EXCERPT FROM:

VALIDATION OF ANALYTICAL METHODS

FOR PHARMACEUTICAL ANALYSIS
BY OONA MCPOLIN

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ISBN 978-0-9561528-1-7
Preface

This book provides guidance on how to perform validation for the analytical methods
which are used in pharmaceutical analysis. Validation of the analytical methods which
are used during drug development and drug manufacturing is required to demonstrate
that the methods are fit for their intended purpose. Additionally, the pharmaceutical
industry around the world is subject to extensive regulations due to the nature of its
products. Validation is a regulatory requirement and the data generated during an
analytical method validation study is included in submissions to regulatory authorities
for clinical trial and marketing applications.

The definitive reference for this topic is the guideline produced by the International
Conference on Harmonisation of Technical Requirements for Registration of
Pharmaceuticals for Human Use (ICH), Q2(R1) ‘Validation of Analytical Procedures:
Text and Methodology’. Sections of the guideline have been reproduced in this book
with the kind permission of the ICH Secretariat. All ICH guidelines are available via
the website, www.ich.org, and thus the validation guideline may be easily checked for
revisions.

The guidance in this book is primarily aimed at analytical methods for small molecules.
Reference is made to validation of methods for pharmaceuticals of biological origin,
such as proteins and peptides. In principle the ICH guidelines should be applied to
these types of compounds, however it is acknowledged they present particular
challenges during validation, in particular relating to biological assays. The references
provided in Chapter 1 provide more information on the validation of analytical
methods for biopharmaceuticals/ biotechnology derived products.

At the back of the book there is a glossary to help the reader become familiar with the
terminology used in analytical method validation. When a new term is introduced it is
shown in bold to indicate to the reader that a definition is available in the glossary. In
the appendix there is a list of abbreviations and also a question section so that the
reader can test their understanding of the content.

Oona McPolin
Contents

CHAPTER 1
INTRODUCTION..........................................................................................................1
Validation in the Pharmaceutical Industry ................................................................................................ 1
Data Quality .............................................................................................................................................. 1
Analytical Instrument Qualification (AIQ) ...............................................................................................2
The Purpose of an Analytical Method .......................................................................................................3
The life cycle of an analytical method ................................................................................................................................ 4
Validation Guidelines ................................................................................................................................5
Types of Analytical Methods .....................................................................................................................6
Universal tests ......................................................................................................................................................................... 6
Analytical methods not included in the ICH guidelines ................................................................................................. 8
Analytical techniques ............................................................................................................................................................. 9
Validation Characteristics........................................................................................................................ 10
Specificity............................................................................................................................................................................... 10
Accuracy ................................................................................................................................................................................ 10
Precision ................................................................................................................................................................................ 10
Detection Limit .................................................................................................................................................................... 11
Quantitation Limit ............................................................................................................................................................... 11
Linearity ................................................................................................................................................................................. 11
Range ...................................................................................................................................................................................... 11
Robustness ............................................................................................................................................................................ 11
References ............................................................................................................................................... 12
Further reading ........................................................................................................................................ 14

CHAPTER 2
VALIDATION CHARACTERISTICS ......................................................................... 17
Validation during Method Development ................................................................................................. 17
Statistics in Analytical Method Validation .............................................................................................. 18
Mean ....................................................................................................................................................................................... 18
Standard deviation ............................................................................................................................................................... 18
Confidence intervals ............................................................................................................................................................ 19
Specificity ................................................................................................................................................ 22
Specificity and selectivity .................................................................................................................................................... 23
Identification methods ........................................................................................................................................................ 23
Assay and impurity methods .............................................................................................................................................. 24
Bioanalytical methods ......................................................................................................................................................... 26
Other methods ..................................................................................................................................................................... 26
Linearity .................................................................................................................................................. 27
Verification of the calibration model ............................................................................................................................... 27
Regression analysis ............................................................................................................................................................... 29
 Assay and impurity methods ..............................................................................................................................................34
Bioanalytical methods ..........................................................................................................................................................34
Range....................................................................................................................................................... 35
Accuracy .................................................................................................................................................. 37
Assay .......................................................................................................................................................................................38
Impurities ...............................................................................................................................................................................40
Other methods ......................................................................................................................................................................41
Precision .................................................................................................................................................. 42
Precision levels ......................................................................................................................................................................43
System precision ...................................................................................................................................................................43
Measurement of precision ..................................................................................................................................................43
Repeatability ..........................................................................................................................................................................44
Intermediate precision .........................................................................................................................................................46
Reproducibility ......................................................................................................................................................................47
Accuracy, trueness and precision.......................................................................................................................................49
Detection Limit ....................................................................................................................................... 50
Quantitation Limit................................................................................................................................... 53
Robustness .............................................................................................................................................. 55
Robustness during method development ........................................................................................................................59
Robustness during the validation study ............................................................................................................................60
Solution stability evaluation ................................................................................................................................................64
System Suitability .................................................................................................................................... 65
References ............................................................................................................................................... 67
Further Reading ...................................................................................................................................... 70

