03/12/09 1

VALIDATION OF ANAYTICAL
METHODS
P.S.RAMANATHAN
DIRECTORCORPORATE ANALYTICAL
OPERATIONS
GHARDA CHEMICALS LTD.
B-29, MIDC(PHASE I), DOMBIVLI(E)-
421203
DIST. THANE, MAHARASHTRA STATE
03/12/09 2
Chemical Analysis as an Integral Process

Chemical analysis of whatever material system
can be described as a chain of decisions,
actions, and procedures. Figure 1 shows the
cyclic nature of many chemical analytical
processes.
03/12/09 3
The last step (interpretation and evaluation of
results of analysis) should eventually
provide an answer to the starting problem,
generally stated by a client of the
laboratory. If the answer is not satisfactory,
the analysis cycle can be followed again,
after a change or adaptation of one or more
steps. Sometimes this leads to a development of
a new method or (part of a) procedure in
order, for example, to achieve better
separation of certain components, or to attain
a lower detection limit for specific compounds.
03/12/09 4
Chemical analysis as a cyclic process
This is illustrated in Fig. 1. Like any chain, a
chain of chemical analysis is only as strong as
its weakest link.
03/12/09 5
Figure 1. Chemical analysis as a cyclic process
03/12/09 6
In general, the weakest links in an analytical
process are not the ones usually being
recognised as parts of chemical analysis, such
as chromatographic separation or
spectrometric detection, but rather the
preceding steps, often taking place outside the
analytical laboratory such as the selection of
object(s) to be sampled, the design of the
sampling plan, and the selection and the use of
techniques and facilities for
obtaining, transporting, and storing samples.
03/12/09 7
When the analytical laboratory is not
responsible for the sampling, the quality
management system often does not even take
account of these weak links in the analytical
process. Furthermore, if the preparation
(extraction, clean-up, etc.) of the samples has
not carefully been carried out, even the most
advanced and quality controlled analytical
instruments and sophisticated computer
techniques cannot prevent that the results of
the analysis become questionable.
03/12/09 8
Finally, unless the interpretation and
evaluation of results have a solid statistical
base, it is not clear how significant these
results are, which in turn greatly undermines
their merit. We, therefore, believe that quality
control and quality assurance should involve
all the steps of chemical analysis as an integral
process, of which the validation of the
analytical methods is only one, though
important, step.
03/12/09 9
In laboratory practice, quality criteria should
concern the rationale of the sampling
plan, the validation of methods, instruments
and laboratory procedures, the
reliability of identifications, the accuracy and
precision of measured concentrations,
and the comparability of laboratory results
with relevant information produced earlier or
elsewhere.
03/12/09 10
03/12/09 11
1)The 1oke of the 1apanese prime minister
talking to President Clinton.
2)Condolence message to a neighbour in the
village, after the death of his wife.
03/12/09 12
The language police(Newspeak.com) have
added more words to their politically correct
lexiconso it is no longer okay to call a
housewife a ~home maker, an ugly woman, ~a
plain 1ane and a handicapped person
~physically challenged.
According to the website that tracks modern
speak and is dedicated and inspired by George
Orwell, ~domestic engineer is the term for
housewife.
03/12/09 13
Visually challenging for the ~ugly.
Handicapable for the physically impaired.
Other examples are:
Broken home: Dysfunctional family
The website compares modern speak with
~Newspeakthe official language of the
totalitarian state of Oceania in Orwell`s classic
184, which deleted all words that went againt
party policy. The state`s propaganda
department was called ~The Ministry of
Truth.
03/12/09 14
The rationaing dept.The ministry of plenty.
Words like ~affirmtive action,
~homophobic, ~peace keepers, ~sexual
harassment and ~the war on drugs, were
nonexistent before this century. They were
fabricated by the government and special
interest groups with the main aim of
misleading the public and swaying public
opinion.
The website also lists words that it claims have
lost their original meaning. ~Patriot -one
who loves the country and cultureNow it
means ~bigot, racist and a possible terrorist.
03/12/09 15
Family now means, ~anyone or more people of
any gender raising any one`s children
anywhere
03/12/09 16
Validation: Element of Laboratory Quality
Assurance

Quality is a relative notion; never high or low,
in an absolute sense. Rather, it is
adequate or inadequate in terms of the extent
to which a product, a process, or a service
meets the requirements specified beforehand
by an objective or a customer.

03/12/09 17
The principal product of an analytical
chemical laboratory is information about
the chemical composition of material systems,
usually in terms of the identity and/or
quantity of one or more relevant components
in samples taken from these materials.
The quality of scientific information, in
general, is evaluated by internationally
accepted standards of objectivity, integrity,
reproducibility, and traceability, in any case,
prior to publication.
03/12/09 18
Essential criteria for the quality of produced
chemical information are the utility
and the reliability, which are closely related to
the margins of uncertainty in the
measurement results regarding both the
identity and the concentration of the target
components.
03/12/09 19
With respect to these correlated criteria,
minimum requirements are generally set
by the customer and usually deduced from a
previously specified purpose. The quality of
produced chemical information is therefore
factually to be acknowledged by the customer
as the end-user of this information.
03/12/09 20
For chemical measurements, this could be a
clinical chemist, who needs to know the
identity of certain isolated compounds from a
biological fluid, a polymer chemist who wishes
to verify the molecular structure of a product
of synthesis, or a health researcher who wants
to know whether the concentration of a certain
toxic compound in certain food is above
certain concentration level.
03/12/09 21
It is not hard to imagine the consequences in terms
of costs, health risks, and so on , when, on closer
examination or statistical evaluation of the
measurement results, a positive finding turns out to
be false, or the uncertainty margin of a measured
concentration appears to be 100 and not the
initially reported 10.
Evaluation and validation of analytical methods
and laboratory procedures are, therefore, of
paramount importance, prominent means being
the use of adequate (preferably certified) reference
materials and participation in interlaboratory
proficiency tests.
03/12/09 22
Quality demands made on the infrastructure,
equipment, operating procedures,
personnel, and organization of the laboratory
are to be deduced from the quality
requirements, that the produced chemical
information should meet. A formal recognition
of this type of quality can be achieved through
accreditation or certification, based on
international quality standards and guidelines,
as issued by International Organization for
Standardization (ISO),
03/12/09 23
Organisation for Economic Co-operation and
Development (OECD), and European
Committee for Standardization (CEN).
Validation of analytical methods is one, though
an essential step in the integral process of
quality assurance and quality control of
chemical measurements in material systems.
03/12/09 24
Validation Concepts in Analytical Chemistry
General Aspects
03/12/09 25
Besides other assignments undertaken by him, an
analytical chemist must use his skill and
judgement to: (a)Study the fundamental
principles of the methods used for analysis,
(b)Develop new analytical methods and
instrumentation, (c) Develop and validate
analytical techniques, (d) Assess the degree of
reliability of the results, prior to reporting the
results and (e) Supervising and training juniors.
03/12/09 26
What is a Validated method?
A good analytical result must have the
potential to withstand the closest scrutiny.
Often, reports are made on cases, where
analytical data have been inadequate.
Unknown interferences and biases all go
towards unreliable data.
03/12/09 27
Validation of a method ensures that the
accuracy of the concentration reported is
known and appropriate for the purpose. All
known interferences and biases will have been
investigated and evaluated and the method will
have been tested against Certified Reference
Materials, where available, and, if possible, by
collaborative studies with peer laboratories.
03/12/09 28
The Validation process in analysis
A good analyst must, therefore, acquire complete
understanding of the method used for analysis, and
its potential applications. He has to ensure
whether the method is capable of answering the
questions put by the customer. He has to ascertain
the limits to the range of applications. He must
have full information about the factors which are
liable to upset the method. All these efforts,
cumulatively, constitute the method validation
process, which is an essential activity if an
analytical measurement is going to be fit for the
purpose for which it was intended.
03/12/09 29
Thus, generating quality analytical results
involves fully understanding and evaluating
the set of procedures that make up the
method, knowing how the result is going to be
used, and maintaining close collaboration with
the customer for that analysis.
03/12/09 30
During the last few decades analytical
measurement has come under very close
scrutiny. The indirectness of the analytical
measurement presents the analyst with unique
problems. In fact, every new sample poses a
different set of challenges, even to the seasoned
analytical chemist.
03/12/09 31
The analytical chemist may use an in-house
method, or a method reported in a standard
(like the Bureau of Indian Standards or
pharmacoepia) or the method may be set out
as a standard method in some piece of
legislation. The fundamental requirement of a
good analytical chemist is to satisfy himself as
to whether the methods he uses actually
measure what they are supposed to measure.
How much is the method affected by outside
interference?
03/12/09 32
Have all the factors affecting the reliability of
the method been fully understood and
investigated? Does the analysis produce
results which are fit for the purpose to which it
is put, and are the results comparable with
those of other laboratories performing similar
analyses? These are the problems faced by a
seasoned analytical chemist, as a matter of
routine.
03/12/09 33
Any Validated analytical measurement
program needs full support from adequate
education and training programs. There must
also be a well designed and organized
program of international collaboration aimed
at the harmonization of analytical
measurement technology.
03/12/09 34
The availability of well characterized reference
materials(ideally with matrices which match
the sample in question) is undoubtedly one of
the corner stones of method validation and
establishing traceability to primary
measurement standards. The procedure
adopted to check that the results are
comparable with those of other
laboratories(performing the same analysis) is
termed ~Proficiency testing program.
03/12/09 35
These schemes are run by authorized
government agencies like the NABL(National
Accreditation Board for Testing and
Calibrating Laboratories) or by some well-
established central co-ordinating laboratories.
Internationally accepted standard
protocols(ISO/IUPAC/AOAC) are available
for operating proficiency testing programs.
03/12/09 36
National and international cooperation are
essential and, in fact, indispensable, for the
harmonization of analytical methods and
protocols, which would pave the way for
increase in the national and international
acceptance of analytical data. Achieving this
acceptance is one of the key steps in the
process of ensuring more freedom and
flexibility for international trade, which will
ultimately lead to greater economic growth
and increased benefits to the customer.

03/12/09 37
Formation of organizations like
EURACHEM(a network of European National
Laboratories) , CITAC(Cooperation on
International Traceability in Analytical
Chemistry), etc., are efforts to harmonise
chemical testing procedures and supporting
infrastructure. The aims and objectives of
EURACHEM and CITAC have been
described in reference.
03/12/09 38
The VAM bulletin, published by the
~Laboratory of the Government Chemist,
UK, is an excellent publication, which provides
information and guidelines on various aspects
connected with Valid Analytical Measurement.
It has been claimed that this publication has a
current circulation of more than 10,000 copies.

