validation of analytical methods -...

of 85 /85
VALIDATION OF ANALYTICAL VALIDATION OF ANALYTICAL METHODS METHODS

Upload: dangque

Post on 13-Apr-2018

220 views

Category:

Documents


4 download

TRANSCRIPT

Page 1: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

VALIDATION OF ANALYTICAL VALIDATION OF ANALYTICAL METHODSMETHODS

Page 2: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation

FDA-guidelines: Validation is establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications and quality attributes

EU-guidelines: Action of proving, in accordance with GMP-principles that any procedure, process, equipment, material, activity or system actually leads to the expected results

Page 3: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

ICH

“Methods validation is the process of demonstrating that analytical procedures are suitable for their intended use”

Page 4: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Why validation?

1. GMP-legislation 2. Good economics3. Good science practices

Page 5: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

The Purpose Of Analytical Method Validation

Identification of Sources and Identification of Sources and QuantitationQuantitation of potential of potential errorserrors

Determination if Method is Acceptable for Intended UseDetermination if Method is Acceptable for Intended Use

Establish Proof that a Method Can be Used for Decision Establish Proof that a Method Can be Used for Decision MakingMaking

Satisfy FDA RequirementsSatisfy FDA Requirements

Page 6: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation guidelines

1. ICH Q2A Text on validation of analytical procedures: Definitions and terminology (March 1995)2. ICH Q2B Validation of analytical procedures: Methodology (June 1997) 3. FDA Guidance for Industry: Analytical procedures and methods validation 4. Pharmacopoeias USP and European Pharmacopoeia

Page 7: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Method Validation Strategy

1. Develop a validation protocol or operating procedure for the validation2. Define the application, purpose and scope of the method3. Define the performance parameters and acceptance criteria4. Define validation experiments5. Verify relevant performance characteristics of equipment6. Qualify materials, e.g. standards and reagents7. Perform pre-validation experiments8. Adjust method parameters or/and acceptance criteria if necessary9. Perform full internal (and external) validation experiments10. Develop SOPs for executing the method in the routine11. Define criteria for revalidation12. Define type and frequency of system suitability tests and/oranalytical quality control (AQC) checks for the routine13. Document validation experiments and results in the validation report

Page 8: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

What methods to be validated?

Defined for: identification quantitative tests for content of impuritieslimit tests for control of impurities quantitative tests for active moiety in drug substances and drug products

Referred to: -dissolution testing

Page 9: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation SOPValidation SOP

What is it?

- It is your laboratory’s documented

procedure/philosophy that describes:

What methods require validation

Who is responsible for performing validation

How much validation will be done for different methods

How the validation process will be documented

Page 10: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation SOPValidation SOPContent

- Purpose

This document describes the procedures for validating analytical methods in the XYZ Pharmaceutical Company

- Scope

This document applies to all analytical methods used for releaseand stability testing

- Responsibilities

State who will

- Prepare the validation protocol/guideline

- Review and approve the protocol

- Prepare the validation report

- Review and approve the validation report

Page 11: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

- DefinitionsDefine any unique of ambiguous terms used within your company

- Compendial, selectivity/specificity, evaluation criteria, etc.

- Validation of non-compendial methodsNoncompendial methods will be evaluated according to the current ICH/FDA guidelines

- Validation of compendial methodsCompendial methods used for their intended compendial material will be tested for sepcificity, intermediate precision,and sample solution stability.Compendial methods used for non-compendial materials will be validated according to the current ICH and FDA guidelines

Page 12: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

-- Revalidation of methodsRevalidation of methods

state your process for state your process for

-- Updating validation for older productsUpdating validation for older products

-- Revalidating when changes in synthesis, formulation, or analyticRevalidating when changes in synthesis, formulation, or analytical al

procedure occurprocedure occur

-- Validation reportsValidation reports

state who is responsible for writing, reviewing, approving, state who is responsible for writing, reviewing, approving,

and archivingand archiving

Page 13: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation ProtocolValidation Protocol

It is a description of…– How specific validation studies will be performed for a given

type of method

– How results will be evaluated for acceptability

It is an opportunity to document your customer’s needs, and their acceptance of your plan

Page 14: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation ProtocolValidation Protocol

Example content:

– Linearity-standard solutions will be prepared and

analyzed at 5 levels over the range of 80 to 120%

of the target concentration. This will be

performed in triplicate.

