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THE ROLE OF REFERENCE MATERIALS AND EQUIPMENT

QUALIFICATION IN METHOD VALIDATION
a b c
Ileana Liliana Popescu , I. Gh. Tanase & Hassan Y. Aboul-Enein
a
National Medicine Agency, Control Department , 48 Av. Sanatescu Street, Bucharest 1,
Romania
b
Faculty of Chemistry , Department of Analytical Chemistry , University of Bucharest , 4-12
Blvd. Elisabeta, Bucharest 3, 703401, Romania
c
Pharmaceutical Analysis Laboratory , Biological and Medical Research, Dept., MBC-03-65 ,
King Faisal Specialist Hospital and Research Centre , P.O. Box 3354, Riyadh, 11211, Saudi
Arabia
Published online: 25 Oct 2011.

To cite this article: Ileana Liliana Popescu , I. Gh. Tanase & Hassan Y. Aboul-Enein (2002) THE ROLE OF REFERENCE MATERIALS
AND EQUIPMENT QUALIFICATION IN METHOD VALIDATION, Analytical Letters, 35:7, 1107-1116, DOI: 10.1081/AL-120005961

To link to this article: http://dx.doi.org/10.1081/AL-120005961

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 ANALYTICAL LETTERS
Vol. 35, No. 7, pp. 1107–1116, 2002

GUEST EDITORIAL

THE ROLE OF REFERENCE MATERIALS

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AND EQUIPMENT QUALIFICATION IN

METHOD VALIDATION

Ileana Liliana Popescu,1 I. Gh. Tanase,2

and Hassan Y. Aboul-Enein3,*
1
National Medicine Agency, Control Department 48
Av. Sanatescu Street, Bucharest 1, Romania
2
University of Bucharest, Faculty of Chemistry,
Department of Analytical Chemistry, 4-12 Blvd.
Elisabeta, 703401 Bucharest 3, Romania
3
Pharmaceutical Analysis Laboratory, Biological and
Medical Research, Dept., MBC-03-65, King Faisal
Specialist Hospital and Research Centre, P.O. Box 3354,
Riyadh 11211, Saudi Arabia

ABSTRACT

The rightness of the measurements performed in analysis and

control laboratories is essential for functioning of the modern
society. Utilization of reference materials and certified reference
materials plays a very important role in obtaining exactness in
analytical measurements. Reference materials and certified
reference materials are used to estimate the performances of
the analytical methods and the laboratory equipment.

*Corresponding author. E-mail: enein@kfshrc.edu.sa

1107
DOI: 10.1081/AL-120005961 0003–2719 (Print); 1532–236X (Online)
Copyright & 2002 by Marcel Dekker, Inc. www.dekker.com
 1108 POPESCU, TANASE, AND ABOUL-ENEIN

Key Words: Reference materials—RM; Certified refer-

ence materials—CRM; Method validation; Equipment
qualification

INTRODUCTION
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A general definition of a reference material is a material or substance

one or more of whose property values are sufficiently homogenous,
stable, and well established to be used for calibration of an apparatus, the
assessment of a measurement method, or for assigning values to materials.[1]
According to National Association for Testing Authorities (NATA),
Australia there are three broad types of matrix reference materials:[2]
– Standard Reference Material (SRM)—a certified reference
material (CRM) issued by the National Institute of Standards
and Technology (NIST);
– Certified Reference Material—a reference material issued and
certified by an organisation generally accepted to be technically
competent;
– In-house Reference Material—a material developed by a labora-
tory for its own internal use.
Certified Reference Material is defined as a reference material, accom-
panied by a certificate, one or more of whose property values are expressed,
and for which each certified value is accompanied by an uncertainty at a
stated level of confidence.[1]
NATA represents Australia in:
– The International Laboratory Accreditation Cooperation (ILAC);
– The Asia Pacific Laboratory Accreditation Cooperation (APLAC);
and
– The OECD Working Group on Good Laboratory Practice.
Two classes of materials are recognised by ISO REMCO (ISO/
REMCO Document N 330 List of producers of certified reference materials,
Information by Task Group 3), namely ‘certified reference materials’
(CRMs) and ‘reference materials’ (RMs). CRMs must by definition be
traceable 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. RMs are of lesser quality
and by definition are materials whose property values are sufficiently
homogeneous and well established to be used for the calibration of an
 CONTROLS IN METHOD VALIDATION 1109

apparatus, the assessment of a measurement method, or for assigning values

to materials.
The following classes of reference materials are also encountered:
– Primary reference material
– Secondary reference materials
– In-house or working reference material, uncertainty decrease
toward primary reference material.
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Other terminology, such as NIST standard reference materials is also

used and a classification (class O—V) based on degree of traceability to SI
has been proposed.[3]

