2. 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.
3. Steps in Validation
REPEATABILITY
PRECISION
ACCURACY Reproducibility
INTERMEDIATE
Sensitivity PRECISION
SPECIFICITY
DETECTION RANGE
LINEARITY QUANTIFICATION
4. Specificit
y
• 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).
5. • Indicates the linear relationship
between concentration and
response of the detector
6. 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.
7. 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
8. Applicable Concentration Range
LOL
Instrument response
LOQ => limit of quantitative
measurement
LOQ LOL => limit of linear
response
Useful range
Concentration
9. 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)
10. 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.
11. Repeatability
• Repeatability expresses the precision under the
same operating conditions over a short interval
of time.
• Repeatability is also termed as intra-assay
precision.
12. 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.
18. 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
19. 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
20. 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.
21. Based on the Standard Deviation of
the 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