– Repeatability precision-a homogeneous

sample will be analyzed 6 times.

Page 15: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation SpecificationValidation Specification

How good to my validation results need to be?

– Agree on the acceptance criteria (specifications) before

beginning the validation.

– Acceptance criteria should be based on

- The intended use of the method

- Sufficient experience with the method

Page 16: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation SpecificationValidation Specification

Examples

– Linearity-standard curves will have correlation

coefficients ≥ 0.99 and y-intercept values less than 2%

of the target level response.

– Repeatability precision-the test results will have a RSD

≤ 2%.

Page 17: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Considerations Prior to Considerations Prior to Method ValidationMethod Validation

Suitability of Instrument Suitability of Instrument

Status of Qualification and Calibration Status of Qualification and Calibration

Suitability of Materials Suitability of Materials

Status of Reference Standards, Reagents, Placebo Lots Status of Reference Standards, Reagents, Placebo Lots

Suitability of Analyst Suitability of Analyst

Status of Training and Qualification Records Status of Training and Qualification Records

Suitability of Documentation Suitability of Documentation

Written analytical procedure and proper approved protocol with pWritten analytical procedure and proper approved protocol with prere--

established acceptance criteriaestablished acceptance criteria

Page 18: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Reference StandardsReference Standards

ICH states that “Well characterized reference

materials, with documented purity, should be

used throughout the validation study.”

Page 19: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Method validation parametersMethod validation parameters

SpecificitySpecificity

PrecisionPrecision–– RepeatabilityRepeatability–– Intermediate PrecisionIntermediate Precision

LinearityLinearity

RangeRange

AccuracyAccuracy

Limit of Detection (LOD)Limit of Detection (LOD)

Limit of Limit of QuantitationQuantitation (LOQ)(LOQ)

RobustnessRobustness

Page 20: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Specificity

Definition:Ability to assess unequivocally the analyte in the presence of components which may be expected to be present (impurities, degradants, matrix).Aspects:-Identification-Purity tests-Assay (Content/potency)

Page 21: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Specificity: Identification Identification

Should be able to discriminate between compounds Should be able to discriminate between compounds closely related in structure.closely related in structure.

Confirmed by obtaining negative results for samples with Confirmed by obtaining negative results for samples with spiked related compounds and positive results for spiked related compounds and positive results for samples with samples with analyteanalyte..

Choice of potential interfering substances should be Choice of potential interfering substances should be based on sensible scientific judgment considering based on sensible scientific judgment considering substances that could likely occur.substances that could likely occur.

Page 22: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Specificity: Impurities/AssaySpecificity: Impurities/Assay

Chromatographic MethodsChromatographic Methods–– Demonstrate Resolution of critical pairDemonstrate Resolution of critical pair

NonNon--specific method use other supporting analytical procedures e.g. specific method use other supporting analytical procedures e.g. titration+TLCtitration+TLC

Impurities/Impurities/DegradantsDegradants Available Available –– Spike with impurities/Spike with impurities/degradantsdegradants and/or and/or excipientsexcipients

–– For assay, compare with For assay, compare with unspikedunspiked samplessamples

–– For impurity test, impurities are separated individually and froFor impurity test, impurities are separated individually and from sample matrixm sample matrix

Impurities/Impurities/DegradantsDegradants NotNot AvailableAvailable–– Stress SamplesStress Samples

–– For assay, Stressed and Unstressed Samples should be compared.For assay, Stressed and Unstressed Samples should be compared.

–– For impurity test, impurity profiles should be compared.For impurity test, impurity profiles should be compared.