Traceability of Reference Materials

Traceability is defined according to the International Vocabulary of

basic and general terms in Metrology (VIM)[1] as a property of a result of a
measurement or the value of a standard whereby it can be related to stated
references, usually national or international standards, through an unbro-
ken chain of comparisons all having stated uncertainties.
Reference materials are important tools for the transfer of measurement
accuracy between laboratories and should, where feasible, be traceable to SI.
Traceability is, however, a relatively new concept in the field of chemical
measurement and as a consequence very few chemical reference materials
are explicitly traceable to SI.
A hierarchy of methods is, however, used for assigning property values
and even if not stated, their traceable can be described as follows:

Measurement Method Traceability

Primary method SI
Method of known bias SI/International standard
Independent method(s) Specified methods
Interlaboratory comparison Specified methods

In the absence of formally stated traceability it will be necessary for the

user to make judgements about implicit traceability, based on the certification
data available in reports and the technical literature. A combination of certi-
fication procedures is sometimes employed, such as a consensus value derived
from an interlaboratory comparison where primary methods were used.
The measurement uncertainty of the certified value of a reference
material employed in a measurement process will contribute to the uncertainty
 1110 POPESCU, TANASE, AND ABOUL-ENEIN

of the final measurement and should be a factor of 3–10 times less than the
required measurement uncertainty. Also, it is important to ensure that chemi-
cal interferences and matrix effects are adequately addressed in arriving at
both the certified value and its uncertainty. Unknown levels of bias are not
uncommon and contribute to poor comparability of measurements.
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Use of Reference Materials in Laboratory

Reference materials are used for the following main purposes in a

laboratory:
– Calibration and validation of methods and instrumentation—
single substance or matrix material;
– To assure the long-term adequacy and integrity of the quality assur-
ance programs and quality control (QC) during method validation;
– Production of secondary reference materials—for routine use;
– Use in standard test methods—to demonstrate that the method
achieves valid results;
– To use as test materials for inter-laboratory comparisons and
proficiency test programs;
– Achievement of traceability of calibration.
Different types of reference materials are required for different func-
tions. For example, a certified reference material would be desirable for
method validation but a working level reference material would be adequate
for quality control.

Types of Reference Materials

Reference materials may be characterized for ‘‘identity’’ (e.g., chemical

structure, fibre type, microbiological species etc.) or for ‘‘measured values’’
(e.g., amount of specified chemical entity, hardness etc). They are produced
to support measurements concerned with chemical composition, biological,
clinical, physical, engineering properties and miscellaneous areas such as
taste and odour. Some commonly encountered types of reference materials
are as follows:
– Pure substances characterized for chemical purity and/or trace
purities
– Standard solutions (mixtures) prepared gravimetrically from pure
substances and used for calibration purposes
 CONTROLS IN METHOD VALIDATION 1111

– Matrix reference materials, characterized for the composition of

specified major, minor trace chemical constituents. Such materials
may be prepared from matrices containing the components of
interest, or by preparing synthetic mixtures
– Physico–chemical reference material characterized for properties
such as melting point, viscosity, and optical density
– Reference objects or artefacts characterized for functional
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properties such as taste, odour, octane number, flash point and

hardness. This type also includes microscopy specimens character-
ized for properties ranging from fibre type to microbiological
specimens.

Assessment of the Suitability of Reference Materials

The use of appropriate reference materials can provide essential trace-

ability and enable analysts to demonstrate the accuracy of results, calibrate
equipment and methods, monitor laboratory performance and validate
methods, and enable comparison of methods by use as transfer (measure-
ment) standards.
The suitability of a reference material for a specified purpose depends
on the appropriateness of the material for the method, sample type, con-
centration range etc. Matrix effects and other factors such as concentration
range can be more important than the uncertainty of the certified value.[4]
The factors to consider include:
– Measurand (analyte),
– Measurement range (concentration),
– Matrix match and potential interferences,
– Sample size,
– Homogeneity and stability,
– Measurement uncertainty,
– Certification procedures (measurement and statistical).
An appropriate reference material must have the following
characteristics:
– Analyte level—appropriate to the analysis,
– Matrix—as similar to the sample as possible,
– Form—similar to the sample,
– Quantity—sufficient for the whole program,
– Uncertainty—known and appropriate for the analysis,
– Stability—known to be consistent.
 1112 POPESCU, TANASE, AND ABOUL-ENEIN

