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Experiment No. : Date:
Title: Analytical Methods Validation
Aim: Documentation of Analytical Methods Validation.
Scope:
1. Noncompendial release assays.
2. Compendial and noncompendial stability assays.
3. Limit tests.
4. Dissolution and uniformity testing.
Procedure:
1. Analytical Methods Validation:
Validation of an analytical methods is the process by which it is established
by laboratory studies that the performance characteristics of the method meet
the requirements for the intended analytical applications. Performance
characteristics are expressed by analytical parameters. Table 1 lists analytical
variable and the categories that are normally required for method validation.
Table 1: Analytical Validation Variables
Parameter Assay Cat. I Assay Cat. II Assay Cat.
III
Quantity. Limit T.
Stability indicating Yes Yes Yes *
Selectivity Yes Yes Yes Yes
Linearity Yes Yes No *
Range Yes Yes * *
Accuracy & recovery Yes Yes No *
Precision Yes Yes * Yes
LOD No Yes Yes *
LOQ No Yes Yes *
Comparative study Yes * * *
Ruggedness Yes Yes Yes Yes
* May be required, depending on the nature of the specific test
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2. Overview and Definitions:
Category I: Analytical methods for the quantitation of major components
of bulk drug substances or active ingredients (including
preservations) in finished products.
Category II: Analytical methods to determine impurities in bulk drug
substances or degradation compounds in finished products.
Category III: Analytical methods of determine performance characteristics,
such as dissolution and drug release.
The validity of an analytical method can be verified only by laboratory
studies. Therefore, documentation of the successful completion of such
studies is a basic requirement for determining if a method is suited for its
intended application.
3. Definition of Analytical Performance Parameters:
a. Stability-indicating Aspects and Selectivity:
The stability-indicating demonstrate baseline separation between the
principal peak and extraneous peaks (related compounds, degradation
products etc). For definition purposes, 80 to 100 percent represents
slight degradation, 50 to 80 percent represents moderate degradation, 1
to 50 percent represents severe degradation and less than 1 percent
represents total degradation.
b. Selectivity:
Selectivity (specificity) may often be expressed as the degree of bias
obtained by analyzing samples containing added impurities,
degradation products, related chemical compounds, or placebo
ingredients against samples without added substances. The bias of the
assay if any is the difference between the two groups of samples.
c. Linearity and Range:
The linearity of an analytical method is its ability to produce test
results that are proportional to the concentration of the analyte in the
sample solutions, within the range of 50 to 150 percent of the working
concentration. Linearity is usually expressed as the variance around
the slope of the regression line. The importance of linearity depends
on how wide ranging the method is intended to be.
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d. Accuracy and Recovery:
The accuracy of an analytical method is the closeness of test results
obtained by that method to the true value. Accuracy may be expressed
as the percent recovery of known, added amounts of analyte. It is a
measure of the exactness of the analytical method.
e. Assay Precision:
The precision of an analytical method is the degree of agreement
among individual test results when the procedure is applied repeatedly
to multiple sampling of a homogenous sample. The precision of an
analytical method is usually expressed as the RSD of the assay results.
f. Limit of Detection:
The LOD is the lowest concentration of analyte in the target matrix
that can be determined from the background to the 95 percent
confidence level (is detectable at the most sensitive instrument
settings). It may not be possible to obtain good quantitative results
near the LOD.
g. Limit of Quantitation:
The LOQ is the minimum level of the analyte in the matrix that can be
quantitated at the 95 percent confidence level. It is a parameter of the
quantitative assay for low levels of compounds in sample matrices
such as impurities in bulk drug substances and degradation products in
finished pharamceuticlas. It is the lowest concentration of analyte in a
sample that can be determined with acceptable precision and accuracy
under the stated experimental conditions.
h. Ruggedness:
The ruggedness of an analytical method is the degree of
reproducibility of test results obtained by analyzing the same sample
sunder a variety of normal test conditions. The method should not be
prone to day-to-day or place-to-place variations. If possible, different
laboratories, analysts, instruments, reagent lots, elapsed assay times,
assay temperatures, days and so on should be used. Ruggedness is a
measure of test results under normal expected operational conditions
from laboratory to laboratory and from analyst to analyst.
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i. Robustness:
The robustness of an analytical procedure is a measure of its capacity
to remain unaffected by small but deliberate variations in method
parameters. If provides and indication of its reliability during normal
usage.
