White Paper

Method Development
and Validation for
Nutraceuticals

Maud Silvent
Technical Specialist

David Neville
Technical Specialist
Method Development and Validation for Nutraceuticals
Introduction
Nutraceutical is a term formed from the
amalgamation of the words “pharmaceutical”
and “nutrition” by Dr Stephen Felice in 1989.
Others have since tried to further define the
term to distinguish and clarify the difference
between functional foods, dietary supplements
and nutraceuticals. There is now some
consensus that a nutraceutical can be loosely
defined as “a functional food or supplement that
aids in the prevention or alleviation of a disease
state or disorder (except for anaemia), and not
just supplement the diet”. Therefore, a
nutraceutical may be incorporated into a food
product/matrix, or into a pill, capsule, tablet
etc. There is no strict legal definition in the US,
Canada or Europe to define nutraceutical, but
other legal requirements do exist to cover
dietary supplements and novel foods within
these markets.
Many of the compounds present in
nutraceuticals that may possess beneficial
health effects are derived from botanical
sources, and will be considered to be natural in
form. These products can be as diverse as
proteins, peptides, lipids, flavonoids and iso-
flavanoids, polyphenols and tannins.
Additionally, vitamins and minerals, sugar
molecules such as glucosamine and chondroitin,
may also be considered as nutraceuticals.
Therefore, the range of nutraceuticals that
require analysis is diverse, both in types of
structures and matrices, and this is matched by
the diversity of techniques and extraction
conditions that need to be employed for
accurate quantitation of the active nutraceutical
of interest. It is also important to note that the
purification, or not, of the nutraceutical from its
natural matrix must also be considered as there
may be contaminants/impurities/adulterants
present that are deleterious to human health,
or whose amounts are regulated by legislation
(heavy metals, allergens, toxins). These must
also be incorporated into a testing regime to
ensure the safety of the product end-user.
This article focuses on the analytical approaches
that must be considered when performing a
method development and validation exercise to
ensure that your test method is fit-for-purpose,
and meets the regulatory need of your
nutraceutical of interest. The ultimate aim is to
accurately quantify your nutraceutical of
interest whether in final product matrix or as a
raw material prior to incorporation into product.
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Due to the inherent complexity of many of the
nutraceutical products that exist on the market,
and to the complexity of the nutraceuticals
themselves, there is no single analytical
approach that can be undertaken. The
extraction of the nutraceutical can be
performed in a number of ways from simple
liquid extraction into water or organic solvent,
to more complex distillation procedures. This
extraction is prior to final analytical procedures
that may be HPLC- or GC-based with
fluorescent, UV, mass spectrometric, refractive
index, FID or other detection techniques.
Additionally, ELISA, enzyme-based kits and
chemical assays can be used. These assays are
often separate from the type of testing regime
that may be necessary to define the physico-
chemical properties of your product of interest.
Therefore, the principles that must be followed
to ensure the assay is accurate, precise,
reproducible, and fit-for-purpose are outlined.
Development of Assay
At the outset of any method development and
validation procedure it is important to
understand the requirements of the assay, how
the data will be used, who will perform the
assay and where the assay will be employed.
Factors such as in-line or near-line testing
(quick and easy), experience of operator,
QC/QA or NPD laboratory (robust), for
regulatory submission, equipment capabilities,
and method transfer will need to be considered.
These findings will help in determining the best
approach that can be taken to ensure that the
method fulfils the business need. The
experience of the laboratory concerned in
analysing similar matrices, a comprehensive
literature search to discover what is currently
available (approved methods from AOAC,
Pharmacopoeias, AACC and ISO, or published
peer-reviewed articles) and the availability of
appropriate standards can be investigated.
Approved methods can form the basis of any
new method that may be required, and are
excellent starting points for development
studies. As many of the analytical methods
currently used are based on HPLC separation
prior to detection by the most appropriate
method for the nutraceutical of interest (UV, RI,
Fluorescence, ELSD, MS etc.), this will be the
focus of this article. However, the principles still
apply to other types of analytical methods that
may be used, such as wet chemistry,
colourimetric or enzyme-based.
Following the initial scoping phase,
development becomes a matter of
experimentation to find the optimal analytical
parameters, such as pH, extraction solution,
