Analytical Method Development

and Validation

Bob Seevers

Importance of Analytical Methods in

Development Stability Studies
Without analytical methods it is not
possible to know what has happened
during stability
Assay
Impurities/Degradation Products
Dissolution
Chiral Purity
Preservative Content

Interactive Role of Analytical

Methods and Stability Data
This is a chicken and egg problem
Need analytical methods to generate stability data
Need stability information to develop methods

Start with preliminary method based on what is

known of drug substance
Can determine content of drug substance
Known or expected degradants are separated

Use preliminary method to analyze stress stability

studies
Stress deliberately creates significant degradation
Can evaluate ability of method to separate degradants

Use this information to modify/improve method

Types of Stress Studies

Photostability
High Temperature
Low Temperature
Oxidation
pH extremes

Selection of Chromatographic
Mode

HPLC
Reversed-phase HPLC
Chiral HPLC
GC
TLC
Electorphoresis

Identifying Degradants
Predicting routes of degradation
Acid/base hydrolysis of esters and amides
Oxidation of thiols, alcohols and amines
Loss of methyl groups

Synthesizing possible degradants

Prepare possible degradant
Known Structure
Test it in potential analytical method
Detectability
Separation from parent peak

Assay Run Time vs.

Detection/Resolution
Long run times for an assay mean
that stability testing will take more
time and cost more
Shorter run times mean that testing
will go faster and be less expensive
but
Lower resolution
Some peaks may not be separated

Validation Protocol
A validation protocol lays out ahead of
time the experimental design that will be
used to establish the validity the
analytical methods
Reagent, solvents
Sample, standard, solution preparation
Identify equipment to be used
Chromatographic conditions
System suitability
Calculations

ICH Q2 Validation of Analytical

Procedures
Types of Analytical Procedures to be
Validated
Identification tests;
Quantitative tests for impurities' content;
Limit tests for the control of impurities;
Quantitative tests of the active moiety in samples
of drug substance or drug product or other
selected component(s) in the drug product.

See also USP General Chapter <1225>,

Validation of Compendial Methods

With Regard to Stability

Types of Analytical Procedures to be
Validated
Identification tests;
Quantitative tests for impurities'
content;
Limit tests for the control of impurities;
Quantitative tests of the active moiety
in samples of drug substance or drug
product or other selected component(s)
in the drug product.
Drug product assay

Typical Validation Characteristics

Accuracy
Precision
Repeatability
Intermediate Precision

Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Robustness

Accuracy
Closeness of agreement between the
value found and either
the value accepted as a conventional
true value
or an accepted reference value

Accuracy for Drug

Substance
Several methods of determining accuracy
are available:
application of an analytical procedure to an
analyte of known purity (e.g. reference
material);
comparison of the results of the proposed
analytical procedure with those of a second
well-characterized procedure, the accuracy of
which is stated and/or defined;
accuracy may be inferred once precision,
linearity and specificity have been established.

Accuracy for Drug Product

Several methods for determining accuracy are
available:
application of the analytical procedure to synthetic
mixtures of the drug product components to which known
quantities of the drug substance to be analysed have been
added;
in cases where it is impossible to obtain samples of all drug
product components , it may be acceptable either to add
known quantities of the analyte to the drug product or to
compare the results obtained from a second, well
characterized procedure, the accuracy of which is stated
and/or defined;
accuracy may be inferred once precision, linearity and
specificity have been established.

Precision
Precision of an analytical procedure expresses the
closeness of agreement (degree of scatter)
between a series of measurements obtained from
multiple sampling of the same homogeneous
sample under the prescribed conditions.
Repeatability
intermediate precision
reproducibility.

The standard deviation, relative standard deviation

(coefficient of variation) and confidence interval
should be reported for each type of precision
investigated.

Repeatability
Repeatability should be assessed
using:
a minimum of 9 determinations covering
the specified range for the procedure
(e.g., 3 concentrations/3 replicates
each);
or
a minimum of 6 determinations at 100%
of the test concentration.

Intermediate Precision
Depends on the circumstances under which
the procedure is intended to be used.
Look at effects of random events on the
precision of the analytical procedure.
Days
Analysts
equipment, etc.