CHAPTER 3
THE VALIDATION PROTOCOL .............................................................................. 73
Contents of the Validation Protocol ........................................................................................................ 73
Validation Characteristics to be Studied ................................................................................................. 74
Experimental Procedure and Acceptance Criteria .................................................................................. 75
Specificity ...............................................................................................................................................................................75
Linearity .................................................................................................................................................................................76
Range ......................................................................................................................................................................................77
Accuracy.................................................................................................................................................................................77
Precision .................................................................................................................................................................................78
Detection Limit and Quantitation Limit ..........................................................................................................................79
Robustness .............................................................................................................................................................................80
Stability ...................................................................................................................................................................................80
Validation Protocol by Type of Method .................................................................................................. 81
Identification .........................................................................................................................................................................81
Assay .......................................................................................................................................................................................81
Impurities ...............................................................................................................................................................................83
Bioanalytical methods ..........................................................................................................................................................86
Other methods ......................................................................................................................................................................87
Validation of methods in Pharmacopoeias ......................................................................................................................87
Automated methods ............................................................................................................................................................87
Validation by Phase of Development ...................................................................................................... 88
Validation Software.................................................................................................................................. 90
Planning and Execution of the Validation Study .................................................................................... 90
Review the timing and resources required....................................................................................................................... 90
Determine the most efficient order of experiments ...................................................................................................... 91
Execute the study ................................................................................................................................................................. 92
Documentation..................................................................................................................................................................... 92
Case Study ............................................................................................................................................... 93
Planning and execution of the study ................................................................................................................................ 99
References ............................................................................................................................................. 102
Further reading ...................................................................................................................................... 104

CHAPTER 4
THE VALIDATION REPORT .................................................................................. 107
Contents of the Validation Report ......................................................................................................... 107
Statistics in Analytical Method Validation ............................................................................................ 108
Statistical significance ........................................................................................................................................................ 108
Comparative studies .......................................................................................................................................................... 108
Outliers ................................................................................................................................................................................ 110
Results for Each Validation Characteristic.............................................................................................112
Specificity............................................................................................................................................................................. 112
Linearity ............................................................................................................................................................................... 113
Range .................................................................................................................................................................................... 113
Accuracy .............................................................................................................................................................................. 113
Precision .............................................................................................................................................................................. 113
Detection Limit and Quantitation Limit ........................................................................................................................ 114
Robustness .......................................................................................................................................................................... 114
Stability................................................................................................................................................................................. 114
Results to include in the validation report ..................................................................................................................... 115
Non-compliance with acceptance criteria...................................................................................................................... 117
Documentation................................................................................................................................................................... 117
Revalidation ............................................................................................................................................117
Case Study ..............................................................................................................................................119
References ............................................................................................................................................. 125
Further reading ...................................................................................................................................... 125

GLOSSARY .................................................................................................................. 129

LIST OF ABBREVIATIONS ...................................................................................... 137
INDEX......................................................................................................................... 141
 Chapter

1
Introduction

V
alidation is defined as ‘finding or testing the truth of something’. When
analytical methods are used to generate results about the characteristics of
drug related samples it is vital that the results are trustworthy: they may be
used as the basis for decisions relating to administering the drug to patients. A
validation study is performed on an analytical method to ensure that reliable results are
always obtained.

Validation in the Pharmaceutical Industry

Analytical method validation is just one type of validation required during drug
development and manufacturing. To comply with the requirements of current Good
Manufacturing Practices (GMP)1-3 pharmaceutical companies should have an overall
validation policy which documents how validation will be performed. This will
include the validation of: production processes, cleaning procedures, analytical
methods, in-process control test procedures, and computerised systems. The purpose
of this validation is to show that processes involved in the development and
manufacture of drugs, such as production, cleaning and analytical testing, can be
performed in an effective and reproducible manner.

The reason that validation is included in cGMP in this way is to ensure that quality is
built in at every step, and not just tested for at the end. ‘Validation is intended to
provide assurance of the quality of a system or process through a quality methodology
for the design, manufacture and use of that system or process, that cannot be found
by simple testing alone.’4

Data Quality
The quality of analytical data is assured by the combination of a number of critical
components as shown in Figure 1. In the data quality triangle5 the components are
layered, each layer adds to the overall quality of the data. The base of the triangle is
Analytical Instrument Qualification (AIQ), this is documented evidence that an
instrument performs suitably for its intended purpose and that it is properly
maintained and calibrated. The next layer is analytical method validation, the subject
of this book. This is documented evidence that demonstrates that the analytical
method is suitable for its intended use. The top layers of the triangle are system

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suitability tests and quality control checks, these are used to demonstrate that the
combination of system and method performed as expected at the time of the analysis.
System suitability tests are commonly used for chemical analyses, which are usually
subject to GMP regulations, and quality control checks are commonly used for
bioanalytical analyses, which are usually subject to GLP regulations. Overall, AIQ and
analytical method validation assure the quality before the analysis is performed and
system suitability tests and quality control checks assure the quality immediately before
or during the analysis.
Figure 1 Data quality triangle, from Basal et al in AAPS PharmSciTech5

Quality
Control Checks

System Suitability Tests

Analytical Method Validation

Analytical Instrument Qualification

Analytical Instrument Qualification (AIQ)

The equipment which is used for all types of validation related to drug development
and manufacture needs to be qualified. In the case of analytical methods this refers to
the analytical instrumentation required to perform the test. Analytical method
validation must be performed on appropriately qualified instruments.