The Choice of a Reference Material for Method Validation

The terms ‘‘validation’’ and ‘‘qualification’’ are used widely and often
to convey the same meaning. The approach taken in this paper is that
validation is application orientated and relates to a specific measurement
method or process, whereas qualification is instrument orientated and
relates primarily to the operational specification of the instrument.
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When a new method is developed or an existing method is modified to

meet special needs it must be validated. The objective of the process is to
identify the performance of the analytical method and to demonstrate that
the analytical system is operating in a state of statistical control.
Validation procedures are a three-part process:[2]

Determination of Key Performance Characteristics

– Limit of detection,
– Accuracy (precision, bias),
– Limit of detection,
– Limit of quantitation,
– Range,
– Selectivity,
– Linearity.

Analysis of Independent Unknown Samples

During this stage of the validation process, reference materials can be

used and make the process very simple if the test samples and the reference
material have a similar macro-composition. Reference materials must be fit
for purpose, ideally standard or certified reference materials that are
matrix matched. The analytical chemist must analyze a sufficient number
of reference materials over the method concentration range and compare
the results to the expected or certified values. An analytical procedure
should be capable of producing results that do not differ from the certified
value more than can be accounted for by ‘‘within laboratory’’ statistical
fluctuations. When a suitable reference material is not available, the
laboratory may use several other approaches. Spiked samples and the
use of surrogates are approaches available to laboratories. These are
less desirable than using reference materials because of the difficulty in
preparation and because artificially added materials like spikes and
 CONTROLS IN METHOD VALIDATION 1113

surrogates may exhibit different matrix effects from naturally occurring

analytes.

Determination of Method Ruggedness

This is a measure of stability of test results produced when steps or

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operational parameters are varied. Such parameters include the environ-

mental factors of temperature, pressure and relative humidity, and chemical
factors such as concentrations of reagents and pH control. By establishing
the relative effects of these parameters, one can estimate tolerances within
which these parameters must be maintained in order to obtain results within
acceptable limits.
Once the new method has been developed and validated, reference
materials can be used by laboratories to gain experience and proficiency
in its application. The use of certified reference materials eliminates ques-
tions of stability and homogeneity that may otherwise complicate interpre-
tation of analytical results from less well characterized samples. Laboratories
can also use reference material to confirm an analyst’s ability to use a new
method, before undertaking analysis of clients’ samples.

Measurement Uncertainty

Estimation of bias (the difference between the measured value and the
true value) is one of the most difficult elements of method validation and
appropriate RMs can provide valuable information, within the limits of the
uncertainty of the RMs certified value(s) and the uncertainty of the method
being validated. Although traceable certified values are highly desirable, the
estimation of bias differences between two or more methods can be estab-
lished by use of less rigorously certified RMs. Clearly the RMs must be
within the scope of the method in terms of matrix type, analyte concentra-
tion etc., and ideally a number of RMs covering the full range of the method
should be tested. Where minor modifications to a well-established method
are being evaluated then less rigorous bias studies can be employed.
Replicate measurement of the RM, covering full range of variables
permitted by the method being validated, can be used to estimate the
uncertainty associated with any bias, which should then normally be
corrected for. The uncertainty associated with an RM should be no greater
than 1/3 to 1/10 of that required for sample measurement.
The successful application of a valid method depends on its correct
use, both with regard to operator skill and suitability of equipment, reagents
 1114 POPESCU, TANASE, AND ABOUL-ENEIN

and standards. RMs can be used for training purposes, for checking
infrequently used methods, and trouble-shooting when unexpected results
are obtained.