It is not considered necessary to study these

effects individually. The use of an
experimental design (matrix) is encouraged.

Reproducibility
Reproducibility is assessed by means
of an inter-laboratory trial.
Reproducibility should be considered
in case of the standardization of an
analytical procedure, for instance, for
inclusion of procedures in
pharmacopoeias. These data are not
part of the marketing authorization
dossier.

Specificity
Specificity is the ability to assess
unequivocally the analyte in the
presence of components which may
be expected to be present. Typically
these might include impurities,
degradants, matrix, etc.
Lack of specificity of an individual
analytical procedure may be
compensated by other supporting
analytical procedure(s).

Identity
Able to discriminate between compounds
of closely related structures which are
likely to be present.
The discrimination of a procedure may be
confirmed by obtaining positive results
(perhaps by comparison with a known
reference material) from samples
containing the analyte, coupled with
negative results from samples which do
not contain the analyte.

Detection Limit
The detection limit of an individual
analytical procedure is the lowest amount
of analyte in a sample which can be
detected but not necessarily quantitated
as an exact value.
Can be determined
Visually
Signal to Noise
Standard Deviation of the Response and the
Slope

Detection Limit: Signal to

Noise
This approach can only be applied to analytical
procedures which exhibit baseline noise.
Comparing measured signals from samples
with known low concentrations of analyte with
those of blank samples and establishing the
minimum concentration at which the analyte
can be reliably detected.
A signal-to-noise ratio between 3 or 2:1 is
generally considered acceptable for estimating
the detection limit.

Detection Limit: Standard Deviation

of the Response and the Slope
The detection limit (DL) may be expressed
as: DL = 3.3 /S
= the standard deviation of the response
S = the slope of the calibration curve

The slope S may be estimated from the

calibration curve of the analyte. The
estimate of may be carried out in a
variety of ways, for example:
Based on the Standard Deviation of the Blank
Based on the Calibration Curve

Quantitation Limit
The quantitation limit of an individual
analytical procedure is the lowest
amount of analyte in a sample which
can be quantitatively determined
with suitable precision and accuracy.
The quantitation limit is a parameter
of quantitative assays for low levels
of compounds in sample matrices,
and is used particularly for the
determination of impurities and/or

Quantitation Limit
Visual Evaluation
Signal-to-Noise
Standard Deviation of the Response and the Slope
quantitation limit (QL) may be expressed as: QL = 10
/S
= the standard deviation of the response

S = the slope of the calibration curve

The estimate of may be carried out in a variety
of ways, for example:
Based on the Standard Deviation of the Blank
Based on the Calibration Curve

Linearity
The linearity of an analytical procedure is its
ability (within a given range) to obtain test results
which are directly proportional to the
concentration (amount) of analyte in the sample.
Test results should be evaluated by appropriate
statistical methods, for example, by calculation of
a regression line by the method of least squares.
correlation coefficient, y-intercept, slope of the
regression line and residual sum of squares should be
submitted
minimum of 5 concentrations is recommended

Range

The range of an analytical procedure is the interval

between the upper and lower concentration (amounts) of
analyte in the sample (including these concentrations) for
which it has been demonstrated that the analytical
procedure has a suitable level of precision, accuracy and
linearity.
The following minimum specified ranges should be
considered:
For the assay of a drug substance or a finished (drug)
product: normally from 80 to 120 percent of the test
concentration;
For content uniformity, covering a minimum of 70 to 130
percent of the test concentration, unless a wider more
appropriate range, based on the nature of the dosage
form (e.g., metered dose inhalers), is justified;
For dissolution testing: +/-20 % over the specified range

Robustness
The robustness of an analytical procedure is a
measure of its capacity to remain unaffected by
small, but deliberate variations in method
parameters and provides an indication of its
reliability during normal usage.
stability of analytical solutions;
extraction time.
influence of variations of pH in a mobile phase
influence of variations in mobile phase composition
different columns (different lots and/or suppliers)
Temperature
flow rate.

Method Validation Report

Provide results of validation effort
Explain
Choice of acceptance criteria
How method was developed

Method Validation Report will be

submitted to regulators

Revalidation May be
Necessary
Changes in the synthesis of the drug
substance;
Changes in the composition of the
finished product;
Changes in the analytical procedure.