Quality of
Analytical Data and
Validation
I GAA Septiari
Instrumental Signals
▪ For quantitation of a given analyte in a certain sample, an instrumental signal is measured for
the analyte.
▪ The instrumental signal normally increases linearly with concentration (amount) of the analyte in
the sample
▪ The classical way to determine the relation is to measure the instrumental signal for a series of
standard solutions containing exactly known concentrations of the analyte and to plot the
signals of these versus concentration.
▪ The resulting linear relationship is termed a standard curve or a calibration curve and the entire
procedure is termed calibration.
Validation
▪ Validation is the process to demonstrate that an analytical procedure is suitable
for its intended purpose.
▪ New analytical methods must be validated before they can be applied.
▪ Analytical procedures:
• Identification tests
• Quantitative tests for impurities
• Limit tests for impurities
• Quantitative tests (assays) of the active moiety in samples of drug substance
or drug product or other selected component(s) in the drug product
Validation
• Analytical method validation forms the first level of
QA in the laboratory
• An analytical result must always be accompanied
by an uncertainty statement, which determines the
interpretation of the result
• Validation is thus the tool used to demonstrate that
a specific analytical method actually measures
what it is intended to measure, and thus is suitable
for its intended purpose
When do you need validation?
▪ Validation is required for any new method -> revalidation
▪ When a new method is being developed
▪ Validation of a new analytical method is typically done at two levels.
▪ The first is pre-validation, aimed at fixing the scope of the validation.
▪ The second is an extensive, ‘‘full’’ validation performed through a collaborative
trial or inter-laboratory study
Validation
▪ The purpose of an analytical method is the delivery of a qualitative and/or quantitative result with an acceptable
uncertainty level -> measuring uncertainty
▪ In practice, method validation is done by evaluating a series of method-performance characteristics, such as
 Precision,
 trueness,
 selectivity/specificity,
 linearity,
 operating range,
 recovery,
 limit of detection (LOD), limit of quantification (LOQ), sensitivity, ruggedness/robustness and applicability
Analytical Characteristics in Validation
Validation
▪ Which performance criteria have to be evaluated depends also on the purpose of the method.
Different ICH/USP guidelines are set up for:
(1) identification tests;
(2) impurity tests; and,
(3) assay tests
▪ An identification test ensures the identity of an analyte in a sample, by comparing it to a known
RM.
▪ An impurity test is intended to confirm the identity of (limit impurity test) or to accurately quantify
(quantitative impurity test) an impurity, defined as an entity ‘which may normally not be present’.
▪ An assay test finally applies to the major component or active ingredient in a sample and
quantifies the drug substance as such, as a whole, or the drug substance in a drug product.
Validation of The Different Types of Analytical
Procedures
Specificity
▪ The specificity of an analytical method is defined as ‘the ability to assess unequivocally the
analyte in the presence of compounds that may be expected to be present (impurities,
degradation products, matrix, etc.)
▪ Analytical methods with high selectivity provide measured instrumental signals solely from the
target analyte.
▪ High (or sufficient) selectivity is vital for analytical methods.
▪ If an analytical method lacks specificity, one or several matrix components in the sample may
contribute to the instrumental signal and the measurements may not provide an exact result.
Specificity
▪ Specificity is tested as part of the validation procedure for identification tests,
determination of impurities, and assay.
▪ For identification methods (qualitative analysis), the ability to select between
compounds of closely related structures that are likely to be present in real samples
should be demonstrated.
▪ This should be confirmed by obtaining positive results (identification) from samples
containing the analyte, coupled with negative results from samples that do not contain
the analyte.
Specificity
▪ For quantitative methods of pharmaceutical ingredients and pharmaceutical
preparations:
▪ This can be done by spiking the pharmaceutical ingredient or preparation
with appropriate levels of impurities or excipients and demonstrate that the
assay result is unaffected by the presence of these extraneous materials.
Specificity
Accuracy
▪ The accuracy of an analytical method is defined as ‘the closeness of agreement
between the value which is accepted either as a conventional true value or an
accepted reference value, and the value found’.
