Test Method Validation : Test Method Validation Muraleedharan CV An ISO 17025 perspective References : References EURACHEM : The Fitness for Purpose of Analytical Methods – A laboratory guide to method validation and related topics : 1998
IUPAC : Harmonized guidelines for the use of recovery information in analytical measurement : 1996
EAL : Validation of test methods : 1997 Validation (ISO 17025 Perspective) : Validation (ISO 17025 Perspective) Validation is confirmation by examination
Laboratory shall validate
Non standard methods
Laboratory designed / developed methods
Standard method used outside their intended scope
Amplifications / modifications of standard methods
The range and accuracy of the values obtained from validate methods shall be relevant to the client’s needs ISO 17025 Method Validation - Revisited : Method Validation - Revisited A process of defining a requirement, and conforming that the method has capabilities to meet with what the application (of the method) requires
Method suitability is the most important parameter ; in the past method validation has tended to concentrate on the process of evaluating a set of performance parameters
Evaluating the set of performance parameters like accuracy, precision, robustness …..is still valid EURACHEM When should methods be validated ? : When should methods be validated ? A new method developed for a particular problem
Established methods revised or extended to a new problem
When QC indicates that established method is changing with time
Established method used in a different laboratory; or with different analyst; or with different instrumentation
To demonstrate the equivalence between two methods EURACHEM Validation parameters : Validation parameters Confirmation of identity
Limit of quantitation (LoQ)
Limit of Detection (LoD)
Linearity (working range estimation)
Ruggedness & Robustness
Repeatability / Reproducibility Precision EURACHEM Confirmation of identity and Specificity : Confirmation of identity and Specificity Confirmation of identity : It is necessary to establish that the signal being produced at the measurement stage, which has been attributed to the analyte, is only due to the analyte and not from the presence of something chemically or physically similar or arising as a coincidence
Specificity: The ability of a method to determine accurately and specifically the analyte of interest in the presence of other components in a sample matrix under stated conditions of test EURACHEM Confirmation of identity and Specificity : Confirmation of identity and Specificity CASE STUDY 1 :
A peak in a chromatographic trace may be identified as being due to the analyte of interest on the basis that a RM containing the same analyte generates a signal at the same point in the chromatogram.
But, is the signal due to the analyte or to something else which coincidently co-elutes ? It could be either or both. EURACHEM Confirmation of identity and Specificity : Confirmation of identity and Specificity CASE STUDY 1 :
A peak in a HPLC trace may be identified as being due to the analyte of interest on the basis that a RM containing the same analyte generates a signal at the same point in the chromatogram.
But, is the signal due to the analyte or to something else which coincidently co-elutes ? It could be either or both.
Validation in such cases may require :
Generating the chromatogram with a column of different polarity, to see whether the test signal and the signal generated by the RM still appear as before.
Alternate techniques could be used for ascertaining the identity of the analyte / specificity of the method (IR with peak matching or GC with detector capable of monitoring peak purity) EURACHEM Confirmation of identity : Confirmation of identity EURACHEM Limit of Detection : Limit of Detection EURACHEM When measurements are made at low analyte or property levels, it is important to know what is the lowest concentration of the analyte or property value that can be confidently detected.
LoD is relevant in qualitative tests also.
LoD is also known in certain documents as minimum detectable net concentration, detection limit, or minimum detectable (true) value. Limit of Detection : Estimation : Limit of Detection : Estimation EURACHEM Limit of Detection in qualitative tests : Limit of Detection in qualitative tests EURACHEM Case Study 2 : Limit of Quantitation : Limit of Quantitation EURACHEM The lowest concentration of an analyte, that can be determined with acceptable precision (repeatability) and accuracy under the stated conditions of the test
Neither LoQ nor LoD represent immeasurable values; but only indicates that below which the measurement uncertainties approach the measured value itself. In other words when the Test Uncertainty Values (TUR) are around or less than unity.
Many agencies specify the LoQ as 6s or 10s values of the standard deviation of the blank mean
LoQ = 2x or 3x LoD Limit of Quantitation : Estimation : Limit of Quantitation : Estimation EURACHEM Working & Linear Range : Working & Linear Range EURACHEM The working range is the range of analyte concentrations over which the method can be employed. The lower limit of the working range may be the LoD (in qualitative estimations) and LoQ (in quantitative estimations). At the upper end of the range, limitations will be imposed by various effects like instrument response.
