Suitability of Analytical Methods
for Stability Testing – Is There
Room for Improvement?
By David R. Rudd, Ph.D.
Glaxo Wellcome Research and Development
❖
O
f all the topic areas which
excellent position to have achieved.
have been addressed by the
With unambiguous guidance avail❝...the general
praiseworthy International
able, we can be confident that, by
philosophy of
Conference on Harmonization (ICH)
following this internationally-agreed
initiative in recent years, arguably
we will gain the approval of
analytical method path,
that of analytical method validation
our benefactors and reviewers (espeis the one which has progressed with
cially the regulatory agencies) when
validation
the least difficulty and in a reasonour work is presented.
employed
able time frame. That is not to say
But is everything as straightforthat there were not problems or areas
ward as we might expect? Like
internationally
of disagreement within the topic
most aspects of life, is the validation
group – or that other topic groups
of analytical methods more comseemed
fairly
were any less efficient or industrious
plex than it seems?
– but rather that the general philosoIn an attempt to answer these
consistent at
phy of analytical method validation
questions, let me allude to two feathe outset. . .❞
employed internationally seemed
tures of the available ICH guidance
fairly consistent at the outset, allowdocuments which are frequently
ing the harmonization process to become more an exer- raised during discussions. The first is one which was
cise of unification of the description of this approach
quite deliberately introduced by the members of the
and not a modification of the approach itself.
topic group during the development of the guidance
As a result, the analytical method validation topic
documents (particularly the “Methodology” text)
group was able to produce two documents which
and is that of flexibility.
assisted in standardizing the terminology used durSome users have been critical of the guidance
ing method validation exercises1 and which provide
documents in this respect, pointing out that even the
some guidance in the practical aspects of conducting
“Methodology” text does not give clear, specific,
these studies.2 These documents are now being ref- step-wise instructions concerning the practical
erenced extensively, both within the pharmaceutical aspects of analytical method validation – and this is
industry and by regulatory agencies, and therefore
quite true. However, this is also quite deliberate,
represent the definitive philosophy which is since the members of the topic group felt strongly
expected to be employed by those engaged in ana- that the guidance documents should allow harmolytical method validation programs.
nization of the philosophy of analytical method valClearly, as an exercise in harmonization, this is an
idation, while recognizing that the practicalities
30
Analytical Method Validation
David R. Rudd, Ph.D.
would depend very much on the circumstances
attempted to do is convey the idea of an analytical
under which the analytical method might be used.
method validation philosophy which will be acceptThese circumstances might encompass aspects such
able internationally if based on the contents of the
as analyte concentration range, sample size, sample
ICH texts, but whose practical implementation will
matrix etc., each of which might influence the pracdepend heavily on the sound scientific judgement
tical aspect of the analytical method validation pro- and rationale provided by the analytical chemist.
gram. In short, it was felt that to attempt to describe This, in fact, is the price that (quite rightly) must be
a single, explicit, step-by-step procedure which
paid if flexibility and the absence of prescriptive
could be followed in all (or, at least, in the majority
acceptance criteria are to be preserved in the guidof) cases was not only naive, but also potentially
ance documents. The analytical chemist must be
misleading to the inexperienced analytical chemist.
prepared to exercise his or her scientific judgement
We live in a complex environment and need to adapt
in order to establish the specific practical steps
to changes, however minor, at all times. Over-simwhich need to be made during the validation proplification can result in a lack of appreciation of subgram, and the appropriate scientific rationale must
tlety and an inability to react, both intellectually and
be provided to justify the approach taken. In short,
practically, to new circumstances.
the analytical method validation philosophy must be
Thus, just as we may choose to furnish a house in
based on good science – nothing more, nothing less.
a “harmonized” way (carpets, drapes, chairs, tables, This is the harmonized philosophy which ICH
beds, etc.), we reserve the right to exercise individpreaches.
ual style or preferences (colors, sizes, etc.) if this
Under these terms, and recognizing the extent to
still enables us to achieve the
required outcome. With ana❝We live in a complex environment
lytical method validation, we
are seeking to demonstrate
and need to adapt to changes,
the suitability of the method
for its intended purpose and,
however minor, at all times.❞
notwithstanding the available
guidance from ICH or any
other reputable source, exactly how this might be
which the ICH guidance has become ingrained into
done remains the prerogative and responsibility of everyday culture within the pharmaceutical industhe analytical chemist.
try’s analytical community, it is worth considering
The second feature which is often highlighted as
whether the process is now complete for the topic of
a shortcoming of the presently available guidance
analytical method validation. Are there major areas
documents is the absence of any acceptance criteria
which would benefit from further development, or is
for results generated during the analytical method this topic no longer one which needs to occupy our
validation program. Again, the topic group largely attention?