4. Determine of Analytical Performance Parameters:
a. Stability-indicating Aspects and Selective (for HPLC assays
using UV detection only).
Before starting forced degradation tests, prepare a 10 times working
concentration solution of the placebo. Inject for at least 30 min and
determine the contribution, if any, of the placebo to the chromatogram.
The stability study consists of the following challenges: exposure to
hydrogen peroxide (H2O2) oxidation, acid and alkali hydrolysis and
exposure to heat and light.
I. As per the method being validated, prepare a standard
solution at 10 times the normal working concentration.
II. Pipette 10.0 ml aliquots of the sample into 5
separate 100.0 ml volumetric flasks. Treat each as
follows:
* Add 20.0 ml of 0.5N HCl and immerse in a boiling
water bath for 1 h.
* Add 20.0 ml of 0.5 N NaOH and immerse in a
boiling water bath for 1 hr.
* Add 10.0 ml 10 percent H2O2 and swirl to mix. Let
stand 30 min.
* Store at 60°C for one week.
* Store under
In addition, prepare a working concentration standard and let stand at
room temperature for 1 week.
b. Dilute each sample, A-E to mark with RO / DI water.
Neutralize the acid sample with 20.0 ml 0.5N NaOH prior to dilute and
the alkali sample with 20.0 ml HCl prior to dilution.
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c. Analyze each sample versus a freshly prepared standard
solution, performing the system suitability test first. Inject all
preparation and allow to run for 30 min each. Report the separation of
any extraneous peaks or degradation products from the analyte.
d. Peak purity techniques can be used to determine the purity of
target analytes after degradation. In lieu of this, knowledge of the
degradation chemistry can be substituted, demonstrating that known
degradation products can be separated from the target analyte peak,
using limits tests such as HPLC or TLC. Peak purity techniques
includes using wavelength ratios, overlay of peak upslope, apex and
downslope spectra and ratiogams (wavelength ratios across a peak
plotted as a function of time).
e. For samples that completely degrade, spike with the actives
and then inject into the system. For acid and alkali stressed samples,
neutrialize and then spike with actives.
f. For direct UV analysis, prepare a solution containing a mixture
of the placebo at the working concentration and also at two times that
concentration. Scan from 200 to 400 nm in order to be sure that the
placebo does not interfere with detection of actives.
5. Linearity and Range:
a. Prepare standard solution containing 50, 75, 100, 125 and 150
percent of the working concentration of analyte by dilution of the 10
times the working concentration standard, prepared as directed in
[Link]. according to the following table:
Linearity Standards ML of 1 Final Volume
(% Working Concentration) 10 x Standard (mL)
50 5.0 100.0
75 15.0 200.0
100 10.0 100.0
125 25.0 200.0
150 15.0 100.0
b. For each of the 5 solutions prepared above make 5 replicate
measurements (e.g. injections) of each solution and obtain the
measurement output (e.g. peak rates).
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c. The signal (e.g., peak area) obtained for each solution is plotted
against its corresponding theoretical concentration and a liner
regression analysis is performed on the 5 coordinates. The resulting
plot should be linear for each analyte at least 0.999 since it describes
the line of most accurate fit to the data or potential assay bias.
d. Calculate the response factor, the residual factor and the
percent residual as follows:
observed signal
Response factor = Kt = concentrat ion (mg/mL)
residual = observed signal – calculated signal
residual
& residual = Observed signal x 100
e. The response factor should demonstrate that a single point
standardization level is sufficient. The residuals should illustrate that
the individual coordinates of the working concentration are small and
randomly distributed.
f. The range of the method is validated by verifying that it
provides acceptable precision, accuracy and linearity when applied to
sample containing analyze at the extremes of the range (e.g. 50 – 150
percent) as well as within the range of normal working concentrations.
6. Accuracy and Recovery:
a. Using the 10 times working concentration standard prepared in
[Link] and the 10 times concentration placebo solution prepared in
5.4.1, prepare a series of matrix-containing standards according to the
following table:
Linearity
Standards mL of mL of Final
(% Working 10 x 10 x Volume
Concentration Placebo Standard (mL)
)
50 10 5.0 100.0
75 20 15.0 200.0
100 10 10.0 100.0
25 20 25.0 200.0
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150 10 15.0 100.0
b. Make 5 replicate measurements (e.g. injections) of each
solution and compare to a freshly prepared standard solution or
standard solution prepared at their normal working concentrations as
per the method under validation.
c. Calculate the recovery and measurement precision (e.g.
injection precision) of each set of measurement as follows:
The RSD for the measurement precision is no more than 2.0 and the
recovery is not more than ± 2.0 percent relative to the freshly prepared
calibration standard or standards.
d. For direct UV analysis, determine the absorbance of each
solution versus a standard solution, similarly prepared at the working
concentration.
