Metabolomics is a relatively new field that provides unprecedented information about an organism’s health by analyzing the metabolites produced by various biological processes or drug administration. From human pharmacodynamics, to preclinical murine studies and medicinal plant metabolomics, metabolites encompass many different classes of compounds, each with different chemical properties.
When coupled with other analytical techniques like NMR and MS, HPLC/UHPLC provides a rapid and quantitative method to analyze metabolite samples with a variety of complexities. However, the components of the HPLC system (detector, column, mobile phase, etc.) must be adapted to each specific application depending on the metabolites of interest.
This HPLC buyer’s guide covers some options, depending on the specific metabolite profile being analyzed, along with literature examples of their use.
Detectors
The detector is one of–if not the–most critical components of the entire HPLC/UHPLC system, as it determines the type of compounds that can be analyzed. Metabolites are small molecules with wildly different chemical structures and bonding patterns, and this must be kept in mind when determining the most suitable detector.
For example, not all compounds absorb in the UV-visible region of the electromagnetic spectrum, or they may have incredibly similar UV absorption spectra, which prevents the use of a UV-vis detector. Similarly, fluorescence detectors may not be suitable because not all compounds show native fluorescence or can be derivatized with fluorescent tags. Therefore, in this section, we’ll provide examples of commonly used detectors for HPLC/UHPLC systems, as well as examples of when (and when not) to use them.
Refractive Index (RI) Detectors
Widely considered to be one of the most universal detectors, refractive index detectors require only a difference in the refractive index between the pure mobile phase and analyte. Furthermore, RI detectors are not pressure-sensitive so pump pulsations don’t tend to cause major issues.1 They can be used to detect analytes that do not produce signals using other detectors, including various sugars, alcohols, fatty acids, cholesterols, and carbohydrates. Unfortunately, RI detectors suffer from several drawbacks and are often unsuitable for gradient elution or trace-level analysis (due to their low sensitivity).
Fluorescence Detectors
This is a popular technique for analyzing metabolites that have native fluorescence such as catechols. They are also suitable for analyzing drug metabolites, such as amino acids, that can be derivatized using fluorescent tags such as o-pthalaldehyde, dansyl chloride, and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC).
Electrochemical Detectors
Electrochemical detectors can be used when metabolites do not absorb UV light or undergo fluorescence. Electrochemical HPLC detectors are much more selective than UV detectors and can be used to measure complex mixtures of metabolites, including redox-active metabolites, such as heavy metals like lead.4
Ultraviolet-Visible (UV-Vis) Detectors
UV-Vis detectors are suitable for the highly sensitive detection of many different metabolites, but this technique requires that the compounds absorb UV light. As such, it can be used to analyze metabolites that include aromatic moieties, conjugated systems, and chromophores. HPLC-UV-Vis has been used for the detection of organic acids such as lactic acid and pyruvic acid.5
Circular Dichroism Detectors
Drugs used to treat disease states often have d- and l- enantiomers that show differential stereoselectivity, which influences their pharmacokinetics and therapeutic effects.6 When analyzing the metabolites of chiral compounds by UV-Visible detectors, enantiomers cannot be distinguished, even if a chiral column is used because their UV-Vis absorption spectra are identical. In this case, chiroptical detectors are necessary to monitor stereochemical changes in blood plasma metabolites to detect disease states such as Alzheimer’s disease and various cancers.
Columns
As with detectors, the type of column must be selected based on the metabolites of interest. In cases with a complex metabolite profile, several different types of columns can be connected in series to achieve the desired separation to capture metabolites with different polarities. For example, a reversed-phase column can be placed before an ion-exchange column, wherein the reversed-phase column separates slightly polar and non-polar compounds, while the ion-exchange column separates charged compounds. This allows for the separation of a variety of clinically-relevant metabolite mixtures that include amino acids and organic acids.7
Reversed-phase columns
Reversed-phase HPLC columns separate hydrophobic metabolites such as lipids, which are difficult to separate using other types of columns. The advantage of many reversed-phase columns is that they are relatively easy to maintain, and some can even be run using 100% water.