sought to avoid being overly prescriptive in this area
In order to address these questions, it would be
for exactly the reasons described earlier – that is, the useful to consider the application of analytical
level of performance deemed acceptable for any par- methodology to formal, long-term stability studies –
ticular analytical method depends heavily on the cirthis area being one in which issues of method percumstances under which it is expected to be used,
formance frequently arise.
and this can only realistically be assessed by the anaIn any application area, there is an important
lytical chemist.
point for the analytical chemist to appreciate – that
So where does this leave us? Can we simply refer
the responsibility for demonstration of suitability of
to the available ICH texts on analytical method valithe method for its intended use is a major feature of
dation, following the guidance when it suits, but
the development and validation of the analytical prolargely “doing our own thing” when it doesn’t? Well,
cedure.
of course not. What these introductory remarks have
It is this point which forms the basis of this artiSpecial Edition
31
David R. Rudd, Ph.D.
cle – the importance of considering the implications
of method performance data generated during
method validation experiments.
So, to revert to the chosen application, what are
the particular issues associated with analytical
methodology used for stability assessment of pharmaceutical materials? There is probably no single
answer to this question which would satisfy everybody, but the following three aspects would usually
be expected to feature somewhere in most replies:
• Specificity (or “selectivity” as many practicing
analytical chemists still prefer to call the level
of discrimination of the method).
• Precision associated with stability data generated (or, more exactly, the confidence interval of
a particular data point).
• The long-term consistency (or robustness) of
the performance of the method.
Let us look at each of these aspects in an attempt
to identify areas which, the author believes, could
still benefit from improvement (albeit to various
degrees). The suggestion here is that if it is agreed
that there is scope for improvement within our current practices, let us try to establish a process which
allows those improvements to be introduced, ideally
in a fully harmonized fashion.
ICH has fulfilled an important role so far. Let us
see if we can extend that process to the common
good (i.e., for the benefit of industry, the regulatory
Agency, and especially the patient).
Specificity
For any analytical method intended to be used to
monitor changes in product quality throughout the
proposed life-to-expiry, it is clearly crucial that this
method has the ability either to detect and quantify
loss of active potency or the increase in levels of
drug-related impurities (degradation products). This
capability reflects the level of discrimination of the
method and is termed within ICH as “specificity.” It
may often prove difficult to achieve all the desired
characteristics of detection, discrimination, and
quantification of the active drug substance and the
drug-related impurities (degradation products) in a
single analytical method. For this reason, ICH (and
32
Analytical Method Validation
good scientific practice) allows the analytical
chemist to arrive at this outcome using a combination of methods. Thus, a long-term stability study
may often be undertaken using an assay method for
the active drug substance where drug-related impurities (perhaps synthetic impurities and/or degradation products) are known not to interfere with the
quantification of the analyte, supported by one or
more additional methods which allow quantification
of the appropriate drug-related impurities (degradation products).
Now, no one would seriously question this practice. The rationale is scientifically sound, and ICH
has rightly highlighted within its guidance texts this
flexibility of approach as being entirely appropriate.
However, the performance requirements of assay
methods and impurity methods are different. In the
former case, the analyte level is relatively high
(compared to levels of drug-related impurities) and,
provided that sufficient specificity is demonstrated
to ensure lack of interference from drug-related
impurities and any other components within the
sample, then that is generally regarded as adequate
discrimination for methods of this type. The fact that
such a method may be unable to detect (for example,
due to inappropriate choice of detection conditions)
certain drug-related impurities (degradation products) is of no consequence as the main purpose of
the method is simply to quantify the level of active
drug substance.
Contrast this with the level of performance
required for drug-related impurity methods. Such
methods, by definition, must be capable of detecting
and quantifying all relevant drug-related impurities
– a requirement made especially more difficult in
view of the wide diversity of chemical structure and
spectroscopic, chromatographic, and physical properties’ variations which are likely to be encountered
with such molecules.
And yet, for most long-term stability studies conducted on pharmaceutical materials, and despite this
extensive range of method performance requirements, both assay and impurity methods are generally developed around a single “core” analytical
technology – that of high performance liquid chromatography (HPLC).
Of course, other approaches are used – and even
the term “HPLC” encompasses a wide range of vari-
David R. Rudd, Ph.D.
ations in separation mode and detection capability.
Nevertheless, reverse-phase HPLC with ultraviolet
(UV) detection (frequently using a single wavelength) accounts for a huge percentage of the stability methodology used in the pharmaceutical industry
today.
Now, this position would be entirely tenable if
such methodology were truly capable of providing
the level of performance we need in all of the aspects
discussed earlier, but can HPLC routinely deliver
this? Frequently “Yes,” but often “No” is the conclusion – and the implications of this will become
apparent later in this article.