The qualification procedure is usually carried out in four stages during which all
actions are documented5,6. These are:
Design Qualification (DQ):
This covers all procedures prior to the installation of the system in the selected
environment. The DQ defines the functional and operational specifications of the
instrument and details the conscious decisions in the selection of the supplier. For
commercial off the shelf (COTS) analytical instrumentation users generally have very
little input into the design of the instrument and thus the DQ will detail the user
requirements and the rationale for the selection of a particular supplier. For custom
designed analytical instrumentation the DQ details the key features of the design and
how they address the user requirements.

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Installation Qualification (IQ):

This covers all the procedures relating to the installation of the instrument in the
selected environment. The IQ establishes that the instrument is received as designed
and specified, that it is properly installed in the selected environment and that this
environment is suitable for the operation and use of the instrument. The IQ may be
carried out by the supplier and/or the user. For some complex instrumentation it may
have to be performed by the supplier.
Operational Qualification (OQ):
This is the process of demonstrating that an instrument will function according to its
operational specification in the selected environment. The OQ usually takes place
after the IQ of a new instrument or after a change to the instrument, such as repair or
change of location. As with the IQ, the OQ may be carried out by the supplier
and/or the user. IQ/OQ is now offered by most suppliers of analytical
instrumentation when a new instrument is purchased.
Performance Qualification (PQ):
This is defined as the process of demonstrating that an instrument consistently
performs according to a specification appropriate for its routine use. PQ may be
offered as part of a service contract for the routine maintenance of analytical
instruments and/or may be carried out by the user. Calibration is part of performance
qualification.

This summary gives an overview of equipment qualification, more information can be

found in the GMP guidelines1-3, also many articles and books are available on this
topic5-7.

The Purpose of an Analytical Method

An analytical method details the steps necessary to perform an analysis. This may
include: preparation of samples, standards and reagents; use of the apparatus;
generation of the calibration curve, use of the formulae for the calculation, etc. The
objective of validation of an analytical method is to demonstrate that the method is
suitable for the intended use.

The use of analytical methods during drug development and manufacturing provides
information on:

• Potency, which can relate directly to the requirement of a known dose.

• Impurities, which can relate to the safety profile of the drug.

• Evaluation of key drug characteristics such as crystal form, drug release, and drug
uniformity, properties which can compromise bioavailability.

• Degradation products, methods need to be stability indicating.

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• Effect of key manufacturing parameters, to ensure that the production of drug

substance and drug product is consistent.

The validation which is performed on the methods which generate this data needs to
demonstrate that they can do so reliably and consistently.

The life cycle of an analytical method

Once a method has been developed and validated it may then be used for routine
analysis, as shown in Figure 2. However, changes may occur which make it necessary
to evaluate whether the method is still suitable for its intended use. The change may
be covered by the existing validation, in which case no further validation is required or
the change may result in revalidation, and in some cases, redevelopment of the
method followed by validation of the new method.
Figure 2 The life cycle of an analytical method

Development of the
method Redevelopment of the method
required due to change

Validation of the
method Revalidation required
due to change

Method in routine use

CHANGE to method:
Change is covered by Evaluate the effect Change is not covered by
existing validation existing validation

Examples of potential changes and the effect are shown in Table 1.

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Summary

1. Validation is an important procedure in the pharmaceutical industry and is

used to ensure that quality is built into the processes supporting drug
development and manufacture.
2. Data quality is assured by the combination of four components: Analytical
Instrument Qualification (AIQ); analytical method validation; system
suitability tests and quality control checks.
3. Validation of an analytical method is intended to demonstrate that it is
suitable for its intended use.
4. Guidelines for analytical method validation as applied to pharmaceuticals are
available from the International Conference on Harmonisation (ICH) of
Technical Requirements for Registration of Pharmaceuticals for Human Use.
5. The type of method and analytical technique used will determine the nature
and extent of the validation studies required. The most common methods
for validation are identification, assay and impurities determination.
6. The characteristics of validation are: Specificity, linearity, range, accuracy,
precision, detection limit, quantitation limit and robustness.

References
1. International Conference on Harmonisation (ICH) of Technical Requirements for
Registration of Pharmaceuticals for Human Use, Topic Q7: Good
Manufacturing Practices for Pharmaceutical Ingredients, 2000, www.ich.org.

2. Current Good Manufacturing Practices for finished pharmaceuticals, 21

CFR, Parts 210 and 211, US Food and Drug Administration, www.fda.gov.

3. EUDRALEX: The Rules Governing Medicinal Products in the European

Community, Volume IV, Good Manufacturing Practice for Medicinal
Products, Annex 15, Qualification and Validation, 2001.

4. R. D. McDowall, Qual Assur J, 9, 3, 196-227, 2005, ‘Effective and practical risk

management options for computerised system validation’.

5. S. K. Basal, T. Layloff, E. D. Bush, M. Hamilton, E. A. Hankinson, J. S. Landy, S.

Lowes, M. M. Nasr, P. A. St. Jean, V. P. Shah, AAPS PharmSciTech 5 (1), Article 22,
2004, ‘Qualification of Analytical Instruments for Use in the Pharmaceutical
Industry: a Scientific Approach’.