7. Assay Precision:
a. Determine the precision of the method by preparing 6 replicate
samples form the same lot of product. Prepare samples according to
the method under validation.
b. Make duplicate measurements of each solution versus freshly
prepared standard solutions, prepared at their normal working
concentration as per the method under validation.
c. Calculate the percent of each analyte recovered from each
sample.
d. The RSD of the 6 results for each analyte should not be more
than 2.0.
8. Limit of Detection:
The LOD of the analytical method is determined by comparing the test results
obtained from samples with known concentrations of analyte against those of
blank samples and establishing the minimum level of analyte that can be
reliably detected. A signal-to-ratio of 3:1 is acceptable.
9. Limit of Quantitation:
The LOQ of the analytical method is determined by analyzing several blank
samples and calculating the RSD of this response. The standard deviation
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multiplied by a factory, usually 10, provides an estimate of the LOQ. The
limit is later validated by the analysis of samples known to be near the LOQ.
NOTE: The LOQ is approximately 3 time the LOD.
10. Ruggedness:
The ruggedness of an analytical method is determined by analyzing aliquots
from homogenous lots in different laboratories on different days by different
analysts, using different instruments and column and using operational and
environmental conditions that may differ but are still within the specified
parameters of the assay. The degree of reproducibility of test results is then
determined as a function of the ass variables.
11. Robustness:
Robustness is determined by observing how a method stands up to slight
variations in normal operating parameters. For HPLC for instance this could
be a change in flow rate of slight variation in mobile phase composition.
12. Validating Changes in Analytical Methods:
When the following changes occur in the method the accompanying
parameters must be revalidated:
a. Changes in the sample and standard concentration:
I. Linearity and range
II. Accuracy and recovery
III. Precision
b. Changes in the diluents solution used for the sample and
standard preparation:
I. Linearity and range
II. Accuracy and recovery
III. Precision
c. Introducing an analyte signal for quantitation, if not previously
validated:
I. Linearity and range
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II. Accuracy and recovery
III. Precision
d. For HPLC, change in the mobile phase proportions of more
than ± 5 percent.
I. Linearity and range
II. Accuracy and recovery
III. Precision
IV. Stability indicating
e. Changes in the sample size, such as injection volumes in
chromatography:
I. Linearity and range
II. Accuracy and recovery
III. Precision
f. For HPLC or UV analysis, if there are changes in the
spectrophotometric wavelength, perform the complete validation
study.
g. Change in mode (e.g. HPLC – isocratic to gradient), perform
the complete validation study.
h. Change in column type (e.g. C18 to C8), perform the complete
validation study.
i. Changes in detector (e.g. UV to refractive index), perform the
complete validation study.
j. Introducing an internal standard, perform the complete
validation study.
13. Validation Report:
A validation report should be prepared and submitted for approval. It should
consist of the following:
a. Summary:
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The summary should contain a simple statement about the results of
the validation study. For example, “The method for assay of Product
XYZ by HPLC was found to be accurate, precise, selective, linear and
stability indicating”.
b. Analytical Validation Data:
Analytical data should be presented in tabular and graphical form for
ease of evaluation. The data presentation should show analytical
results for all validation parameters described in Section 5.4, plus
residuals and all calculations used to derive results from laboratory
data.
c. Discussion:
The discussion should describe the outcome of the validation in detail.
It should deal with any problem that were encountered and should
include a rationale for accepting or rejecting the validation. Any
experiments or any failing results that were repeated and then accepted
need to be explained and justified.
Any deviations from acceptance criteria must be explained and the
conditions under which the method may be used (method limitations)
should be clearly defined, such as only linear from 75 to 125 percent
of the working concentration or meets all acceptance criteria and can
be used throughout the ranges tested in the validation.
14. Documentation and Acceptance:
1. Protocol:
The validation protocol (Sections 5.1-5.5) must be approved prior to
beginning a validation study.
2. Report:
The validation report must be approved prior to use of the method
under validation.
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