Conducting a successful metabolomics study can be a challenging undertaking. This brief eBook will walk you through the five (5) main components of a study necessary for optimal outcomes.

1. The Five Key Elements of a
Successful Metabolomics Study

2. The 5 Key Elements of a Successful Metabolomics Study 1
2
Metabolomics: Completing the Biological Picture
Metabolomics is offering new insights into
systems biology, empowering biomarker
discovery, and advancing research across
the life sciences.
Metabolomics is the systematic measurement
of the low molecular weight biochemicals
(~ 50-1500 Da) known as metabolites.
Nearly every variable, from genes and
microbiota to disease and diet, exerts
influence by subtly altering metabolite levels.
In this way, metabolites are sensitive
indicators of homeostatic imbalances.
Measuring metabolites using metabolomics
improves our understanding of how disease,
drug exposure, genetics, the microbiome,
diet, and lifestyle all influence the phenotype.

3. Leveraging Metabolomics to Advance Research
Researchers across multiple areas of the
life sciences are using metabolomics to
answer key research questions and
advance programs in translational science,
biomarker identification, and the study of
disease and treatment mechanisms.
While metabolomics has proven itself to be
an incredibly valuable research tool,
metabolomics studies also have unique
challenges to consider.
This brief e-book will provide you with the
Five Key Elements of a successful
metabolomics study:
1. Define a Clear Objective
2. Use Strong Study Design Elements
3. Power Your Study for Success
4. Choose The Right Profiling
Technology for Your Goals
5. Have a Data Interpretation Plan in
Place
The 5 Key Elements of a Successful Metabolomics Study 2

4. 1. Define a Clear Objective
The first and most critical step in any successful scientific study is to clearly define
the study’s objective.
Are you seeking general information to help you form a hypothesis?
Do you have a hypothesis in mind that you wish to validate?
Are you hoping to discover biomarkers for a disease?
Do you want to understand the MOA of a potential drug candidate?
The Metabolon Advantage:
Metabolon offers every investigator study design
assistance from an experienced Ph.D. scientist.
The 5 Key Elements of a Successful Metabolomics Study 3

5. A clear objective is critical to
identifying the ideal metabolomics
approach for your study.
For example, if you want to understand
a variable’s impact on the subject’s
entire metabolic system, you probably
want to employ a discovery
metabolomics approach, which
surveys all of the metabolites from
across the whole metabolome.
On the other hand, if you already have
some understanding of the metabolites
implicated in your study, you may want
to narrow your focus and home in on
specific metabolite classes (such as
lipids), specific metabolic pathways
(such as the glycolysis pathway), or a
specific metabolite biomarker.
• Whole-metabolome Profile
• DiseaseCharacterization
• Target Discovery/Validation
• Biomarker Discovery
• Translational Studies
Discovery Metabolomics
• Pathway Regulation
• Markers of Efficacy
• Monitor Individual
Response
• Translational Studies
Focused Profiling
Biomarker Assays
• Trial Enrollment
• Clinical Diagnostics
Define Your Approach
The 5 Key Elements of a Successful Metabolomics Study 4

6. Strong study design elements are central to uncovering biologically significant results.
Select the appropriate sample matrix (or combination of matrices)
Collect adequate exposures (dose and time of collection)
Employ controls for each tested variable – don’t skimp!
Take steps to minimize excess variation – maintain consistency!
A Tip from Metabolon: The more inherent variation
you can control for, the fewer samples that are
required for the study.
2. Use Strong Study Design Elements
The 5 Key Elements of a Successful Metabolomics Study 5

7. Sample Matrix & Collection
Collect Adequate Exposures
Sampling at only a single exposure point
is a gamble and may result in too few
metabolic changes to derive any
biomarkers or enhanced understanding.
At the other extreme, a single point
sample may reveal so many changes
that it can be difficult to assign cause
and effect.
Multi-exposure point studies, such as a
time-course study or a multi-dose study,
increases your chance of seeing
meaningful variations.
A good rule of thumb is to collect
samples at “mild,” “moderate” and
“severe” exposures to ensure that all
salient cause and effect changes are
captured.
SelectYour Sample Matrix
Depending on what you are hoping to
uncover, select an appropriate sample
matrix or matrices.
Try to use the matrix or combination
of matrices that are “closest” to the
system you are attempting to study.
For example, when studying the effect
of drug exposure on the brain, a CSF
sample will provide significantly more
information than a urine sample. On the
other hand, urine would be an appropriate
matrix if you are attempting to study a
treatment effect on kidney function.
When using animal models it may be
advisable to also include organ
samples, which could improve
understanding of the physiology’s precise
site of action.
The 5 Key Elements of a Successful Metabolomics Study 6

8. A Control for Every Variable
Sometimes our desire for robust results around a
research project makes us overly ambitious. It’s
important to avoid the temptation of testing
multiple variables unless you are prepared to
have each variable accompanied by rigorous
controls.
Embedding too many questions in a study without
sufficient controls built around each questions can
leave you with a lot of data and few meaningful
answers. Only incorporate as many questions and
variables as you can realistically test with
adequate controls.
Variables & Controls
Minimize Excess Variation
For every study, you need to consider how
much variance is inherent in the cohort or
study samples. For example, inbred rodent
model have significantly less variability than
human study subjects with different ages,
genders, genotypes, BMIs and lifestyles.
To accommodate the off-target variables, collect
adequate samples, employ strong controls, and
attempt to balance the groups where appropriate.
Also, consistency in sample collection and
handling is essential. Having rigorous collection
and handling protocols minimizes variance so that
you can obtain meaningful results with fewer
samples.
Remember: The more inherent variation you
can control for, the fewer samples that are
required for the study.
The 5 Key Elements of a Successful Metabolomics Study 7

