermediate

Precision

Reproducibility

Ruggedness

Robustness

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Analytical Method Validation 50

Precision Terms

Instrument Precision

Repeatability

Intermediate Precision

Reproducibility

Ruggedness

Robustness

- 10 Std. Injections

- One Analysis (6 preps)

- Two Analyses

- Two Lab.

- Many Variables

- Intentional Changes

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Analytical Method Validation 51

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

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Analytical Method Validation 52

Robustness-Mobile Phase Change

MeOH/

Water

Retention

Time 1

Retention

Time 2Resolution

75:25 11.94 16.41 7.39

80:20 8.47 11.17 6.17

85:15 7.81 10.18 5.93

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Analytical Method Validation 53

ICH/USP System Suitability

ICH

Definition: evaluation of

equipment, electronic,

analytical operations and

samples as a whole

Determination: repeatability,

tailing factor (T), capacity

factor (k’), resolution (R), and

theoretical Plates (N)

USP 23 <621>

System Suitability Requirements

Parameters Recommendations

K’ In general k’ ≥ 2.0

R

R > 2, between the peak of interest and the closest potential interferent (degradant, internal STD, impurity, excipient, etc…..)

T T ≤ 2

N In general N > 2000

Repeatability RSD ≤ 2.0% (n ≥ 5)

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Analytical Method Validation 54

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.”

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Analytical Method Validation 55

References

‘Analytical Methods Validation for FDA Compliance’ 교육교재 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.