Your Guide to Metabolomics

Chapter 9—Designing a Metabolomics Study

As you’ve learned throughout this guide, metabolomics analyses are powerful additions to any scientific study, from basic research to toxicology studies. Agencies around the world are also working to make metabolomics an important component to uphold existing regulatory standards and inform the development of new ones. But achieving a controlled and insightful research outcome is highly dependent on the quality of your study preparation. In this final installment of our metabolomics guide, we’ll walk you through the fundamentals of how to design and execute a robust metabolomics study to answer your research question(s).

What’s Your Study Goal?

The most important part of any metabolomics study is defining the scope of your study. Two approaches may be leveraged: global or targeted. Global, or untargeted metabolomics approaches are semi-quantitative and consider all output metabolite data, while targeted studies utilize panels of known metabolites to quantify the amounts of those specific metabolites in the sample(s) of interest. Which approach you choose, or whether you use a combination, depends on your research question.

For those interested in identifying novel biomarkers or seeking to understand the presence of a broad set of metabolites, global metabolomics is the better choice. If, however, you know what you’re looking for and want to know how much of a specific metabolite or groups of metabolites are present in your sample, you’ll need a targeted approach. Targeted approaches can facilitate pretty much any scientific question; at Metabolon, we provide targeted panels for amino acids, bile acids, central carbons, complex lipids, fatty acids, impaired glucose tolerance, insulin resistance, short chain fatty acids, and more. And, of course, we can help you develop custom target panels specific to your research goals.

Due to their nature, targeted panels necessarily have limited analytical coverage compared to global panels, but they are more sensitive and reproducible than untargeted approaches. It can also be difficult to identify unknown compounds using global panels, mostly due to the limited availability of analytical standards and spectral information in public databases.1 Often, researchers opt for a two-pronged approach to get the best of both worlds: starting with a global approach and then using targeted metabolomics to further understand key analytes of interest.

Regardless of the approach or combination of approaches you choose, it’s important to define this study parameter at the outset, as this will inform the sample types and quantities required to make data collection as straightforward as possible.

Which Technology is Best for Your Study?

As briefly discussed in previous chapters of this guide, there are two main technologies used to obtain metabolite data: nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS).

  • NMR: NMR exposes samples to a magnetic field and radio frequency pulse to identify compounds based on the resonance energy of electromagnetic radiation absorbed and re-emitted by cellular nuclei. Specifically for metabolomics, proton atoms from small molecules are typically analyzed (1H-NMR). It’s a highly reproducible method capable of measuring tens to one hundred metabolites from various sample types.
  • LC-MS: Mass spectrometers measure small molecules by ionizing (adding charge to) them and then detecting them as they move through a spectrometer. By using liquid chromatography (LC) to physically separate the molecules present in a sample prior to ionization in the mass spectrometer, researchers can work with the very small sample sizes (down to 0.1 μL) supported by LC to detect thousands of metabolites from diverse sample types.2-5

Each method has its advantages and disadvantages. The main advantage of NMR is that it is non-invasive and doesn’t destroy the sample,6 meaning you can re-use your samples for additional experimental investigation (including complementary LC-MS, if you wish).

Samples aren’t destroyed during NMR because the technology doesn’t require any sample pre-processing or separation prior to identification, unlike LC-MS, making sample preparation for NMR much simpler.6 Additionally, NMR is highly reproducible and quantitative, because electromagnetic peak intensities are directly proportional to the number of nuclei in the sample.6 It is also a powerful method for identifying unknown metabolites,6 making it a great tool for novel biomarker discovery.7

LC-MS is a much more sensitive method than NMR, however, and it is better suited for targeted metabolomics analyses.7 Unlike NMR, LC-MS can measure thousands of metabolites at a time and is often used to analyze different classes of metabolites from the same sample, achieving a wider coverage of the metabolome than NMR.6 Currently available MS techniques are incredibly sensitive and accurate and offer a high mass resolution, meaning that more than 80% of the pathways present in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database can be detected using this method.1

Do It Yourself or Outsource?

The next thing to determine before you even begin collecting samples is whether you will execute your project in-house or outsource. Asking this question up front may save you a lot of time and money, and it will help you focus your research objectives appropriately considering the limitations, risks, and costs associated with each option.


  • Pros: no risk of samples becoming lost or damaged in transit, can begin processing samples immediately, you know exactly when your data are coming back
  • Cons: need to purchase or rent instrumentation and access to analysis software, need your own research standards and reference libraries, requires know-how for confident data interpretation

While some kits that can be purchased to facilitate in-house metabolomics projects, these are typically semi-quantitative and therefore don’t support targeted studies. They also may not have the best coverage, meaning you might miss important scientific discoveries. Unless you’re going to be performing metabolomics analyses on a regular basis, the financial investment into instruments, software, and personnel trained in handling your samples, running the instruments, and data analysis might not be worth it.