Calibration of Instrumentation
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The primary requirement for all equipment used in analytical labora-

tories is that it must be fit for its intended purpose.[5] The equipment
qualification (EQ) process must therefore establish that an instrument’s
operational specification is appropriate for its intended use and that the
instrument performs according to that specification.[6] EQ must also
establish that an instrument is, and will be, kept in a state of maintenance
and calibration consistent with its use.
The EQ process is based on four stages of ‘‘qualification’’: design
qualification (DQ), installation qualification (IQ), operational qualification
(OQ), and performance qualification (PQ).
DQ is the planning part of EQ process. IQ, OQ and PQ are the
implementation stages of the EQ process and provide an assurance that
the instrument is installed properly, that it operates correctly, and that its
ongoing performance remains within the limits required for its actual use.
EQ provides important evidence of an instrument’s satisfactory
performance and its fitness for the purpose for which it is used. However,
EQ is just one of a range of activities which contribute towards achieving
and demonstrating reliable and valid data.
Many quality standards and enforcement authorities stipulate that
‘‘where possible calibration should be traceable to national or international
standards’’ in order to ensure accuracy. Tests to verify the accuracy of
critical parameters (e.g., wavelength accuracy) will necessitate the use of
traceable standards and calibrated apparatus.
As a bare minimum users must demonstrate that the precision and
linearity of the instrument are satisfactory prior to routine use. For some
instruments, calibrating with a series of standard solutions should assess
linearity, which covers the range of anticipated results, plus a safety
margin. This type of calibration should be performed before sample analysis
or at an interval specified in a standard operating procedure, the frequency
of which should, as a minimum, be based on the period over which the
instrument has previously been found to remain within calibration.
The precision should be determined from the relative standard deviation
(RDS) of responses to replicate determinations with standard solution.
For HPLC technique, a RSD greater than 1% is generally unacceptable.
 CONTROLS IN METHOD VALIDATION 1115

Reference materials are used as calibrating standards by a number of

different measurement techniques. For some types of measurements it is
sufficient simply to calibrate the instrument using pure reference materials
for their physical properties. However, many analytical techniques are a
complex sequence of operations, and to calibrate the total analysis system
a matrix CRM needs to be used that simulates all the steps and procedures
that would be carried out on a routine test sample.
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The National Institute of Standards and Technology (NIST) pro-

vides SRMs that define the practical pH scale as well as SRMs for fixed
temperature points.[2] When analyzing ores and metals the X-ray fluores-
cence (XRF) spectrometry technique is commonly calibrated using
reference materials. For example, the ASTM Standard Method E322 for
low alloy steels recommends 1220 and 1700 series low-alloy steel SRMs as
calibrants.
The precision of the XRF technique is normally very high while, on
the other hand, accuracy depends heavily on two factors: the attenuation-
enhancement correction procedure used to correct matrix effects caused by
concomitant elements, and calibration procedures. Analyses of metals using
emission spectrometry also use reference materials for calibration, and these
are often a combination of both certified metal alloys as well as standards
developed in-house.
The uncertainty associated with RM purity will contribute to the total
uncertainty of the measurement. For example, a RM certified as 99.9%
pure, with a U (k2) of 0.1% will result in an uncertainty component of
0.1% in the overall measurement uncertainty budget. In the case of trace
analysis, this level of uncertainty will rarely be important but for assay
work, it can be expected to be significant.

CONCLUSIONS

The use of reference materials allows the analyst to demonstrate the

validity of an analysis. Analysts should select the most appropriate reference
material for the analysis they are undertaking. Wherever possible, certified
reference materials should be used. Where certificated reference materials
are not available, the analyst should use the best reference material
available, and be able to demonstrate its validity.
Increasingly, both analytical laboratories and regulatory authorities
are acknowledging that, under some circumstances, both method validation
and equipment qualification are important prerequisites for obtaining
reliable data.
 1116 POPESCU, TANASE, AND ABOUL-ENEIN

REFERENCES

1. International Vocabulary of Basic and General Terms in Metrology

(VIM), Second Edition, 1993.
2. Eames, J. Use of Reference Materials in the Laboratory, 1993.
http://www.nata.asn.au/fs_publications.htm
3. Standard Reference Materials—Handbook for SRM Users; NIST
Downloaded by [University of California Santa Cruz] at 07:25 01 April 2015

Publication, 1993; 260–300.

4. EURACHEM/CITAC Guide, Guide to Quality in Analytical
Chemistry, Annex B—The Selection and Use of Reference Materials,
A Basic Guide for Laboratories and Accreditation Bodies, February
2001.
5. National Measurement System 1997–2000 Valid Analytical Measure-
ment (VAM) Programme—Guidance on Equipment Qualification of
Analytical Instruments: High Performance Liquid Chromatography,
June 1998.
6. Bedson, P.; Sargent, M. The Development and Application of
Guidance on Equipment Qualification of Analytical Instruments.
Accreditation and Quality Assurance 1996, 1(6), 265–279.

Received February 20, 2002

Accepted March 11, 2002