▪ In the case of the assay of a pharmaceutical ingredient, accuracy may be
determined by application of the analytical procedure to a substance of known
purity, for example a certified reference substance or a substance with a similar
high quality.
Accuracy
▪ In an assay of API in a pharmaceutical preparation, accuracy may be determined
by application of the analytical procedure to synthetic mixtures of the preparation
(drug-free) to which known amounts of the drug substance (e.g. a CRS) have
been added.
▪ Another possibility of establishing accuracy is to compare the results with another
independent well-characterized method for which accuracy has been documented
CRS : Chemical Reference Substance is a highly purified and well-defined quality of the
analyte.
Precision
▪ The precision of an analytical procedure is defined as ‘the closeness of
agreement (degree of scatter) between a series of measurements obtained from
multiple sampling of the same homogenous sample under the prescribed
conditions’
▪ Precision is usually expressed as the standard deviation (s) or the relative
standard deviation (%RSD) of the mean (x) of a series of measurements:
Precision
Another measure of precision is the confidence interval, in which all
measurements fall with a certain probability or confidence level 1-a (a is often
0.05, giving a probability here of 95%)
Precision
▪ Precision is normally considered at three different levels:
• Repeatability
• Intermediate precision
• Reproducibility
Precision
▪ The repeatability is the precision under the same operating conditions over a short
interval of time.
▪ Normally, the same operator with the same equipment carries out the measurement
repeatedly within one day within the same laboratory.
▪ Intermediate precision expresses within-laboratories variations of measurements, as
on different days, or with different analysts or equipment.
▪ Reproducibility expresses the precision of a procedure between different laboratories
in a collaborative study.
Detection Limit
▪ The detection limit (DL) of an analytical method is defined as ‘the lowest amount of
analyte in a sample that can be detected, but not necessary quantitated as an exact
value’ under given experimental conditions.
▪ The detection limit is usually expressed as analyte concentration in the sample.
▪ The terms limit of detection (LOD) and lower limit of detection (LLOD) are used as
alternatives to detection limit.
▪ For instrumental methods, calculation of the detection limit can be based on the
signal-to-noise ratio (S/N).
Detection Limit
▪ The detection limit (DL) is defined as the concentration providing an instrumental
signal two, three, or three-point-three times the noise level, thus providing a
signal-to-noise ratio (S/N ratio) of 2, 3, or 3.3.
▪ The latter is the factor used in the ICH guideline:
DL = 3.3 × 𝜎/S
▪ 𝜎 is the standard deviation of the response (representing the noise level)
▪ S is the slope of the calibration curve, which is used to convert the signal
to a concentration
Quantitation Limit
▪ The quantitation limit (QL), also termed the limit of quantitation (LOQ) or the lower limit of
quantitation (LLOQ) of an analytical method, is defined as ‘the lowest amount of analyte in a
sample, which can be quantitatively determined with suitable precision and accuracy’.
▪ This parameter should be estimated for assays for low levels of analytes and particularly for the
determination of impurities and/or degradation products. Quantitation should not be performed
below this limit. The quantitation limit is defined as the concentration of analyte providing a
signal-to-noise ratio of 10:
▪ QL = 10 × 𝜎/S
Linearity and Range
▪ The linearity of an analytical method is defined as ‘its ability (within a given range)
to obtain test results, which are directly proportional to the concentration (amount)
of analyte in the sample’
Linearity and Range
▪ For establishing linearity, standard solutions or calibration samples covering the
entire range are analysed
▪ For assay (quantitation) of pharmaceutical ingredients and preparations, linearity
should be established in the range of 80–120% of expected concentration,
▪ Quantitation of impurities the linearity should be established from 50% to 120% of
acceptance criteria
Robustness
▪ The robustness of an analytical method is defined as ‘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’
▪ In the case of LC, examples of typical variations are:
• Influence of variations of pH in the mobile phase
• Influence of variations in the mobile phase composition
• Different columns (different lots/or suppliers)
• Temperature of the column
• Flow rate of the mobile phase
Robustness
▪ Thus, the performance of the method is tested with small changes in the
parameters listed above, and the analytical results should be unaffected