The linear range is the range of analyte concentrations over which the method gives test results proportional to the concentration of the analyte Working Range INPUT OUTPUT Linear Range Working & Linear Range : Working & Linear Range EURACHEM Accuracy : Accuracy EURACHEM Accuracy expresses the closeness of result with the TRUE value or CONVENTIONAL TRUE value
Accuracy is expressed as BIAS. ie, the difference between the mean value of the method and the mean value of the TRUE VALUE or CONVENTINAL TRUE VALUE
Certified Reference Materials (CRM) can be used for establishing the bias of the method
Alternate method for estimation of bias is the use of any other STANDARDISED METHOD, that could be used dfor the estimation of the same result. Accuracy : Accuracy EURACHEM Precision : Precision EURACHEM Most common precision measures are REPEATABILITY and REPRODUCIBILITY
Repeatability will provide the dispersion of results when a method is performed by a single person, on one measurement system, over a short period of time (time interval being comparable to the duration of the test)
Reproducibility refers to the dispersion of results when there occurs following conditions
Different measurement systems (instruments)
Larger time / spatial gap between measurements
Some times intermediate measures of REPRODUCIBILITY by varying any one or combinations of measurement conditions are also considered SD, RSD (CV) as measures of precision : SD, RSD (CV) as measures of precision EURACHEM Normally standard deviation of the mean (SD) is estimated to represent the measure of precision
But, the standard deviation of the mean may some times depend on the mean itself. In such cases, relative standard deviation or coefficient of variation could be a better measure of precision
A Precision estimation plan could have three distinct objectives
Estimation of Repeatability precision
Estimation of Intra Laboratory reproducibility precision
Estimation of Inter Laboratory reproducibility precision Precision - Estimation : Precision - Estimation EURACHEM Measurement Uncertainty : Measurement Uncertainty EURACHEM Measurement uncertainty is a parameter that could be attributed to the dispersion of the measurement.
Uncertainty estimation of the measurement shall take into account,
Over all precision of the method
Bias and its uncertainty (method)
Any other significant factors and their uncertainty that might affect the results (Eg. Environmental conditions, sample preparation, sampling …..) Robustness / Ruggedness : Robustness / Ruggedness EURACHEM The robustness of a method is a measure of its capability to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
Ruggedness Testing : A inter laboratory study to assess the behavior of a method when small changes in the environmental and / or operating conditions are made (which signifies the changes that could arise between different laboratories)
Robustness is normally evaluated during the method development (one laboratory) and subsequently, ruggedness estimation is attempted using an inter laboratory study. Robustness / Ruggedness : Robustness / Ruggedness EURACHEM Recovery : Recovery EURACHEM Test for analytes do not always measure the analyte present in the sample. Analytes may be present in different forms; either bound or unbound conditions, some of which during the extraction process does not contribute towards the result.
Since it is not possible to know how much analyte is present in a sample, it is difficult to be certain how successful the method is in extracting the analyte.
Estimation of efficiency of recovery is an acceptable way to ascertain the suitability of extraction method. Recovery Efficiency : Recovery Efficiency EURACHEM Recovery (%) = (1/C3)*(C1-C2)*100
C1 = Concentration determined in fortified sample
C2 = Concentration determined in unfortified sample
C3 = Concentration of fortification
Recoveries from fortified samples or matrix blanks will usually be better than real samples in which the analyte is more closely bound.
Another method used for establishing the recovery efficiency is using CRMs (where recovery for a specific method will be certified) Choice of validation parameters : Choice of validation parameters What sort of answer is required ?
(Qualitative or Quantitative)
Confirmation of identity
LoD EURACHEM Choice of validation parameters : Choice of validation parameters Is the analyte present in more than one form ? Are we interested in extractable, free or total analyte ?
Confirmation of identity
Recovery EURACHEM Choice of validation parameters : Choice of validation parameters What are the analytes of interest ? And their likely levels present (%, ppm, ppb)
Confirmation of identity
Linearity (Working and linear range) EURACHEM Choice of validation parameters : Choice of validation parameters How accurate and precise the answer must be ? What degree of uncertainty is allowed during measurement ?
Reproducibility precision EURACHEM Choice of validation parameters : Choice of validation parameters What are the likely interferences to the analytes (s) ?
Specificity EURACHEM Choice of validation parameters : Choice of validation parameters Do the results need to be compared with the results from other laboratories ?
Reproducibility Precision EURACHEM Choice of validation parameters : Choice of validation parameters Do the results need to be compared with External Specifications ?
Reproducibility Precision EURACHEM