Method validation is an important requirement in the practice of chemical analysis to ensure quality and reliability of the results for all analytical applications, so that a laboratory's clients can have confidence in the results produced by its application, Therefore (Jatto and Okhamafi 2002) outlines that Validation in itself does not improve process but confirm that the processes have been properly developed and are under control.
The necessity for laboratories to use a `fully validated' (has been studied in a collaborative trial) method of analysis is now universally accepted or required within many sectors of analysis. (Wood 1999).
according to (Eurachem Guide 1998) there is little awareness from analytical chemists to its importance, why it should be performed and when, and exactly what needs to be performed.
Nowadays, the validation characteristics needed for the various test procedures and their general requirements are well understood (Ermer 2001), despite this imprtance the (Eurachem Guide 1998) Suggests that The "analyst's understanding of method validation is inhibited by the fact that many of the technical terms used in processes for evaluating methods vary in different sectors of analytical measurement, both in terms of their meaning and also the way they are determined".
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Some laboratory analysts believe that method validation cannot be done unless the laboratories are collaborating with other and therefore they do not do it (Eurachem Guide 1998)
The purpose of this essay is to discuss the issues related to method validation and enhance the understanding of what is involved, the extent and reason of its importance, and give some idea of how it can be achieved.
What is method validation?
There are many definitions for method validation
The FDA in its most recent publication, Guidance for Industry on Analytical Procedures and Methods Validation, states:
"Methods validation is the process of demonstrating that analytical procedures are suitable for their intended use. The methods validation process for analytical procedures begins with the planned and systematic collection by the applicant of the validation data to support analytical procedures" (Bliesner, 2006)
The ISO definition of validation is "the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled" (ISO/IEC 17025:2005 cl. 22.214.171.124; Wong, 2009)
The validation of analytical procedures, can be defined as the assurance or confirmation of its suitability for the intended purpose, and is "an important part of the registration application for a new drug" (Ermer 2001).
Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an "integral part of any good analytical practice". (Huber, 2007). Validation is thus an confirmative tool used to check that if an analytical method is really fit for purpose (Taverniers et al. 2004) and as (Bliesner, 2006) said " Methods validation is part science, part art, and a lot of bookkeeping and accounting ".
according to (Huber, 2007) 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
whenever the method is changed and the change is outside the original scope of the method
in addition to that methods need to be validated for other reasons (Eurachem Guide 1998)
to prove the equivalence between a new method and a standard one.
when quality control indicates an established method is changing with time.
The process of validating analytical methods can be divided into four steps according to (Bliesner, 2006) These steps include:
1. Method evaluation and further method development
2. Final method development and trial methods validation
3. Formal methods validation
4. Formal data review and report issuance.
The first 2 steps is regarded as Pre-validation Qualification Phase which covers all activities relating to product research and development (Jatto and Okhamafi, 2002)
Replication of The development and evaluation process continues until the method is considered capable of meeting the requirement. (Eurachem Guide 1998).
While step 4 is Known as Validation Maintenance Phase, which needs "frequent review of all process related documents, including validation of audit reports, to assure that there have been no changes, deviations, failures and modifications to the production process and that all standard operating procedures (SOPs), have been followed". (Jatto and Okhamafi, 2002).
The Relationship of methd validation with analytical quality assurance (AQA)
Always on Time
Marked to Standard
Although validation and quality assurance (QA) terms are widely used, (Taverniers et al. 2004),believes that many analysts and laboratories "do not know the exact meaning neither the difference nor the relationship between the two terms". As mentioned previously validating a method is checking whether method is fit for the analytical purpose, which means that analytical results can be obtained with an acceptable accuracy level. Analytical method validation forms the first step of QA in the laboratory (Taverniers et al. 2004).
According to (Jatto and Okhamafi 2002) Validation is" an integral part of quality assurance"; it involves the study of systems, facilities and processes targeted for checking whether they achieve their intended functions adequately as specified. Adequate validation is beneficial to the manufacturer in many ways:
Â· It deepens the understanding of Processes; minimizing the risk of problems and assuring smooth running of the process.
Â· It reduce the risk of defect costs.
Â· It reduce the risk of regulatory noncompliance.
Â· A fully validated process may require less in-process controls and end product testing.
Approaches for evaluating acceptable methods of analysis
There are different number of method validation protocols which specify the performance characterstics for validating mthods ,According to (Huber 2007) the ISO/IEC 17025 includes a chapter on the validation of methods enumerating nine validation characterstics while The ICH has developed a harmonized text on the validation of analytical procedures. The document includes definitions for eight validation parameters.