6. P. Bedson, M. Sargent, Accred. Qual. Assurance 1, 265-274, 1996, ‘The

development and application of guidance on equipment qualification of
analytical instruments’.

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 Chapter

2
Validation Characteristics

V
alidation of an analytical method is performed by consideration of a number
of characteristics as introduced in Chapter 1. The nature of the analytical
method will determine which characteristics should be investigated. In this
chapter each characteristic is considered separately in the order in which they are listed
in the ICH guidelines1. The guidance for each as written in the ICH guidelines is
provided together with a discussion of the interpretation of the guidance and of the
associated practical considerations.

Validation during Method Development

When validation is performed for analytical methods which are used to analyse drug
related samples the nature of the investigation is usually confirmatory. During the
development of the method many of the validation characteristics have already been
considered although probably not in a formal way. For example:

• The concentration of the samples used for the analysis will have been selected so
as to achieve a suitable response, e.g. in the case of a HPLC method to determine
impurities using a UV detector, the concentration will be selected to fall within the
linear range of the detector and to have a suitable response for the low
concentrations required.

• The specificity will have been considered during the initial method development,
e.g. in any chromatographic method the separation of the components of interest
is the basis of the method development process and thus the retention time of
components of interest and potential interferences are evaluated during the
development of the method.

• An evaluation of the robustness of the method is usually commenced as soon as

the method is developed. This will include identification of the critical parameters
of the method and an evaluation of the stability of test solutions.

The result of this information gained during method development is that the
validation experiments are expected to comply with the selected acceptance criteria
and major problems are not usually anticipated. However, it is always possible that the
validation study may identify problems that could result in changes to the method.

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Statistics in Analytical Method Validation

Statistical analysis of the data produced during a method validation study is required to
demonstrate the validity of the method. The statistical procedures which are
commonly used are described during the discussion of each validation characteristic in
this chapter and also in Chapter 4. In this preceding section the use of the mean,
standard deviation and confidence intervals are considered.

Mean
The mean of a dataset is the total sum of all the values in the dataset divided by the
number of values in the dataset. It is denoted by x (x bar) and is calculated using the
following equation:

1
x =
n
∑ xi
Consider as an example the analysis of a pharmaceutical sample to determine the
amount of active pharmaceutical ingredient present. A single determination or
measurement will provide an estimation of the true value. If another determination is
performed then the result is likely to be different from the first determination but
which is the best estimate of the true value? The mean of the results obtained for the
two determinations is normally calculated to provide a better estimate for the true
value. As the number of determinations increases the calculated sample mean will be
nearer to the true value. In this situation the true value is the population mean,
denoted by µ.

Standard deviation
The standard deviation of a dataset is a measure of the spread of the values in the
dataset. It is calculated by measuring the difference between the mean and the
individual values in the dataset. The standard deviation for a sample is determined
using the following equation:

Standard deviation, s = s2

Where:

1
s2 = ∑
n −1 i
(
xi − x
2
)
The calculation for standard deviation assumes a normal distribution of the data. This
distribution is shown in Figure 3 and is typical for the results of most physico-
chemical analysis. The data is centred about the mean with the majority of the
observations near to the mean value. Fewer and fewer observations occur the further
they are away from the mean. 68.3 % of the observations lie within 1 standard
deviation of the mean, 95.5 % of the observations lie within two standard deviations
and 99.7 % lie within 3 standard deviations. When using small data sets, typical in

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pharmaceutical analysis, a normal distribution may not be apparent on inspection of

the data but this distribution can usually be assumed.
Figure 3 Normal distribution curve
Number of observations

-3s -2s -1s x 1s 2s 3s

68.3%
95.5%
99.7%

If the population standard deviation, σ, is defined as the standard deviation calculated

for a large data set, and the sample standard deviation, s, is defined as the standard
deviation of a small subset of this large data set, then it is found that the value
calculated for the sample standard deviation on a number of subsets may vary
considerably due to random variability in the data. The smaller the number of data,
the higher is the variability of the sample standard deviation. As a result of this, it is
recommended that the standard deviation should only be calculated for data sets
where n>3.

Confidence intervals
Confidence intervals are used to indicate the reliability of an estimate. In the example
quoted previously, where the amount of active pharmaceutical ingredient present in a
pharmaceutical sample is analysed, the mean result obtained is an estimate of the
actual amount present. A confidence interval provides limits around the
experimentally determined value of the mean within which the true value (or
population mean, µ) lies with a given degree of probability, usually 95%.