03/12/09 39
It should be noted that ~validation does not
guarantee that a method is free from error. It
just confirms that the method is adequate to
meet the specification prepared for the
analysis. This confirmation will usually
require the laboratory to carry out an
experimental study but, in certain
circumstances, the experience of the analyst,
together with validation data obtained
previously or elsewhere, may be sufficient.
03/12/09 40
Whatever the approach used, it is important
that the analyst notes in the laboratory`s
records the agreed analytical specification, in
terms of various performance characteristics
of the method, and the evidence or other
reasons for believing that the specification has
been met. This should be done before any
samples are analysed and, where the method is
used routinely, checked periodically.
03/12/09 41
It must be emphasized that reliable chemical
analysis will always ultimately depend on the
skill of the analyst. From the wealth of
literature available for guidance, it is the duty
of the analytical chemist to select from it a
proper approach in order to provide a method
which is fit for a specific purpose. 1udged
from this angle, it can be seen that the method
validation for the everyday work of the
laboratory is both essential and realistic.
03/12/09 42
Checking the validity of procedures for
environmental monitoring, food analysis, etc.,
has, now, become a matter of utmost
importance because of the need to regulate
environmental pollution and contamination of
food articles.
It is worth mentioning here that the validity of
analytical measurements is of such key
importance to many aspects of business and
social activities that it should be high on the
agenda of those who teach science in colleges
and universities.
03/12/09 43
It may not be out of place to mention here that
in an intercomparison of results from expert
international laboratories, (available in the
literature and provided in the bar chart form),
the results of different laboratories showed
significant variability.

03/12/09 44
The average percentage of error varies from
less than 1 to about 13( out of the results
provided by 16 laboratories). The results
provided are for relatively straightforward
analyses of inorganic elements in solution.
Such practical problems must be kept in mind
while understanding the subject of Valid
analytical measurements and its practical
importance.
03/12/09 45
PT schemes provide laboratory analysts and
their customers with valuable information
about the way in which they carry out their
analyses. A major development is the
publication an International Harmonised
Protocol on PT. This is the result of
collaboration of scientists from many
countries, under the joint auspices of ISO,
IUPAC and AOACI.

03/12/09 46
Valid Analytical Measurements

A chemist can generate a number, and call it a
result!! A standard analyst must be able to
stand up in a court of law and say that, if the
analysis is repeated, the result will always
come within the expected range. Another
analyst, applying the same or different method
to the same sample, should be able to come up
with a comparable result.
03/12/09 47
Professional analysts do not generate
numbers---they perform Valid analytical
measurements.
03/12/09 48
Globalisation
Q: What is the truest definition of
Globalisation?
Answer: Princess Diana`s death.
Q: How come?
A: An English princes with an Egyptian
boyfriend crashes in a French tunnel, driving
a German car with a dutch engine, driven by
a Belgian who was drunk on Scottish whisky,
followed closely by Italian paparazzi, on
1apanese motorcycles, treated by an
American doctor, using Brazilian
medicines...
03/12/09 49
..sent to you by an American, using Bill
Gate`s technology and you are probably
reading this on your computer, that uses
Taiwanese chips and a Korean monitor,
assembled by Bengaladeshi workers in a
Singapore plant, transporeted by Indian
lorry drivers, hijacked by Indonesians,
unloaded by Sicillian longshoremen and
trucked to you by Mexican illegals..
That, my good friends, is real globalisation!!

03/12/09 50
The importance of validated analytical
measurements
Several billion analyses are carried out per
year, in various countries. They form a vital
part of ensuring the quality of goods and
commodities. They assist the government in
policy development, and the proper
enforcement of regulations. Analytical
measurements of proven validity pervades all
aspects of the economy and everyday life.
03/12/09 51
Obtaining the necessary accuracy and
precision in analysis is important (a) in terms
of the quality of industrial goods, (b) R&D, (c)
the health, safety and welfare of the
community, (d) protection of the environment
and (e) economic well-being of the nation.
03/12/09 52
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Method validation process includes, among other
things:
(a)Ensuring that the method offers satisfactory
answers to the needs of the customer,
(b)Understanding the limits to the range of
application and (c) Identifying the factors that
may upset the results.
03/12/09 55
The VAM(Valid Analytical
Measurement) Initiative, is a
programme funded by the U. K. Dept.
of Trade and Industry. The VAM
Initiative seeks to improve the quality
of analytical data and to facilitate the
mutual recognition of analytical
results by promoting 6 key principles
of good analytical practice.
03/12/09 56
The Six VAM Principles
The six VAM principles enable organisations
to implement best practice, and make valid
measurements. They are designed to control
all factors that might affect the reliability of
analytical results, thereby reducing the cost
and risk of unreliable measurements.
03/12/09 57
Principle 1:Analytical measurements should
be made to satisfy an agreed requirement.
Principle 2:Analytical measurements should
be made using methods and equipment, which
have been tested to ensure, they are fit for
purpose.
Principle 3:Staff, making analytical
measurements, should be, both qualified and
competent, to undertake the task.
03/12/09 58
Principle 4:There should be a regular
independent assessment of the technical
performance of a laboratory.
Principle 5:Analytical measurements made in
one location should be consistent with those
elsewhere.
Principle 6:Organisations making analytical
measurements should have well defined
quality control and quality assurance
procedures.
03/12/09 59
In other words, key principles of valid
analytical measurements include,
(1) Measurements should be made using
properly validated methods. (2) Reference
materials should be used to ensure traceability
of measurements. (3) Laboratories should seek
independent assessment of their performance
by participating in national and international
proficiency testing schemes. (4) Labortories
should seek independent approval of their
quality system, preferably by accreditation or
licensing, to a recognised quality standard.
03/12/09 60
When VAM are not realised, data of poor
quality are reported by a lab. In such
circumstances, the existing problems and
costs for the end-user of the analytical
data can be substantial and are associated
with consequences such as :
1)the costs involved in repeat
measurements to correct poor data; (2)the
faulty decisions making that ensues when
invalid results are acted upon; (3)damage
to reputation and credibility that results
when an end-user is associated with poor
data;
03/12/09 61
(4)possible loss of business where the end-
users customer is compromised by poor
data; and (5)any legal and financial
liability incurred from the use of poor data.

It is important that those who commission
labs to undertake analytical measurements
on their behalf appreciate the critical need
to select only competent labs.
03/12/09 62
To sum up, validation is "establishing
documented evidence, that a system does what
it purports to do".
Validation master plan, in any organisation,
includes, among other things, analytical
methods for products and low level detections,
standardisation of equipments and related
facilities, and validated documentation.
03/12/09 63
HR Manager`s loveletter
Ever wonder how an HR manager would write a
love letter to his girl friend?
Dearest 1uliet:
I am very happy to inform you that I have fallen in
love with you, since the October 14, 2005,
(Saturday), 12 noon to be precise.
With reference to the meeting held between us on
the 13
th
of October at 15.00 hrs., in the HRD
Conference room, I would like to present myself as
a prospective lover of your good self.
03/12/09 64
Our love affair would be kept confidential, and
maintained on probation for a period of three
months. Depending on compatibility, and your
detailed medical examination by a lady doctor,
and appropriate physical examinations by me, it
would be made permanent. Of course, upon
completion of probation, there will be continuous
on-the-job training and performance appraisal
schemes, leading upto promotion from lover to
spouse.
The expenses incurred for coffee and
entertainment would initially be shared equally
between us.
03/12/09 65
Later, based on your performance and inherent
skills I might take up a larger share of the expense.
However, I am broad-minded enough to be taken
care of, on your expense account as well.
I request you to respond to this offer within 30
days of receiving this letter, failing which, this offer
would be cancelled, without further notice, and I
shall have no alternative other than considering
some other suitable candidate for the position
indicated above.
If due to any reason, you do not wish to take up
this offer, I request you to forward this letter to
your sister, who is also under my consideration for
the said post.
03/12/09 66
Please acknowledge receipt of this letter. As a
token of your having received and accepted this
offer, please sign the duplicate copy of this letter
and send the same to the undersigned by courier
service or Regd. Post, eventhough hand delivery, in
the absence of others in my cabin, may be the most
welcome approach.
Wishing you all the best, and looking forward to
get a favourable response, before the date
stipulated above, and thanking you in anticipation,
Yours sincerely
HRD Manager
03/12/09 67
Method validation is a major challenge for
analytical chemists in the present global
scenario.
The reasons include, (a) The increasing
importance of accurate analytical
measurements, (b) Concern about the existing
deficiencies in the quality of data, (c) The onus
to demonstrate the validity of its data is on the
analytical community, (d) Sole dependence on
the skill and experience of the analyst to ensure
the quality of analytical results is no longer
accepted, and (e) Evidence is needed, which can
be provided by systematic QA protocol.
03/12/09 68
The Food and Drug Administration of USA
has defined Validation as, ~The documented
program providing high degree of assurance
that, specific process or equipment, will
consistently produce product, meeting
predetermined specification and quality
attributes.
03/12/09 69
The documentation process consists of the
following steps.
(1)A trained and knowledgeable person
prepares a written document, (2)Review by a
higher or more qualified person, (3) Approval
by the Head of the Department and
(4)Filing and control of the prepared
document.
03/12/09 70
Validation of results obtained by any
analytical method is a process to confirm that,
the analytical procedure employed for a
specific test is suitable for its intended use.
Methods need to be validated or revalidated,
a) before their introduction into routine use, b)
whenever the conditions change for which the
method has been validated, e.g., instrument
with different characteristics and c) whenever
the method is changed, and the change is
outside the original scope of the method.
03/12/09 71
A laboratory, applying a specific method,
should have documentary evidence that, the
method has been appropriately validated.
~The responsibility remains firmly with the
user, to ensure that the validation documented
in the method is sufficiently complete to meet
his or her needs."
03/12/09 72
This holds for standard methods, for example,
from Environmental Protection Agency(EPA),
American Society for Testing
Materials(ASTM), International Organisation
for Standardisation(ISO) or US
Pharmacoepia(USP), as well as for methods
developed in-house.
03/12/09 73
If standard methods are used, it should be
verified that, the scope of the method and
validation data, for example, sample matrix,
linearity, range and detection limits, etc.,
comply with the laboratory`s analyses
requirements; otherwise, the validation of the
standard method should be repeated, using
the laboratory`s own criteria. The laboratory
should demonstrate the validity of the method
in the laboratory`s environment.
03/12/09 74
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03/12/09 76
For example, in the case of analytical methods for
pesticide formulations, these characteristics are
defined by CIPAC(Collaborative International
Pesticides Analytical Council)
and AOAC(Association of Official
Analytical Community) protocols and
EU(European Union) directives. Since an
interlaboratory study is not conducted,
estimation of the laboratory effect (bias) can be
made, either by use of the Horwitz equation, or
by the analysis of Certified Reference Material
(CRM), where the combined laboratory and
method bias are assessed.
03/12/09 77
Method Validation makes use of a set of tests
that both 1) test any assumptions, on which the
analytical method is based and 2) establish and
document the performance characteristics of a
method.