  • Pros: access to metabolomics experts who run and interpret metabolomics data daily, access to the best available technology for the highest accuracy and confidence, fully validated methods for reproducibility and reliability across cohorts
  • Cons: may not be feasible for urgent analyses due to shipping and data turnaround times this, fee-for-service may not be in scope for every project or budget

Outsourcing your metabolomics studies can be nerve-wracking, especially if you’re working with limited samples that are hard to obtain, which is why it is essential that you choose a trustworthy metabolomics partner with expertise you can count on. Metabolon offers several different models for working with you to meet your research needs, from sample processing and data only to complete analysis and interpretation. Our 20 years of experience optimizing protocols and working with hundreds of sample types combined with our reference library containing over 5,400 metabolites means that we are fully equipped to help you design your study, collect the right samples the right way, and obtain the highest quality data possible with meaningful, definitive answers to your research questions.

Sample Collection, Preparation, Storage, and Shipment

As we briefly discussed in Chapter 4 of this guide, several different sample types can be analyzed using metabolomics protocols, including cells in culture, tissue, feces, blood, urine, and sweat. The sample source you used depends on your research goal; for example, you’ll select stool if you want to study the impact of the gut microbiome on host nutrient acquisition, but you’ll work with tissue samples if you want to identify biomarkers that differentiate primary tumors and metastases. Planning ahead will help you map out your budget early and avoid the unexpected need to add more samples later on or request funding extensions.

Each sample type requires different handling and preparation, which is also impacted by whether you’ve selected a targeted or untargeted approach and whether you’re using NMR or LC-MS. If you are doing your project in-house, you’ll need to make sure you know how to handle your samples appropriately; and for particularly difficult samples, outsourcing is often the easier and cheaper option–especially if you’re working with limited sample material. With every project, Metabolon offers a free Study Success Sample Handling Kit, customized specifically for your study. With the kit, we include barcoded tubes specific to your matrix (ie, biofluids, solid samples, or cell pellets) with instructions for mailing your samples to us to ensure they arrive safely. If you store large quantities of samples in a biorepository or biobank, we’ll even work directly with personnel there to prepare and ship your samples.

Data Interpretation

Data interpretation is often the most difficult part of any metabolomics project. Instruments don’t produce easy-to-read summaries with beautiful graphics; instead, you get tables with endless columns and values that you must transform into understandable insights using any one of a number of available software tools (some of which are free, others of which are not). If no one on your team is trained in analyzing NMR or LC-MS data, you may lack confidence in your interpretation and the likelihood of missing important, actionable observations is high. Outsourcing data analysis and interpretation can save you a lot of time and money and prevent you from having to re-do experiments.

Working with Metabolon, you’ll never be left in the dark when interpreting your data. Global panels come with continuous access to the MyMetabolon Portal, where you can access customized interpretation by our PhD-level study directors, statistical tests they performed, and fully customizable data visualizations. And while targeted panels don’t have the same statistical analysis and visualization as global panels, we do provide a formatted data table that is ready for import and analysis with third-party tools such as R and Python.

For those research groups that want to outsource metabolite identification and quantification but not data interpretation, we also offer data-only. This can be a good option for labs with the necessary expertise for data analysis but who don’t necessarily have access to or funds for appropriate instrumentation or who are working with sample types outside of their expertise.

What’s Next?

After going through this guide, you’re ready to start executing your own metabolomics studies and experience the scientific power they have to offer. We’re here to help you on your journey, whether you simply need us to process your samples or work with you one-on-one from start to finish to design and execute an insightful metabolomics study. Contact us today to get started!

Continue to Chapter 10 - The End of the Guide

Here’s a quick review of the main points of discussion in each chapter that you can use to help you find what you need quickly.


  1. Bedia C. Metabolomics in environmental toxicology: Applications and challenges. Trends in Env Anal Chem. 2022;34:e00161. doi: 10.1016/j.teac.2022.e00161
  2. Silva Siqueira Sandrin V, Moraes Oliveira G, Weckwerth GM et al. Analysis of Different Methods of Extracting NSAIDs in Biological Fluid Samples for LC-MS/MS Assays: Scoping Review. Metabolites. 2022;12(8):751. doi: 10.3390/metabo12080751
  3. Thevia M, Krug O, Geyer H et al. Expanding analytical options in sports drug testing: Mass spectrometric detection of prohibited substances in exhaled breath. Rapid Commun Mass Spectrom. 2017;31(15):1290–1296. doi: 10.1002/rcm.7903
  4. Frank N, Bessaire T, Tarres A et al. Development of a quantitative multi-compound method for the detection of 14 nitrogen-rich adulterants by LC-MS/MS in food materials. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2017;34(11):1842–1852. doi: 10.1080/19440049.2017.1372640
  5. Zwiener C and Frimmel FH. LC-MS Analysis in the aquatic environment and in water treatment – a critical review. Part I: Instrumentation and general aspects of analysis and detection. Anal Bioanal Chem. 2004;378(4):851–861. doi: 10.1007/s00216-003-2404-1
  6. Nagana Gowda GA and Raftery D. NMR Based Metabolomics. Adv Exp Med Biol. 2021;1280:19–37. doi: 10.1007/978-3-030-51652-9_2
  7. Emwas AH, Roy R, McKay RT et al. Recommendations and Standardization of Biomarker Quantification Using NMR-Based Metabolomics with Particular Focus on Urinary Analysis. J Proteome Res. 2016;15(2):360–373. doi: 10.1021/acs.jproteome.5b00885

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