Method Validation - ICH /USP Validation, Linearity and Repeatability

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Method Validation Prepared by : Santram Ra-jput (Technical Man-ager)

Sigma Test & Research Centre

European and International regulatory bodies and their guidelines on different aspects of QA

Body Full name Guidance on

Eurachem Focus for Analytical Chemistry in Europe Method validation

CITAC Cooperation of International Traceability in Analytical Chemistry

Proficiency testingQuality Assurance

EA European Cooperation for Accreditation Accreditation

CEN European Committee for Normalization Standardization

IUPAC International Union of Pure & Applied Chem. Method validation

ISO International Standardization Organisation Standardisation

AOAC

ILAC

Association of Official Analytical Chemists

International Laboratory Accreditation Cooperat.

Internal qual. ControlProficiency testingAccreditation

FDA US Food and Drug Administration Method validation

USP United States Pharmacopoeia Method validation

ICH International Conference on Harmonization Method validation

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Method Validation Validation of analytical procedures is the process of determining the

suitability of a given methodology for providing useful

analytical data.

J. Guerra, Pharm. Tech. March 1986

Validation is the formal and systematic proof that a method compiles

with the requirements for testing a product when

observing a defined procedures.

G. Maldener, Chromatographia, July 1989

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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

substances in products.

Method validation is primarily concerned with:

identification of the sources of potential errors

quantification of the potential errors in the method

An method validation describes in mathematical and quantifiable

terms the performance characteristics of an assay

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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, AAS, XRD, ICP-MS,

AAS, XRF, etc

Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC,

etc.

Automated Analytical Methods - Robots, Automated Analysis.

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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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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 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.

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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 Requirements

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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/ Drift

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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 10.0%

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System Suitability

Sample

Validation

MethodAnalyst

CalibrationPumpDetector

InjectorData System

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Method Life Cycle

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Validation

Development Optimization

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Verification vs. Validation

Compendial vs. Non-compendial Methods

Compendial methods-Verification

Non-compendial methods-Validation requirement

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Published Validation Guidelines 1978 Current Good Manufacturing Practices (cGMPs) 1987 FDA Validation Guideline 1989 Supplement 9 to USP XXI

1994 CDER Reviewer Guidance:

Validation of Chromatographic Method 1995 ICH Validation Definitions:

Q2A, Text on Validation of Analytical procedures 1997 ICH Validation Methodology:

Q2B, Validation of Analytical Procedures: Methodology 1999 Supplement 10 to USP 23 <1225>: Validation of Compendial Methods 1999 CDER “Bioanalytical Method Validation for Human Studies” 2000 CDER Draft “Analytical Procedures and Method Validation”

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The objective of validation of an analytical procedure is to demonstrate that it is suitable

for its intended purpose

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ICH Guideline for Industry Q2A, Text on Vali-dation of Analyti-cal ProceduresMarch 1995

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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.

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ICH Guideline for In-dustry Q2B, Validation of Ana-lytical Procedures: Methodology

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Today’s Validation Requirements

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ICH/USP

GMPs(legal) FDA

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ICH/USP Validation Requirements & Parameters

Specificity Linearity Range Accuracy Precision

Repeatability Intermediate Precision Reproducibility

Limit of Detection Limit of Quantitation

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ICH Specificity Linearity and Range Accuracy Precision Limit of Detection Limit of Quantitation Ruggedness Robustness

USP

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USP Data Elements Required For Assay Validation

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Analytical PerformanceParameter

Assay Category

1

Assay Category 2Assay

Category 3

Quantita-tive Limit Tests

Accuracy Yes Yes * *Precision Yes Yes No YesSpecificity 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.

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ICH Validation Characteristics vs. Type of Analytical Procedure

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Type of Ana-lytical

ProcedureIdentifica-

tion

Impurity testing

AssayQuantita-tive Limit Tests

Accuracy No Yes No YesPrecision Repeata-bility No Yes No Yes

Interm. Prec. No Yes No Yes

Specificity Yes Yes Yes YesLOD No No Yes NoLOQ No Yes No NoLinearity No Yes No YesRange No Yes No Yes

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Specificity/Selectivity Ability of an analytical method to measure the analyte free from

interference due to other components.

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.

Purity Tests: to ensure that all the analytical procedures performed allow an accurate

statement of the content of impurities of an analyte, i.e. related substances test, heavy

metals, residual solvents content, etc.

Assay (content or potency): to provide an exact result which allows an accurate statement

on the content or potency of the analyte in a sample.

Selectivity describes the ability of an analytical method to differentiate various substances in a sample

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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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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

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Linearity

Ability of an assay to

elicit a direct and

proportional response

to changes in analyte

concentration.

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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

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Range The specified range is normally derived from linearity studies and depends on the intended application of

the procedure. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the extremes of the specified range of the analytical procedure.

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

From MDL to 100% of Impurity Specification Limit

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Accuracy

Closeness of the test

results obtained by the

method to the true value.

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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

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Accuracy Data Set (1 of 3)

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Amount Added (mg)

AmountFound (mg)

Percent Re-covery

0.0 0.0 ---50.2 50.4 100.579.6 80.1 100.699.9 100.7 100.8120.2 119.8 99.7150.4 149.7 99.5

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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.302016

Precision… Considered at 3 Levels

Repeatability

Intermediate Precision

Reproducibility

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Repeatability

Express the precision

under the same

operating conditions

over a short interval of

time.

Also referred to as

Intra-assay precision

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Should be as-sessed using min-imum of 9 deter-minations

(3 concentra-tions/ 3 repli-cates) or Minimum of 6 de-terminations at the 100% level.2016

Intermediate Precision

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Express within-labo-

ratory variations.

Expressed in terms of

standard deviation,

relative standard de-

viation (coefficient of

variation) and confi-

dence interval.

Depends on the cir-

cumstances under

which the procedure

is intended to be

used.

Studies should in-

clude varying days,

analysts, equipment,

etc.2016

Repeatability & Intermediate Precision

Day 1 Day 2100.6 99.5100.8 99.9100.1 98.9100.3 99.2100.5 99.7100.4 99.6

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GrandMean = 100.0RSD = 0.59%

Mean = 100.5RSD = 0.24%

Mean = 99.5RSD = 0.36%

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Reproducibility

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

Lab 1 Lab 2 Lab 3

Day 1

Day 2

Day 1

Day 2

Day 1

Day 2

Man 1

Man 2

Man 1

Man 2

Man 1

Man 2

3 Prep

3 Prep

3 Prep

3 Prep

3 Prep

3 Prep

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Detection Limit (LOD)/ Quantitation Limit (LOQ)

LOD

Lowest amount of analyte in a

sample that can be detected

but not necessarily

quantitated.

Estimated by Signal to Noise

Ratio of 3:1.

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LOQLowest amount of an-

alyte in a sample that

can be quantified with

suitable accuracy and

precision.

Estimated by Signal to

Noise Ratio of 10:1. 2016

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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LOD and LOQ Estimated by

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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 38

DL =

3.3sQL =

10s

S S

LOD and LOQ Estimated by

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Ybl

LOD LOQ

Statistical estimate of LOD & LOQ

LOD = 3.3 Sbl / b LOQ = 10 Sbl / b

Y = b X + a

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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

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Robust-ness

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Ruggedness

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

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Reference Sites

www.fda.gov

www.fda.gov/cder/

www.waters.com

www.usp.org

www.ich.org

www.aoac.org

www.pharmweb.net

Thankyou

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