03/12/09 78
The term ~Method validation, in all
definitions given by various international
organizations (Food and Agricultural
Organisation(FAO), International Atomic
Energy Agency(IAEA), Eurachem,
International Union of Pure and Applied
Chemists(IUPAC), AOAC), comprises of two
key parts: 1) establishment of performance
characteristics and 2) fitness for purpose.
03/12/09 79
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Different levels of validation have occurred, due
to the different needs for application of each
particular method, and the different levels of
quality required from the results. Methods, that
are to be applied in a wide variety of instruments,
in many different countries worldwide, under
different climatic conditions, by analysts of
different levels of knowledge and expertise, have
to be ~fully validated.
03/12/09 82
On the other hand, methods, that are to be
used for a specific purpose in a specific
laboratory by personnel of a particular degree
of training, can be validated at a lower level,
what is known as ~in-house validation, or
more accurately ~Single laboratory
validation.
03/12/09 83
~Full validation for an analytical method is
usually taken to comprise an examination of
the characteristics of the method in an
interlaboratory method performance study
(also known as collaborative study or
collaborative trial). This study could be viewed
as the co-ordinated ~Single laboratory
validation study of the analytical method, in
usually 10-15 laboratories.
03/12/09 84
From the accumulated results of the
interlaboratory performance of the method,
some characteristics, that cannot be assessed
within a single laboratory, are taken into
account, and quantified.
03/12/09 85
Depending on the filed of application of a
method, the performance characteristics
required for validation vary. For example,
there is no need for determining the limit of
detection for a method, that is used for quality
control of pesticide formulations, while it is
imperative for a method for the determination
of residues of a pesticide in any matrix.

03/12/09 86
In their excellent article Balayiannis et al have
summarized the requirements for ~Single
laboratory validation of analytical methods,
for the determination of the chemical
composition of pesticide formulations. Special
attention have been drawn by them to the
requirements demanded by the CIPAC and
EU Directive 91/414 ~Concerning the placing
of plant protection products on the market.
03/12/09 87
Method validation is a key element in the
establishment of reference methods and in the
assessment of a laboratory`s competence in
producing reliable analytical data. Hence, the
scope of the term, ~Method validation. is wide,
especially if one bears in mind the role of
Quality Assurance/Quality Control (QA/QC).
Validation has been put in the context of the
process, generating chemical information. Basic
performance parameters, included in the
validation processes, have been well discussed in
the literature, including evaluation of current
approaches to the problem.
03/12/09 88
Validation parameters
Validation of results / Method validation has
received considerable attention in the
literature, and from industrial committees and
regulatory agencies. ~The Guidance on the
Interpretation of the EN 45000 Series of
Standards and ISO/IEC 17025 (2005)
includes information on the validation of
methods, with a list of nine validation
parameters.
03/12/09 89
The International Conference on
Harmonization (ICH) of Technical
Requirements for the Registration of
Pharmaceuticals for Human Use has developed
a consensus text on the validation of analytical
procedures. The document includes definitions
for eight validation characteristics. The United
States Food and Drug Administration (US FDA)
has proposed guidelines on submitting samples
and analytical data for methods validation. The
United States Pharmacopoeia (USP) has
published specific guidelines for method
validation for compound evaluation
03/12/09 90
Based on the various guidelines available, one
can summarise that Validation of the
analytical system includes, among other things,
verification of the appropriateness of the test
method(Assay validation), System suitability
test, Hardware validation(Design stage, Design
qualification, Installation, Inspection and
Regular inspection) and Software validation.
Validation and testing are not the same.
03/12/09 91
According to the Directive 91/414/EEC 4 ],
methods for the quantification of the
active substance in formulated pesticide
products are required to be robust, accurate
and precise. Validation studies of quantitative
analytical methods, according to CIPAC and
EU guidelines, should determine the following
performance characteristics:
03/12/09 92
(i)Applicability
(ii)Specificity (a definition of the species being
determined)
(iii)Selectivity (a demonstration of no
interference from excipients).
(iv)Linearity of response for the analyte in the
method.
(v)A demonstration of the accuracy of the
procedure.
(vi)Trueness (Bias)
(vii)An estimation of the precision of the
procedure (repeatability and reproducibility).
03/12/09 93
However, according to FAO/IAEA, some
additional parameters have to be assessed, in
accordance to AOAC International:
(viii)Calibration
(ix)Range
(x)Sensitivity
(xi)Ruggedness
(xii)Practicability
03/12/09 94
Brief explanations of the parameters,
connected with validation, and the
requirements for each individual parameter,
taken from the available guidelines, are given
below.
03/12/09 95
Applicability
According to the IUPAC after validation, the
documentation should provide, in
addition to any performance specification,
information about the identity of the analyte
(e.g., ~fenthion), specification of the range of
matrices of the test material covered by the
validation (e.g., ~pesticide formulations),
concentration range (e.g., ~0-50 ppm).
03/12/09 96
The intended application, and its critical
uncertainty requirements, (e.g., ~The analysis
of pesticide formulations for screening
purposes) and, finally, a protocol, describing
the equipment, reagents, procedure (including
permissible variation, unspecified instructions,
e.g., ~heat at 100 5C for 305 min),
calibration and quality procedures, and any
special safety precautions required.
03/12/09 97
Specificity
According to CIPAC Guidelines 3], the
specificity of the method is a definition of
the species giving rise to the signal, used for
quantification. It shows that the detected signal is
due to the analyte, and not due to another
compound.
Specificity is a quantitative indication of the
extent to which a method can distinguish between
the analyte of interest and interfering substances
on the basis of signals produced under actual
experimental conditions. Random interferences
should be determined using representative blank
samples.
03/12/09 98
Selectivity (interference)

The capability of an analytical method, to
reliably discriminate among chemically or
physically related substances. It is sometimes
quantified as cross sensitivity.
According to IUPAC, selectivity is the degree
to which a method can quantify the analyte
accurately, in the presence of interferents.
03/12/09 99
The terms specificity and selectivity are often
used simultaneously, to describe the same
phenomenon. However they have specific
meaning. Specificity describes the
performance of detection (rise of signal), while
selectivity is used for characterising the
chromatographic separation (discrimination
between the signals of two substances).
03/12/09 100
According to chromatographic theory 8 ]
R
s
1.18 (t
R2
-t-
R1
)/(W
h1
+ W
h2
)
where Rs resolution for two consecutive peaks
t
R1
retention time of first peak
t
R2
retention time of second peak
W
h1
peak width at half peak height of the first
peak
W
h2
peak width at half peak height of the
second peak
and the acceptable values are Rs>1.0 (and
preferably >1.54 from the practical point of
view).
03/12/09 101
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.
03/12/09 102
The USP monograph(REFERENCE) defines
selectivity of an analytical method as its ability
to measure accurately an analyte, in the
presence of interference, such as synthetic
precursors, excipients, enantiomers and
known (or likely) degradation products, that
may be expected to be present in the sample
matrix. Selectivity in Gas chromatography is
obtained by choosing optimal columns and
setting chromatographic conditions, such as
column temperature and detector.
03/12/09 103
Linearity
The linearity of a test procedure is its ability
(within a given range), to obtain test results,
proportional to the concentration (amount) of
analyte in the sample.
According to EU and CIPAC Guidelines, the
linearity of response to the analyte should be
demonstrated, at least over the range: nominal
concentration 20. At least three
concentrations should be measured, with
duplicate measurements for each.
03/12/09 104
Linearity can be tested informally, by
examination of a plot of residuals, produced by
linear regression of the responses, on the
concentrations, in an appropriate calibration
set. The linearity of an analytical method is its
ability to elicit test results that are directly, or
by means of well-defined mathematical
transformations, proportional to the
concentration of analytes in samples, within a
given range.
03/12/09 105
Linearity is determined by a series of three to
six injections of five or more standards, whose
concentrations span 80-120 percent of the
expected concentration range. The response
should be directly, or by means of a well defined
mathematical calculation, proportional to the
concentrations of the analytes. A linear
regression equation, applied to the results,
should have an intercept, not significantly
different from zero. If a significant nonzero
intercept is obtained, it should be demonstrated
that, there is no effect on the accuracy of the
method.
03/12/09 106
Frequently, the linearity is evaluated
graphically, in addition or alternatively to,
mathematical evaluation. The evaluation is
made by visual inspection of a plot of signal
(height or peak area) as a function of analyte
concentration. Because deviations from
linearity are sometimes difficult to detect, two
additional graphical procedures can be used.
The first one is to plot the response against the
concentration. For linear ranges, the
deviations should be equally distributed
between positive and negative values.
03/12/09 107
Another approach is to divide signal data by
their respective concentrations yielding the
relative responses. A graph is plotted with the
relative responses on the Y-axis, and the
corresponding concentrations on the X-axis,
on a log scale. The line obtained should be
horizontal over the full linear range. At higher
concentrations, there will typically be a
negative deviation from linearity. Parallel
horizontal lines are drawn in the graph
corresponding to, for example, 95 percent and
105 percent of the horizontal line.
03/12/09 108
The method is linear up to the point, where the
plotted relative response line intersects the 95
percent line. Fig. 3.1 shows a comparison of
the two graphical evaluations, carried out in a
typical GC evaluation.
03/12/09 109
Figure 3.1. Graphical presentations of a typical GC estimation.
Plotting the sensitivity (response/amount) gives clear indication of
the linear range. Plotting the amount on a logarithmic scale has a
significant advantage for wide linear ranges. Rc Line of constant
Response
03/12/09 110
The linearity range for examination depends
on the purpose of the test method
Under most circumstances acceptable
regression coefficient (r) is 0.999.
Intercept and slope should be indicated.

