Evaluation of An Analytical Method

INTERNATIONAL JOURNAL OF PHARMACEUTICAL CHEMISTRY RESEARCH
Review Article
EVALUATION OF AN ANALYTICAL METHOD

Shah, K.*1, Kumar, S.2, Upmanyu, N.3 and Mishra, P.1
1
Institute of Pharmaceutical Research, GLA University, Mathura, UP, India, 281406
2
Department of Pharmacy, Barkatullah University, Bhopal M.P. India
3
RKDF College of Pharmacy, Bhopal M.P. India
Email: kamal0603@gmail.com
Abstract:
Pharmaceutical analysis plays an important role right from testing of raw materials, inprocess quality checks to the analysis of finished products. Pharmaceutical analysis is
considered to determine identity, strength, quality and purity of drug samples. Pharmacists
have made important contributions in the field of medicinal chemistry, both in discovering or
isolating new therapeutic agents and in developing methods for standardizing and controlling
medicinal. In small laboratories the responsibility for performing analysis may be delegated
entirely to pharmacist staff members. But whether or not pharmacists may have occasion to
conduct analysis, they at least should understand the basic principles involved in the
standardization and control of the medicinal agents dispensed . The paper is focusing on the
parameters required to validate an analytical method.
Keywords : analytical chemistry, validation, limit of detection, limit of quantification,
robustness and ruggedness.
INTRODUCTION:
Analytical chemistry is as old as chemistry
itself. In many respect analytical chemistry
acts as a foundation for other branches of
chemistry. In fact, the science of chemistry
came into being as a result of human
inquisitiveness to understand the nature of
an extraordinary variety of matter that
surrounds him. In analytical chemistry it is
Volume 1 Issue 1 2012
of prime importance to gain information

about the qualitative and quantitative
compositions of substances and chemical
species, that is, to find out what a
substance is composed of and exactly
how much. Analytical chemistry may be
defined as the science and art of
determining the composition of materials
www.earthjournals.org
in terms of elements or compounds

contained.[1]The resolution of a chemical
compound into its proximate or ultimate
parts. The determination of its elements or
of the foreign substances it may contain.
Thus reads a dictionary definition.[2] In
general terms, then pharmaceutical
analysis comprises those procedure
necessary to determine the identity,
strength, quality and purity of drugs.[3]
Analytical Chemistry can also be said that
it deals with the methods for determining
the chemical composition of samples of
matter. A qualitative method yields
information about the atomic or molecular
species or the functional groups that exist
in the sample; a quantitative method in
contrast, provides numerical information
as to the relative amount of or more of
these compounds [4-6].
Method validation can be defined as (ICH)
Establishing documented evidence, which
provides a high degree of assurance that a
specific activity will consistently produce
a desired result or product meeting its
predetermined specifications and quality
characteristics.
possess quality, safety and efficacy must

be designed to build into the product.
Each step of the manufacturing process
must be controlled to maximize the
probability that the finished products
meet all quality and design specification.
Method validation is an integral part of the

method development; it is the process of
demonstrating that analytical procedures
are suitable for their intended use and that
they support the identity, quality, purity,
and potency of the drug substances and
drug products. Simply, method validation
is the process of proving that an analytical
method is acceptable for its intended
purpose.
USFDA: According to this Validation

is the process of establishing
documented evidence which provides a
high degree of assurance that a specific
process will consistently produce a
product meeting its predetermined
specifications and quality attributes.
WHO:
Defines Validation as an action of
providing any procedure, process,
equipment, material, activity or system
actually leads to the expected results.
OBJECTIVE OF VALIDATION [7]

The primary objective of validation is to
form a basis for written procedure for
production and process control which are
designed to assure that the drug products
have the identity, strength, quality and
purity they purport or are represented to
Benefits of Validation
a) Produces quality products
b) Helps in process improvement
technology transfer, related product
validation, failure investigation, and
increased employee awareness.
c) Cost reduction by increasing
efficacy,
few
reject,
longer
equipment life, production of cost
effective products
d) Helps in optimization of process or
method.
e) Regulatory
affairs-produces
approved products and increased
ability to export.
The US Food and Drug Administration
(FDA) [8-9] and US Pharmacopoeia
(USP) [10] both refer to ICH guidelines.
Validation as Defined by Different
Agencies[11]
EUROPEANCOMMITTEE:
Defines Validation as an action of
providing in accordance with the
principles of GMP that any procedure.
Process, material, activity or system

actually leads to expected results.
This process consists of
establishment
of
the
performance
characteristics and the limitations of the
method. Method Validation is required
when A new method is been developed.
Revision established method. When
established methods are used in different
laboratories and different analysts.
Comparison of methods.
When quality control indicates method
changes. Method Validation, however, is
generally a one-time process performed
after the method has been developed to
demonstrate that the method is
scientifically sound and that it serves the
intended analytical purpose [12-16].
Performance characteristics examined
when carryout method validation are:
Accuracy / Precision.
Repeatability / Reproducibility.
Linearity / Range.
Limit of detection (LOD)/ Limit of
quantification (LOQ).
Selectivity / Specificity.
Robustness / Ruggedness.
Accuracy
It relates to the closeness of test results to
true value i.e. measure of exactness of
analytical method.
Determination of accuracy
The accuracy may be determined by
application of the analytical method to an
analyte of known purity (example:
reference standard) and also by comparing
the results of the method with those
obtained using an alternate procedure that
has been already validated.
Accuracy is calculated as the percentage
of recovery by the assay of the known
added amount of the analyte in the sample
or the difference between the mean and
accepted true value together with

