Method Validation 2370

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Method validation and

verification

W. W. Wong

Senior Accreditation OfficerHKAS

20 November 2009


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Method Validation

What is it?

When is it required?

Why is it necessary?

What are required? How much is adequate?

How should it be done?


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Method Validation what are required

Validation of methods (cl. 5.4.5.3 )

The range and accuracy of the valuesobtainable from validated methods (e.g.

Uncertainty of results

Detection limit Selectivity Linearity Repeatability Reproducibility Robustness/cross-sensitivity


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HOKLAS SC No. 37 Food TestCategory Chemical Testing

Being commented by AAB WP on Food(today is the last day for comment)

Will be published very soon Contains criteria specific to food testing With a view to assist labs

In particular, method validation and samplepreparation


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HOKLAS SC No. 37 Section5.3 Validation of methods

Screening methods:


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Confirmation of identity

Mass spectrometric detection

+50%+50%10% to 20%+25%+15%>20% to 50%

+20%+10%>50%

CI-GC-MS, GC-

MSn

, LC-MS,LC-MSn

EI-GC-MS

(relative)

Relative intensity

(% of base peak)


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Limit of DetectionxLD = 3sO +xBl

WherexLD is the limit of detection

sO is the SD of the outlier free result of a matrix blank sample.

should be based on at least 10 independent completedeterminations of analytes concentration in a typical matrix

blank or low-level material, with no censoring of zero or

negative results.

xBl is the mean concentration of the matrix blank


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Limit of Quantification

The limit of quantification represents a

concentration of the determinand that can

reasonably be determined with an acceptable

level of accuracy. Usually it is arbitrarily taken

as a fixed multiple of the detection limit.


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Limit of Quantification

Estimated by:

xLQ = 3xLD

The factor k=3 corresponds to a relative result

uncertainty of approximately 33%.


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Limit of QuantificationAOAC

smallest amount of analyte in a test sample that can bequantitatively determined with suitable precision and

accuracy under previously established method conditions.

It is often taken as the blank value plus 10 times the std

dev.


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Reporting limits

Set at a level at which quantitativeresults may be obtained with a

specified degree of confidence

Limits of detection and reportingshall be verified.


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Reporting limits

ISO/TS 13530:2009

RL is a specific concentration at orabove the limit of quantification that isreported to the client with a certain

degree of confidence. It is often definedon a project-specific basis. If the RL isset below the limit of quantification by

the client, method modification isrequired.


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Reporting limits

ISO/TS 13530:2009

For verification of limit of detectionand limit of quantification, spiked

blank matrix samples at theseconcentration levels and blank

matrix samples shall be analysed inthe same manner as real samples.


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Reporting limits

ISO/TS 13530:2009

If the uncertainty of results for thesamples spiked at the limit of

quantification level is smaller thanor equal to the relative precision

corresponding to the factork, thelimit of quantification is verified.


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Reporting limits

ISO/TS 13530:2009

x = 1xLQ k

where

x = t .s/n

kis the factor for calculating the limit of

quantification as a multiple of detection,here: k=3


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Reporting limits

In essence, the laboratory needs to provide

supporting data that, at the reporting limit,the measurement uncertainty (including biasand precision) meets the acceptance criteria.

The direct method is to find the recovery andRSD at the reporting limit and compare themwith the acceptance criteria.


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Recommended LOD and LOQ

ML= specified maximum and/or

minimum level

For ML 0.1 mg/kg, LODML X 1/5


LOQ


For ML

0.1 mg/kg, LOD

ML X 1/5

LOD


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Recommended Recovery

40-1201g/kg

60-11510g/kg

80-110100g/kg80-1101mg/kg

80-11010mg/kg

90-107100mg/kgRecovery (R)95-105

0.1% (1mg/g)

97-103 1% (1g/100g)

98-102 10% (10g/100g)

98-102100%(100g/100g)

Recovery (%)Unit


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Precision

AOAC Official Methods of Analysis (2005)

Appendix E: Laboratory Quality Assurance

Common values of within lab precision

2010ppb

101ppm

50.01%

21%

1100%

RSDr

Concentration


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Estimation of standard deviation by meanrange

s =R/d2

d2is 1.128 for duplicate

R is the mean range of the duplicates


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Range of food matrices

If a method is to be accredited undergeneral foodstuffs, satisfactoryvalidation data shall be obtained for atleast five different food matrices(protein, carbohydrate, oil, dietary fibre

and water) with at least three food typesrepresentative of each food matrix.


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0%

%FAT

%PROTEIN

%CARBOHYDRATES0%

0%

100%

100%

100%

J. AOAC, 83 (2), 413 (2000)The Referee, AOAC Intl, July 1993


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The range of matrices shall also in line

with those listed in relevant regulations.Due consideration shall also be taken

for the food matrices with potentialinterferences, e.g. high chloride effect

on the ICP-MS determination.


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General foodstuffs all foods Test procedure shall document the food matrices used in

the validation studies

Labs need to assess and determine the applicability oftheir methods to the food samples received.


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Method verification

Released early 2008

http://www.aoac.org/alacc_guide_2008.pdf


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The following are some examples of useful references

Harmonised guidelines for single-laboratoryvalidation of method of analysis (IUPAC Technical

Report), 2002

How to meet ISO 17025 Requirements for MethodVerification, AOAC, 2007

ISO/TS 21748:2004 Guidance for the use of

repeatability, reproducibility and trueness estimatesin measurement uncertainty estimation


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CRMs

How many replicate results are required for propercomparison of the certified reference value and the

actual analysis result?

Is it necessary for the mean of test result for a CRM tolie within the uncertainty range of the certified value?

Is a result outside the uncertainty range acceptable ?


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Useful references- ISO Guide 33: 2000

Uses of certified reference materials


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Comparison of results with another laboratory(ISO 5725-6:1994 clause 7.2.4.3)

- the means of the two laboratories are compared

if ,

then statistically no significant difference

where = repeatability standard deviation

= reproducibility standard deviation

222

22

21r

Ryy

2

r

2

R


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How should it be done - verification

Worked examples of method verification areavailable from AOAC

Determination of Total, Saturated, andMonosaturated Fats In Foodstuffs by Hydrolytic Extraction and Gas ChromatographicQuantitation: Collaborative Study

Determination of Low-Level Glucose andFructose in Raw and Refined Crystalline Sugarby High-Performance Anion ExchangeChromatography


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Common deficiencies

Number of food matrices used not adequate

Number of food types for each matrix notadequate

Concentration levels used not adequate

Reporting limits estimated but not verified Food samples chosen not representative of the

food matrices


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Common deficiencies

Confirmation of identity technique not availableor adequate Validation/verification data analyses not done

correctly

Measurement uncertainty not available/notestimated correctly

Equipment and/or test procedures notconforming to the test std requirements


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Common deficiencies

Lack of or inappropriate sampling procedure QC plan acceptance limits not appropriate


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Thank you

Documents

Method Validation 2370