The size of the confidence interval depends on the value of the standard deviation, s.
The confidence interval determined for a sample where the standard deviation

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Linearity
The section on linearity in the ICH guidelines1 is as follows:

ICH

2. Linearity
The linearity of an analytical procedure is its ability (within a given range) to obtain test results
which are directly proportional to the concentration (amount) of analyte in the sample.
A linear relationship should be evaluated across the range (see section 3) of the
analytical procedure. It may be demonstrated directly on the drug substance (by
dilution of a standard stock solution) and/or separate weighings of synthetic
mixtures of the drug product components, using the proposed procedure. The
latter aspect can be studied during investigation of the range.
Linearity should be evaluated by visual inspection of a plot of signals as a
function of analyte concentration or content. If there is a linear relationship, test
results should be evaluated by appropriate statistical methods, for example, by
calculation of a regression line by the method of least squares. In some cases, to
obtain linearity between assays and sample concentrations, the test data may
need to be subjected to a mathematical transformation prior to the regression
analysis. Data from the regression line itself may be helpful to provide
mathematical estimates of the degree of linearity.
The correlation coefficient, y-intercept, slope of the regression line and residual
sum of squares should be submitted. A plot of the data should be included.
In addition, an analysis of the deviation of the actual data points from the
regression line may also be helpful for evaluating linearity.
Some analytical procedures, such as immunoassays, do not demonstrate linearity
after any transformation. In this case, the analytical response should be described
by an appropriate function of the concentration (amount) of an analyte in a
sample.
For the establishment of linearity, a minimum of 5 concentrations is
recommended. Other approaches should be justified.

Verification of the calibration model

If an analytical method is going to be used to generate quantitative results then a
method of calibration is required. The purpose of the validation characteristic referred
to as ‘linearity’ is to verify the calibration model. The term ‘linearity’ is slightly
misleading because it implies that the relationship between the test results and the
concentration of the analyte should be linear. This is not always the case as is
acknowledged in the penultimate paragraph of section 2 in the guidelines,
immunoassays being quoted as an example. In the FDA guidelines4 on bioanalytical
method validation the term ‘Calibration/Standard Curve’ is used instead of linearity.

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In reality a large number of pharmaceutical analytical methods are based on linear

relationships and the terminology is appropriate in these situations. Therefore the
term ‘linearity’ is used in the remainder of this book, but it is assumed that the reader
understands that a linear calibration may not always apply.
Single point calibration
The most common type of calibration model encountered in the analysis of drug
substances and drug products is known as a single point calibration, where the
standard is prepared at one concentration level only. In fact this model defines a two
point calibration line where one point equals zero and the other the standard
concentration. A single point calibration line is shown in Figure 5. The unknown
concentration of an analyte in a sample is calculated by entering the response obtained
for the analyte when analysed by the method into the equation of the calibration line,
y = mx + c. In order to force the line through zero, and thus use a single point
calibration, the value of ‘c’ must be negligible.

To verify the single point model it is necessary to demonstrate that the value of the ‘c’
(the intercept) is negligible. This is achieved by measuring the response due to a
number of different concentration levels across the range of the method and
calculating the intercept for the best fit straight line. Since the aim of the investigation
is to show that the line goes through zero it is not appropriate to include zero as a
point in the investigation unless it is a measured value. For example, the absence of a
peak in a chromatographic analysis cannot be interpreted as a zero result. However
the measurement of the blank in a UV assay is a measured value and may be included
in an investigation of linearity. This applies to all calibration models, not just single
point.
Figure 5 Example of a single point calibration

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Robustness
The section on robustness in the ICH guidelines1 is as follows:

ICH

8. Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by
small, but deliberate variations in method parameters and provides an indication of its
reliability during normal usage.
The evaluation of robustness should be considered during the development
phase and depends on the type of procedure under study. It should show the
reliability of an analysis with respect to deliberate variations in method
parameters.
If measurements are susceptible to variations in analytical conditions, the
analytical conditions should be suitably controlled or a precautionary statement
should be included in the procedure. One consequence of the evaluation of
robustness should be that a series of system suitability parameters (e.g.,
resolution test) is established to ensure that the validity of the analytical
procedure is maintained whenever used.
Examples of typical variations are:
- stability of analytical solutions;
- extraction time.
In the case of liquid chromatography, examples of typical variations are:
- influence of variations of pH in a mobile phase;
- influence of variations in mobile phase composition;
- different columns (different lots and/or suppliers);
- temperature;
- flow rate.
In the case of gas-chromatography, examples of typical variations are:
- different columns (different lots and/or suppliers);
- temperature;
- flow rate.

Robustness is not included in the tabular summary of required validation

characteristics to be tested in the ICH guidelines, however it is expected that it is
‘considered at an appropriate stage in the development of the analytical procedure’.1
Robustness is included in the summary table provided in the draft FDA guidelines24
(see Table 14). Evaluation of the robustness of an analytical method is usually

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performed in two phases. Initial robustness testing is part of the method development
process. A more formal study is then performed during validation of the method.

The factors which are investigated in robustness studies are method related, they are
parameters which are defined in the method, e.g. temperature. Altering the value of
these factors is a deviation from the method. In contrast, the factors which are
investigated in intermediate precision (sometimes referred to as ruggedness) are non-
method related factors, e.g. the analyst performing the analysis. When investigating
these factors the method is followed in full without any deviations. Robustness
factors are sometimes referred to as internal factors and those relating to intermediate
precision as external factors. When an analytical method has been shown to have
acceptable intermediate precision and robustness through the validation study,
confidence is gained that the method can be used successfully in routine analysis. Also
included in robustness testing is the evaluation of the stability of test solutions which
are used in the analysis.