–– Demonstrate peak purity (HPLCDemonstrate peak purity (HPLC--DAD, mass)DAD, mass)

Page 23: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Stress conditionsStress conditions

Heat Heat High Temperature (50 to 60 High Temperature (50 to 60 ooCC))

Humidity Humidity HumidityHumidity (70 to 80%)(70 to 80%)

Acid Hydrolysis Acid Hydrolysis (0.1 N (0.1 N HClHCl))

Base Hydrolysis Base Hydrolysis (0.1 (0.1 NaOHNaOH))

Oxidation (Oxidation (3 to 30% H3 to 30% H22OO22))

LightLight

Intent is to create 10 to 30 % Degradation

Page 24: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

LinearityDefinition:Ability (within a specified range) to obtain test results which are directly proportional to the concentration of analyte in the sample

Aspects:– Test across the range (at least 5 concentrations)– Evaluate linearity by– visual inspection of the plot and by– statistical techniques

Calculate corr. coefficient, y-intercept, slope and residualsResponse factor

Page 25: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Range

Definition: Interval between upper and lower concentration of the analyte in the sample for which it has been demonstrated that the procedure has a suitable level of precision, accuracy and linearity Aspects:– Defined from linearity study– Depends on the application of the method

assay (80-120 %)content uniformity (70-130%)dissolution test(± 20 % over the specified range)Impurity from specs to 120 % of the specs

Page 26: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

AccuracyDefinition: Expresses the closeness of agreement between the value found and the true value.Methods: Drug substance – use of reference standard with known purity– comparison with independent, well-characterized procedure – may be inferred once precision, linearity and specificity are

establishedMethods: Drug product– Analysis of synthetic mixture– comparison with independent, well-characterized procedure– Standard addition method

Methods: Drug product– Spiking– comparison with independent, well-characterized procedure

Page 27: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Accuracy

Recommended data – Assessed by 9 determinations over a minimum of 3

concentration levels covering the specified range – To be reported as percent recovery or difference

between mean and true value with confidence interval

Page 28: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Precision

Definition – Closeness of agreement (‘scatter’) between a series

of measurements obtained from multiple sampling of the same homogeneous sample.

Aspects – Repeatability – Intermediate precision – Reproducibility

Page 29: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

PrecisionPrecisionRepeatabilityRepeatability–– Precision under the same conditions over a short interval of timPrecision under the same conditions over a short interval of time.e.–– MethodMethod

9 determinations covering the specified range 9 determinations covering the specified range or: 6 determinations at 100% of the test concentrationor: 6 determinations at 100% of the test concentration

Intermediate precision– Expresses within laboratory variations. – Method

Should include variations in days, analysts, columns

Reproducibility– Precision between laboratories – Method

-Dependent on usage of method -Should include interlaboratory study

Recommended dataRecommended data–– SD, RSD and confidence intervalSD, RSD and confidence interval

Page 30: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Detection limit

Lowest amount of an analyte in a sample which can be detected but not necessarily quantitated.Method

1. Based on visual evaluation2. Based on signal-to-noise ratio (3:1)3. Based on st.dev. (SD) of response and slope

(DL=3.3xSD/S)Recommended dataRecommended data–– DL and relevant chromatograms for 1&2DL and relevant chromatograms for 1&2–– For 3, Estimated DL is validated by analysis of For 3, Estimated DL is validated by analysis of

samples at the DLsamples at the DL

Page 31: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Quantitation limit

Lowest amount of an analyte in a sample which can be quantitatively determined with a suitable precision and accuracy Method– Based on visual evaluation– Based on signal-to-noise ratio (10:1)– Based on st.dev. (SD) of response and slope

(DL=10xSD/S)Recommended dataRecommended data–– QL and relevant chromatograms for 1&2QL and relevant chromatograms for 1&2–– For 3, Estimated QL is validated by analysis of For 3, Estimated QL is validated by analysis of

samples at the DLsamples at the DL

Page 32: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Signal-to Noise-Ratio

Page 33: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Robustness

Measure of the capacity of a method to remain unaffected by small variations in method parameters.– Aspects

To be considered during developmentTo be used for establishment of system suitability criteriaInclude testing of stability of solutionsTo be tested by introducing small variations in method parameters

Page 34: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

System Suitability Test

ICHICH

–– Definition: evaluation of Definition: evaluation of

equipment, electronic, equipment, electronic,

analytical operations and analytical operations and

samples as a wholesamples as a whole

–– Determination: repeatability, Determination: repeatability,

tailing factor (T), capacity tailing factor (T), capacity

factor (kfactor (k’’), resolution (R), ), resolution (R),

and theoretical Plates (N)and theoretical Plates (N)