confidence intervals.
The ICH guidelines recommended to
take minimum of 3 concentration levels
covering the specified range and 3
replicates of each concentration are
analyzed
(totally
3*
3=9
determinations.)
It is expressed as percent recovery by the
assay of known/added amount of analyte
in the linearity range. Accuracy can also
be determined by comparing the results
with those obtained using an alternative
method which has already been
validated.
Precision
It expresses as degree of agreement
among individual test results when
method/procedure
is
applied
to
homogenous sample usually expressed
as SD/RSD. It is measure of degree of
repeatability or reproducibility under
normal conditions.
Determination of precision
The procedure is applied repeatedly to
separate identical samples drawn from
the homogeneous batch of material and
measured by the scatter of individual
results from the mean and expressed as
the standard deviation or as the
coefficient of variation (relative standard
deviation).
Precision may be the measure of either
the degree of reproducibility or of
repeatability of the analytical method
under normal operating conditions.
According
to
ICH
guidelines
repeatability should be assessed using
minimum of 9 determinations covering
the specified range for the procedure.
Precision should be measured for
repeatability (intra-assay precision),
intermediate
precision
and
reproducibility.
Linearity and Range

Linearity
Linearity is the ability of the method to
obtained test results that are directly
proportional to the analyte concentration
within a given range.
Range of analytical procedure is the
interval between the upper and lower
concentration of analyte in the sample
(including concentrations) for which it has
been demonstrated that the analytical
procedure has a suitable level of precision,
accuracy, and linearity.
Measurement
A range of standards should be prepared
containing
at
least
5
different
concentrations of analyte which are
approximately evenly spaced and span 50150% of the label claim.
At least 6 replicates per concentration to
be studied. Plot a graph of concentration
(on x - axis) Vs mean response (on Y axis). Calculate the regression equation,
Y- intercept and correlation coefficient.
Plot another graph of concentration (on Xaxis) Vs response ratio (replicate response
divided by concentration, on Y-axis).
The range of the method is validated by
verifying that the analytical method
provides acceptable precision, accuracy
and linearity when applied to samples
containing analyte at the extreme of the
range as well as within the range.
Specificity
ICH document divides specificity into two
categories.
a) Identification tests
b) Assay / impurity tests
ICH defines specificity of an assay is its
ability to measure accurately and
specifically the analyte of interest in the
presence of other components that might
be expected to present in the sample
matrix.
It is the degree of interference from

excipients, impurities or degradation
products ensuring that a peak response is
due to a single component only i.e., no
co-elutions.
Determination of Specificity
When chromatographic procedures are
used representative chromatograms
should be presented to demonstrate the
degree of selectivity and peak should be
appropriately labeled.
Peak purity test (e.g. using diode assay
or mass spectrometry) may be useful to
show that the analyte chromatographic
peak is not attributable to more than one
component.
Selectivity
It is a procedure to detect qualitatively
the analyte in the presence of
components that may be expected to be
present in the sample matrix or the
ability of a separative method to resolve
different compounds. It is the measure of
the relative method location of two
peaks.
Determination of Selectivity
It is determined by comparing the test
results obtained on the analyte with and
without
addition
of
potentially
interfering
material.
When
such
components are either unidentified or
unavailable a measure of selectivity can
be obtained by determining the recovery
of a standard addition of pure analyte to
a material containing a constant level of
the other components.
Ability of the method to measure
accurately and specifically the analyte of
interest in presence of matrix and other
components likely to be present in the
sample matrix as impurities, degradation
products and other related substances.
For this, one may compare the test
results of analysis of sample containing
other ingredients, impurities, degradation
product, related substances, placebo

ingredients with those obtained from
analysis of sample without these, i.e., the
method must allow distinct analytical
measurement of analyte of interest and
exclusion
of
all
other
relevant
interferences. If the impurities/degradation
products or potential contaminants are not
available, one can apply a proposed
method to the strained and stressed (heat,
light, humidity) samples, degree of
agreement among results will explain
specificity of the method.
Limit of Detection (LOD) and Limit of
Quantitation (LOQ)
Lowest concentration of the analyte in the
sample that the method can detect but not
necessarily quantify under the stated
experimental conditions simply indicates
that the sample is below as above certain
level. The LOD will not only depend on
the procedure of analysis but also on type
of instrument.
Measurement is based on
1.
Signal to noise ratio
2.
Visual evaluation (relevant
chromatogram acceptable)
3.
The standard deviation of
the response and the slope.
LOD =
3.3
S
= Standard deviation of
the response.
S = Slope of the calibration
curve of the analyte
Limit of Quantification
The LOQ is the lowest concentration of
analyte in a sample which can quantitative
determined that may be measured with an
acceptable level of accuracy and precision
under the stated operational conditions

