Analytical Method Validation

BY
Dr. Alka N Choudhary
Division of Pharmaceutical Sciences
S.G.R.R.I.T.S., Patel Nagar, Dehradun (UK)
 WHAT IS VALIDATION?

“Validation of an analytical procedure is the process by which it is established, by

laboratory studies, that the performance characteristics of the procedure meet the
requirements for its intended use.”

WHY VALIDATE ANALYTICAL PROCEDURES?

There are many reasons for the need to validate analytical procedures. Among
them are regulatory requirements, good science, and quality control
requirements.

The Code of Federal Regulations (CFR) 311.165c explicitly states that “the
accuracy, sensitivity, specificity, and reproducibility of test methods employed by
the firm shall be established and documented”
 CYCLE OF ANALYTICAL METHODS
The analytical method validation activity is not a one - time study. This is illustrated
and summarized in the life cycle of an analytical procedure in Figure 1.
Validation Parameter

Typical validation characteristics which should be considered are:.

1) Accuracy
2) Precision
3) Specificity
4) Linearity
5) Range
6) Detection Limit
7) Quantitation Limit
8) Robustness
9) Ruggedness
10) Noise
11) Trueness
12) Sensitivity
 ACCURACY

“Accuracy is the closeness of an individual test result to the true value.”

 Measures exactness of the analytical method developed.

 Expressed as percent recovery by the assay of a known amount of analyte
added.
 Determined by

applying the method to samples + known amount of analyte added both above &

below normal levels expected in the samples.

 Calculated from
The test results as the percentage of the analyte recovered by the assay. Dosage
form assays commonly provide accuracy within 3-5% of the true value.

 The ICH recommend that accuracy should be assessed using a minimum of nine

determinations over a minimum of three concentration levels, covering the

specified range
PRECISION

“Precision is a measure of the degree of reproducibility / repeatability of the

analytical method under normal operating circumstances.”

 Repeatability involves analysis of replicates by the analyst using the same

equipment, method and conducting the precision study over short period
of time while reproducibility involves precision study at
 Different Occasions,
 Different Laboratories,
 Different Batch of Reagent,
 Different Analysts,
 Different Equipment.
Determination of Repeatability
 Procedure when repeated by same analyst under the same operating
conditions (same reagents, equipment, settings and laboratory) over a
short interval of time. (intra-day)

Determination of reproducibility 
 Carried out by different analysts under different conditions (different reagents,
equipment, laboratories or by carrying out the analysis at different times also
provide valuable information) on separate, putatively identical samples taken
from the same homogenous batch of material. (inter-day)
 SELECTIVITY

“A measure of the discriminating power of a given analytical procedure in

differentiating between the analyte and other components in the test sample.”
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, degrades, matrix, etc. Lack of specificity of an individual procedure may
be compensated by other supporting analytical procedure(s)”
 SELECTIVITY & SPECIFICITY

Selectivity: Specificity:

If an analytical procedure is able to If the method determines or measures

separate and resolve the various quantitatively the component of
components of a mixture and detect interest in the sample matrix without
the analyte qualitatively separation

Restricted to qualitative detection of Restricted to quantitative

the components of a sample measurement of one or more analyte
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”

It may be demonstrated directly on the drug substance (by dilution of a standard

stock solution) and/or separate weighing of synthetic mixtures of the drug
product components, using the proposed procedure.

Linearity should be evaluated by visual inspection of a plot of signals as a

function of analyte concentration or content.

If there is a linear relationship, test results should be evaluated by appropriate

statistical methods, for example, by calculation of a regression line by the
method of least squares.
The correlation coefficient, y-intercept, slope of the regression line and residual
sum of squares should be submitted. A plot of the data should be included. In
addition, an analysis of the deviation of the actual data points from the regression
line may also be helpful for evaluating linearity.
For the establishment of linearity, a minimum of 5 concentrations is
recommended.
Under normal circumstances, linearity is acceptable with a coefficient of
determination (r 2 ) of >/=0.997
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 specified range is normally derived from linearity studies and depends on the
intended application of the procedure.
 LIMIT OF DETECTION

“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. “
Several approaches for determining the detection limit are possible, depending on
whether the procedure is a non-instrumental or instrumental. Approaches other
than those listed below may be acceptable.
 Based on Visual Evaluation
 Based on Signal-to-Noise
 Based on the Standard Deviation of the Response and the Slope
The detection limit (DL) may be expressed as:
DL = 3.3 Sa / S
Where Sa = the standard deviation of the response
S = the slope of the calibration curve
QUANTITATION LIMIT

The quantitation limit of an individual analytical procedure is the lowest

concentration of analyte in a sample which can be quantitatively determined with
suitable precision and accuracy.”
Several approaches for determining the quantitation limit are possible, depending
on whether the procedure is a non-instrumental or instrumental. Approaches
other than those listed below may be acceptable
 Based on Visual Evaluation
 Based on Signal-to-Noise
 Based on the Standard Deviation of the Response and the Slope

The quantitation limit (QL) may be expressed as:

QL =10 Sa / S
Where, Sa = the standard deviation of the response
S = the slope of the calibration curve
 ROBUSTNESS

“Measure of its capacity to remain unaffected by small but deliberate variation

in method parameters and provides an indication of its reliability during
normal usage.”
Determination requires that methods characteristic are assessed when one or
more operating parameter varied.
Examples of typical variations are:
- stability of analytical solutions;
- Extraction time.
In the case of liquid chromatography, examples of typical variations are:
- 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.
In the case of gas-chromatography, examples of typical variations are:
- different columns (different lots and/or suppliers);
- temperature;
- Flow rate.
RUGGEDNESS
“Degree of reproducibility of test results obtained by the analysis of the same
samples under a variety of normal test  conditions within the specified
parameters of the assay.”
 Determination involves the degree of reproducibility of test result is
determined as function of the assay variable.
 This reproducibility may be compared to the precision of the assay under
normal condition to obtain a measure of the ruggedness of the analytical
method.
 According to USP, ruggedness is determined by analysis of aliquots from
homogeneous batches in different laboratories, by different analysts, using
operational and environmental conditions prescribed for the assay. The
degree of reproducibility is then evaluated by comparison of the results
obtained under varied conditions with those under standard conditions.
 NOISE

“A phenomenon defined as fast changes in the intensity and frequency of a

measured signal irrespective of the presence or absence of the analyte.” The
speed of change is significantly different from the normally expected detector
response.
A measure of noise is the measured difference between the highest and
lowest value of the measured signal with no analyte present, observed in a
relatively short time-span, as compared to the time-span necessary for
measurement of the analyte.
TRUENESS

The closeness of agreement between the average values obtained from a

large series of test results and an accepted reference value. The measure of
trueness is usually expressed in terms of bias.
SENSITIVITY
The change of the measured signal as a result of one unit change in the
content of the analyte.
The change is calculated from the slope of the calibration line of the analyte
REFERENCE

 U.S.Pharmacopoeia 2007,validation of compendia procedure,chapter-

1225.,pp 680-681

 ICH Q2 (R1): Validation of Analytical Procedures:

Text and Methodology, ICH Harmonized Tripartite Guideline, Current Step 4
version

 S.Iyer, Validation of analytical Procedure, Guidelines on CGMP and Quality

of Pharmaceutical Products, D. K. Publications, chapter-8, pp145-150.