T T ≤≤ 22TT

RSD RSD ≤≤ 2.0% (n 2.0% (n ≥≥ 5)5)RepeatabilityRepeatability

In general N > 2000In general N > 2000NN

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

RR

In general kIn general k’’ ≥≥ 2.02.0KK’’

RecommendationsRecommendationsParametersParameters

USP 23 <621>USP 23 <621>–– System Suitability RequirementsSystem Suitability Requirements

Page 35: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Recommended Validation characteristics of various Types of Tests

*YesYesYesYesRuggedness

***YesYesRange**NoYesYesLinearity**NoYesNoLOQ**YesNoNoLOD

Yes*YesYesYesSpecificity*YesNoYesYesPrecision***YesYesAccuracy

Assay Assay Category Category

IVIV

Assay Assay Category Category

IIIIII

Assay Assay Category IICategory IILimit testLimit test

Assay Assay Category IICategory II

QuantitativeQuantitative

Assay Assay Category ICategory I

AnalyticalAnalyticalPerformancePerformance

ParameterParameter

Page 36: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

483 Observations483 Observations

There was no inadequate method validation specificity data to demonstrate that each method was capable of distinguishing the active ingredient from its impurities and degradation products.Specificity studies did not include the minimum stress conditions of acid and base hydrolysis, oxidation, thermal degradation and photolysis, degradation schematic for the active ingredient that ident i f ies the major degradat ion products

was not included for each product.

Page 37: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

483 Observations483 Observations

Stress studies conducted as part of method validation do not target a minimum amount of degradation. … a standard period of two hours as commonly used for stress studies with no

justification…Spreadsheets used to calculate linearity, percent recovery, and final assay results for the cleaning validation of …were not validated and the data transcribed from chromatographs to the s p r e a d s h e e t s w e r e n o t c h e c k e d f o r a c c u r a c y .

Page 38: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

FDA Waning LetterFDA Waning Letter

On addition to the example of modifying both compendialmethods and customer supplied methods, we also observed the use of unvalidated in-house methods as well as unvalidated

modifications to in-house methods.A statement indicating that the method has not been validated in the particular formulation was included in the certificate ofanalysis for…use of this statement does not absolve…from u s i n g v a l i d , a c c u r a t e , a n d

reproducible methods. (June 2000)

Page 39: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

FDA Warning LetterFDA Warning Letter

Change control procedures in the laboratory failed to document test method changes to assure accurate, reliable, and reproducible results. The test method did not state whether a helix was to be used during dissolution testing. A … was reportedly used during method development, validation and daily method runs, but there is no documentation of a … being

used in any of the documents.

There is no assurance that qualification or maintenance of the laboratory equipment can consistently produce valid and accurate analytical results in that numerous examples of test data were invalidated due to instrument malfunction.

(December 2000)

Page 40: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

FDA Warning LetterFDA Warning Letter

Attempts to corroborate data in the validation report with suppoAttempts to corroborate data in the validation report with supporting rting

raw data in the laboratory were difficult and frustrating for thraw data in the laboratory were difficult and frustrating for the FDA e FDA

p e r s o n n e l c o n d u c t i n g t h e i n s p e c t i o n .p e r s o n n e l c o n d u c t i n g t h e i n s p e c t i o n .

OOS accuracy results reported by analyst 3 were never submitted OOS accuracy results reported by analyst 3 were never submitted in in

the final report. Repeat analysis performed in a different systethe final report. Repeat analysis performed in a different system m

p a s s e d s p e c i f i c a t i o n s a n d t h e s e r e s u l t s w e r e p a s s e d s p e c i f i c a t i o n s a n d t h e s e r e s u l t s w e r e

submitted in the report.submitted in the report.