Some examples of robustness factors from available literature1,25,26 for a variety of

analytical techniques are listed below:

High Performance liquid Chromatography (HPLC)

• Influence of variations of pH in a mobile phase

• Influence of variations in mobile phase composition

• Different columns (different lots and/or suppliers)

• Temperature

• Flow rate

• Buffer concentration (ionic strength)

• Additive concentration

• Gradient slope

• Initial mobile phase composition

• Final mobile phase composition

• Injection volume

• Sample preparation (pH of solutions, reagent concentrations, etc.)

• Equilibration time

• Column age

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 Chapter

3
The Validation Protocol

T
he planning and design of a validation study involves a number of steps. First,
the appropriate validation characteristics, as described in Chapter 2, are
selected for the analytical method being studied. Next, the experiments which
are required for each characteristic are defined including the concentration levels and
the number of replicates to be tested. Then the required results and acceptance
criteria are defined for each test.

Contents of the Validation Protocol

The validation protocol details the design of the validation study. Its purpose is to
provide information on which characteristics will be tested during the study, how the
experiments will be performed, and what results will be calculated. Typical
information in a validation protocol may include:

• Details regarding the analytical method (or methods) to be validated.

• The validation characteristics which will be investigated for the method(s).

• Details on how the experiments will be performed including: the type and number
of solutions to be prepared; how the solutions should be prepared; how
measurements should be performed.

• Details of the results which will be calculated (with directions if appropriate).

• The acceptance criteria which will be applied to the results.

• Details of the reference materials which will be used in the validation study.

• Details of batches of the material used during the validation study. In the case of
precision studies, particular representative batches may be selected.

• Details of the equipment which will be used in the study.

• Details of responsibilities and required signatures.

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Validation Characteristics to be Studied

A tabular summary which details the validation characteristics that should be applied
for different types of methods is included in the ICH guidelines1. This table is
extended in the draft FDA guidelines2 on ‘Analytical Procedures and Method
Validation’ to include specific tests and robustness, and is reproduced below, see
Table 14.
Table 14 Recommended validation characteristics of the various types of tests2

Type of analytical Identification Testing for impurities Assay Specific Tests

procedure
Dissolution
(measurement
only),
Characteristic Quantitative Limit Content/potency

Accuracy - + - + +4

Precision

Repeatability - + - + +4

Intermediate - +1 - +1 +4
precision

Specificity +2 + + +5 +4

Detection Limit - -3 + - -

Quantitation Limit - + - - -

Linearity - + - + -

Range - + - + -

Robustness - + -3 + +4

NOTE:
- Signifies that this characteristic is not normally evaluated.
+ Signifies that this characteristic is normally evaluated.
1 In cases where reproducibility has been performed, intermediate precision is not needed.
2 Lack of specificity for an analytical procedure may be compensated for by the addition of a second analytical
procedure.
3 May be needed in some cases.
4 May not be needed in some cases.
5 Lack of specificity for an assay for release may be compensated for by impurities testing.

The ‘Type of analytical procedure’ in the table, i.e. identification, assay, testing for
impurities and specific tests, was defined previously in Chapter 1. The table lists the

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validation characteristics which are regarded as most important for the different types
of methods. The ICH guidelines1 state that ‘the list should be considered typical for
the analytical procedures cited but occasional exceptions should be dealt with on a
case-by-case basis’. Laboratories which perform analytical method validation typically
have in-house guidelines or standard operating procedures to describe the approach
which should be taken for validation studies. These documents are usually based on
the ICH guidelines and provide additional information on the organisational policy
regarding which validation characteristics should be investigated for particular types of
analytical methods.

Experimental Procedure and Acceptance Criteria

When the relevant validation characteristics have been identified, the experimental
procedure which will be used to investigate those characteristics needs to be defined.
Minimum requirements for the number of determinations and replicates which should
be used are provided in the ICH guidelines. Also required is the rationale for
assessing the results which are generated from the study. The acceptance criteria
selected may be based on an absolute value of practical relevance or a statistical test
may be used. The experimental procedure for performing a validation study and the
relevant acceptance criteria are discussed for each validation characteristic below. The
values quoted are suggestions provided for the purposes of orientation. Therefore
they should be used with caution and may not be appropriate for a particular method.

Specificity
The aim of the specificity investigation is different for identification methods when
compared to the aim for assay and impurity methods, due to the difference in the
nature of the methods. For identification, specificity demonstrates that the method
does not give a positive response for samples other than the one of interest.
However, for assay and impurity methods, specificity demonstrates that the response
due to the analyte of interest in the sample is not affected by potential interferences
which may also be present in the sample. The result of this is that the approach used
for the experimental part of the investigation and the acceptance criteria applied for
each type of method differs.
Experimental
For identification methods samples will be selected which could be mistaken for the
sample of interest. This selection requires knowledge of the analytical method, the
manufacturing process for the material in question and knowledge regarding other
processes using the same plant. The samples may include materials which are
structurally similar or closely related to the analyte, e.g. isolated intermediates for the
drug substance. Samples should be prepared in a similar way to the sample of interest
and analysed as per method.

For assay and impurity methods the samples tested will contain materials which are
potentially present during routine analysis and may interfere with the result, e.g.