mobile phase, type of liquid chromatography
column, and retention time, for the method.
The optimisation of each parameter will require
careful attention to detail. This will ensure that
maximum recovery of nutraceutical and
accurate quantitation of the analyte of interest
are achieved. Adequate time must be taken for
each separate phase as these conditions will be
used to determine the final, optimised and
streamlined procedure in the next stage of the
method validation. Additionally, it is the
optimisation of each stage (see Figure 1) that
ensures that progress through method
validation is as easy as possible. It is important
that the source and quality of reagents used,
the supplier of the HPLC system (and model
number of component parts) used, and
instrument settings are specified within the
method to ensure that the method can be
reproduced by other operators and laboratories.
Figure 1
HPLC method development single
considerations
It is important to note that method
development and validation can be undertaken
to analyse a component, but within a number of
different matrices. As the nutraceutical may be
incorporated into different product matrices, a
decision must be made whether the
development and validation is for one or
multiple matrices. It is often better to develop
and validate for multiple matrices rather than
go through an extension-of-scope exercise.
This, of course, is highly dependent on the
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initial product development cycle, and the
amount of analytical time available to perform
the necessary experiments.
Validation of Assay
Validation is defined by the International
Organization for Standardisation (ISO) as
“verification, where the specified requirements
are adequate for an intended use”. Additionally,
the term verification is defined as “the
confirmation by examination and the provision
of objective evidence that the particular
requirements for a specific intended use are
fulfilled (ISO/IEC 17025:2005 cl. 5.4.5.1)”.
The method validation starts only when the
method parameters have been finalised as part
of the method development. In majority of
cases, a validation protocol will be written, and
agreed upon by the relevant people (analyst,
laboratory manager, QC/QA department, clients
etc.), following the ICH guidelines that are
routinely based on ISO guidelines. The
acceptance criteria will be set using data
generated during the method development
stage but will also depend of the agreed
compound specification tolerance. Additionally,
as previously mentioned, the source and quality
of reagents used, and the supplier of the HPLC
system (and model number of component
parts) used are defined. Final instrument
settings that are optimal for the nutraceutical of
interest (column temperature, eluent gradient
conditions, and detector settings) are also
defined, and used, as part of the validation
exercise. It is important to note that not all
laboratories are equipped with the same HPLC
systems. Therefore, part of the validation may
incorporate a study to test the equivalence of
results using different HPLC systems (see
reproducibility below).
Why is it important to validate a method once it
has been developed? The answer is to be
confident in your results and to ensure the
method is fit-for-purpose for that specific
compound in that specific matrix. When would it
be required? At any stage of discovery,
development, and manufacture of
pharmaceuticals or food products, but also to
perform stability studies, and to submit a
dossier for claim substantiation or novel food
ingredient. For example, knowing the
uncertainty of the method would give you an
indication if a trend in stability results is
genuine or not. The purpose of a method
validation is to confirm that the method is
specific, precise, accurate and robust. The level
and extent of the validation will evolve with the
nutraceutical development phase. Typical matrix. Working with CRMs would increase the
method parameters recommended by FDA, confidence in accessing the efficiency of the
USP, and ICH are as follows: extraction procedure of the compound(s) of
interest from the sample matrix. A second
 Specificity, approach would be to spike the sample
 Linearity range extraction solution with a known concentration
 Accuracy of the compound(s) of interest and check how
 Method precision (incorporating method much is recovered. This approach will not
repeatability, intermediate precision and investigate the efficiency of the extraction
method reproducibility) procedure on a sample embedded in a matrix,
 Limit of detection (LOD) and limit of but only how the sample matrix may affect the
quantitation (LOQ) spiked sample extraction procedures.
 Solution stability Investigation of different levels of spiking is the
 Robustness best approach when CRMs are not available. It
is important that the level of spiking should
Each parameter will give specific indication of cover the concentration of the compound(s) of
the method performance, and each topic is interest in your sample matrix over time.
covered in greater detail below.

