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
Analytical Characteristics in Validation
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
▪ 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