03/12/09 111
Range
Range is defined as the difference between the
largest and the smallest observed value of a
quantitative characteristic. In practical terms,
this means that the ~range is the interval of
concentrations, within which the analytical
procedure demonstrates a suitable level of
precision and accuracy. The analytical range
may result from an analytical curve, that is
linear or not linear.
03/12/09 112
The range of an analytical method is the
interval between the upper and lower levels
(including these levels) that have been
demonstrated to be determined with precision,
accuracy and linearity, using the method as
written. The range is normally expressed in
the same units as the test results (e.g.
percentage, parts per million) obtained by the
analytical method.
03/12/09 113
The chemists normally employ two terminologies
with respect to the analysis carried out. One is
the working range and the second is the linear
range.
For any quantitative method, there is a range of
analyte concentrations over which the method
may be applied. At the lower end of the
concentration range, the limiting factor is the
value of the limit of detection and/or limit of
quantification. At the upper end of the
concentration range, limitations will be imposed
by various effects, depending on the detection
mechanism.
03/12/09 114
Within this ~working range, there may exist a
~linear range, within which the detection
response will have a sufficiently linear relation
to analyte concentration. The working and
linear range may differ in different sample
types, according to the effect of interferences
arising from the sample matrix.
03/12/09 115
It is recommended that, in the first instance,
the response relationship should be examined
over the working range, by carrying out a
single assessment of the response levels to at
least six concentration levels. To determine the
response relationship within the linear range,
it is recommended that three replicates are
carried out at each of at least six concentration
levels.
03/12/09 116
Accuracy
Accuracy is the closeness of agreement
between a test result, and the accepted
reference or true value of the property being
measured. Accuracy is a qualitative concept
13 ], and cannot be given a numerical value,
but can be expressed on an ordinal scale such
as ~poor, ~fair and ~good.
03/12/09 117
Trueness (Bias)
The closeness of agreement between the
average value obtained from a large series of
test results, and the accepted reference or true
value of the property being measured.
Trueness is a measure of systematic error. The
measure of trueness is usually expressed in
terms of bias. As ihe true value cannot be
determined by chemical analysis, it has been
replaced by the ~accepted reference value or
~the most probable value.
03/12/09 118
Bias is the difference between the mean value
(expectation) of the test results, and an
accepted reference value. The bias is the total
systematic error.
The accuracy of an analytical method is the
extent to which test results, generated by the
method, and the true value agree. The true
value for accuracy assessment can be obtained
in several ways. One alternative is to compare
results of the method, with results from an
established reference method. This approach
assumes that the uncertainty of the reference
method is known.
03/12/09 119
Secondly, accuracy can be assessed by
analyzing a sample with known
concentrations, for example, a certified
reference material, and comparing the
measured value with the true value, as
supplied with the material. If such certified
reference material is not available, blank
sample matrix of interest can be spiked with a
known concentration by weight or volume of
the pure material and analysis carried to
evaluate the recovery.
03/12/09 120
Accuracy refers to closeness of agreement
between the true value of the analyte
concentration and the mean result obtained by
applying experimental procedure to a large
number of homogeneous samples. It is related
to systematic error and analyte recovery.
Systematic errors can be established by the use
of appropriate certified reference materials
(matrix-matched) or by applying alternative
analytical techniques.
03/12/09 121
Recovery is expressed as the amount/weight of
the compound of interest, analyzed, as a
percentage to the theoretical amount present
in the medium.
Accuracy is the measure of how close the
experimental value is to the true value.
Accuracy studies for drug substance and drug
product are recommended to be performed at
the 80, 100 and 120 levels of label claim.
03/12/09 122
Recovery data, at least in triplicate, at each
level (80, 100 and 120 of label claim) is
recommended. The mean is an estimate of
accuracy, and the RSD is an estimate of
sample analysis precision.
The concentration should cover the range of
concern, and should, particularly, include one
concentration close to the quantitation limit.
03/12/09 123
The expected recovery depends on the sample
matrix, the sample processing procedure, and
on the analyte concentration. The AOAC
Manual for the Peer Verified Methods
program(REFERENCE) includes a table
(Table 3.1), with estimated recovery data, as a
function of analyte concentration.
03/12/09 124
Table 3.1. Analyte recovery at different concentrations
Active Ingred. ] Unit Mean recovery ]
100 100 98-102
>10 10 98-102
>1
1 97-103
>0.1 0.1 95-105
0.01 100 ppm 90-107
0.001 10 ppm 80-110
0.0001 1 ppm 80-110
0.00001 100 ppb 80-110
0.000001 10 ppb 60-115
0.0000001 1 ppb 40-120
03/12/09 125
Limit of Detection and Quantitation
The limit of detection is the point at which, a
measured value is larger than the uncertainty
associated with the measurement. It is the
lowest concentration of analyte in a sample,
that can be detected, but not necessarily
quantified. In chromatography, the detection
limit is the injected amount, that results in a
peak, with a height at least three times as high
as the baseline noise level.
03/12/09 126
The limit of detection is usually expressed as
the analyte concentration corresponding to the
sample blank plus three sample standard
deviations, based on 10 independent analyses
of sample blanks.
03/12/09 127
The limit of quantitation is the minimum
injected amount, that gives precise
measurements. If the required precision of the
method at the limit of quantitation has been
specified, then the following approach can be
used. A number of samples with decreasing
amounts of the analyte are injected six times.
The calculated RSD of the precision is
plotted against the analyte amount. The
amount, that corresponds to the previously
defined required precision. is equal to the limit
of quantitation.
03/12/09 128
Figure 3.2. Limit of quantitation
03/12/09 129
The limit of quantification is the lowest
concentration of analyte that can be
determined with an acceptable level of
uncertainty or, alternatively, it is set by
various conventions to be five, six, or ten
standard deviations of the blank mean. It is
also sometimes known as the limit of
determination.
03/12/09 130
Sensitivity
Sensitivity is the measure of the change in
instrument response, which corresponds to a
change in analyte concentration. Where the
response has been established as linear with
respect to concentration, sensitivity
corresponds to the gradient of the response
curve.

03/12/09 131
Sensitivity is the capability of an analytical
procedure, to reliably discriminate between
samples, having differing concentrations, or
containing differing amounts of an analyte.
The sensitivity of a method is correctly defined
as the slope of the calibration curve.
03/12/09 132
Precision (repeatability and reproducibility).
Precision is the measure of how close the data
values are to each other for a number of
measurements under the same analytical
conditions. ICH has defined precision to
contain three components: repeatability,
intermediate precision and reproducibility.
The precision of a method is the extent to
which the individual test results of multiple
injections of a series of standards agree.
03/12/09 133
The measured standard deviation can be
subdivided into three categories: repeatability,
intermediate precision and reproducibility.
Repeatability is obtained when the analysis is
carried out in one laboratory by one operator
using one piece of equipment over a relatively
short time span. At least 5 or 6 determinations
of three different matrices at two or three
different concentrations should be done and
the relative standard deviation calculated. The
acceptance criteria for precision depend very
much on the type of analysis.
03/12/09 134
While for compound analysis in agrochemical /
pharmaceutical quality control precision of
better than 1 RSD is easily achieved, for
biological samples the precision is more like
15 at the concentration limits and 10 at
other concentration levels. For environmental
and food samples, the precision is very much
dependent on the sample matrix, the
concentration of the analyte and on the
analysis technique. It can vary between 2
and more than 20.
03/12/09 135
Precision is the closeness of agreement
between independent test results obtained
under stipulated conditions. The measure of
precision is usually expressed in terms of
imprecision, and computed as a standard
deviation or relative standard deviation of
the test results. Precision is a measure of
random errors, and may be expressed as
repeatability and reproducibility. These
terms are defined in ISO 5725-1986E:

03/12/09 136
For single laboratory validation, two sets of
conditions are relevant 7]: a) Precision
under repeatability conditions, describing
variations observed during a single run and b)
precision under run-to-run conditions,
describing variations in run bias.
Usually, both of these sources of error are
operating on individual analytical results. The
two precision estimates can be obtained most
simply by analysing the selected test material,
in duplicate, in a number of successive runs.
03/12/09 137
The combined precision can be estimated
directly, by the analysis of the test material
once, in successive runs, and estimating the
standard deviation from the usual equation.
03/12/09 138
Repeatability

Repeatability is the closeness of agreement
between mutually independent test results,
obtained with the same method, on identical
test material, in the same laboratory, by the
same operator, using the same equipment,
within short intervals of time. The measure of
repeatability is usually expressed in terms of
Relative Standard Deviation, RSD
r
.
03/12/09 139
Reproducibility
Reproducibility is the closeness of agreement
between test results, obtained with the same
method, on identical test material, in different
laboratories, with different operators, using
different equipment. The measure of
reproducibility is usually expressed in terms of
Relative Standard Deviation, RSD
R
.
03/12/09 140
Theoretical repeatability and reproducibility
values can be calculated from the Horwitz
equation. Horwitz trumpet and equation is
RSD
R
2
(1-0.5logC)
where C is the concentration of analyte in the
sample, expressed as a decimal fraction.
03/12/09 141
Horwitz has derived the equation, after
studying the results from many (~3,000)
collaborative trials. The equation is an
approximation of the possible precision that
can be achieved. The data points from all the
~acceptable collaborative trials lie within a
range of one half to twice the values derived
from the equation. Their graphical
representation is an idealised smoothed curve,
independent of the analyte, method, matrix,
laboratory and time (state of the art).
03/12/09 142
In general, the values taken from this curve
are indicative of the precision that is
achievable and acceptable of an analytical
method, by different laboratories. Its use
provides a satisfactory and simple means of
assessing method precision acceptability.
The assumption that RSD
r
0.67 RSD
R
was
made by Horwitz, after he noted that values
for RSD
r
were, usually, between half and two
thirds of that of RSD
R
.

03/12/09 143
It should be noted that the equation has been
recalculated in the light of recent collaborative
trials. This has now been published by
Thompson, who recommends that, for values
less than 120 g/kg, a constant value for the
relative standard deviation of 22 should be
used. However, for many purposes, e.g.,
mycotoxins and pesticide residues, the original
form is still applicable, in many cases.
03/12/09 144
The AOAC manual for the Peer Verified
Methods program includes a table (Table 3.2),
with estimated precision data, as a function of
analyte concentration.
03/12/09 145
Table3.2. Analyte concentration versus precision within or
between days
Analyte Unit RSD ()
100 100 1.3
10 10
2.3
1 1 2.7
0.1 0.1 3.7
0.01 100 ppm 5.3
0.001 10 ppm 7.3
0.0001 1 ppm 11
0.00001 100 ppb 15
0.000001
10 ppb 21
0.0000001 1 ppb 30
03/12/09 146
Intermediate precision was previously known
as part of ruggedness. The attribute evaluates
the reliability of the method in a different
environment, other than that used during
development of the method.
03/12/09 147
The objective is to ensure that the method will
provide the same results, when similar samples
are analyzed once the method development
phase is over. Depending on time and
resources, the method can be tested on
multiple days, analysts, instruments, etc.
Intermediate precision in the test method can
be partly assured by good system suitability
specifications. Thus, it is important to set tight,
but realistic, system suitability specifications.
03/12/09 148
Reproducibility, as defined by ICH,
represents the precision obtained between
laboratories. The objective is to verify that
the method will provide the same results in
different laboratories. The reproducibility
of an analytical method is determined by
analyzing aliquots from homogeneous lots
in different laboratories, with different
analysts, and by using operational and
environmental conditions that may differ
from, but are still within the specified
parameters of the method (inter laboratory
tests).
03/12/09 149
Validation of reproducibility is important, if
the method will be used in different
laboratories.
Both repeatability and the reproducibility are
expressed in terms of standard deviation, and
are generally dependent on analyte
concentration. It is, therefore, recommended
that the repeatability, and within-laboratory
reproducibility, are determined at different
concentrations across the working range, by
carrying out 10 repeated determinations, at
each concentration level.
03/12/09 150
As stipulated by Horwitz and Albert (10), the
variability among laboratories is the
dominating error component in
the world of practical ultratrace analysis. They
conclude that a single laboratory cannot
determine its own error structure, except in
the context of certified reference materials or
consensus results from other laboratories.
03/12/09 151
Standard deviation
The standard deviation is usually understood
in terms of a hypothetical normal (Gaussian)
distribution of the error.
03/12/09 152
Ruggedness/robustness
Ruggedness is the ability of a chemical
measurement process, to resist changes in
results, when subjected to minor changes in
environmental and/or operating conditions,
similar to those likely to arise in different test
environments. It is a measure of how
effectively the performance of the analytical
methods stands up to, less than perfect
implementation.