The traditional 'criteria approach' or performance-based approach is to identify specific performance characteristics and to set numeric threshold values to these parameters so in order for the method to be acceptable it should achieve these threshold values (Taverniers et al 2004).
When these different characteristics are being evaluated individually, general evaluation of analytical methds is done as such where the input is the purified or isolated analyte and the output is the analytical result (Taverniers et al 2004).
. The extent of validation
The extent of validation depend on the type of the analytical method whether it is qualitative or quantitative ( i.e the conditions in which the method is going to be used), therefore the analytical requirement will determine the validation plan (Eurachem Guide 1998; Taverniers et al. 2004).
Table1. Validation characteristics normally evaluated for the different types of test procedures and the minimum number of determinations required (if applicable) ICH (Ermer 2001, p756).
Parameters for Method Validation
An attempt was made through the The International Conference on the Harmonization of the Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) for harmonizing different definitions among the different organizations where representatives from the industry and regulatory agencies from the United States, Europe and Japan defined parameters, requirements and, to some extent, methodology for analytical methods validation ( Huber, 2007).
method validation is done by evaluating a series of method-performance characteristics, such as precision, trueness, selectivity/specificity, linearity, operating range, limit of detection (LOD), limit of quantification (LOQ), sensitivity, robustness and applicability. Calibration and traceability have been mentioned also as performance characteristics of a method.
no official guidelines can be followed to determine the sequence of validation experiments, and the best sequence can depend on the method itself. ( Huber, 2007)
Accuracy can also be described as the closeness of agreement between value found and the value that is adopted, either as a conventional, true or accepted reference value (Huber, 2007). According to this, the true value of accuracy assessment can be determined by different ways including:
Comparison with well defined and characterized procedure ,Other alternative involve application of reference material , To check accuracy using a reference material, the mean and standard deviation can be determined by performing a series of replicate tests, and compare with the characterized value for the reference material (Eurachem Guide 1998 ) according to (Huber, 2007 ) this approach assumes that the uncertainty of the reference method is known . In addition to that spiking experiments in which, as ( Nash and Wachter 2003 p554) a "blank sample matrix of interest can be spiked with a known concentration by weight or volume" can be performed to investigate recoveries between analyte and matrix is regarded as usual way to estimate accuracy.
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accuracy of results can be assessed by quantifying both systematic and random effects on results, therefore, Accuracy is studied as two components: 'trueness' and 'precision' (Eurachem Guide, 1998). Trueness is expressed in terms of bias while 'Precision' is a measure of how close results are to each other, and is usually expressed by measures such as standard deviation, which describe the spread of results.
The precision of an analytical procedure expresses the closeness of agreement between independent test results obtained under prescribed conditions (AMC, 2003).
Precision and bias studies , are the most important validation criteria, According to (Taverniers et al., 2004) Precision measures can be divided into 3 levels:
1. repeatability precision measures SD and RSD
2. intra-laboratory reproducibility precision or 'intermediate precision' measures, SD and RSD
3. inter-laboratory reproducibility precision or SD and RSD
Both repeatability and reproducibility are generally dependent on analyte concentration and therefore it should be tried to establish the relationship between precision and analyte concentration (Eurachem Guide, 1998)
Repeatability precision express the degree of agreement among test results under the same operating conditions over ashort period of time (AMC, 2003) meanwhile, the term reproducibility is representing the precision among laboratories while the intermediate precision is a parameter that depend on additional random error within laboratories depending on specified purpose of the method (Ermer and Miller, 2005)
Precision can be computed as a standard deviation of the test results and is usually expressed in terms of imprecision by other words the larger the standard deviation the less the precision (AMC, 2003).
coefficients of variation, is additional parameters of high value in the assessment of precision , data obtained from precision estimation can be documented in control charts, such as Shewhart control charts (Taverniers et al., 2004).
Trueness and bias studies
Trueness is expressed in terms of bias or percentages of error Bias is the difference between the mean value determined for the analyte of interest and the accepted true value or known level actually present (Isabel Taverniers et al., 2004)
bias of a measurement result can be attributable to many factors and these can be the bias of the method itself , laboratory bias and the bias caused by particular analytical run (Eurachem Guide , 1998)
Replicated analysis of samples with known concentrations, such as reference materials (RMs) is the best way to assess bias practically. The ideal RM is a matrix CRM, because it resemble the samples of interest . nevertheless, this matrix matching with the unknown samples does not guarantee that the results of the latter will be correct with other matrix compositions . (Traverniers et al., 2004)
SPECIFICITY AND SELECTIVITY
The terms selectivity and specificity are often used interchangeably (Huber, 2007) those 2 terms are given different difinition by some authors while for others they regarded identical although inconsistent with ICH the term specific according to (Taverniers et al., 2004; Huber, 2007) refers to a "method that produces a response for a single analyte only", while the term selective refers to a "method which provides responses for a number of chemical entities that may or may not be distinguished from each other" and it should be taken into consideration that specificity is taken in account at the beginning of method development with respect to the properties of both analyte and sample (Ermer and Miller, 2005).