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Table 17 Suggestions for inclusion in a validation protocol for a quantitative impurities method

Validation Experimental details Acceptance criteria

characteristic

Specificity Analyse samples containing potential No interference observed for response

interferences, e.g. impurities, excipients. due to analyte or impurities of interest.
Prepare samples by: Chromatographic:
Spiking drug substance/drug product No peaks interfering with the analyte
with the potentially interfering peak or the peaks due to the
material. impurities of interest observed.
Prepare samples of the potentially Minimum resolution between peaks of
interfering materials. interest and neighbouring peaks = 1.5.
Perform stress studies on drug No co-elution detected from peak
substance/drug product. purity investigation.
Chromatographic:
Investigate peak purity of analyte peak.

Linearity A least 5 concentrations over the range Calibration model valid.

QL-120% of the specification limit.
Residual plot shows random scatter and
e.g. 25, 50, 75, 100 and 120% specification no systematic trends.
limit of the impurity, the values will
95% confidence interval of intercept
depend on the values of the specification
includes zero, or intercept is less than 2%
limit and the quantitation limit for a
of nominal response.
particular method.

Range The range is defined by the results Linearity, accuracy and precision
obtained for linearity, accuracy and demonstrated over the range.
precision.

Accuracy At least 9 determinations over 3 Mean recovery within 90-110%.

concentration levels.
Individual recoveries within 70-130%.
e.g. 3 at quantitation limit, 3 at 50% of
Regression analysis of known vs.
specification limit, and 3 at 120% of
estimated:
specification limit.
Slope within 0.9-1.1
95% confidence interval of slope
includes 1

Precision System precision assessed by 6 replicate %RSD ≤ 2%

measurements/injections.

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 Chapter

4
The Validation Report

W
hen the experiments detailed in the validation protocol have been
performed the next stage of the validation study is the interpretation of the
results. The calculations and statistics associated with each validation
characteristic are carried out and the results obtained are assessed against the
acceptance criteria to decide if the method meets the validation requirements.

Contents of the Validation Report

The validation report details the results of the validation study. Its purpose is to
provide information on which characteristics were tested during the study, the results
obtained, and the interpretation of those results. Typical information in a validation
report may include:

• Details of the validation protocol.

• Details regarding the analytical method (or methods) validated.

• The validation characteristics which were investigated for the method(s).

• The results which were calculated for each validation characteristic.

• A discussion of the interpretation of the results and how they compare to the
acceptance criteria.

• Any relevant validation information which was obtained during method

development, e.g. solution stability, robustness, stress studies, etc.

• Details of the reference materials used in the validation study.

• Details relating to batch numbers, etc. of the materials used in the validation study.

• Details of the equipment used in the study, e.g. identifiers and qualification details.

• References to the laboratory notebooks, or equivalent used to record the raw data
obtained during the validation study.

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• Details of responsibilities and required signatures.

Statistics in Analytical Method Validation

The statistics required for the interpretation of validation results include: the
calculation of the mean, standard deviation, confidence intervals and relative standard
deviation for data sets obtained; regression analysis for evaluation of linearity and
accuracy (these were discussed in Chapter 2); comparative studies; and assessment of
the significance of outliers. Validated statistical software packages2,3 are normally used
for the calculation of all statistics associated with analytical method validation.

Statistical significance
A statistically significant difference means that there is statistical evidence that a
difference exists which is unlikely to have occurred by chance. It does not mean that
the difference is necessarily large or important and thus may not be of practical
relevance. Statistics are very useful to support analytical method validation but the
results should be used with caution. Sound scientific judgement is required for
interpretation of statistical results.

Comparative studies
Examples of comparative studies which could be performed during method validation
are:

• Specificity and accuracy, to compare the results obtained using the method being
validated against a second well characterised method.

• Precision, to compare the results obtained during intermediate precision and

reproducibility studies.

• Robustness, to compare the effects due to the factors under evaluation.

• Stability of solutions, to compare the results obtained from solution which have
been stored against the original results or results obtained for a freshly prepared
sample.
Student’s t-test
Student’s t-tests are one of the most commonly used statistical significance tests
applied to small data sets. A t-test may be used to compare two sets of data which are
each characterised by their mean, standard deviation and number of data points,
provided that the distribution of the data can be assumed to be normal. The outcome
of a t-test is that the null hypothesis is accepted or rejected. The null hypothesis is
that any differences between the data sets are due to random and not systematic
errors, i.e. both methods give the same results, or both samples contain the same
amount of analyte.

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Case Study
A validation protocol for the analytical method presented in Chapter 3, ‘Analytical
Method for Determination of the Assay and Degradation Products of ‘MiracleCure’
25 mg Tablets by HPLC’ (Figure 12), was designed previously (refer to Table 19). The
results from this study are presented in a tabular summary format in Table 25.
Table 25 Summary validation report for the analytical method and validation protocol detailed in Chapter 3
(refer to Figure 12 and Table 19)

Validation Results Acceptance criteria

characteristic

Specificity Chromatograms obtained for the No peaks interfering with the peaks due to
specificity solutions showed that there are MiracleCure, Impurity X, DP 1 or DP 2.
no interfering peaks.
Minimum resolution between peaks of
Retention times: interest and neighbouring peaks = 2.
MiracleCure 10.3 minutes
Impurity X 12.4 minutes
DP 1 15.0 minutes
DP 2 37.2 minutes
Excipients no peak detected

Critical pair resolution = 3.2

(MiracleCure and Impurity X)

Linearity – Assay Calibration model is valid Calibration model valid.

