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HPTLC_Method Validation by Priyanka Singh

Method Validation Process
Method Validation – is in-separable process of method development.
Method validation is the process used to confirm that the analytical procedure
employed for a specific test is suitable for its intended use.
It starts from -
Pre-validation
 Defined analytical goal
 Method selection
 Optimization
 Development
2

After performing all the experiments described in the validation protocol →
obtained data are evaluated and compared with the acceptance criteria → If
all criteria are met, the method can be awerded as valid.
3

 Establishes; that test data obtained is
reproducible , reliable & accurate.
 Confirms; that the method is suitable for a
particular system
 Determine; if Method is Acceptable for
Intended Use.
 Identification; of source of potential errors.
 Validation of analytical methods is also
required by most regulations.
4

Validation parameters typically monitored are - As per the QMS
• Selectivity - (to detect analyte)
• Stability - (before, during, and after development)
• Linearity - (of the calibration graph)
• Range - (of levels within which the analyte can be quantified)
• LOD & LOQ - (limits of detection and accurate and precise quantification)
• Accuracy - (indication of systematic errors),
• Precision - (indication of random errors),
• Sensitivity - (ability to measure small variations in concentration),
• Ruggedness - (results of the method when used by different analysts in a
variety of locations).
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 Ability of the developed method is to detect analyte quantitatively in
the presence of other components which are expected to be present in the
sample matrix.
 Results are expressed as Resolution.
 The spot for the sample is confirmed by comparing retardation factor
(Rf) values of the spot with that of the standard.
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 Ability of the method within a given
range to obtain test results in direct
proportion to the concentration of analyte
in the sample – calibration curve for the
analyte.
 Sensitivity of the method is determined
with respect to limit of detection (LOD)
and limit of quantification (LOQ).
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 The LOD of an analytical procedure is the lowest amount of analyte in
sample which can be detected.
 Limit of detection (LOD) is determined on the basis of signal to noise ratio.
 Noise can be determined by scanning blank spot (solvent) six times.
 Series of concentrations of drug solutions are applied on plate and analyzed
to determine LOD and LOQ.
 LOD is calculated as three times the noise level.
 LOD is the amount of applied sample producing a peak area which is equal
to the sum of mean blank area and three times standard deviation.
 LOD = 3.3 × Standard Deviation/ Slope
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 The LOQ is the lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracy
 LOQ is calculated as ten times the noise level.
 LOD and LOQ are experimentally verified by diluting the known
concentrations of sample until the average responses are
approximately 3/10 times the standard deviation (SD) of the
responses for six replicate determinations.
 LOQ = 10 × Standard Deviation/ Slope
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• Linearity of the method is evaluated by
constructing calibration curves at different
concentration levels.
• The calibration curve is developed by plotting
peak area vs. concentrations with the help of the
vision-CATS software.
11

