method validation notes

7/29/2019 Method Validation Notes

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Introduction

An introduction to analytical method validation

Mr. Bhavadip B. Tanna

For Pharmaceutical drug analysis analytical method validation is veryimportant parameter. So, there is a need for the study of validationparameters.

The analytical procedure refers to the way of performing the analysis. Analytical method validation isrequired for herbal procedure,new process and reaction,new molecules, active ingredients,residues,impurity profiling and component of interest in different matrices.An analytical methodology consists of thetechniques,method , procedure and protocol.this methodology the required data for a given analyticalproblem, required sensitivity,required accuracy,required range of analysis and required precision to theanalyst.It is required for assuring quality,achiving acceptance of products by the international agencies,

mandatory requirment purposes for accreditation as perISO 17025guidelines,mandatory requirment forregistration of any pharmaceutical product or pesticide formulation.The main objective is to demonstratethat the procedure is suitable for its intended purpose.the method validation studies for the developedmethods for various parametersas per protocol and guidelines which are EN 45000 series ofstandards,ISO\ IEC Guide 25, Internation conference on harmonization (ICH) , US EPA,USP, publishedlitreture.There are 4 types of analytical procedures which includes identification testes, impurity tests, limittests and potency tests.Identification testes which normally compares that sample under evalution with aknown reference sample standard with spectrographic or chromatographic methods. Impurity tests maybe eitherquantitative or limit test and different validation requirements applied. For limit testes, specificityand detection limits only may required.For assay of actives or other key components of drug productspotency tests is performed.

Validation of an analytical method is the process by which it is established, by

laboratory studies, that the performance characteristics of the method meet therequirements for the intended analytical applications Methods validation means establishing, through documented evidence, a high degree

of assurance that an analytical method will consistently yield results that accuratelyreflect the quality characteristics of the product tested.

The objective of any analytical measurement is to obtain consistent, reliable and accurate data.

Validated analytical methods play a major role in achieving this goal. The results from method

validation can be used to judge the quality, reliability and consistency of analytical results, whichis an integral part of any good analytical practice. Validation of analytical methods is also

required by most regulations and quality standards that impact laboratories

Method validation is the process used to confirm that the analytical procedure employed for aspecific test is suitable for its intended use. Results from method validation can be used to judge thequality, reliability and consistency of analytical results; it is an integral part of any good analyticalpractice.

Analytical methods need to be validated or revalidated

before their introduction into routine use; whenever the conditions change for which the method has been validated (e.g., an

instrument with different characteristics or samples with a different matrix); and
http://pharmaceuticalvalidation.blogspot.in/2010/02/introduction-to-analytical-method.htmlhttp://pharmaceuticalvalidation.blogspot.in/2010/02/introduction-to-analytical-method.htmlhttp://www.ich.org/http://www.ich.org/http://www.ich.org/http://www.usp.org/http://www.usp.org/http://www.usp.org/http://www.usp.org/http://www.ich.org/http://pharmaceuticalvalidation.blogspot.in/2010/02/introduction-to-analytical-method.html


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whenever the method is changed and the change is outside the original scope of themethod.

What is the purpose of

Analytical 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 FDA Requirements

What are the Benefits of

Analytical Method Validation?

Regulatory Compliance Assurance that Test data from Methods are Reliable

Establishment that Test Data are Reproducible, Accurate, Specificity

The USP has published specific guidelines for method validation for compound evaluation (7). USPdefines eight steps for validation:

Accuracy Precision

Specificity Limit of detection Limit of quantitation Linearity and range Ruggedness Robustness

The validation parameters that should be considered during validationof analytical procedures are shown as:

Specificity: It conforms the ability of the methods to evalute the desired analyte in the presence ofknown other components like degradants, impurities, potential contamination and excipients.It is notpossible to demonstrate that an analytical procedures is specific for a particular analyte. In such case acombination of two or more analytical procedure is recommended to achieve the necessary level ofdiscrimination.lack of specificity of an individual analytical procedure may be compensated by othersupporting analytical procedures or tests.

Accuracy: The accuracy of analytical procedure expresses the closeness of agreement between thevalues which is accepted either as conventional true value or an accepted reference value in the value


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found which is sometimes called as trueness.It should be established across the specified range ofprocedure.

Precision: The procedure of an analytical procedure expresses the closeness of agreement (degree ofscatter) between a series of measurements obtained from multiple sampling of the same homogeneoussample under the prescribed condition.

It may be considered at 3 levels:

~~Repeatability: It expresses the precision under the same operating conditions over a short interval oftime .It is also known as inta-assay precision.