Raw data and calculations were not checked by a second responsiRaw data and calculations were not checked by a second responsible ble

individuals required by your procedures. Inaccurate calculationsindividuals required by your procedures. Inaccurate calculations were were

n o t e d i n t h e r e p o r t . ( N o v e m b e r 1 9 9 7 ) n o t e d i n t h e r e p o r t . ( N o v e m b e r 1 9 9 7 )

Page 41: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

FDA Warning LetterFDA Warning Letter

The process validation samples were assays using an HPLC method that had not been validated. The method validation used for bothproducts … did not include a protocol that included specification and acceptance criteria. … The method validation was not reviewed and approved until during the current inspection. Lots of both products were released for distribution prior to completion of the method v a l i d a t i o n . ( A u g u s t 2 0 0 1 )

Method validation for the product Sennosides is inadequate in that the data does not assess all variables, such as different mobile phase concentrations and analytes, to demonstrate that the method can s u s t a i n v a r i a n c e . ( N o v e m b e r 2 0 0 1 )

Page 42: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Recent 483s and warning lettersRecent 483s and warning letters

HPLC SST did not include limits for HPLC SST did not include limits for RsRs, T, N., T, N.Limits of Limits of quantitationquantitation and detection not and detection not established for individual bulk drug impurities.established for individual bulk drug impurities.Impurity peaks in HPLC assay not identifiedImpurity peaks in HPLC assay not identifiedSample dilution not recordedSample dilution not recorded

Page 43: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation of the assay method of active compounds

by HPLC

Page 44: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Selectivity

Tests – Inject solutions of standard, product, impurities,

known degradation products, excipients; – Inject solutions of degraded/stressed products and

placebo 2 hours art. daylight (70-90 klux)1 week at 75°C/amb. humidity and 75°C/100% RH24 hrs 3% H2O2, 1 mol/L HCl, 1 mol/L NaOH

– Demonstrate separation – Demonstrate peak purity

Page 45: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Selectivity

Criteria – Separation between relevant peaks of at least Rs > 2.0 – Peak of analyte should be pure

Documentation – Chromatograms of all solutions – retention times – peak purity results – data of contents of active substance and degradation products in

stress samples

Page 46: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Linearity

Tests – Inject solutions of 25%, 50%, 75%, 100%, 125% and 150% of

expected concentration in duplicate;

– Calculate by statistical techniques the order of function (first orsecond), significance of intercept and correlation coefficient

– In case of second order and/or significant deviation ofintercept from zero: determine the degree of linearity in the range of 70-130%.

Page 47: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Linearity

Criteria Use of one reference concentration is acceptable when:

– regression line is linear (lack of fit test) – true zero is within 95% conf. interval of calculated intercept

or in case of second order curve:-if experimental rel. response at 70% and 130% does not deviate by more than 1% from the calculated values

– -Linear when corr. coefficient > 0.9990

Page 48: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Linearity

Documentation Plots of peak height and peak areas

Statistical results (equations, significance of intercept,, response factor, corr. coefficient)

Plots of peak area and peak heights residuals

Page 49: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Accuracy

Test Prepare placebo sample Prepare spiked placebo samples: 3 replicates over 3 concentration levels (e.g. 70%, 100%, 130% of theoretical strength)Carry out the method Calculate mean percent recoveries and rel. standard deviation (RSD) from both peak area and peak height responses.

Page 50: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

AccuracyCriteria – The average result of the mean for each level should be 98.0 -102.0%– Range for response of placebo within -1% and +1%– RSD of pooled results should be < 2%

Documentation

– Details on sample preparations

– Individual results (peak areas and peak heights)

– Calculated % recovery and pooled RSD

Page 51: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Repeatability of system

Test Inject in six-fold one of the 100% solutions from the accuracy experiment Calculate RSD for both peak height and peak area

Criterion RSD < 1.5%

Documentation Results and statistical calculation

Page 52: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Repeatability of method Test

Test– Analyze within one day by one operator with one column 6

– times a homogeneous sample of the product -Calculate the RSD for results of both peak height and peak area

Criterion – RSD < 2%

Documentation – Results and statistical calculation

Page 53: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Intermediate precision

Test -Same as for repeatability of the method but by at least 2 analysts, more days, different labs, different (batches of) columns -Calculate the RSD on overall results

Criterion RSD < 2.5%

Page 54: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Intermediate precision