of the method. LOQ can vary with the
type of method employed and the nature
of the sample. Based on the standard
deviation of the response and the
quantitation limit QL may expressed as
LOQ =
10
S
= Standard deviation of
the response.
S = Slope of the
calibration curve of the analyte
Measurement
For instrumental and non instrumental
methods the quantitation limit is
generally determined by the analysis of
the samples with known concentration of
the analyte and by establishing the
minimum level at which the analyte can
be determined with acceptable accuracy
and precision.
In case of instrumental methods that
exhibit back ground noise the ICH
document
describes
to
compare
measured signals from samples with
known concentration of analyte with
those of blank samples.
A typically acceptable signal to noise
ratio is 10:1
Ruggedness
Degree of reproducibility of test results
obtained by analyzing the same sample
under variety of normal test conditions
such as different.
Analysts
Instruments
Days
Reagents
Columns and TLC plates

i.e. lack of influence of environmental
variables on the method. Comparison of
reproducibility of test results to the
10
precision of assay is the direct measure of

ruggedness of the method.
Robustness
It is the measure of the capacity of the
analytical method to remain unaffected by
small but deliberate variations in
procedure to provide an indication about
variability of the method during normal
laboratory conditions.
Simplicity
Simplicity refers to when the analysis can
be carried out in a minimum number of
step and need only easily available
reagents and equipments. As with the
increase in number of steps, there are more
chances of personal errors creeping in.
Cost of Analysis
Cost of analysis is based on the simplicity
of method, number of steps involved, time
required, cost of reagent, requirement of
instrument and the skilled and experienced
analyst etc. These are the some of the
factors that contributes to the cost of
analysis.
Validation of the proposed analytical
method and its acceptability depends on
the above factors.
REFERENCES
1. Jeffery GH, Bassett J, Mendham J,
Denney RC, In : Vogels Text
book of Quantitative Chemical
Analysis, 5th edn., Longman Group
UK Ltd., England; 1989. P. 3.
2. Sharma BK, In : Instrumental
Methods of Chemical Analysis, 20th
edn., Goel Publishing House,
Meerut; 2001. P. 3.
3. Beckett AH and Stenlake JB, In :
Practical Pharmaceutical Chemistry,
3rd edn., Vol. II, CBS Publishers
and Distributors, New Delhi ; 1986.
P. 131.
4. United State Pharmacopoeia,

National
Formulary,
US
Pharmacopial conention Inc, MD
2006. p. 2109,107.
5. British Pharmacopoeia, Volume I,
(2005). p. 106-107.
6. Harsono
T,
Yuwono
M,
Indrayanto G, Journal of AOAC
International. Vol. 88 (4), 2005,
1093-1098.
7. FDA, "Analytical Procedures and
Methods Validation: Chemistry,
Manufacturing and Controls
Documentation;
Availability,"
Federal
Register
(Notices)
65(169), 5277652777 (2000).
8. www.fda.gov/cder/guidance/cmc3
.pdf
9. USP 25NF 20, Validation of
Compendial Methods Section
(1225)
(United
States
Pharmacopeal
Convention,
Rockville, Maryland, USA, 2002)
p 2256.
10. Skoog DA, Holler FJ and Crouch,
SR, Pharmaceutical Analysis, 5th
edition, Brooks/cole ; 2007. 32-35
11. Shabir
A,
Journal
of
Chromatography A. Vol. 987 (12), 2003, 57-66.
12. Sethi PD, In : Quantitative
Analysis
of
Drugs
in
Pharmaceutical Formulations, 3rd
edn., CBS Publishers and
Distributors, New Delhi, 1997.xi
and 53-57.
13. ICH
Harmonised
Tripartite
Guidelines (Nov 2005) Validation
of Analytical Procedures: Text and
Methodology Q2 (R1).
14. Willard HH, Merritt LL and Dean,
JA, Settle, FA. In : Instrumental
11
Methods of Analysis, 4th edn.,

Affiliated East West Press Pvt. Ltd.,
New Delhi; 1986. P. 30-32.
15. Crosby NT, Merritt LL, Dean JA,
Hardcastle WA, Holcombe DG,
Treble RD, Prichard FE, Quality in
Analytical Chemistry Laboratory.
John Wiley and Sons; 1995. P. 6777.
16. Christian,
G.D.,
Analytical
th
Chemistry, 6 edn., John Wiley and
Sons; 2004. p. 126-132.
12

Evaluation of An Analytical Method

Caricato da

Informazioni sul documento

Titolo originale

Copyright

Formati disponibili

Condividi questo documento

Condividi o incorpora il documento

Opzioni di condivisione

Hai trovato utile questo documento?

Questo contenuto è inappropriato?

Copyright:

Formati disponibili

Evaluation of An Analytical Method

Caricato da

Copyright:

Formati disponibili

INTERNATIONAL JOURNAL OF PHARMACEUTICAL CHEMISTRY RESEARCH