Equation of line, y = 1219x + 691 Residual plot shows random scatter and
no systematic trends.
Correlation coefficient = 1.000
95% confidence interval of intercept
Residual sum of squares = 150186
includes zero, or intercept is less than 2%
95% confidence interval for the intercept of nominal response.
includes zero, -105 to 1487, intercept is
0.56% of nominal response.
The plot of residuals shows random
scatter and no systematic trends.

Linearity – DP 1 Calibration model is valid Calibration model valid.

Equation of line, y = 1303x – 3.17 Residual plot shows random scatter and
no systematic trends.
Correlation coefficient = 0.998
95% confidence interval of intercept
Residual sum of squares = 4318
includes zero, or intercept is less than 2%
95% confidence interval for the intercept of response at 50% SL (0.25% nominal).
includes zero, -18.47 to 12.12, intercept is
0.95% of response at 50% SL.
The plot of residuals shows random
scatter and no systematic trends.

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Glossary

A
Acceptance criteria The criteria which are applied to the results obtained
from a validation study, if the results comply with the
criteria then it is concluded that the analytical method is
fit for the intended purpose.
Accuracy The closeness of agreement between a test result and the
accepted reference value.
Active Pharmaceutical The ‘active’ or the ‘active pharmaceutical ingredient’ is
Ingredient (API) the substance in a drug preparation that is
pharmaceutically active.
Analyte The compound of interest to be analysed.
Analytical Instrument Documented evidence that an analytical instrument
Qualification (AIQ) performs suitably for its intended purpose and that it is
properly maintained and calibrated.
Assay An analytical method to analyse or quantify a substance
in a sample.

B
Bioanalysis The chemical analysis of biological samples, e.g. plasma,
urine etc.
Bioassay A biological test, measurement or analysis to determine
whether compounds have the desired effect either in a
living organism, outside an organism, or in an artificial
environment.
Bioavailability The amount of drug absorbed into the body.
Bioequivalence The comparison of the expected in vivo biological
equivalence of two proprietary preparations of a drug. If
two products are said to be bioequivalent it means that
they would be expected to be, for all intents and
purposes, the same.
Biopharmaceutical A drug produced by biotechnology.
Biotechnology The application of scientific and engineering principles to
the processing of materials by biological agents.

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List of Abbreviations

AIQ Analytical Instrument Qualification

API Active Pharmaceutical Ingredient

CDS Chromatography Data System

COTS Commercial Off The Shelf

DAD Diode Array Detector

DL Detection Limit
DOE Design of Experiments
DQ Design Qualification

EMEA European Medicines Agency

EP European Pharmacopoeia

FDA Food and Drug Administration (US)

GC Gas Chromatography
GFC Gel Filtration Chromatography
GLP Good Laboratory Practice
GMP Good Manufacturing Practice

HPLC High Performance Liquid Chromatography

ICH International Conference on Harmonisation

IMPD Investigational Medicinal Product Dossier
IND Investigational New Drug
IQ Installation Qualification

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Index

Acceptance criteria, 73, 75 selection of factors and levels, 60

Accuracy, 10, 37, 49, 77, 113 Forced degradation, 25
acceptance criteria, 78 Good Manufacturing Practices, 1
assay methods, 38 Guidelines, validation, 5
experimental procedure, 77 FDA, 6
ICH guideline, 37 ICH, 5
impurities methods, 40 Hetereoscedasticity, 33
results, 113 Intermediate precision, 10, 46
sample preparation, 40 factors, 46
Analytical method ruggedness, 46
assay, 7 Limit of detection. See Detection limit
automated, 87 Limit of quantification. See Quantitation
bioanalytical, 9 limit
cleaning samples, 41 Linearity, 11, 27, 76, 113
identification, 6 100% standard methods, 34
impurities, 7 acceptance criteria, 77
life cycle, 4 assay and impurity methods, 34
microbiological, 9 bioanalytical methods, 34
pharmacopoeial, 87 experimental procedure, 76
purpose, 3 ICH guideline, 27
reporting level, 36 results, 113
specific tests, 8 Peak purity, 25
transfer, 47 Precision, 10, 42, 49, 78
types, 6 acceptance criteria, 79
universal tests, 6 experimental procedure, 78
water determination, 41 Horwitz Trumpet, 45
Calibration ICH guideline, 42
model, 27, 41 Intermediate precision. See Intermediate
multi-level, 29 precision
non-linear models, 33 measurement, 43
single point, 28 repeatability. See Repeatability
Coefficient of determination, 32 reproducibility. See Reproducibility
Confidence intervals. See Statistics results, 113
Correlation coefficient, 32 system precision, 43
Data quality, 1 Protocol, validation, 73
Detection limit, 11, 50, 79, 114 assay methods, 81
acceptance criteria, 80 bioanalytical methods, 86
experimental procedure, 79 case study, 93
ICH guideline, 50 choosing characteristics for study, 74
results, 114 contents, 73
Experimental design, 46, 60 execution of, 90
fractional factorials, 60 experimental procedure, 75
Plackett-Burman, 60 identification methods, 81

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