According to (Eurachem Guide, 1998) specificity and selectivity reflect the same characteristic and are very closely related to each other in such a way that specificity means 100% selectivity (i.e a method can only be specific if it is for 100% selective) so practically there is no such specific method and the term selectivity is more appropriate ( Nash and wachter , 2003).as a result sensitivity can als be defined as ability to measure analyt concentratio accurately in the presence of interference such as excipients , enantiomers and degradation products that may be present in the sample matrix (Huber, 2007)
DETECTION LIMIT (LOD)
Although this analytical term has great variety of terminology and formulation (Taverniers et al., 2004), it has been defined by all official organization as "the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value".by other words the detection limit represent the amount of analyte in sample allowing for identification of analyte qualitatively without accurate and precise quantification (Taverniers et al., 2004).
The LOD is often defined [2, 4] as the sample
For validation purposes it is necessary to provide an indication of the level at which detection becomes possible (Eurachem Guide, 1998). So in order to achieve this level LOD can be defined as "the sample concentration which produces a peak with height 3 times the level of the baseline noise" (Altria and Rudd, 1995 p 326), from this definition it can be concluded that detection limit can be measured my spiking sample blanks with the analyte at a range of concentration levels.
At each concentration level, it is necessary to measure (6 - 10) independent replicates but randomization of the replicates measurement at the various levels should be done (Eurachem Guide, 1998).
LIMIT OF DETERMINATION (LOQ)
Limit of determination According to (ICH, 2005)is the lowest amount or concentration of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.it is also known as 'Limit of Quantitation '
The limit of deterimination (LOQ) is always higher than the limit of detection (LOD) and mostly is recommended to quote the LOQ as a fixed multiple (3 times) of the LOD (Taverniers et al., 2004).the practical assessment of determination limit (LOD) is done in similar way to that performed in assessment of detection limit by measuring 10 independent sample blanks then calculate the standard deviation and then the lowest signal corresponding to (LOD) is calculated by mulitplying stndard deviation by 10. (Taverniers et al., 2004).
The linearity of an analytical method is its ability to obtain test results which are directly proportional to the concentration of analyte in the sample within a given range (Huber, 2007).
(Thompson et al., 2002; LGC, 2003) have recommended a protocol for establishing linearity of an analytical method which involve spacing of six calibration standards or more over the concentration range of interest (concentrations span 80-120 percent of the expected concentration range) also thy recommend repeating standard calibration for 3 or more runs in a random order. ( Taverniers et al., 2004) suggest an alternative approach to establish linearity by dividing the signal data by the respective concentrations and to plot these 'relative responses' as a function of the concentration, on a log scale to obtain horizontal line over the full linear range.
(Huber, 2007) states that There should be direct proportion between response and the concentrations of the analytes.
The range of an analytical method is the interval between the upper and lower concentrations (including these concentrations) of the analyte in a sample that have been demonstrated to have a suitable level of precision, accuracy and linearity (Huber, 2007).
The specified range is normally derived from linearity studies and depends on the intended application of the procedure (ICH, 2005).
Definitions for linearity, range, LOQ, LOD (Huber, 2007).
The robustness of an analytical procedure is "a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage" (ICH, 2005).so robustness measure the effect of modifying operational parameter on the analytical results (Huber, 2007) and due to this importance of this parameter on method performance robustness shoud be tested during method development (HSA, 2004).
For example if a method states an operating procedure at PH 5.5 Will acceptable performance be maintained at PH 6 or PH 5? According to (Altria and Rudd, 1995) robustness can be assessed or measured either by testing each parameter sequentially "one-by-one" in this approach each parameter may be varied by 5-10 % above and below, the value set in the method.another approach is the multi variate analysis which assess for the robustness of analytical method by testing the effect of simulatneous changing of different factors .
Method validation should be regarded as part of life cycle of analytical procedure to ensure quality, efficacy and safety of analytes to be determined by analytical procedure (Ermer and Miller, 2005) as a conclusion due to this critical importance of validation in analytical methods, this put the analyst in a responsibility position to pay great attention to the different method performance parameters and the possible approaches for their assessment and expression.