~~Inter mediate precision: It express within laboratory variations like different analysts, days andequipments.

~~Reproducibility: It expresses the precision between laboratories.There are variations like differences inroom temperature and humidity, operating with different expertience and thoroughness,variation inmaterial and instrument condition , equipment and consumable of different ages affecting a methodsreproducibility.

Limit of Detection (LOD): The detection of an individual analysis procedure is the lowest amount ofanalyte in a sample which can be detected but not necessarily quantify as an exact value. It may bedetermined by the analysis of sample with known concentrations of the analyte and by establishing theminimum level at which the analyte can be reliably detected.

Based on standard deviation of the response and the slope it is shown as:

DL= 3(SD/Slope)

Limit of quantification(LOQ) :The quantification limit of an individual analytical procedure is the lowestamount of analyte in a sample which can be quantitatively determine with suitable precision and accuracy

.It is used particularly for the determination of impurities and degradation products. It is shown as:

DL=10(SD/Slope)

Linearity : The linearity of an analytical procedure is its ability to obtain test results which are directlypropotional to the concentration of analyte in the sample.Minimum 5 concentrations are recommended forthe establshiment of linearity.

Range: The range of an analytical procedure is the interval between the upper and lower concentrationof analyte in the sample for which it has been demonstrated that the analytical procedure has a suitablelevel of precision , accuracy and linearity.

Robustness : The robustness of analytical procedure is a measure of Its capacity to remain unaffectedby small,but deliberate variations(e.g.stability of analytical solutions, extraction time ,etc.) in methodparameters and provide an indication of its reliability during normal usage.

System Suitability Testing: It is an integral part of many analytical procedure.The tests are based in theconcept that equipment electronics , analytical operations and sample to be analysed constitute anintegral system that can be evaluated as such.

Revalidation:


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operating ranges should be defined for each method based on experience with similar methods. Theavailability of such operating ranges makes it easier to decide when a method should be revalidated.It isnecessary whenever a method is changed and the new parameters is outside the operating range.Forexample , a revalidation is necessary if a high performance liquid chromatography method has beendeveloped and validated on a pump with a delay volume of 5ml and the new pump only has 0.5ml

SPECIFICITY The ability of the analytical method to differentiate (and quantify) the analyte in the

presence of other components in the sample (to amplify only the Sequence ofinterest.)

Specificity usually is defined as the ability to detect the analyte of interest in the presence of interferingsubstances. Specificity can be shown by spiking known levels of impurities or degradants into a sample with aknown amount of the analyte of interest.

For both examples UV spectra are taken at the peak upslope and downslope,normalized and compared. In the example on the left, the spectraare identical indicating that the peak consists of single compound, or thepeak is spectrally pure. In the example on the right, the peak is clearlyimpure which is demonstrated by two different UV spectra. Moderndiode array detectors compare the spectra automatically and print a match factor for each peak. This, in

combination with the graphical visualization helps to assess selectivity without any additional workload. LINEARITY AND RANGE

Linearity: The ability of the assay (within a given range) to obtain test results whichare directly proportional to the concentration/amount of the analyte

Range: The interval between the upper & lower concentrations of an analyte forwhich the assay has suitable levels of precision, accuracy & linearity.

The linearity of an analytical procedure is its ability, within a given range, to obtain test results that are directlyproportional to the concentration of analyte in the sample. De facto, the range is the smallest and largestconcentration that maintains a linear relationship between the concentration and the response of the method.


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ICH defines linearity of an analytical procedure as its ability (within agiven range) to obtain test results that are directly proportional to theconcentration (amount) of analyte in the sample.Linearity may be demonstrated directly on the test substance (by dilutionof a standard stock solution) or by separately weighing synthetic mixturesof the test product components.Linearity is determined by a series of five to six injections of five or morestandards whose concentrations span 80120 percent of the expectedconcentration range. The response should be directly proportional to theconcentrations of the analytes or proportional by means of a well-definedmathematical calculation. A linear regression equation applied to theresults should have an intercept not significantly different from zero. If asignificant nonzero intercept is obtained, it should be demonstrated thatthis has no effect on the accuracy of the method.Frequently, the linearity is evaluated graphically, in addition to or as analternative to mathematical evaluation. The evaluation is made by visuallyinspecting a plot of signal height or peak area as a function of analyteconcentration. Because deviations from linearity are sometimes difficultto detect, two additional graphical procedures can be used. The first isto plot the deviations from the regression line versus the concentration

or versus the logarithm of the concentration if the concentration range

covers several decades. For linear ranges, the deviations should beequally distributed between positive and negative values.Another approach is to divide signal data by their respective concentrations,yielding the relative responses. A graph is plotted with the relativeresponses on the y-axis and the corresponding concentrations on thex-axis, on a log scale. The obtained line should be horizontal over the fulllinear range. At higher concentrations, there will typically be a negativedeviation from linearity. Parallel horizontal lines are drawn on the graphcorresponding to, for example, 95 percent and 105 percent of the horizontalline. The method is linear up to the point where the plotted relativeresponse line intersects the 95 percent line. Figure 5 shows a comparison

of the two graphical evaluations using HPLC.