03/12/09 153
In any method there will be certain parts,
which will severely affect the method
performance, unless they are carried out with
sufficient care. These aspects should be
identified and, if possible, their influence on
the method performance should be evaluated,
using the ruggedness tests, sometimes also
called robustness tests.
03/12/09 154
The ruggedness/robustness tests provide
important information for the evaluation of
the measurement uncertainty. The
methodology for evaluating uncertainty
given in the ISO Guide relies on identifying
all parameters that may affect the result
(that is, the potential sources of
uncertainty), and on quantifying the
uncertainty contribution from each source.
03/12/09 155
This is very similar to procedures used in
robustness tests, which identify all the
parameters, likely to influence the result, and
determine the acceptability of their influence
through control. If carried out with this in
mind, the robustness tests can provide
information on the contribution to the overall
uncertainty, from each of the parameters
studied.
03/12/09 156
Robustness is the ability of a chemical
measurement process, to resist changes in
results, when small but deliberate variations,
similar to those likely to arise during normal
usage, are applied to method parameters.
Robustness test examines the effect of
operational parameters on the analysis results.
03/12/09 157
For the determination of a chromatographic
method`s robustness, a number of
chromatographic parameters, for example,
flow rate, column temperature, injection
volume, are varied, within a realistic range,
and the quantitative influence of the variables
is determined.
03/12/09 158
If the influence of the parameter is within a
previously specified tolerance, the parameter
is said to be within the method`s robustness
range. Obtaining data on these effects will
allow to judge whether a method needs to be
revalidated, when one or more of parameters
are changed, for example, to compensate for
column performance, over time.
03/12/09 159
ICH defines robustness as a measure of the
method's capability to remain unaffected by
small, but deliberate variations in method
parameters. Robustness can be partly assured
by good system suitability specifications. Thus,
it is important to set tight, but realistic, system
suitability specifications.
03/12/09 160
In chromatographic procedures one has to
include System suitability tests(SST), which
will consist of the following investigations. (a)
Capacity factor, (b) Resolution, (c)Tailing
factor and (d) Theoretical plate number.
Details of these would be given in one of the
following sections.
03/12/09 161
Practicability
The ease of operation (in terms of sample
throughput and costs), the ability to achieve
the required performance criteria and,
thereby, meet the specified purpose.
Conclusions

In conclusion it can be stated that, in modern
laboratories, methods can be ~single
laboratory validated and, thus, produce
reliable results, without necessarily
undergoing wide interlaboratory trials.
03/12/09 162
Single laboratory method validation is
appropriate in several circumstances. One case
is the initial estimation of the performance of a
method, prior to a costly interlaboratory trial.
Another case is, when the correct use of ~off
the shelf validated methods is ensured.
Finally, when evidence of reliability is
provided in the case of methods, with no
collaborative studies available, or where the
conduct of such studies is not possible.
03/12/09 163
Single laboratory validation of methods,
developed for the determination of active
ingredients` and impurities` content of
pesticide formulated products, requires the
determination of specific performance
characteristics, defined specifically by CIPAC
and EU official guidelines. FAO/IAEA have
defined some additional parameters to be
assessed, in accordance with the general
specifications of AOAC International.
03/12/09 164
Methods should not be validated as a one-time
situation, but methods should be validated by
the developer or user, to ensure ruggedness or
robustness throughout the life of the method.
The variations, due to the laboratory sample
preparation procedure and the instrument
performance, contribute to the accuracy of the
data obtained for a particular analysis.
03/12/09 165
With proper validation and tight
chromatographic performance (system
suitability) criteria, an improvement in the
reliability of the data can be obtained. Only
with good reliable validated methods, can data
that are generated for purity, stability, and
pharmacokinetics be trust-worthy.
03/12/09 166
Control charts
Validation studies often demonstrate the
performance of an analytical method, before
its routine application. The validity of the
assessed performance for the routine
measurements can be controlled by repeated
analyses of control samples. The results are
monitored in a control chart, with warning
and action limits. Application of a stable
control sample also provides necessary
information for the interpretation of long
term, trend studies.


03/12/09 167
Method Validation approach in a typical
Chromatographic procedure
An outline of the validation approach in a
typical chromatographic analytical procedure
is given below.

Chromatographic methods are to be validated
before first routine use. Variables to be
considered are: Sampling procedure, sample
preparation, chromatographic separation,
detection and data evaluation, using the same
matrix as that of the intended sample.

03/12/09 168
The proposed procedure must go through a
rigorous process, verified by laboratory studies.
All validation data must be documented.
There are no general rules for validation--Only
guidelines are available. What is appropriate
for one application, in one laboratory, may not
be appropriate for another application in the
same or a different laboratory.
One has to demonstrate that, the performance
characteristics of the method are fit for the
intended purpose, and the evidence for this is
statistically valid.

03/12/09 169
System Suitability Specifications and Tests for
chromatographic methods

The accuracy and precision of data collected
begin with a well-behaved chromatographic
system. The system suitability specifications
and tests are parameters that provide
assistance in achieving this purpose.
03/12/09 170
1.Capacity factor

Capacity factor (K`) is a measure of where the
peak of interest is located with respect to the
void volume, i.e., elution time of the non-
retained components.
The peak should be well-resolved from other
peaks. Generally the value of k' is > 2.
03/12/09 171
2. Resolution (R)
R is a measure of how well two peaks are
separated. For reliable quantitation, well-
separated peaks are essential for quantitation.
This is a very useful parameter, if potential
interference peak(s) may be of concern. The
closest potential eluting peak to the analyte should
be selected. R is minimally influenced by the ratio
of the two compounds being measured.
R of > 2, between the peak of interest and the
closest potential interfering peak (impurity,
excipient, degradation product, internal standard,
etc.), is desirable.

03/12/09 172
3. Tailing factor (T)
The accuracy of quantitation decreases with
increase in peak tailing, because of the
difficulties encountered by the integrator in
determining where/when the peak ends, and,
hence, the calculation of the area under the
peak. Integrator variables are preset by the
analyst for optimum calculation of the area for
the peak of interest. If the integrator is unable
to determine exactly, when an upslope or down
slope occurs, accuracy drops.

03/12/09 173
4. Theoretical plate number (N)
Theoretical plate number (N) is a measure of
column efficiency. N is fairly constant for each
peak on a chromatogram with a fixed set of
operating conditions. H, or HETP, the height
equivalent of a theoretical plate, measures the
column efficiency per unit length (L) of the
column.

The theoretical plate number, in general,
should be > 2000.
03/12/09 174
System suitability testing is essential for the
assurance of the quality performance of the
chromatographic system. The amount of
testing required will depend on the purpose of
the test method. For acceptance, release,
stability, or impurities/degradation methods
using external or internal standards, k'
(capacity factor), T (Tailing factor), R
(Resolution) and RSD are recommended as
minimum system suitability testing
parameters.
03/12/09 175
In practice, each method submitted for
validation should include an appropriate
number of system suitability tests, defining the
necessary characteristics of that system.
The validation procedure should have data
demonstrating (a) Suitable accuracy, precision
and linearity over the range of interest (b)
Specificity of the method and the
determination of the limits of
detection/quantitation for all the components
including degradation products/impurities
03/12/09 176
(c) Recovery from the sample matrix
(d) That neither fresh nor degraded placebo
interferes with the method (e) Reproduction of
chromatograms and instrumental recordings
(f) Ruggedness of data: Characterising day-to-
day, lab.-to-lab, analyst-to-analyst and
column-to-column variability (g) System
suitability tests for chromatographic assays.
03/12/09 177
The evaluation of robustness should be
considered during the development phase, and
depends on the procedure under study. It
should show the reliability of an analysis with
respect to deliberate variations in the method
parameters. If measurements are susceptible
to analytical conditions, they should be
suitably controlled or a precautionary
statement should be included in the procedure.
03/12/09 178
Typical variations are: Stability of analytical
solutions, Extraction time, pH variations in
HPLC, Mobile phase composition,
temperature, flow rate, etc.

Modern chromatographic systems are
provided with GLP support softwares, which
help in method validation, and system
suitability evaluation. The parameters
evaluated include those already specified.
03/12/09 179
The System suitability test is performed, every
time, daily, when analysis is carried out. For
SST, in chromatographic assays, analysis is
made, using the standard sample for
calibration, control sample for repeatability or
accuracy check, unknown sample, and again
the control sample/standard sample. System
suitability standarsises the integrated system
for a given method.
03/12/09 180
Failing SS requirement invalidates the use of
the integrated system for the intended method
application.

When all the results of evaluation are satisfied,
the results of quantitative analysis of the
unknown are approved as valid data. Any
partial data obtained cannot be handled
separately
03/12/09 181
The case of some aspects of validation, carried
out for Isoproturon analysis in our laboratory,
will be illustrated. They include (a) Linearity
of response (b) System precision and (c) Assay
accuracy and precision.
Similar experiments for the impurities are to
be carried out. One, then, carries out the five
batch analysis, getting a closure as close to 100
as possible.
Structure of active ingredient and impurities
are to be given. Methods followed are to be
given.

03/12/09 182
Linearity should be evaluated by visual
inspection of signal vs concentration. If there
is a linear relationship, test results should be
evaluated by appropriate statistical
methods(e.g., by calculation of a regression
line, by the method of least squares,
correlation coefficient, Y-intercept, slope of the
regression line, etc.)

To establish linearity, a minimum of five
concentrations are required.
03/12/09 183
The range depends on the intended application
of the procedure. For assay of the major
component: 80-120 of the concentration. For
content uniformity: 70-130

For precision a minimum of 6 determinaltions
at 100 of the test concentration.

Accuracy should be assessed using a minimum
of 9 determinations over a minimum of 3
concentration levels covering the specified
range. Accuracy should be reported as percent
recovery.
03/12/09 184
ICH(International Conference on
Harmonisation) Guidelines: Minimum nine
determinations over a minimum of three
concentration levels covering specified
range(for e.g., 3 concns. With 3 replicates each


AOAC Guidelines given in Table 3.1 provide
Estimated Recovery at various concentration
levels.

03/12/09 185
Precision: ICH Guidelines: Minimum of 9
determinations for 3 concentration levels
covering specified range(3 levels with 3
replicates each)

AOAC Guidelines given in Table 3.2 give
estimated precision at various concentration
levels.
The data related to HPLC method validation
of isoproturon are given in Tables 3.3 to 3.9.
03/12/09 186
Table 3.3: Linearity of response for the determination of isoproturon

S.No. Nominal wt.


(mg/ 50 ml)

Concn. nominal

Peak areas`
1) 36.3

72.6

1734511



1750430
2) 40.2


80.4

1923978

1947210


3) 45.4





90.8

2238330
2165342
4) 50.5

101.0

2437310




2423821
5) 55.5


111.0

2683268



2640086
6) 60.6

121.2

2891407



2856872
7) 65.6


131.2

3120371



3124240
* Normalised with respect to IS
03/12/09 187
Table 3.4
System precision for the determination of Isoproturon

S.No. Determined
peak areas`



Mean Concn.
(mg/ml)
Coeff. of
Variation
(!
"! 2406344
#! 2407694
3! 2405475
$! 2405442 2403779 1.01 0.24
%! 2400166
&! 2392064
'! 2409267

`Normalised with respect to IS
03/12/09 188
Table 3.5
Assay accuracy and precision of Isoproturon active estimation


Nominal
concn.
(mg/50ml)

Determined
concn.
(mg/50ml)



recovery Mean
recovery
()
Coeff. of
variation



()
1)


40.2
100.8



40.6
40.5
40.5


101.0
100.7
100.7
100.7 0.17

2)
45.4

45.9
45.9
45.9

101.1
101.1
101.1

101.1

0.0
3)
50.5



50.9
50.9
50.9


100.8
100.8
100.8
100.8 0.0

4)

55.5




55.4

55.9
55.9


99.8
100.9
100.9
100.5 0.63
5)
60.6


60.1

60.1

60.1


99.2

99.2
99.2

99.2 0.0

Overall assay accuracy: 100.5;
Overall assay precision:0.16
03/12/09 189
Table 3.6
Linearity response for impurity 1




S.No. Nominal

wt. mg/ml


Concn.
nominal

Determined
peak areas

Coeff. Of
correlation
1) 0.0019


0.09 22410, 22023
2) 0.0040


0.20 49652, 49836
3) 0.0060


0.30 76170, 79460

0.9965
4) 0.0080


0.40 100814, 100716
5) 0.0100

0.50 116455, 118623
6) 0.0119


0.60 139046, 138684

03/12/09 190
Table 3.7
System precision for the determination of impurity 1



S. No. Determined


peak areas


Mean Concn.
(mg/ml)
Coeff. of
variation
1) 137835
2) 139955
3) 139046
4) 138684

138091 0.0119 1.6
5) 133854
6) 140420
7) 136846


03/12/09 191
Table 3.8
Assay accuracy and precision of impurity 1


Nominal
concn.
(mg/ml)




Determined
concn.
(mg/ml)


recovery Mean
recovery
Coeff. Of
variation
0.0019

0.0018
0.0018
0.0017


94.7
94.7
89.5
93.0 3.2
0.0040

0.0040

0.0040
0.0040

100.0
100.0
100.0
100.0 1.5
0.0061

0.0061
0.0064
0.0058



100.0
104.9
95.1

100.0 4.9

0.0080

0.0081
0.0081
0.0080


101.3
101.3
100.0
100.9 0.74

0.0100

0.0093

0.0095
0.0101

93.0
95.0
101.0
96.3 4.3

0.0119


0.0111

0.0111

0.0110


93.3
93.3
93.0

93.3 0.56
Overall assay accuracy : 97.1
Overall assay precision : 2.5
03/12/09 192


`Normalised with respect to IS
Table 3.(
)soproturon % batch analysis

Batch no.