Specificity
The specificity for the compound(s) of interest
needs to be evaluated to understand the ability
of the method parameters to separate any
potential interferences coming from the sample
matrix like other excipients, potential
degradation products and the mobile phase.
Ideally a placebo sample with the same
Figure 2
ingredients/excipients but without the
Precision and accuracy
compound(s) of interest will be available to
analyse to determine the specificity of the
Precision
method. A forced degradation study can also be
The method repeatability is the ability of an
performed to artificially stress the product and
analytical method to produce consistent results.
obtain potential degradation compounds that
The precision of a method is determined by its
could interfere with the analysis of the
repeatability, intermediate precision, and
compound(s) of interest.
reproducibility. A simplified diagram to illustrate
accuracy and precision is shown in Figure 2.
Linearity
The repeatability will be measured to define the
The linearity will assess the proportional range
consistency of results when analysed by the
of the method response, i.e. the concentration
same analyst, using the same instrument under
range over which the method can accurately
the same analytical conditions. In most cases, it
determine the compound concentration. A
is assessed by preparing and analysing the
minimum of five standard concentration levels,
same sample six to ten times. The percentage
covering the concentration range in the final
standard deviation will be assessed and
dilution of the sample matrix to be analysed, is
compared to the acceptance criteria laid out in
recommended for investigation. In the case of a
the validation protocol. The intermediate
chromatography technique, the different
precision will be measured to define the
standard solutions will be injected and analysed
consistency of results when samples are
in ascending and descending order to assess if
prepared and analysed by a second analyst
the method is affected by sample carryover into
using the same analytical conditions. Where
the next injection/analysis. It is also at this
possible, it is also best practice to perform the
stage that the standardisation parameters (use
new analysis using a second column of the
of internal or external standards for accurate
same specification as the first, and a second
quantitation of analyte) are investigated.
HPLC system (may or may not be the same
manufacturer/model of HPLC system). This will
Accuracy
evaluate the intra-laboratory precision. The
The accuracy is the capability of an analytical
percentage standard deviation will be assessed
method to determine the correct measurement,
and compared to the acceptance criteria laid
or exactness between a measured value and a
out in the validation protocol. The
theoretical value. The best approach is to work
reproducibility evaluates the consistency of
with certified reference materials (CRMs).
results between different laboratories or inter-
However, those are not always available for the
laboratory precision. It is worthwhile to note
compound(s) of interest or for the specific