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3ICH defines the range of an analytical procedure as the interval from theupper to the lower concentration (amounts) of analyte in the sample (includingthese concentrations) for which it has been demonstrated that the analyticalprocedure has a suitable level of precision, accuracy and linearity.The range of an analytical method is the interval from the upper to thelower levels (including these levels) that have been demonstrated to bedetermined with precision, accuracy and linearity using the method as written.The range is normally expressed in the same units as the test results (forexample percentage, parts per million) obtained by the analytical method.For assay tests, ICH requires the minimum specified range to be 80 to

120 percent of the test concentration. It also requires the range for thedetermination of an impurity to extend from the limit of quantitation orfrom 50 percent of the specification of each impurity, whichever isgreater, to 120 percent of the specification. Figure 6 shows graphicaldefinition of linearity and range.


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ACCURACYAccuracy is the difference between the measured value and the true value.

Accuracy can also be described as the extent to which test resultsgenerated by the method and the true value agree.The true value for accuracy assessment can be obtained in several ways.One alternative is to compare the results of the method with results froman established reference method. This approach assumes that the uncertaintyof the reference method is known.Secondly, accuracy can be assessed by analyzing a sample with knownconcentrations (for example, a control sample or certified referencematerial) and comparing the measured value with the true value as

supplied with the material. If certified reference materials or controlsamples are not available, a blank sample matrix of interest can bespiked with a known concentration by weight or volume.

The ICH document on validation methodology recommends accuracy tobe assessed using a minimum of nine determinations over a minimum ofthree concentration levels covering the specified range (for example,three concentrations with three replicates each).

PRECISION Variation among response within an assay

ICH defines the precision of an analytical procedure as the closeness

of agreement (degree of scatter) between a series of measurementsobtained from multiple sampling of the same homogeneous sample underthe prescribed conditions. Precision may be considered at three levels:repeatability, intermediate precision and reproducibility.Repeatability expresses the precision under the same operatingconditions over a short interval of time. Repeatability is also termedintra-assay precision.Intermediate precision expresses variations within laboratories, suchas different days, different analysts, different equipment, and so forth.Reproducibility expresses the precision between laboratories (collaborative


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studies usually applied to standardization of methodology).

The most important part of any analytical method validation is precision analysis. The ICH guidelines breakprecision into two parts: repeatability and intermediate precision. Repeatability expresses the precision under

the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.Intermediate precision expresses within-laboratory variations: different days, different analysts, differentequipment, etc. Additionally, the ICH Q2A guideline defines reproducibility as the precision among laboratories

(collaborative studies, usually applied to standardization of methodology).4 This lab-to-lab precision could becombined into the estimate of intermediate precision, because it is possible that a particular test method couldbe run in more than one laboratory. The suggested testing consists of a minimum of two analysts on twodifferent days with three replicates at a minimum of three concentrations. If lab-to-lab variability is to beestimated, the experimental design should be performed in each lab.

Intermediate precision is determined by comparing the results of amethod run within a single laboratory over a number of days. A methodsintermediate precision may reflect discrepancies in results obtained from:different operatorsinconsistent working practicedifferent instrumentsstandards and reagents from different supplierscolumns from different batches

a combinationThe objective of intermediate precision validation is to verify that in the same laboratory

the method will provide the same results once the development phase is over.

The objective of intermediate precision validation is to verify that in thesame laboratory the method will provide the same results once the developmentphase is over.The objective of reproducibility is to verify that the method will providethe same results in different laboratories. The reproducibility of an analyticalmethod is determined by analyzing aliquots from homogeneous lots in

Reproducibility (Table 5), as defined by the ICH (4), represents the precision obtained between

different laboratories. The objective is to verify that the method will provide the same results indifferent laboratories. The reproducibility of an analytical method is determined by analyzing aliquotsfrom homogeneous lots in different laboratories with different analysts, and by using operational andenvironmental conditions that may differ from, but are still within, the specified parameters of themethod (interlaboratory tests). Validation of reproducibility is important if the method is to be used indifferent laboratories.