Documentation– Description of preparation of homogeneous sample– Description of experimental conditions – Results and statistical evaluation

Page 55: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Detection and quantitation limit

Determination not necessary Only applicable for impurities and degradation products

Page 56: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Range

No specific test:Normally a range of 70-130% is acceptable, unless

a wider range is required based upon the nature of the dosage form (e.g. metered dose inhalers)

Page 57: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Robustness

Test on stability of solutions– Prepare 2 sample and 2 reference standard solutions -Store in

refrigerator and at room temperature – Analyze at zero time and after at least 24 and 72 hours storage -

Calculate differences between samples

Criterion– Storage period is defined by period with no more than 1%

difference between room temperature and refrigerator

Documentation–– Individual results Individual results –– Calculations, difference between room and refrigerator samplesCalculations, difference between room and refrigerator samples

Page 58: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Robustness (2) Test on variations– Vary relevant analytical parameters e.g.

composition and/or pH of mobile phasecolumn temperature different column (other batch or brand/supplier) stability of chromatographic system

Criteria– Chromatographic results meet system suitability criteria – Typically plate count should not decrease by more than 50%

DocumentationRelevant chromatogramsCalculations and results of system suitability parameters

Page 59: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

System Suitability Testing Test

– Collect all data from previous experiments with regard to -number of theoretical plates

-tailing factor -relative retention -resolution factor -precision of the system

– Include information on minimum resolution between analyte and most-difficult-to-resolve impurity/degradation product

Page 60: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

System Suitability Testing

Criteria – Criteria dependent on development and validation

results. – Evaluate and optimise defined criteria when more

experience is gained with the method. Documentation-Summary of data on individual parameters-Calculations and relevant chromatograms

Page 61: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation Of The Determination Of An Impurity In A Drug Product By HPLC

Page 62: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Selectivity

Tests and documentation – Same as for determination of active substance.

Criteria – -Assay of impurity should not be influenced by any other peak

originating from other components in the sample solution.– Resolution factor between 2 peaks should be at least > 1.5. – Resolution between active substance and impurity should be >

2.

Page 63: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Linearity

Tests – Inject solutions of 10%, 50%, 100%, 150%, and 200% of

expected concentration in duplicate (concentration based upon registered limit; if not defined then 1%);

– Calculate by statistical techniques the order of function (first or second), significance of intercept and correlation coefficient

– In case of second order and/or significant deviation of intercept from zero: determine the deviation in the relative response of the 10% and 200% points.

Page 64: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Linearity

Criteria – Use of one reference concentration is acceptable

when:regression line is linear (lack of fit test)true zero is within 95% conf. interval of calculated intercept

– or in case of second order curve:

if deviation of the rel. response of 10% point isd less than 20% and of the 200% point is less than 5%values-Linear when corr. coefficient > 0.995

Page 65: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Linearity

Documentation (same as for DS) – Plots of peak height and peak areas

– Statistical results (equations, significance of intercept, lack-of-fit test, rel. responses, corr. coefficient)

– -Plots of peak area and peak heights residuals

Page 66: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Range

No specific test:Normally a range of 10-200% is acceptable. In most cases 100% is 1% relative to the drug substance.

Page 67: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Accuracy

Test – Prepare placebo sample– Prepare spiked placebo samples: 3 replicates over 3

concentration levels (e.g. 2 x QL, 100% and 200% of 1% of the drug substance concentration)

– Perform analysis – Calculate mean percent recoveries and rel. standard

deviation (RSD) from both peak area and peak height responses.

Page 68: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Accuracy

Criteria -The average result of the mean for 100% and 200% level should

be 90-110% and for 2xQL 70-130%– RSD of 100 and 200%: < 5% and 2xQL level: < 15%

Documentation– Details on sample preparations– Individual results (peak areas and peak heights)– Calculated % recovery and pooled RSD

Page 69: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Repeatability of system

Test – Inject in six-fold one of each of the strengths of the reference

solutions from the accuracy experiment – Calculate RSD for both peak height and peak area

Criterion – RSD (2xQL) < 15%; RSD (100% and 200%) < 5%

Documentation – Results and statistical calculation

Page 70: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Repeatability of method

