Learning objectivesLearning objectives

• ValidationValidation

• Steps in validation, and Steps in validation, and understanding of such stepsunderstanding of such steps

Validation?Validation?• Validation is the assessment of a Validation is the assessment of a

process or instrument to assure that the process or instrument to assure that the process and instrument is suitable for process and instrument is suitable for its intended use (FDA, 1987). its intended use (FDA, 1987).

• Validation enables an efficient and Validation enables an efficient and productive use of the process and productive use of the process and instrumental variables. instrumental variables.

• A new assay method, change in A new assay method, change in operator, laboratory and equipment operator, laboratory and equipment than the one in previous method than the one in previous method requires validation. requires validation.

Steps in Validation Steps in Validation

PRECISION

Sensitivity

ACCURACY

LINEARITY

RANGE

SPECIFICITY

REPEATABILITY Reproducibility

QUANTIFICATION

INTERMEDIATE PRECISION

DETECTION

SpecificiSpecificity ty • Specificity is the ability to assess Specificity is the ability to assess

unequivocally the analyte in the presence of unequivocally the analyte in the presence of other components such as impurities, other components such as impurities, degradants and matrix etc.degradants and matrix etc.

• Lack of specificity of an assay procedure, may Lack of specificity of an assay procedure, may be compensated by other supporting assay(s). be compensated by other supporting assay(s).

• Indicates the linear relationship between concentration and response of the detector

EVALUATION OF EVALUATION OF LINEARITYLINEARITY

• Linearity is evaluated by visual inspection of a plot of Linearity is evaluated by visual inspection of a plot of signals as a function of analyte concentration. signals as a function of analyte concentration.

• If there is a linear relationship, the data is evaluated by If there is a linear relationship, the data is evaluated by appropriate statistical methods, for example, linear appropriate statistical methods, for example, linear regression. regression.

• In some cases, to obtain linearity, the test data may need In some cases, to obtain linearity, the test data may need mathematical transformation prior to the regression mathematical transformation prior to the regression analysis. analysis.

• For the establishment of linearity, a minimum of 5 For the establishment of linearity, a minimum of 5 concentrations are recommended. concentrations are recommended.

RANGE RANGE • The specified range is normally derived from The specified range is normally derived from

linearity studies and depends on the intended linearity studies and depends on the intended application of the procedure. application of the procedure.

• It confirms that the assay will provide an It confirms that the assay will provide an acceptable accuracy and precision when acceptable accuracy and precision when applied to samples containing analyte, within applied to samples containing analyte, within or at the extremes of the specified range or at the extremes of the specified range

Applicable Concentration RangeApplicable Concentration Range

Useful range

LOQ

LOL

Inst

rum

ent r

espo

nse

Concentration

LOQ => limit of quantitative measurement

LOL => limit of linear response

ACCURACY ACCURACY

• The accuracy of an assay expresses the The accuracy of an assay expresses the closeness or agreement between the true value closeness or agreement between the true value and the value found. This is sometimes termed and the value found. This is sometimes termed as truenessas trueness

• Within day accuracy Within day accuracy ((Repeatability)Repeatability)

• Between days accuracy Between days accuracy ((Reproducibility)Reproducibility)

PRECISIONPRECISION• The precision of an assay expresses the degree of The precision of an assay expresses the degree of

scatter between a series of measurements scatter between a series of measurements obtained from multiple sampling of the same obtained from multiple sampling of the same homogeneous sample under the prescribed homogeneous sample under the prescribed conditions. conditions.

• Precision may be considered at three levels:Precision may be considered at three levels:– RepeatabilityRepeatability– Intermediate precision Intermediate precision – ReproducibilityReproducibility

• The precision is usually expressed as the The precision is usually expressed as the

variancevariance, , standard deviation or coefficient of standard deviation or coefficient of variancevariance of a series of measurements of a series of measurements. .

RepeatabilitRepeatabilityy

• Repeatability expresses the precision under the Repeatability expresses the precision under the same operating conditions over a short interval same operating conditions over a short interval of time. of time.

• Repeatability is also termed as intra-assay Repeatability is also termed as intra-assay precision.precision.

Intermediate precisionIntermediate precision

• Intermediate precision expresses within-Intermediate precision expresses within-laboratories variations, laboratories variations, different daysdifferent days, , different analystsdifferent analysts, , different equipmentdifferent equipment, etc. , etc.

• Typical variations to be studied include days, Typical variations to be studied include days, analysts, equipment etc. analysts, equipment etc.

Reproducibility Reproducibility

• Reproducibility is assessed by Reproducibility is assessed by means of an inter-laboratory trial, means of an inter-laboratory trial, and between days assay. and between days assay.

Accuracy vs precisionAccuracy vs precision

Good accuracy and precision

Accuracy vs precisionAccuracy vs precision

• Poor accuracyPoor accuracy

• Good precisionGood precision

Accuracy vs precisionAccuracy vs precision

• Poor precisionPoor precision

• Good accuracyGood accuracy

DETECTION LIMITDETECTION LIMIT

• The detection limit of an analyte in an assay is The detection limit of an analyte in an assay is the lowest amount of analyte in a sample the lowest amount of analyte in a sample which can be detectedwhich can be detected but not necessarily but not necessarily quantitated as an exact value quantitated as an exact value

• Several approaches for determining the Several approaches for determining the detection limit are used, depending on whether detection limit are used, depending on whether the procedure is a non-instrumental or the procedure is a non-instrumental or instrumental instrumental

Based on Visual Evaluation Based on Visual Evaluation

• Visual evaluation may be used for non-Visual evaluation may be used for non-instrumental methods but may also be used instrumental methods but may also be used for instrumental methods.for instrumental methods.

• The detection limit is determined by the The detection limit is determined by the analyzing samples having known analyzing samples having known concentrations of analyte and by establishing concentrations of analyte and by establishing the minimum level at which the analyte can the minimum level at which the analyte can reliably be detected reliably be detected

Based on Signal-to-Noise Based on Signal-to-Noise • This approach is applied to analytical procedures This approach is applied to analytical procedures

which exhibit baseline noise.which exhibit baseline noise.

• Determination of the signal-to-noise ratio is Determination of the signal-to-noise ratio is performed by comparing measured signals of performed by comparing measured signals of samples having known low concentration of samples having known low concentration of analyte with those of blanks , and establishing the analyte with those of blanks , and establishing the minimum concentration at which the analyte can minimum concentration at which the analyte can reliably be detected. reliably be detected.

• A signal-to-noise ratio between A signal-to-noise ratio between 3:1 or 2:13:1 or 2:1 is is generally considered acceptable for estimating the generally considered acceptable for estimating the detection limit.detection limit.

Based on the Standard Based on the Standard Deviation of the Response and Deviation of the Response and the Slopethe Slope

The detection limit (DL) may be The detection limit (DL) may be expressed as:expressed as:

DL = 3.3 DL = 3.3 /S/S

where where = the standard deviation = the standard deviation of the interceptof the intercept

S = mean of the slope of the S = mean of the slope of the calibration curvecalibration curve

RobustnessRobustness

Small changes do not affect the Small changes do not affect the parameters of the assayparameters of the assay