Differences in room temperature and humidity Operators with different experience and thoroughness Equipment with different characteristics, e.g. delay volume of an HPLC system Variations in material and instrument conditions, e.g. in HPLC, mobile phases composition,

pH, flow rate of mobile phase Variation in experimental details not specified by the method Equipment and consumables of different ages Columns from different suppliers or different batches Solvents, reagents and other material with varying qualit


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DETECTION AND QUANTIFICATION LIMITS

1. Limit of detection (LOD), The minimum amount of target analyte that can bedetected with a given level of confidence

2. Limit of quantification (LOQ), The lowest amount of target analyte that can

be quantified with acceptable levels of precision and accuracy.

The detection limit of an assay is the lowest concentration that can be detected but necessarily quantified; thequantification limit is the lowest concentration that can be quantified with acceptable precision. Thequantification limit is the lowest level of analyte that can be reported. The ICH guidelines suggest three differentmethods for determining the detection and quantification limits. These are: visual determination, signal-to-noisedetermination, and standard deviation and slope method. Each method will give different results. The signal-tonoisemethod is the most logical, because it is based on comparing low levels of the analyte to a blank orbackground sample.Determination of the signal-to-noise ratio is performed by comparing measured signals from samples withknown low concentrations of analyte with those of blank samples, and subsequently establishing the minimumconcentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3:1 and 2:1 isgenerally considered acceptable for estimating the detection limit.3

Figure 6. Definitions for linearity, range, LOQ, LODICH defines the detection limit of an individual analytical procedure asthe lowest amount of analyte in a sample which can be detected but notnecessarily quantitated as an exact value.The limit of detection (LOD) is the point at which a measured value is largerthan the uncertainty associated with it. It is the lowest concentration of analytein a sample that can be detected but not necessarily quantified. Thelimit of detection is frequently confused with the sensitivity of the method.The sensitivity of an analytical method is the capability of the method todiscriminate small differences in concentration or mass of the test analyte.In practical terms, sensitivity is the slope of the calibration curve that isobtained by plotting the response against the analyte concentration or mass.In chromatography, the detection limit is the injected amount that resultsin a peak with a height at least two or three times as high as the baselinenoise level. Besides this signal-to-noise method, the ICH5 describes threemore methods:Visual evaluation: The detection limit is determined by the analysis ofsamples with known concentrations of analyte and by establishing theminimum level at which the analyte can be reliably detected.


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Standard deviation of the response based on the standard deviationof the blank: Measurement of the magnitude of analytical backgroundresponse is performed by analyzing an appropriate number of blanksamples and calculating the standard deviation of these responses.Standard deviation of the response based on the slope of thecalibration curve:A specific calibration curve is studied using samplescontaining an analyte in the range of the limit of detection. The residualstandard deviation of a regression line, or the standard deviation of y-interceptsof regression lines, may be used as the standard deviation. Figure 7illustrates the graphical evaluations of LOD and LOQ via signal-to-noise.

ICH defines the limit of quantitation (LOQ) of an individual analytical procedureas the lowest amount of analyte in a sample which can be quantitatively determinedwith suitable precision and accuracy. The quantitation limit is a parameterof quantitative assays for low levels of compounds in sample matrices, and isused particularly for the determination of impurities or degradation products.The quantitation limit is generally determined by the analysis of sampleswith known concentrations of analyte and by establishing the minimumlevel at which the analyte can be quantified with acceptable accuracyand precision. If the required precision of the method at the limit ofquantitation has been specified, 5 or 6 samples with decreasing amountsof the analyte are injected six times. The amounts range from the knownLOD as determined above to 20 times the LOD. The calculated relative standard deviation (RSD) percent of the precision ofsix repetitive injections is plotted against the analyte amount. The amountthat corresponds to the previously defined required precision is equal to thelimit of quantitation. It is important to use not only pure standards for thistest but also spiked matrices that closely represent the unknown samples.Figure 8 shows required experimental steps and a typical graph.


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For chromatographic methods the LOQ can also be determined throughcomparing measured signals from samples with known low concentrationsof analyte with those of blank samples. This establishes the minimumconcentration at which the analyte can be reliably quantified. A typical

signal-to-noise ratio is 10:1.Any results of limits of detection and quantitation measurements must beverified by experimental tests with samples containing the analytes atlevels across the two regions. It is equally important to assess othermethod validation parameters, such as precision, reproducibility andaccuracy, close to the limits of detection and quantitation. Figure 6 illustratesthe limit of quantitation (along with the limit of detection, range,and linearity). Figure 7 illustrates both the limit of detection and the limitof quantitation based on signal-to-noise.