Test – -Inject in six-fold one of each of the strengths of the samples

used in the accuracy experiment – -Calculate the RSD for results of both peak height and peak area

Criteria – RSD (2xQL) < 15%;– RSD (100% and 200%) < 5%

Documentation– Results and statistical calculation

Page 71: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Intermediate precision

Test and documentation– Same as for assay but tested on spiked samples at

1% level

Criterion RSD < 10%

Page 72: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Detection and quantitation limit

Test – Determine peak-to-peak distance of baseline at the

position of the analyte in a blank sample. Calculate noise as 0.5 times this distance

– Calculate the detection limit as 3 times noise and quantitation limit as 10 times noise

– Verify the calculated DL and QL by injecting at least one solution with a concentration at or near the DL and QL.

Page 73: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Detection and quantitation limit

Criterion – The Quantitation Limit is, by preference, less than

0.1% relative to the drug substance. Documentation– Chromatograms used for calculations -Chromatogram of sample

at a concentration near DL and QL

Page 74: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Robustness (1)

Test and documentation on stability of solutions– Same as for assay

Criterion– Storage period is defined by period with no more

than 5% difference between samples stored at room temperature and in the refrigerator

Page 75: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Robustness

Test, Criteria and Documentation on variations – Same as for assay

Page 76: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

System Suitability Testing

Test, Criteria and Documentation – Same as for Assay

Page 77: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

“Analytical Method Transfer Procedure”

Page 78: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Select labs- Prepare protocol including:– detailed description of analytical method– samples to be tested– items to be checked: assay, precision (reproducibility & intermediate

precision), SST values– calculation formulas– way of reporting

- Carry out analyses and report results- Perform statistical analysis on results and report on conclusions

Page 79: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

c1 I B 33

c2 II A 32

c1 I A 31

HPLC column HPLC apparatus

Analyst Number of analyses

Day

Test schedule and criteria for transfer

No stat. No stat. signifsignif. . difference or < 2%difference or < 2%

Max. difference of meanbetween labs

< 15%< 3.0%< 3.0%Reproducibility< 10%< 2.5%< 2.5%Intermediate precision< 5%< 2.0%< 2.0%Method repeatability

DegradationDegradationproductsproducts

AssayAssayCriteria for method transfer

Page 80: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Specificity

<0.1 0.20.2<0.1 0.998191.40%2 hrs 3% H2O2

1.253.40.10.4ndnd2 hrs 1M NaOH

0.790<0.1 ndndnd2 hrs 1M HCl

nd12.33.81.5-4.60%1 wk 75°C/100% RH

0.110.922.10.999383.60%1 wk 75°C/amb. RH

0.30.20.3<0.1 0.998580.30%2 hrs art. daylight

nd0.20.1<0.1 0.9985100%Non stressed

Total Contentothers

Content OM 38

ContentOM 06

Pur. factor OM 08

ContentOD 14

Condition

Results of stress-testing

Page 81: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation Report

ContentsContents–– TitleTitle–– SummarySummary–– Validation dataValidation data–– DiscussionDiscussion–– Conclusion Conclusion

Page 82: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation Report

Summary– Simple statement about the results of the validation

study. – Example, “The method for the assay of product XYZ

by HPLC was found to be accurate, precise, selective, linear and stability indicating.”

Page 83: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation Report

Analytical Validation Data– Analytical data should be presented in tabular and

graphical form.– Copies of raw data (chromatograms and notebook

pages)– Analytical results for each validation parameter, plus

all calculations used. Be sure to include all graphs, curves and).

Page 84: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

Validation Report

Discussion– Describe the outcome of the validation in

detail. – Problems encountered and rationale for

accepting or rejecting the validation. – Repeated experiments. – Deviations from acceptance criteria must be

explained. – Restrictions.

conclusions

Page 85: VALIDATION OF ANALYTICAL METHODS - …community.learnaboutgmp.com/uploads/db7093/original/1X/71d1a7f7cdd...current ICH/FDA guidelines ... – For impurity test, ... ICH – Definition:

8585

References References 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. Marcel Dekker, Inc. New York, 1997.

USP 23 <1225>

http://www.waters.com