Precision
What level of precision is needed in an assay
method used in a long-term stability study? This
question is best answered by reconsidering the problem. During most stability studies, we are looking to
detect and quantify any long-term change in drug
content using our assay method. So what level of
change needs to be detected? Well, this is clearly
product dependent and will be based on the manufacturing and life-to-expiry specifications, but let us
take a simple, yet typical case. If a three-year study
was being conducted, for example, on a typical
tablet product, it would be important to have confidence that a change in assay of two to three percent,
for example, could be detected over this period.
(More exactly, it would be of great concern if the
assay method was unable to detect such a change.)
Figure 1 shows a simulated data set (derived statistically using Microsoft Excel® and representing a
population with a specified mean value and a specified standard deviation) which may be regarded as
typical in form of that generated using an HPLC
assay method during a long-term stability study. By
universal acclaim, and opinions have been sought
from a large number of experienced people within
the pharmaceutical industry and from regulatory
agencies, this data set is regarded as being far too
variable or irreproducible to allow meaningful conclusions to be drawn.
Nevertheless, this data set is nothing more than a
series of individual values drawn from two normally
distributed populations with defined characteristics.
Each of the individual data points represents an estimate of the true population mean, with the variation
in results indicative of the level of precision of the
measurement method rather than any inhomogeneity
in the tablet batch. The data points assigned to the
25ºC/60%RH storage condition come from a population of mean = 100 and with a standard deviation
= 2.0, whereas those from the 40ºC/75%RH storage
condition have the same standard deviation (2.0), but
a mean = 98.0.
In stability terms, this equates to a batch of product losing 2.0% of its active content instantly (rather
than gradually over the full duration of the stability
study) and being monitored using an assay method
with a level of reproducibility of 2.0% (a typical relative standard deviation for HPLC). And yet, by universal agreement, such a change (arguably even
more distinct than if the 2.0% loss occurred gradually over the full duration of the stability study) cannot reliably be detected using methodology with a
level of precision typical for HPLC.
The startling, and somewhat ominous, conclusion
is that many HPLC assay methods used during stability studies within the pharmaceutical industry are
unlikely to be capable of detecting significant levels of
change of active content in pharmaceutical products.
Such methods are clearly inadequate for their
intended purpose, and it is this situation which, the
author believes, must be rectified with immediate effect.
By taking this simulation work further, we have
established that assay precision levels of 1.0% RSD (or
Figure 1
Simulation of Assay Data Obtained at Two Storage Conditions
During a 12-Month Stability Program
Storage Period (Months)
25ºC / 60% RH
40ºC / 75% RH
0
100.0
100.0
1
99.2
94.6
3
99.9
100.3
6
97.9
97.3
9
104.7
97.5
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99.1
99.4
33
David R. Rudd, Ph.D.
better) are necessary to achieve the level of performance
required in the chosen example and, realistically, this is
beyond the capabilities of most HPLC methods currently applied routinely to long-term stability studies.
So, does this mean that HPLC is wholly inappropriate as a principal assay technique for long-term
stability studies involving pharmaceutical materials?
Obviously, this conclusion would be an over-reaction to the shortcomings discussed previously, but it
does indicate that either highly precise, yet equally
selective, assay methods are required in order to
achieve the level of “change detection” necessary in
stability studies or that emphasis must be placed on
quantification of degradation products as more subtle indicators of changes in product quality.
Interestingly, Figure 2 (representing a hypothetical impurity data set such as might be obtained using
an HPLC impurity method during a stability study)
reveals unequivocally that changes in product stability have occurred – even though the relative accuracy
and precision associated with the impurity data
(compared to that obtainable using an HPLC assay
method) is quite poor. (As an example, an accuracy
of +/- 10% and a relative standard deviation of 5% to
10% is generally regarded as an acceptable level of
performance for an HPLC impurity method). This is
indeed a fortuitous situation and suggests how valuable impurity assessment can be as the principal indicator of change, while not imposing excessive
demands on method performance (such as would be
required if reliance were placed mainly on the use of
assay data as we have seen earlier).
So, the conclusion seems to be that in the absence
of a highly precise, highly selective assay technique
(at least, better than most HPLC approaches used at
present), the assessment of product quality during
stability studies is best carried out using estimates of
increase in levels of drug-related impurities (degradation products). Modern methodology (even based
on HPLC) seems to render this approach feasible –
with one important caveat. That is, if emphasis is to
be placed primarily on monitoring changes in impurity levels, we need to be confident that all impurities are being detected. This is another major stumbling block for HPLC-based impurity methods using
single wavelength UV detection: First, not all impurities have identical spectroscopic characteristics;
second, not all impurities exhibit a UV chromophore; and, third, not all impurities elute from an
HPLC column. Each of these features could contribute to an underestimation of total impurity content by HPLC and, hence, lead to erroneous assessment of product quality and stability.