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Validation PlanningSuccessful validation requires the cooperative efforts of several departmentsof an organization including regulatory affairs, quality assurance,quality control and analytical development. Therefore, it is important thatthe process follows a well-defined validation master plan for analyticalmethods. The plan documents a companys approach and steps formethod validation and serves two purposes. When implemented correctly,it ensures consistent and efficient execution of validation projects. Inaddition, it answers an inspectors questions regarding the companysapproach for all aspects of analytical method validation. The master plan isalso an ideal training tool for all employees affected by method validation.The master plan should include:

1. Purpose and scope2. Glossary3. Responsibilities, such as user departments, management, QA4. Method performance characteristics and approaches for testing5. Steps for method validation6. Selection of tests and acceptance criteria7. Approach and parameters for system suitability testing

48. Modification and revalidation of methods


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9. Verification of compendial and standard methods10. Transfer of analytical methods11. List of required Standard Operating Procedures12. Approval process, documentation and archiving13. Templates for project plans, test protocols and validation reportsFor each individual validation project a project plan should be developed.It outlines what is to be done in order to get a specific method orprocedure validated. This plan should be derived from the master plan.The plan should include a time table with specific tasks, deliverables andowners.Scope and MethodSpecifications

4The scope of the method and its validation criteria should be definedearly in the process. Defining a scope is a cooperative effort of severaldepartments including business development, analytical development,quality control and quality assurance. Questions include:What samples should be analyzed?What analytes should be measured?

What are the expected concentration levels?What are the sample matrices?Are there interfering substances expected, and, if so, should they bedetected and quantified?Are there any specific legislative or regulatory requirements?Should information be qualitative or quantitative?What are the required detection and quantitation limits?What is the expected concentration range?What precision and accuracy is expected?How robust should the method be?Which type of equipment should be used? Is the method for onespecific instrument, or should it be used by all instruments of thesame type?

Will the method be used in one specific laboratory or should it beapplicable in all laboratories at one side or around the globe?What skills do the anticipated users of the method have?Defining the scope of a method is important to find the optimal effortfor testing. For example, including equipment from different vendors willincrease the test effort but also a laboratorys flexibility to use differentinstruments in routine analysis. If the method is to be run only on a specificinstrument, there is no need to use instruments from other vendors in thevalidation experiments. In this way, the experiments can be limited towhat is really necessary. Similarly, including different locations in the validationstudy will increase the test effort but it will also allow easy transferof the method to sites that have been part of the validation studies.

Selecting ValidationParameters and LimitsFor an efficient validation process, it is important to specify the rightvalidation parameters and acceptance criteria. The methods performanceparameters and limits should be based on the intended use of themethod. It is not always necessary to validate all analytical parametersavailable for a specific technique. For example, if the method is to beused for qualitative trace level analysis, there is no need to test andvalidate the methods limit of quantitation, or the linearity over the full dynamic range of the equipment. The more parameters, the more time it


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will take to validate. It is not always essential to validate every analyticalperformance parameter, but it is necessary to define the ones that arerequired. The selection of validation parameters and acceptance criteriashould be based on business, regulatory and client requirements andshould be justified and documented.

Validation Report and other Documentation

Once the method has been developed and validated, a validation reportshould be prepared. The report should include sufficient information sothat an experienced analyst can repeat the validation study. Typically itshould include the following:Purpose and scope of the method (applicability, type)Summary of methodologyResponsibilities

Type of compounds and matrixAll chemicals, reagents, reference standards, QC samples with purity,grade, their source, or detailed instructions on their preparationProcedures for quality checks of standards and chemicals usedSafety precautionsA plan and procedure for method implementation from the methoddevelopment lab to routine analysisCritical parameters taken from robustness testingDetailed parameters and conditions on how the experiments were


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conducted, including sample preparation and method parametersStatistical procedures and representative calculationsProcedures for QC in routine analyses, such as system suitability testsRepresentative plots, such as chromatograms, spectra and calibrationcurves including raw dataMethod acceptance limit performance dataExpected uncertainty of measurement resultsCriteria for revalidationQualification records of the individuals who developed and validatedthe methodother DocumentationReferences, if necessaryDeviations from the validation plan and protocolSummary and conclusionsApproval with names, titles, date and signatures of those responsiblefor the review and approval of the analytical test procedure.

Published Validation Guidelines

1978 Current Good Manufacturing Practices (cGMPs)

1987 FDA Validation Guideline

1989 Supplement 9 to USP XXI

1994 FDA Reviewer Guidance

1995 ICH Validation Definitions

1997 ICH Validation Methodology

1999 Supplement 10 to USP 23

2000 FDA Draft Validation Guidance

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