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that The International Conference for
Harmonisation (ICH) addresses “ruggedness” as
intermediate precision and reproducibility. The
percentage standard deviation will be
determined and compared to the acceptance
criteria laid out in the validation protocol. The
repeatability, intermediate precision, and
reproducibility are used to determine the
method uncertainty or relative uncertainty.
Limit of Detection (LOD) and Limit of
Quantitation (LOQ)
The limit of detection (LOD) is commonly
defined as the lowest concentration of the
compound(s) of interest that can be detected
under given method conditions using the
defined instrument parameters. In
chromatographic analysis a signal-to-noise
(S/N) approach may be taken, where there is a
detected baseline noise in the method. The LOD
may be calculated as being of three times the
signal-to-noise (S/N) ratio. This is, in essence,
an instrumental detection limit. It is important
to note that though the compound can be
detected, it may not necessarily be within the
linearity of the assay. Therefore, the parameter
limit of quantitation must be defined.
Figure 3
The relationship between LOQ, LOD and linear
range of assay
The limit of quantitation (LOQ) is the lowest
concentration of the compound(s) of interest
that can be accurately and precisely quantified
under given method and conditions. The LOQ
may be calculated as being the sample
concentration that gives a ten times the signal-
to-noise ratio, and lies within the linear range of
the assay. The relationship between LOQ, LOD
and the linear range of the assay are illustrated
in Figure 3.
Solution Stability
There may be times when samples cannot be
immediately analysed, or there is a time delay
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between sample preparation and actual HPLC
analysis, or prepared samples need to be
reanalysed. Different conditions of temperature
and times-of-storage of prepared samples and
standards are assessed to determine if these
are detrimental to sample quantitation.
Robustness
The robustness will assess the method ability to
obtain consistent results when small variations
in the analytical parameters are applied. The
parameters under scrutiny could be column
temperature, mobile phase concentration, flow
rate, pH value, sample and standard solution
stability, mobile phase stability, any other
parameters specific to a particular technique
that could change over time and could
potentially affect the results.
Control
Once the method has been validated, it may be
useful to perform continuous quality control on
the assay to ensure that it is performing within
specifications. Control (or Shewhart) charts are
useful for large volume or continuous work.
They require starting with at least 20 – 30
values to calculate a mean and a standard
deviation (SD), which form the basis for control
values equivalent to the mean ± 2 * SD
(warning limits) and the mean ± 3 * SD
(rejection limits). At least one replicate test
portion of a stable in-house reference material
and a blank are run with every batch of test
samples. The calculated mean and standard
deviations (or range of replicates) of the
controls are continuously assessed. These
values are used for ongoing determination of
warning and rejection limits.
The analytical process is “in control” if not more
than 5% of the values fall in the warning zone.
Any value falling above the rejection limit or
two consecutive values in the warning region
requires investigation and corrective action.
Additionally, trends in the values obtained can
indicate that further investigation of the
analytical method may be required.
Challenges
A major challenge for nutraceutical analysis in
both method development and validation is the
availability of standard or certified reference
materials. In the case of a compound where no
previous method exists, it can be difficult to
obtain a reference material, or considerable
cost can be incurred in synthesising reference
material. Additionally, because the compounds
of interest may be endogenous to a matrix
(such as a plant extract), the level of analyte
often varies, depending on the agronomic
conditions. Therefore, developing and
optimizing methods for endogenous compounds
usually requires a great deal of experimentation
for the extraction and isolation phase of the
method. Ensuring that all of the analytes of
interest have been extracted from the matrix is
critical for the analysis of complex matrices,
such as processed foods. There is less difficulty
where the compound of interest is embedded
within clean matrices (tablet, capsule etc.) with
known excipients.
Considerable expense can be incurred when
developing and validating new methods. This
expense may be warranted where it can be
foreseen that the method will be used for
multiple samples over an extended time period.
Both the business need, and return-on-
investment (ROI), must be taken into
consideration when assessing the feasibility of
developing and validating any new method. It is
also worth noting that as instrumental
technology and detection methods evolve, new
methods may be necessary to meet the
demands of speed and efficiency. Additionally,
regulatory requirements may force the
adaptation of new methods that are more
sensitive, both in terms of compound
determination and identification of
impurities/adulterants within the sample
matrices.
Conclusion
Method development and validation is
necessary to ensure that the analytical
procedures for a nutraceutical are fit-for-
purpose, and can accurately quantify the
compound of interest. The process can be time
consuming and expensive, both in terms of
man-hour and equipment/consumables costs.
The work performed is carefully planned and
must be documented at all stages to ensure
that due process is followed. Once the method
is developed, a validation protocol is agreed
and, deviations from this procedure can occur
unless agreed upon and approved by the
relevant authorities. This does give some scope
for limited further method development, but
ideally this would be minimal. The purpose of
the development phase is to try and ensure
that the validation is performed using as close
to as final a method as is possible. The
validated final method can be specific for single
or multiple matrices, but each matrix must be
tested as part of the method development and
validation exercise. Before the method can be
put into routine use, the validation report must
be issued and approved. Subsequently, the
method can be transferred to the laboratory
where appropriate training of personnel can be
undertaken. When the required level of
proficiency is attained, the method can be
placed into routine use.

David Neville – Technical Specialist

David is a Senior Associate Principal Scientist at Reading Scientific Services Ltd (RSSL). His expertise is in the
analysis of sugars, carbohydrates, bioactive molecules and proteins in both food and biological matrices, and
has published widely on these topics. Additionally, he has developed and validated a number of methods for
detection of bioactive compounds within varied food matrices.

Maud Silvent – Technical Specialist

Maud is a Senior Scientist at Reading Scientific Services Ltd (RSSL). Her expertise is in ion chromatography-
based analysis of sugars in varied food matrices. Many of the analyses are novel and require, mostly
performing method development and method validation.

For further information, please contact Customer Services on enquiries@rssl.com

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