Single+Laboratory+Validation

8/13/2019 Single+Laboratory+Validation

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Definitions

Validation is the processof demonstratingor confirming the performancecharacteristicsof a method of analysis.

A process of evaluating methodperformance and demonstrating that itmeets a particular requirement.

Validation applies to a specific operator,laboratory, and equipmentutilizing themethod over a reasonable concentration

rangeand period of time.


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Why Method Validation?

To minimize analytical and instrumental errors

To give reliable and reproducible results in

accordance with the given specifications of the

test method

To ensure the quality of the test results

To meet accreditation requirement

Objective evidence for defense againstchallenges

To be assured of the correctness of results


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When Method Validation Not

Required? Standard methods on condition that

used within their scope of applicability(e.g. matrices, ranges, etc)

without modifications (including QA planand reporting)

Otherwise, required


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Ask yourself

What analytes should be detected?

What are the expected concentration levels? What are the sample matrices? Are there interfering substances expected, and, if so, should they be

detected 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 one specific

instrument, or should it be used by all instruments of the same type? Will the method be used in one specific laboratory or should it be

applicable in all laboratories at one side or around the globe?

What skills do the anticipated users of the method have?

Before start to validate a method


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Parameters for Method Validation

Accuracy

Precision

Specificity Limit of detection

Limit of quantitation

Linearity and range Ruggedness

Robustness


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Strategy for Method Validation

1. Develop a validation protocol, an operatingprocedure or a validation master plan for thevalidation.

2. For a specific validation project define owners andresponsibilities.

3. Develop a validation project plan.

4. Define the application, purpose and scope of themethod.

5. Define the performance parameters and acceptance

criteria.6. Define validation experiments.

Source: LabCompliance (2007). Validation of Analytical Methods and Procedures:

Tutorial. http://www.labcompliance.com/tutorial/methods/default.aspx?sm=d_d


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Strategy for Method Validation

Major

compounds

Major

compounds

Traces Traces

quantitative qualitative quantitative qualitative

Limit of

detection

Limit of

quantitation

Linearity Range Precision Accuracy Specificity Ruggedness


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Method validation tools

Linear-data plotter can be used with data from a linearity experiment to

assess the reportable range of a method.

provides a plot of the average of a group of replicatetest results on the y-axis versus the assigned value(in % or concentration units) on the x-axis.

SD Calculator

can be with data from a replication experiment tocalculate the mean, standard deviation (SD orsmeas), and coefficient of variation (CV).

a histogram display of the data is also available.


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Method validation tools

Paired-data Calculator

can be used with data from a comparison of methodsexperiment to calculate linear regression statistics(slope, y-intercept, and standard deviation about theregression line, sy/x), and the correlation coefficient(r, Pearson product moment correlation coefficient); t-test statistics (average difference between twomethods or biasmeas; SDdiff, standard deviation ofthe differences between the two methods).

can also be used to provide a "comparison plot" thatshows the test method results on the y-axis versusthe comparative method results on the x-axis, as wellas a "difference plot" that displays the differencebetween the test minus comparative results on the y-axis versus the comparative method result on the x-axis.


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Sampling

Sample preparation

Analysis

Calibration

Data evaluation

Reporting


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Sampling

Analysis starts with sampling

For trace analysis: sampling becomes amajor source of error

Differs from matrix to matrix


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Sampling Method

When, where and howto collect samples

Sample transportationto laboratory

Samplingequipment Sample containers

Sample-treatment procedures(drying,

mixing, etc. prior to measurements) Sub-sampling procedures

Storageduring sampling


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Sampling Method

To obtain a representative sample

Types of sampling

car or bin sampling

stratified sampling

random sampling

2 steps sampling interval sampling


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Problems during sampling and storage

of samples

Losses from volatilization

Decomposition by means of:

temperature

UV irradiation

microbial activity chemical reactions with oxygen, samplecontainer, etc.


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ISTA Validation Process

ISTA method validation is a five-stepprocess:

1. Test method selection and/or development.

2. Validation through either multi-laboratorycharacterization of the test method performance, peerverification of the test method, or verification ofperformance claims for the test method.

3. Review of data.

4. Approval of the test method by the relevant ISTATechnical Committee, publication in ISTA MethodValidation Reports and preparation of a Rules proposal

for the test method.5. Final acceptance by the ISTA voting members andpublication of the test method in the ISTA Rules.

