Learning objectives• Validation• Steps in validation, and understanding of such steps
Validation?• Validation is the assessment of a process or instrument to assure that the process and instrument is suitable for its intended use (FDA, 1987).• Validation enables an efficient and productive use of the process and instrumental variables.• A new assay method, change in operator, laboratory and equipment than the one in previous method requires validation.
Steps in Validation REPEATABILITY PRECISION ACCURACY Reproducibility INTERMEDIATE Sensitivity PRECISIONSPECIFICITY DETECTION RANGE LINEARITY QUANTIFICATION
Specificity• Specificity is the ability to assess unequivocally the analyte in the presence of other components such as impurities, degradants and matrix etc.• Lack of specificity of an assay procedure, may be compensated by other supporting assay(s).
• Indicates the linear relationship between concentration and response of the detector
EVALUATION OF LINEARITY• Linearity is evaluated by visual inspection of a plot of signals as a function of analyte concentration.• If there is a linear relationship, the data is evaluated by appropriate statistical methods, for example, linear regression.• In some cases, to obtain linearity, the test data may need mathematical transformation prior to the regression analysis.• For the establishment of linearity, a minimum of 5 concentrations are recommended.
RANGE• The specified range is normally derived from linearity studies and depends on the intended application of the procedure.• It confirms that the assay will provide an acceptable accuracy and precision when applied to samples containing analyte, within or at the extremes of the specified range
Applicable Concentration Range LOLInstrument response LOQ => limit of quantitative measurement LOQ LOL => limit of linear response Useful range Concentration
ACCURACY• The accuracy of an assay expresses the closeness or agreement between the true value and the value found. This is sometimes termed as trueness• Within day accuracy (Repeatability)• Between days accuracy (Reproducibility)
PRECISION• The precision of an assay expresses the degree of scatter between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.• Precision may be considered at three levels: – Repeatability – Intermediate precision – Reproducibility• The precision is usually expressed as the variance, standard deviation or coefficient of variance of a series of measurements.
Repeatability• Repeatability expresses the precision under the same operating conditions over a short interval of time.• Repeatability is also termed as intra-assay precision.
Intermediate precision• Intermediate precision expresses within-laboratories variations, different days, different analysts, different equipment, etc.• Typical variations to be studied include days, analysts, equipment etc.
Reproducibility• Reproducibility is assessed by means of an inter-laboratory trial, and between days assay.
Accuracy vs precision Good accuracy and precision
Accuracy vs precision • Poor accuracy • Good precision
Accuracy vs precision • Poor precision • Good accuracy
DETECTION LIMIT• The detection limit of an analyte in an assay is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value• Several approaches for determining the detection limit are used, depending on whether the procedure is a non-instrumental or instrumental
Based on Visual Evaluation• Visual evaluation may be used for non- instrumental methods but may also be used for instrumental methods.• The detection limit is determined by the analyzing samples having known concentrations of analyte and by establishing the minimum level at which the analyte can reliably be detected
Based on Signal-to-Noise• This approach is applied to analytical procedures which exhibit baseline noise.• Determination of the signal-to-noise ratio is performed by comparing measured signals of samples having known low concentration of analyte with those of blanks , and establishing the minimum concentration at which the analyte can reliably be detected.• A signal-to-noise ratio between 3:1 or 2:1 is generally considered acceptable for estimating the detection limit.
Based on the Standard Deviation ofthe Response and the Slope The detection limit (DL) may be expressed as: DL = 3.3 σ /S where σ = the standard deviation of the intercept S = mean of the slope of the calibration curve
Robustness Small changes do not affect the parameters of the assay