The solution? Either alternative methodology
which fails to exhibit these shortcomings (and
many practicing analytical chemists believe that
the combination of capillary electrophoresis with
mass spectroscopic detection currently offers the
best hope of high selectivity, adequate precision,
and something approaching “universal” detection
capability) or a method validation program which
addresses the performance issues raised. Clearly it
is in this latter area, at least until viable alternative
methodology becomes routinely available, that the
author believes that progress, in an internationallyharmonized fashion, should and must be made.
Figure 2
Simulation of Impurity Data Obtained at Two Storage Conditions
During a 12-Month Stability Program
Storage Period (Months)
25ºC
40ºC
34
Principal Impurity
0
0.5
1
0.5
3
0.5
6
0.4
9
0.6
12
0.6
Secondary Impurity
0.2
0.3
0.2
0.2
0.2
0.3
Total
Principal Impurity
1.3
0.5
1.4
0.5
1.5
0.5
1.2
0.7
1.5
0.8
1.6
0.9
Secondary Impurity
0.2
0.3
0.2
0.5
0.6
0.8
Total
1.3
1.5
1.5
2.0
2.1
2.4
Analytical Method Validation
David R. Rudd, Ph.D.
Consistency of Performance
This final aspect is considered largely on the
basis that long-term stability studies can typically
last for periods of three years or longer. During this
time, consistent method performance must be
assured if relatively subtle changes in product stability are not to be overshadowed by (larger) variations
in the quantitative aspects of the measurement
method. How can this be ensured?
There are two ways: First, by designing robustness (consistency of performance by virtue of insensitivity to typical variations in operating conditions)
into the analytical method during its development;
and, second, by providing appropriate system suitability tests which will allow confirmation of
method performance each time the method is used.
Both of these aspects of “method quality assurance” may be studied and then implemented using
an appropriate validation program and, hence, provide (in the author’s opinion) another opportunity
for rational, harmonized development of suitable
strategies and philosophies.
ble of quantifying all relevant drug-related impurities (degradation products) – and that no relevant impurities are lost, either through inappropriate sample treatment or through on-column
retention (for example) in the case of HPLC.
• Development of meaningful system suitability
tests, based on carefully designed method robustness studies, with appropriate acceptance criteria
for key aspects of method performance, to ensure
that a consistent level of operation is achieved
throughout the duration of the stability study.
While there may be other aspects of method performance which merit equal consideration, the
author believes that substantial advances may be
made in improving data quality (and, hence, knowledge of product quality) by addressing the issues
raised. In keeping with the ICH philosophy, however, the plea is not for prescriptive, inflexible guidance documents, but rather for an appreciation of the
issues raised (and, hence, the shortcomings in our
current practice) and a concerted, internationally
harmonized approach to establishing ways in which
these points may be addressed. ❏
Conclusion
The ICH process has substantially advanced the
harmonization of method validation philosophy
within the pharmaceutical industry. Adoption of the
principles expounded in available guidance, combined with sound, scientific judgement will generally allow the practicing analytical chemist to
address most of the issues likely to be encountered
under normal circumstances.
When it comes to applying such methods to longterm stability studies, however, the author believes
there are a number of aspects of method performance
which remain to be addressed and which, therefore,
offer the opportunity for further development of suitable validation strategies and philosophies.
Such aspects include:
• Suitability of assay methods (or assay techniques, in general) with respect to the level of
precision required in order to detect defined levels of change during the life-to-expiry of the
pharmaceutical material.
• Demonstration that impurity methods are capa-
About the Author
David R. Rudd, Ph.D. heads the Process Improvement and Automation Group within Pharmaceutical Development (Europe) at Glaxo Wellcome
Research and Development in the U.K. He has been
actively involved in all aspects of pharmaceutical
development for over 20 years and was a key member of the ICH working group which produced the
definitive guidance texts on analytical method validation. David can be reached by phone at 44-1920882367, by fax at 44-1920-882295, and by e-mail at
drr1605@glaxowellcome.co.uk
Please note that the opinions expressed in this article are those of the author and do not necessarily
reflect those of Glaxo Wellcome Research and
Development Ltd.
References
1.
ICH harmonized tripartite guideline for Q2A: Validation of
Analytical Procedures: Definitions and Terminology – available at http://www.fda.gov/cder/guidance/index.htm.
2. ICH harmonized tripartite guideline for Q2B: Validation
of Analytical Procedures: Methodology – available at
http://www.fda.gov/cder/guidance/index.htm.
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