Source: Hampton, J. (2005), ISTA Method Validation, Issues of Technical Common Interest,

Seed Testing International No. 130, p. 22, October 2005

http://www.seedtest.org/upload/cms/user/130_p22.pdf


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Accuracy

To determine the closeness of the testresults obtained to the true value of the

standard used

To measure the systemic error of the

analysis


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Accuracy

Method used:

Purchase standardfrom the supplier witha known tolerance given or standard

recommended by a particular reference

method. Working standard can be used as a

standard addition(spiked analysis).

Perform the method with more than 2determinationsat low, middle and high

concentrations.


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Accuracy

Method used:

For methods using an instrument with a knownstandard calibration curve, selection of the rangecan be made.

For methods without the above, the range canbe selected from a knowledge of theconcentration of samples analyzed.

Based on the results obtained, calculate theaverage of the standard values obtained andcompare them with recommended values(Table1) or

Compare the result obtained with certifiedreference material(CRM).


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Accuracy

Analyte (%) Unit Mean Recovery (%)

100 100% 98-102

10 10% 98-102

1 1% 97-103

0.1 0.1% 95-105

0.01 100 ppm 90-107

0.001 10 ppm 80-110

0.0001 1 ppm 80-110

0.00001 100 ppb 80-110

0.000001 10 ppb 60-115

0.0000001 1 ppb 40-120

Table 1: Acceptable Recovery Percentages

Source: AOAC (2002). AOAC Requirements for Single Laboratory Validation of

Chemical Methods. DRAFT 2002-11-07,\AOACI\eCam\Single-

Lab_Validation_47.doc. http://www.aoac.org/Ag_Materials/additives/aoac_slv.pdf.
http://www.aoac.org/Ag_Materials/additives/aoac_slv.pdfhttp://www.aoac.org/Ag_Materials/additives/aoac_slv.pdf


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Precision

To determine the degree of agreementwithinthe test results for a particular sample.

This is a measurement of the random errorsofan analysis.

3 types of precision measurement(1) Repeatability(one single operator, single laboratory,

short time span)

(2) Intermediate precision(internal reproducibility:between operators, single laboratory)

(3) Reproducibility (proficiency testing/collaborative

studies between laboratories)


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Precision

Repeatability (one single operator, single laboratory,

short time span).

Refers to the degree of agreement of results whenconditions are maintained as constant as possible.

Select one sample for every matrix and performreplicationfor each matrix within the same day.

If resources permit, run the same sample for 3different days (within 7 days).

Calculate the standard deviation (SD) and relativestandard deviation(RSD) for each matrix.

Compare the precision results with the RSD% inTable 2.


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Precision

Intermediate precision (internal reproducibility: between

operators, single laboratory). To evaluate the degree of agreement between different

operatorsfor a particular sample.

Measure the random errorsinherent when different

analysts perform the same analysis with the samesample.

Use 2-tailed F-test to determine whether there is anysignificant difference in the results between the

precisions of two operators. Calculate the sample variance for each operator.

Calculate the precision for each operator according tothe types of samples and compare the RSD(%).


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Precision

Reproducibility (proficiency testing/collaborative

studies between laboratories). Only applicable when resourcesare available. The blind sample(s) will be analyzed at least

three timesby using the routine method and/orother standard methods.

Calculate the average value of the replicatesand SDif more than three replicates areperformed.

Compare the resultsobtained from otherlaboratoriesin terms of the z-score, mean valeuand median once these results are madeavailable.


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Analyte (%) Unit RSDr(%) RSDR(%)

100 100% 1 2

10 10% 1.5 3

1 1% 2 4

0.1 0.1% 3 6

0.01 100 ppm 4 8

0.001 10 ppm (g/g) 6 11

0.0001 1 ppm 8 16

0.000001 10 ppb (g/kg) 15 32

Table 1: Acceptable Recovery Percentages

AOAC (2002). AOAC Requirements for Single Laboratory Validation of Chemical

Methods. DRAFT 2002-11-07,\AOACI\eCam\Single-Lab_Validation_47.doc.

http://www.aoac.org/Ag_Materials/additives/aoac_slv.pdf.

Precision


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The value of RSD% can be calculatedfrom HORRAT formula:

RSDr= C0.15(Repeatability)

RSDR= 2C0.15

(Reproducibility)

HORRAT = RSD (found)/RSD (calculated)

Acceptable values for this ratio aretypically 0.5 to 2.