Component
A,(w/w)
Component
D,(w/w)
Total
1)

99.1 0.58 99.7
2)

98.8 0.64 99.4
3)

98.6 0.70 99.4
4)

99.1 0.70 99.8
5)

98.9 0.64 99.5

Impurities B, C, E and F are less than 0.05
The structres of the active ingredient and impurities are to be presented.
A description of each method used in the study is to be presented.

03/12/09 193
GLP COMPLIANCE OF GC and HPLC
The following parameters are to be specified
by the user of GC/HPLC.

(a) Limit of detection (b) Limit of quantitation,
(c) Selectivity, (d) Detector sensitivity and
linearity and (e) Accuracy.
03/12/09 194
Automatic performance measurement
generally includes, plate number of a column,
capacity factor, peak width at half height,
tailing factor, resolution between two peaks,
selectivity relative to preceding peaks,
precision of peak retention time, peak areas,
heights or amounts, baseline noise and S/N,
baseline drift and linearity.
03/12/09 195
The quality of the analytical data can be
supported by keeping records of the actual
instrument conditions at the time of
measurement. Precolumn pressure and
temperature of the column compartment are
recorded before, during and after the run and
are stored together with chromatographic raw
data.
03/12/09 196
The system suitability test includes testing of
sample injection, chromatographic separation
and associated data handling. A system
suitability test should be designed so that it
proves whether the method achieves the same
or better accuracy/precision on the current
system as shown during validation.

03/12/09 197
Substituted benzenes have peaks at 205 nm for
low wavelength checks. Anthracene shows
absorbance peaks at 250 and 360 nm for
calibration checks at mid and near UV
wavelengths. Far UV transparent solvents like
acetonitrile or water must be used for
wavelength calibration checks in the far UV,
mid UV and near UV regions.
03/12/09 198
The universal test mixture used consists of 4
alkyl benzenes(Toluene, ethyl benzene,
isopropyl benzene and 1-butyl benzene) and
anthracene.
The relative standard deviations in area and
retention time can be arrived at by overlaying
6 replicate injections of known amounts(say 5
microlitres).

Flow rate sensitivity can be evaluated by
carrying out HPLC with 0.02-0.03 ml/min.
increments(In all the five cases of analytes).
03/12/09 199

Injection sequence for holistic validation

procedure
03/12/09 200

03/12/09 201
CURRENT APPROACHES TO VALIDATION

In a recent review, van der Voet and co-workers
discuss current approaches to validation of
analytical chemical methods, identifying some
shortcomings of existing validation schemes, such
as insufficient coverage of variability in space or
time and mismatches between validation criteria
and intended use of the method, giving an
example of regulatory control.
03/12/09 202
The authors make an attempt to link
validation concepts used in different fields,
such as measurement uncertainty, and the
prediction error. They recommend general
statistical modelling approach for combining
different aspects of validation, and illustrate it
with an example. This type of modelling
should be the basis for the development of new
statistically underpinned validation schemes,
which integrate current validation and quality
assurance activities.
03/12/09 203
It is stated that validation includes the initial
assessment of performance characteristics,
several types of inter-laboratory testing, and
quality control. Validation is thus concerned
with assuring that a measurement process
produces valid measurements; this has also
been called measurement assurance.
03/12/09 204
The validation of an analytical method as a
concept may be understood in (at
least) three senses. In the narrow and
traditional sense, the term denotes validation
of a chemical method, as described in a
standard operating procedure (SOP). In a
wider sense, validation may be concerned with
a method of analysis (e.g., in an ISO standard)
which explicitly leaves freedom to adapt the
procedure to the infrastructure, in a specific
situation. In this case, there are more SOPs, all
in conformity with the master method.
03/12/09 205
Finally, in a still wider and perhaps
unconventional sense, validation of analytical
methods may be considered from the
perspective of those, who use analytical results
for other purposes. The method of analysis for
end-users of analytical results amounts to the
specification of an analytical result (e.g.,
clenbuterol in liver.), with the implied
statement: analysed by any reasonable
method.
03/12/09 206
Accordingly, a specific method of analysis in the
analytical chemical sense can be considered as
just one realisation of the class of all methods,
currently applied to measure component X in
matrix Y.

03/12/09 207
03/12/09 208
The second edition of the ISO 5725 standard has
much in common with the IUPAC/ISO/AOAC
Protocol for design, conduct and interpretation of
collaborative studies. Important contributions to
some of the problems mentioned above were
made in two other protocols on proficiency
testing (PT), and on internal quality control
(IQC).
03/12/09 209
The harmonised international protocol for the
proficiency testing of (chemical) analytical
laboratories considers laboratory performance
studies, in which each laboratory uses its own
analytical method, as opposed to the method-
performance studies of ISO 5725.
03/12/09 210
Although the primary purpose of proficiency
testing is often the evaluation or improvement
of laboratory performance, it is also
reasonable to consider it as a method-
performance validation in the wider sense of
the definition. This would solve problems of
different laboratories having different SOPs,
and of SOPs changing every now and then in
each laboratory.
03/12/09 211
The prescribed repetition in proficiency testing
schemes considers reasonable, the frequencies
of once every two weeks to once every four
months. This solves the problem with the static
nature of ISO 5725 validation, and, by varying
the test materials, the problem of not assessing
matrix variability.
03/12/09 212
Despite all the advantages, proficiency testing
according to the IUPAC guidelines (16) cannot
be considered as a complete validation
methodology on its own. First of all, it does not
provide for SOP-specific validation. More
importantly, the scheme requires repeated
interlaboratory studies, which severely
restricts the amount and variety of samples
that can be analysed.
03/12/09 213
Therefore, proficiency testing is an extensive
validation methodology. Finally, the current
protocol has limited consideration of
performance to laboratory bias, most often in
the form of a z-score. This information alone
may be insufficient to evaluate a method`s
fitness for the purpose.
03/12/09 214
It has been shown that an effective
measurement assurance requires validation
at different scales. Newly developed or
implemented methods are usually first
validated through in-house validation. This
type of validation should be supplemented by
ongoing internal quality control validation in
each laboratory, and by participation of the
laboratory in interlaboratory schemes.
03/12/09 215
Considering the complex nature of many
modern methods of analysis, proficiency
testing schemes, allowing laboratory-specific
SOPs, are more to the point than the method-
evaluating schemes like ISO 5725.
03/12/09 216
Currently, the three validation schemes, in-
house validation, internal quality control, and
proficiency testing, are not sufficiently linked.
The development of integrated validation
approaches is possible from the various
guidelines available.
03/12/09 217
A recent 1oint FAO/IAEA Expert
Consultation on validation of analytical
methods
for food control defined the .ideal validated
method. as follows:
The ideal validated method is one that has
progressed fully through a collaborative
study in accordance with internationally
harmonised protocols for the design, conduct
and interpretation of method performance
studies.
03/12/09 218
This usually requires a study design involving
a minimum of 5 test materials, the
participation of 8 laboratories reporting valid
data, and most often includes blind replicates
or split levels, to assess within-laboratory
repeatability parameters.

Limiting factors for completing ideal multi-
laboratory validation studies include
high cost, lack of expert laboratories available
and willing to participate in such studies, and
overall time constraints.
03/12/09 219
Validation by using different analytical
methods.
Above all, the described validation strategies
assume that methods are applied on a
routine basis in various laboratories. In a
research environment, a rather unique method
might be developed and validated for the use
in only one or a few studies. Then, of course,
one would like to establish the same amount of
information on the validity of the method.
03/12/09 220
However, some of the usual tools, like
participation in proficiency schemes and the
use of reference materials are probably not
possible.
Hogendoorn and co-workers discuss a number
of practical examples such asscreening and
analysis of polar pesticides in environmental
monitoring programmes by coupled-column
liquid chromatography and gas
chromatography-mass spectrometry (GC-MS).
03/12/09 221
One example is a study on the levels of
ethylenethiourea (ETU) in groundwater. In the
validation of both methods, the calibration
procedure is very important, and provides
information for several of the criteria. The
calibration is based on spiked samples, which are
comparable to samples of groundwater to be
analysed which, by comparison with the analysis
of standards, in itself gives the recovery data.
03/12/09 222
The calibration samples are analysed, in time,
around and between the real samples.
Possible influence of the conditions during the
analysis (ruggedness) of the samples
is automatically captured in the calibration
sequence.

To determine the working range of both methods,
the calibration data are evaluated
by the Calwer spreadsheet application.
03/12/09 223
The application enables an extensive evaluation
of the calibration curve with respect
to the appropriate calibration model , showing
that the assumption of equal variance over the
working range is nearly always severely violated.