9. 3. Power Your Study For Success
Strong study design can deliver biologically significant results, but a
well-powered study can provide statistically significant results.
Cell
Culture
Small
Animals
Human
Studies
Optimal >7 >10 >50
Rigorous 6-7 8-10 40-50
Acceptable 4-5 6-7 25-40
Fewer Required
• Strong phenotype or treatment effect
(toxicology study)
• Repeated sampling from the same
subject
• Multiple time points
• Multiple doses of a drug/inhibitor
More Required
• Subtle phenotype or treatment effect
(dietary supplements, exercise-induced
changes
• Mixed populations of subjects (mixed
gender, wide-ranging age or BMI)
• Multiple-site collections
The 5 Key Elements of a Successful Metabolomics Study 8
Study Subjects Per Group

10. Tissues:
100 mg
Biological Fluids:
100 μl
Cells:
100 μl pellet
Sample Quantities
Recommended for Optimal
Results:
Cell-Based Studies t1 t2
Drug Dose 2 5 5
Vehicle Control 5 5
Drug Dose 1 5 5
t3
5
5
5
Small Animal Studies Chow HFD
Overexpression 8 8
WT 8 8
KO 8 8
Human Studies ♂ ♀
Control 30 30
Case 30 30
Suggested Sample Type & Amount for
Adequate Power
The 5 Key Elements of a Successful Metabolomics Study 9

11. 4. Choose the Right Technology for Your Goals
Metabolomics requires a different and more
sophisticated profiling platform than many
other types of “omic” research (such as
genomic or proteomic research).
Metabolites, which are small and highly abundant,
are also extremely chemically diverse, and they
vary substantially in their molecular weight and
solubility. This makes it difficult for a single
analytical method to accurately measure all of the
metabolites present in a sample.
In order to have meaningful study results, you
should select a metabolomics “platform” (screening
technology) with both advanced instrumentation
and sophisticated informatics.
Sensitive and precise instrumentation is necessary
to detect all of the low-level metabolites present in
a sample, while sophisticated informatics help
rapidly and accurately identify and quantify these
metabolites.
Profiling
Platform
The 5 Key Elements of a Successful Metabolomics Study 10

12. 12
The Right Instrumentation
There are many approaches to generating
metabolomics data. Some of the most popular
approaches include liquid or gas chromatography
coupled to mass spectrometry (LC/MS, GC/MS),
nuclear magnetic resonance (NMR)
spectroscopy, and capillary electrophoresis (CE)
chromatography mass spectrometry methods.
Each approach has pros and cons, and some
may be more suited to your study than others.
NMR, for example, does not destroy the sample,
but it has very limited breadth of metabolite
coverage. CE allows you to profile with smaller
sample quantities and great resolving power, but
it has limitations for detecting “neutral”
metabolites.
UPLC/MS technology is among the most
sensitive and highly-resolving metabolomics
technologies. LC-MS offers a broad and
sensitive screening of most metabolite classes,
while GC-MS and LC-MS+ HILIC are able to
detect certain polar metabolites.
For a metabolomics discovery study, you
generally have the best chance of capturing
the full range of metabolites present in a
sample if you select a multi-system approach
that runs multiple detection instruments in
tandem.
The 5 Key Elements of a Successful Metabolomics Study 11

13. Informatics: the Key to Good Data
Regardless of the instrumentation used, a single
metabolomics study is likely to identify hundreds
of metabolites and produce thousands to tens of
thousands of data points.
Attempting to identify metabolites and derive
biological significance from the mountain of data
collected can be time-consuming and resource-
intensive.
Many metabolomics technologies use an “ion-
centric” approach. This approach requires
manual identification of all of the metabolites
using ion features. This method has a high
potential for false discovery and ambiguous
identification, leading to substantially fewer
accurately identified metabolites.
On the other hand, an automated,
“chemocentric” approach incorporates
sophisticated software tools that distills the
thousands of ion features, eliminate noise, and
automatically classify metabolites by key ion
features (mass, retention time and peak area).
This approach requires an exhaustive chemical
reference library of metabolite standards and
sophisticated informatics software that can
identify the metabolites.
When this approach is used for broad, “whole
metabolome” profiling, it ensures a more accurate
measurement of the metabolites and their
perturbation. It also allows for faster, easier and
more accurate statistical analysis.
The 5 Key Elements of a Successful Metabolomics Study 12

14. Nearly any metabolomics profiling technology
will produce large amounts of complex data.
You must have a plan in place for translating
that data into meaningful biological insight.
Your data interpretation plan should be
driven by the “deliverables” you expect to
receive from your metabolomics study.
If you will receive only the raw study data, be
prepared to dedicate substantial time and
resources to verifying the metabolite
assignments, performing statistical analysis,
and researching the biological implications of
any metabolic perturbations. Be aware that
this process can be time-consuming and
challenging, particularly if your analyst does
not provide you with specialized interpretation
tools. Make sure to budget your resources
accordingly.
On the other hand, if your metabolomics study
provider offers specialized interpretation tools or
verified statistical analysis and pathway
interpretation, your resources can be devoted to
conducting any recommended follow-up studies
and preparing your work for publication.
5. Have a Data Interpretation Plan in Place
The 5 Key Elements of a Successful Metabolomics Study 13

15. To review, the five key elements of a successful metabolomics study are:
1. Define a Clear Objective
2. Use Strong Study Design Elements
3. Power Your Study for Success
4. Choose the Right Technology for Your Goals
5. Have a Data Interpretation Plan in Place
When all of these components are present in your study, you dramatically
improve your chances of deriving meaningful findings that translate into
actionable results.
To learn how metabolomics is advancing research in your field, visit
www.metabolon.com/pubs, or access our illustrated Publication Index.
Summary: The 5 Key Elements of Success
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