Precision


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Limit of Detection

It is the smallest amountor concentration of an analytethat can be estimatedwith acceptable reliability.

An alternative definition of the limit of detection and limitof determination is based upon the variability of the blank.

The blank value plus three times the standard deviationof

the blank is taken as the detection limit and the blankvalue plus 10 times the standard deviationof the blank istaken as the determination limit.

The detection limit is only useful for control of undesirableimpuritiesthat are specified as not more than a specifiedlow level and for low-level contaminants.

Limits of detection and determination are unnecessary forcomposition specificationsalthough the statistical problemof whether or not a limit is violated is the same near zero

as it is at a finite value.


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Instrument Detection Limit

To determine the lowest concentrationof analytein a sample that can be detected. It is important for trace analysis. Perform several determinations and calculate

the SD of the blank LOD = x + 3 SD(x = average blank reading) or Perform several determinations at the lowest

acceptable concentration using a standard

sample (fortified blank sample) and calculate theSD.

LOD = x + 3 SD (x = average sample blankreading)


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Method Detection Limit

It is based on a sample, which have gone through theentire sample preparationprior to analysis.

Method detection limit is approximately 4 instrumentdetection limit.

Perform several determinationseach at low, middle andhigh range concentrations using a sample.

Calculate SD at each limit.

Plot SD against concentration of the analytes.

Extrapolate the graph until the y-axis is reached. At zero concentration, record the SD0.

Method detection limit = 3 SD0 It is unique for a particle sample matrix.


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Signal to Noise Ratio

Source: LabCompliance (2007). Validation of Analytical Methods and Procedures:Tutorial. http://www.labcompliance.com/tutorial/methods/default.aspx?sm=d_d


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Source: International Conference on Harmonization (ICH) of Technical

Requirements for the Registration of Pharmaceuticals for Human Use,

Validation of analytical procedures: Methodology, adopted in 1996, Geneva


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Specificity/Selectivity

The terms selectivity and specificity are oftenused interchangeably.

To determine the purity of the peak areain

chromatogram. Difficult to ascertain whether the peaks within a

sample chromatogram are pureor consist ofmore than one compound.

The analyst should know how many compoundsare in the sample or whether procedures fordetecting impure peaks should be used.


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Examples of pure and impure HPLC peaks

Source: LabCompliance (2007). Validation of Analytical Methods and Procedures:Tutorial. http://www.labcompliance.com/tutorial/methods/default.aspx?sm=d_d


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Linearity

To determine the degree of varianceabout thecalibration curve

Perform several determinations using astandard sample at low, middle and high range.

Calculate the SD of each concentration group.

Calculate the best-fit line of the calibration curveand the correlation coefficient(r2) of the curve.

Use the F-test to determine the significantdifference in variance of the curve for each

concentration group.


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Ruggedness

Ruggedness is defined by U.S. Pharmacoepia as thedegree of reproducibility of resultsobtained under avariety of conditions, such as different laboratories,analysts, instruments, environmental conditions,

operators and materials. Ruggedness is a measure of reproducibilityof test resultsunder normal, expected operational conditions fromlaboratory to laboratory and from analyst to analyst.

Ruggedness is determined by the analysis of aliquotsfrom homogeneous lots in different laboratories.

Refer to the examples of Ruggedness Trial in the AOACRequirements for Single Laboratory Validation ofChemical Methods (DRAFT 2002-11-07).


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Robustness Robustness examine the effect that operational

parametershave on the analysis results. These parameters are pH, flow rate, column

temperature, injection volume, detection wavelength ormobile phase composition.

If the influence of the parameter is within a previouslyspecified tolerance, the parameter is said to be withinthe methods robustness range.

Obtaining dataon these effects helps to assesswhether a method needs to be revalidatedwhen one

or more parameters are changed, for example, tocompensate for column performance over time.

It is recommended to consider the evaluation of amethods robustness during the development phase,and any results that are critical for the method should

be documented.


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Sources

ISTA guide:

http://www.seedtest.org/upload/cms/user/130_p22.pdf

EURACHEM guide:http://www.farmacia.ufmg.br/lato/eurachem%20guide%

20validation%20(1).pdf

AOAC guide:http://www.aoac.org/Ag_Materials/additives/aoac_slv.p

df

Other guide:http://www.labcompliance.de/documents/FDA/FDA-

Others/Laboratory/f-505-method-validation-draft.pdf


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Single+Laboratory+Validation