Precision provides an indication of random error and evaluated in terms of
intraday and interday precisions.
Intraday precision -is determined by analyzing sample solutions of analyte
from formulations at three levels covering low, medium, and higher
concentrations of calibration curve for five times on the same day
Interday precision - is determined by
analyzing sample solutions of analyte at
three levels covering low, medium, and
higher concentrations over a period of 7
days.
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Precision under same conditions Precision under different conditions
Same analyst Different analyte
Same laboratory Different laboratory
Same instrument ---
Short interval of time – same day Different days
Identical reagents using the same sample Reagents from different sources using the same
sample
Measurement of peak area: RSD should not be
greater than 1%
Peak position: RSD should not be greater than 2%
Sample application: equal volume applied as
seven spots and RSD should not be greater than 3%
RSD should not be greater than 10% within
laboratory reproducibility.
The peak areas obtained are used to calculate mean and % RSD (relative SD) values.
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 Accuracy of an analysis is determined by systematic error involved.
 Accuracy of the method is evaluated by carrying the recovery study
at three levels.
 Recovery experiments are performed by adding three different
amounts of standard drug, i.e., 80, 100, and 120% of the drug, to the
pre-analyzed formulations, and the resultant is reanalyzed six
times(n=6).
 The accuracy is acceptable - if the difference between the true value
and mean measured value does not exceed the RSD values obtained
for repeatability of the method.
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 Analyte should not decompose during development of the chromatogram
and should be stable in solution and on the sorbent for at least 30 and 15
min, respectively.
 The intensity of the spot on the chromatogram should be constant for at
least 60 min.
 Chemical reaction of the solvents and their impurities may produce extra
spot/peak, thus leading to false assay values.
 Other important factor is pH of the aqueous phase used for
extraction/purification which may lead to hydrolysis, oxidation and
isomerization.
 The complete removal of organic solvent should be avoided.
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• Repeatability of measurement of peak area is
determined by analyzing different amount of
analyte covering low, medium, and higher
ranges of the calibration curve seven times
without changing the position of plate.
• Repeatability of sample application is assessed
by spotting samples covering similar range of
calibration curve seven times and analyzing
them once.
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Ruggedness & Robustness is reliability of an analysis with respect to
deliberate variations in method parameters. A slight change in external
and internal factors on the results are examined.
 Robustness of the method is determined in triplicate and the mean and %
RSD of peak area are calculated.
 ruggedness of the method is determine by Chromatographic analysis
of analyte in different concentrations by two analysts and analyzed
for recovery and %RSD values.
 Parameters that are affected by the changes in chromatographic conditions
are retardation factor (Rf) and peak purity.
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Sample preparation: composition, quantity of solvent, ph, shaking time, temperature and
number of extractions;
Sample application: volume applied, spot shape and size, band and spot stability;
Separation: at least on three different plates;
Chromatographic conditions: chamber saturation, eluent composition, eluent volume,
temperature, humidity and development distance;
Spot visualization: postchromatographic derivatization, spraying, dipping, reaction
temperature and time;
Quantitative evaluation: drying of plates, detection and wavelength.
19

• Retardation factor (Rf) is defined as the amount of separation due to the
solvent migration through the sorbent layer as shown in the formula.
• It depends on time of development and velocity coefficient or solvent front
velocity.
Rf = Migration distance of substane
Migration distance of solvent front from origin
20

The purity of the peak is determined by comparing the spectra at three
different levels:
 peak start (s)
 peak maximum (m)
 peak end (e)
During the purity test,
the spectrum taken at the first-peak slope is correlated with the spectrum of
peak maximum and the correlation of the spectra taken at the peak maximum
with the one from the down slope or peak end which is used as a reference
spectra for statistical calculation.
Basic acceptance criteria for evaluation of various
validation paramerers are summerized
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Type of analytical
Procedure
Characteristics
Identification Assay
in vitro release
study
dissolution study
Content
potency
Testing for
impurities
Quantitative Limit
test
Linearity
- + + -
Range
- + + -
Specificitya
+ + + +
Accuracy
- + + -
Precision
Repeatability
- + + -
Intermediate
precision - + + -
Limit of detection
- - - +
Limit of
quantification - - + -
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Characteristics
parameter
Assay Testing for impurities
Acceptance criteria
Linearity
No trend No trend
Residual plot
Correlation coefficient r ≥ 0.998 r ≥ 0.99
Y-axis intercept ≤2% ≤25%
RSD residuals ≤ 1.5% ≤10% - Impurity level ≤ 0.5%
≤5% - Impurity level ≥0.5%
Range 80–120% From limit of quantification to 120% of the specified
limit of impurity
Precision
Repeatability RSD≤2% (n ≥6) RSD ≤ 20% - Impurity level 0.1–0.2%
RSD ≤ 10% - Impurity level 0.2–0.5%
RSD ≤ 05% - Impurity level ≥0.5%
Intermediate precision RSD ≤ 3% (n ≥ 6) 1.5 RSD of repeatability
Accuracy RSD ≤3% RSD ≤ 10% (n=3) - Impurity level ≤0.5%
RSD ≤ 5% - Impurity level ≥0.5%
Limit of detection - RSD ≤10–20%
Limit of quantification - RSD ≤ 20–50%
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HPTLC_Method Validation by Priyanka Singh

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