The strategy of variance models, weighted
regression in combination with the maximum
likelihood criterion, is formalised for method
comparison calibration/validation in ISO.
03/12/09 224
The selectivity of the method is checked by
comparing the shape and the retention
of real samples and calibration samples with
the chromatogram of standard
solutions. More details are available in the
paper cited.
03/12/09 225
Interlaboratory method validation

Another approach to validation is by
interlaboratory tests. An example of the
validation of GC determination of
chlorophenols in water has been summarized.
Details can be found in reports of
Hoogerbrugge and co-workers.
The results obtained complied with the general
variation in interlaboratory studies as found
by Horwitz.
03/12/09 226
03/12/09 227
03/12/09 228
Accreditation
Accreditation of test laboratories, and adoption
of certified Quality systems by manufacturing
industries, can eliminate repeated checks on
quality of the same product. Mutual recognition
of test results between organisations would
automatically emerge, if both adopt quality
systems. Accreditation provides independent
endorsement of a laboratory`s quality systems.
In the field of chemical testing, it is recognised as
an essential ingredient in achieving valid
analytical measurements.
03/12/09 229
OTHER USEFUL ELEMENTS IN IN-
HOUSE VALIDATION PROCEDURE

A Comprehensive Discussion of the Role of
Certified Reference Materials(CRM) in
Chemical Analysis

At this point, it would be worthwhile to
provide a comprehensive coverage of CRMs
used in chemical analysis.
03/12/09 230
It must be emphasized that standard samples,
which have been analysed by a number of
skilled analysts, are commercially available.
These include, certain primary
standards(sodium oxalate, potassium
hydrogen phthalate, arsenic(III) oxide and
benzoic acid) and ores, ceramic materials,
irons, steels, steel -making alloys and non-
ferrous alloys.
03/12/09 231
Reference materials play an important role in
analytical chemistry, where they are used by
analysts for a variety of purposes, including:
checking and calibrating instruments;
validating methods and estimating the
uncertainty of analytical measurements;
checking laboratory and analyst performance;
and internal quality control.
03/12/09 232
Literature provides adequate guidance and
information for the users of Certified
reference materials (CRMs), explaining how
they can best be used to achieve valid
analytical measurements, and improve quality
in the analytical laboratory. General
information on CRMs, and how they are
produced, have been well documented. The
main uses of CRMs, and statistics relating to
CRM use, are also available.
03/12/09 233
If relevant reference materials are available,
they are a powerful tool in the assessment and
control of the accuracy of the performance of
the applied analytical method. In practical
laboratory application, one should, however,
realise that, additional control experiments are
often necessary since, reference materials
usually do not reflect the complete range of
application of the method with respect to
concentration, matrix effects, and possible
inhomogeneity of samples.
03/12/09 234
All chemical measurements, whether
qualitative or quantitative, depend upon and
are, ultimately, traceable to a CRM, or
standard material of some sort. Qualitative
measurements of identity based(e.g., retention
time measurement in gas chromatography or
spectroscopic properties), require a reliable,
authentic, reference material to calibrate the
particular instrument or test used.
03/12/09 235
Quantitative determination have the
additional requirement, that the instrument is
calibrated with an accurately known amount
of the reference material concerned. This is
because, an instrument generates only a signal
, and it is the responsibility of the analytical
chemist to convert the signal into
concentration.
03/12/09 236
An instrument converts a chemical
information into an observable form. (i) It
generates a signal. (ii) Transduction, (iii)
Amplification, and (iv) Display of results.
Developments in electronics have influenced
chemical instrumentation in a bewildering
manner(Fig. 3.3).
03/12/09 237

Fig.3.3. Parts of a typical instrument
SIGNAL GENERATOR

ANALYTICAL SIGNAL

DETECTOR
ELECTRICAL/

MECHANICAL
INPUT SIGNAL
SIGNAL PROCESSOR


METER RECORDER DIGITAL
(ANALOGUE PRINTER-PLOTTER/ READ
SIGNAL) INTEGRATOR OUT



03/12/09 238
Emphasis on CRM in the Six VAM Principles
The key role of CRMs in analytical chemistry
is recognized in the six Valid Analytical
Measurements (VAM) principles indicated
earlier.
The testing of methods and equipment,
referred to in VAM Principle 2, is most
effectively accomplished by the use of
appropriate CRMs.
03/12/09 239
For example, the accuracy of the wavelength
scale of UV-Visible HPLC detector can be
verified by the use of a CRM, comprising a
solution, that has absorption peaks, with well
characterised reference values for the
wavelengths of maximum absorbance.
Likewise, the accuracy of an entire analytical
method can be checked by the use of a CRM.
03/12/09 240
VAM Principle 5 emphasises the importance
of comparability between analytical data,
produced in different laboratories and
locations. One effective means of achieving
this is, to ensure that all analytical data are
traceable to reliable CRMs.
A laboratory can check the repeatability of its
data, by setting up Internal Quality
Control(IQC) procedures, to provide evidence
of day-to-day consistency in its results, but a
laboratory relying exclusively on IQC
procedures could conceivably be producing
consistent, but biased results.
03/12/09 241
The use of CRMs, as measurement bench
marks, can provide the essential reference or
anchor points, against which analysts can
achieve comparability of their measurements.
When several laboratories can achieve the
same analytical result(within the uncertainty
specified), for a given CRM, they will have
demonstrated the comparability of their
measurements.
03/12/09 242
Instrument
Performance
Verification
Instrument
Calibration
Laboratory
Performance
Verification
REFERENCE
MATERIALS
Method
Validation
Internal Quality
Control
Uncertainty
Estimation
Analyst Performance
Verification
The areas in which Reference materials would find use
are depicted in Fig. 3.4.
Figure 3. 4. Areas in which Reference materials would find use
03/12/09 243
The Reference Material as a Transfer
Standard in the Traceability Chain
Most results of chemical analytical
measurements are directly related to reference
materials, which are used for the calibration of
the measurement process. Two decisive
preconditions must be fulfilled in order to
obtain a result, that is traceable to the SI
(Systme International d`Units). First, the
reference material itself must carry an SI-
traceable value, and an attached uncertainty.
03/12/09 244
Second, the whole measurement procedure -
from sampling to calculation of the result -
must be fully validated, and the uncertainty
must be evaluated according to common rules
While the analyst has to validate his
procedure, and to evaluate his measurement
uncertainty by himself, he may take for
granted, that the value declared on the label of
the reference material is traceable to the SI.
Thus, the reference material plays a most
important role, as it serves as a transfer
standard in the traceability chain.
03/12/09 245
One of the key factors affecting laboratories`
capabilities to produce reliable test data is the
availability of standard materials, with property
values, that can be relied upon by their users.
The quality of a result is dependent on the quality
of standards used to validate a method, or
calibrate an analytical instrument. Every day,
standards are used to calibrate instruments, or
validate test methods, e.g., pH meters,
Conductivity systems, Auto-titrators, Karl-Fisher
titrators, Chromatographs, etc. If the standard is
not accurate, this inaccuracy will be reflected in
the test result.
03/12/09 246
~No matter how skilled the analysts or how
sophisticated the analytical technique used, if
the calibration of the system is in error, the
analytical result will always be wrong.
The definitions of various kinds of Reference
materials used by analysts, and some related
aspects, published by NIST, are summarized
below.
03/12/09 247
Certified Reference Material (CRM) *
Reference material, accompanied by a
certificate, one or more of whose property
values are certified by a procedure, which
establishes traceability to an accurate
realization of the unit in which the property
values are expressed, and for which, each
certified value is accompanied by an
uncertainty at a stated level of confidence.
03/12/09 248

In other words, a CRM is a reference material,
one or more of the property values of which
are certified by a technically valid procedure,
accompanied by a certificate(See Annexure
III), or other documentation, which is issued
by a certifying body. This certificate will
provide detailed information on the analyte
values, their associated uncertainties, methods
of analysis, and traceability. Their production
and certification is expensive. Therefore, they
are not used for routine analysis.
03/12/09 249
Reference Material (RM) - Material or
substance, one or more of whose property
values are sufficiently homogeneous and well
established, to be used for the calibration of an
apparatus, the assessment of a measurement
method, or for assigning values to materials.
Reference Material Certificate - Document
accompanying a certified reference material,
stating one or more property values and their
uncertainties, and confirming that the
necessary procedures have been carried out to
ensure their validity and traceability.
03/12/09 250
NIST Standard Reference Material (SRM) -
A CRM, issued by NIST, that also meets
additional NIST-specific certification criteria,
and is issued with a certificate or certificate of
analysis, that reports the results of its
characterizations, and provides information
regarding the appropriate use(s) of the
material (NIST SP 260-136).
03/12/09 251
Note: An SRM is prepared and used for three
main purposes: (1) to help develop accurate
methods of analysis; (2) to calibrate
measurement systems used to facilitate
exchange of goods, institute quality control,
determine performance characteristics, or
measure a property at the state-of-the-art
limit; and (3) to ensure the long-term
adequacy and integrity of measurement
quality assurance programs.
03/12/09 252
NIST Reference Material - Material issued by
NIST, with a report of investigation, instead of
a certificate to: (1) further scientific or
technical research; (2) determine the efficacy
of a prototype reference material; (3) provide
a homogeneous and stable material so that
investigators in different laboratories can be
ensured that they are investigating the same
material;
03/12/09 253
and (4) ensure availability, when a material
produced and certified by an organization
other than NIST, is defined to be in the public
interest, or when an alternate means of
national distribution does not exist. A NIST
RM meets the ISO definition for a RM, and
may meet the ISO definition for a CRM
(depending on the organization, that produced
it).
03/12/09 254
NIST Traceable Reference Material
TM
(NTRM
TM
) - A commercially-produced
reference material with a well-defined
traceability linkage to existing NIST standards
for chemical measurements. This traceability
linkage is established via criteria and protocols
defined by NIST to meet the needs of the
metrological community to be served (NIST
SP 260-136). Reference materials producers,
adhering to these requirements, are allowed
use of the NTRM trademark. A NIST NTRM
may be recognized by a regulatory authority,
as being equivalent to a CRM.
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NIST Certified Value - A value reported on an
SRM certificate or certificate of analysis, for
which NIST has the highest confidence in its
accuracy in that, all known or suspected
sources of bias have been fully investigated or
accounted for by NIST. (NIST SP 260-136)
NIST Reference Value - A best estimate of the
true value provided on a NIST certificate,
certificate of analysis, or report of
investigation, where all known or suspected
sources of bias have not been fully investigated
by NIST. (NIST SP 260-136)
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NIST SRM Certificate or Certificate of
Analysis - In accordance with ISO Guide 31:
2000, a NIST SRM certificate is a document
containing the name, description, and intended
purpose of the material, the logo of the U.S.
Dept. of Commerce, the name of NIST as a
certifying body, instructions for proper use
and storage of the material, certified property
value(s) with associated uncertainty(ies),
method(s) used to obtain property values, the
period of validity, if appropriate, and any
other technical information deemed necessary
for its proper use.
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A Certificate is issued for a SRM, certified for
one or more specific physical or engineering
performance properties, and may contain
NIST reference, information, or both values, in
addition to certified values. A Certificate of
Analysis is issued for a SRM, certified for one
or more specific chemical properties. Note:
ISO Guide 31 is updated periodically; ISO
provides the updated version.
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NIST Certificate of Traceability - Document
stating the purpose, protocols, and
measurement pathways, that support claims
by an NTRM to specific NIST standards or
stated references. No NIST certified values are
provided, but rather the document references
a specific NIST report of analysis, bears the
logo of the U.S. Department of Commerce, the
name of NIST as a certifying body, and the
name and title of the NIST officer, authorized
to accept responsibility for its contents.
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NIST RM Report of Investigation - Document
issued with a NIST RM, that contains all the
technical information necessary for proper use
of the material, the logo of the U.S.
Department of Commerce, and the name and
title of the NIST officer, authorized to issue it.
There are no NIST certified values provided,
and authorship of a report's contents may be
by an organization, other than NIST.
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NIST Report of Analysis (ROA) - Document
containing the certification of the material,
and including such information as the base
material used, how the SRM was
manufactured, the certification method(s) and
description of procedures, outside
collaborators, instructions for use, special
instructions for packaging, handling, and
storage, and plan for stability testing. The
ROA is intended for internal NIST use only.
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The goal of any analytical measurement is to
get accurate, reliable and consistent data.
Prerequisites for achieving accurate results in
analytical laboratories are correct sampling,
correct weighing of the sample and standards,
use of well-maintained and calibrated
equipment, qualified operators, validated
methods and procedures for data validation.
Most important is the use of accurate
standards or Certified Reference Materials.
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Eventhough the chief role of reference
material is to ensure accuracy for a specific
method, there is another, equally important
use of such materials: they enable the
laboratory and a specific user to verify the
performance of equipment, procedures and
operators at any time.
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Agreement in analysis results with the certified
value proves that, not only the method is right,
but also the equipment and the chemicals used
for sample preparation are right, and that the
operator did a good job. The laboratory can
conclude that, the data generated for this
particular procedure are correct.
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All laboratories obtaining the same results are
'intercalibrated', and in line with the
technically competent organization, which
certified the material. Any disagreement
between the certified value and the value
determined by the laboratory indicates a
problem with the analysis, which requires a
thorough follow-up.
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Commercially available Reference Materials

Reference materials are available from a large
number of producers. Among them are private
companies, federal research laboratories, and
metrology institutes. ISO has set up several
rules to assure a suitable quality of reference
materials which should be clearly indicated in
a certificate(See Annexure III).
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Producers of CRM`s include
National Institute of Standards and
Technology NIST (USA)`
Commission of the European Communities
BCR (Belgium)
Laboratory of the Government Chemist LGC
(UK)
National Institute for Environmental Studies
NIES (1apan)
Laboratoire National d`Essais LNE, (France)
and many more. A comprehensive list of ref.
standard suppliers is given in Annexure I.
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Criteria for all Standard Materials
+tability
The stability of a standard material is defined
as its ability to maintain its property / value
over a specified period of time. The aim of
stability testing is to determine whether the
standard maintains its reference value from
the time of production to time of use. The
frequency of stability testing depends on the
risk of any change in reference value with
time.
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Resulting from a well defined stability trial,
one should be in a position to
(i) Identify suitable packaging materials,
Determine shelf life of the product,
(ii) Determine optimal storage conditions, for
the standard material being investigated.

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The ideal standard material should be
physically and chemically stable. Most
materials change due to evaporation, or
chemical reaction initiated by temperature,
light, air, or humidity. Precipitation,
bacteriological activity, and interaction with
storage container may lead to instability. The
producer of the standard should carry out
extensive stability studies, and assign expiry
dates, based on these studies.
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,omo-eneity
The Standard must be homogeneous; i.e., the
difference between representative sample
measurements must be less than the overall
uncertainty limits of the measurement. A
material can only be said to be homogeneous
at or above the weight / volume of the
representative samples analysed.
Accuracy of .eference Value
Accuracy of the value / property of a standard
material may be defined as the closeness of the
agreement between the reported value and the
true value of the analyte.
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/ncertainty of .eference Value
The reference value of the material must be
determined with rigorous estimates of
uncertainty, the value being as close as
possible to the true value. Uncertainty of
Measurement is defined as the parameter
associated with the result of a measurement
that characterizes the dispersion of the values,
that could reasonably be attributed to the
measurand.
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Application of Standards
Standards provide vital ingredients in every
day lab. analysis; these include,
Instrument calibration, ensuring the analytical
device is giving a correct result over the
analyte range of interest. It is always advisable
to check operator`s manual for the
manufacturer`s requirements for frequency of
"actual calibration" of an instrument.
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The broad areas of application of standards
include, Validation of test methods, Analytical
QC, Production of working standards,
Establish traceability, Check equivalence of
methods and Comparability.
By the use of Standards, the analyst can
demonstrate the validity of an analysis,
the concept of measurement traceability can
be realized and the comparability of analytical
data between laboratories can be improved.
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Selection of a Standard

When deciding on the most appropriate
standards for a particular use, one must first
decide on the accuracy of result required. The
use of Primary Standards / CRMs are
appropriate to work of highest accuracy, or
when a specific procedure requires their use.

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Secondary standards are more appropriate to
routine analysis. They provide known
accuracy, traceability, certification, and
economy.

When selecting a Standard, the analyst must
consider, the accuracy of standard required,
stability of standard, matrix of standard and
concentration of analyte (value of standard).
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General Guidelines for Handling Standard
Materials.
Standards, when supplied, are accurate,
homogeneous, and stable. The guidelines to
handle and store standards are given below.
Store in original container, Store in
accordance with accompanying instructions,
Check the Expiry Date before use, Replace cap
when standard is not in use, Avoid contact
with fumes, Avoid the possibility of
contamination during removal of sample.
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Dispose of any unused` sample of Standard
material; do not return to the original
container. Standards are chemicals, in some
cases they may be classed as hazardous
substances. One must handle, and dispose of,
in accordance to local safety regulations.
Most importantly they must be accompanied
by appropriate certification.
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Standard samples can be obtained from
various sources. Annexures 1 and II give
Sources of reference materials, and important
guidelines available for Certification of
reference standards, respectively. In Annexure
III a typical Certificate issued by NIST(for
Hydrogen sulfide) is reproduced, just to
illustrate the various aspects covered in such a
certificate.
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Assigning the traceable value to the CRMs
There have been several discussions on how
the traceable value has to be assigned to the
reference material, and who is responsible for
the assignment. However, there is still no
generally accepted guideline at present. In
fact, one has to face a contradictory situation
today. On the one hand, there is a growing
demand for reference materials, which carry
traceable values. On the other hand, assigning
a traceable value means putting much effort
into a primary measurement.
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For a private company, this may not be
economically feasible. Thus, not all the
produced reference materials will carry
traceable values.
Several studies at LNE (Laboratoire National
d`Essais, France) and EMPA (Swiss Federal
Laboratories for Materials Testing and
Research) have shown that even the values
declared for monoelemental standard solutions,
which are used for calibration and which are
relatively simple materials with respect to their
matrices, are often not traceable.
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An Effective Solution

As the need for reference materials will keep
growing, a division of tasks may be the most
effective solution. Federal institutes and
private companies will focus on their unique
skills and core activities. While a private
company may be able to produce a reference
material very efficiently on a large scale, they
may not be
equipped for the assignment of a traceable
value.
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In many cases, the declared value has been
regarded as a traceable value by most analysts.
Federal institutes, especially the metrological
institutes, are well-equipped to assign a
traceable value, because, normally, it is one of
their core tasks to provide the link to the SI, by
performing a primary measurement. However,
they often may not be equipped well enough
for an efficient production.
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Regarding the large number of reference
materials that are already available, one could
state, that there is already enough production
capacity. There is, however, a lack of capacity
for a traceable value assignment. As a
consequence, it would be more effective to
combine the different skills in a
complementary way.
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Concept for the Value Assignment of
Reference Materials

The way, described above, has been chosen by
several well reputed organizations. A suitable
quality assurance system is required from each
partner. An ISO DIS 9001:2000 certification is
in place at EMPA, and FLUKA. The
production and certification of a
monoelemental standard solution follows an
acceptable flow chart, with different well
defined steps.

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If several fundamentally different methods of
analysis for a given constituent are available,
e.g., gravimetric, titrimetric,
spectrophotometric or spectrographic, the
agreement between atleast two methods of
essentially different character can, usually, be
accepted, as indicating the absence of an
appreciable systematic error in either( a
systematic error is one which can be evaluated
experimentally or theoretically).
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Primary Standards(Primary reference
materials)
A Primary Reference Material is one, whose
value is accepted without reference to other
standards of the same quality. The standard
must be pure, stable, have high equivalent, be
soluble under the conditions in which it is to be
used, react with the standard solution
instantaneously and stoichiometrically.
Primary Standards are expensive, but offer the
highest accuracy.
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Many other relevant information regarding
primary standards given in Ref. (36A) and are
summarized in several literature sources.
A primary standard is a compound of
sufficient purity from which a standard
solution can be prepared by direct weighing of
quantity of it, followed by dilution to give a
defined volume of solution. The solution
produced is then primary standard solution.
The substances commonly employed as
primary standards are given below.
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Acid - Base reactions: Sodium carbonate
Na
2
CO
3
, Sodium tetraborate Na
2
B
4
O
7
,
Potassium Hydrogen phthalate KH (C
8
H
4
O
4
),
Potassium Hydrogen Iodate KH (IO
3
)
2
.
Complex formation reactions: Pure metals
(e.g. Zinc, magnesium, copper and manganese)
and salts, depending upon the reaction used.
Precipitation reactions: Silver, Silver nitrate,
sodium chloride, potassium chloride and
potassium bromide.
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Oxidation - Reduction titrations: K
2
Cr
2
O
7
,
KBrO
3
, KIO
3
, KH (IO
3
)
2
, Potassium Oxalate,
Na
2
C
2
O
4
, arsenic (III) oxide As
2
O
3
and pure
iron.

Hydrated salts, as a rule, do not make good
standards; this is because it is difficult to dry
them efficiently. However, those salts which do
not effloresce, such as sodium tetraborate
Na
2
B
4
O
7
. 10 H
2
O and copper sulfate CuSO
4
.
5H
2
O are found by experiment to be
satisfactory secondary standards.
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Secondary Standards
A secondary standard is a substance, which may
be used for standardization, and whose content of
the active substance has been found by
comparison against a primary standard. It
follows that, a secondary standard solution is
solution in which the concentration of dissolved
solute has not been determined from the weight
of the compound dissolved but by reaction
(titration) of a volume of the solution against a
measured volume of primary standard solution.
A secondary standard is traceable to a primary
standard, or a CRM.
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Working Standard

A Working Standard is used as an alternative
to a secondary standard. It may be
characterized by, either primary or secondary
methods. Typical applications would include
internal quality control, and instrument
performance checks. Typically it might be
obtained by dilution of a Secondary Standard.

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Certification of Secondary Standards
As the reported test results forms the basis for
the Certificate of analysis` of the standard,
the testing procedure is critical to the reported
value. All tests must be carried out using
Validated methods. Such methods must be
technically valid, i.e.,
i) have a valid and well described practical
foundation, (ii) have been experimentally
evaluated so that reported results have
negligible systematic errors, (iii) have a high
level of precision and (iv) have high reliability.
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Where possible, a primary method should be
used, i.e., titrimetry, gravimetry, coulmetry
and depression of freezing point.
The term traceability is used to describe the
reliability of measurements, but it is not
always clear exactly what that means(36H). In
the science of chemical measurements
comparability of results is a primary
requirement, and traceability is a tool to help
achieve comparability.
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Definition of traceability provided in ISO 8402
is ~Traceability is the ability to trace the
history, application or location of an entity by
means of recorded identifications.
Nowadays, there is great emphasis, not only
on the precision of the results obtained using a
specified method, but also on their traceability
to a defined standard or SI unit. Traceability
refers to the completeness of the information
about every step in a process chain.
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The formal definition of Traceability is the
~ability to chronologically interrelate the
uniquely identifiable entities in a way that
matters. The term is, for example, used to
refer to an unbroken chain of measurements,
relating an instrument`s measurements to a
known standard. Traceability can be used to
certify an instrument's accuracy, relative to a
known standard.
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