Hydrophilic interaction liquid chromatography (HILIC) columns
Although reversed-phase separation is the most popular technique for metabolomics studies,2 the columns are hydrophilic and show improved retention of polar compounds that are only weakly retained by reversed-phase columns. Hydrophilic interaction liquid chromatography (HILIC) shows better retention of polar metabolites such as catecholamines and their metabolites by engaging in hydrogen bonding.8
Ion-exchange columns
Ion-exchange columns can be used to capture basic metabolites that are often incompatible with the mobile phases of typical HPLC approaches, such as nucleotide tetra- and pentaphosphates such as ppGpp and ppGppp, which act as indicators of stress responses in bacteria.9
HPLC/UHPLC Column Ovens
Day-to-day or even hour-to-hour variations in ambient laboratory temperature may have drastic effects on your analysis and may compromise reproducibility. The use of HPLC column ovens for metabolomics studies can help avoid this by ensuring a constant temperature, which is especially useful in situations when small temperature changes may significantly affect the analysis (e.g., when using a refractive index detector, where a 0.001 °C change in temperature changes the RI by 10−6 units1).
Using a column oven can also reduce the time needed for separation—but don’t overdo it! An excessively high temperature may degrade compounds, volatilize solvent (thus damaging the column), or not give the adsorbates enough time to interact with the stationary phase. The peak shape, retention time, and selectivity are affected by the temperature gradient within a column, which can be controlled using a column oven.
Conclusion
This guide provides readers with some products and literature examples to get them started with using HPLC for metabolomics. There are a wide variety of HPLC/UHPLC detectors and columns available depending on the specific metabolites of interest. Although there is no truly universal method for detecting all possible metabolites, the use of two or more columns in-series with an appropriate detector may help develop a metabolite profile to minimize the number of runs necessary.
About the author: Brandon Sharp, Ph.D., is a freelance technical content writer with experience designing photoresists and other organic materials for advanced lithography applications.
References
1. Robards K, Haddad PR, Jackson PE. 5 - High-performance Liquid Chromatography—Instrumentation and Techniques. In: Robards K, Haddad PR, Jackson PE, eds. Principles and Practice of Modern Chromatographic Methods. Academic Press; 2004:227-303. doi:10.1016/B978-0-08-057178-2.50008-X
2. Kanamori T, Funatsu T, Tsunoda M. Determination of catecholamines and related compounds in mouse urine using column-switching HPLC. Analyst. 2016;141(8):2568-2573. doi:10.1039/C5AN02617B
3. Tomita R, Todoroki K, Machida K, et al. Assessment of the Efficacy of Anticancer Drugs by Amino Acid Metabolomics Using Fluorescence Derivatization-HPLC. ANAL SCI. 2014;30(7):751-758. doi:10.2116/analsci.30.751
4. Sudama G, Zhang J, Isbister J, Willett JD. Metabolic profiling in Caenorhabditis elegans provides an unbiased approach to investigations of dosage dependent lead toxicity. Metabolomics. 2013;9(1):189-201. doi:10.1007/s11306-012-0438-0
5. Gómez-Mingot M, A. Alcaraz L, A. MacIntyre D, et al. Development of a novel analytical approach combining the quantification of amino acids , organic acids and glucose using HPLC-UV-Vis and HPLC-MS with screening via NMR. Analytical Methods. 2012;4(1):284-290. doi:10.1039/C1AY05610G
6. Ou F, Zhou Y, Lei J, et al. Development of a UHPLC-MS/MS method for the quantification of ilaprazole enantiomers in rat plasma and its pharmacokinetic application. Journal of Pharmaceutical Analysis. 2020;10(6):617-623. doi:10.1016/j.jpha.2019.09.002
7. Le A, Mak J, Cowan TM. Metabolic profiling by reversed-phase/ion-exchange mass spectrometry. Journal of Chromatography B. 2020;1143:122072. doi:10.1016/j.jchromb.2020.122072
8. Wang J, Tan G, Liu X, Huang Y. Expanding the Coverage of Metabolome Using Multiple Liquid Chromatography Modes. https://assets.thermofisher.com/TFS-Assets/CMD/posters/PN-64420-LC-MS-Multiple-Modes-Metabolome-ASMS2015-PN64420-EN.pdf
9. Girel S, Guillarme D, Fekete S, Rudaz S, González-Ruiz V. Investigation of several chromatographic approaches for untargeted profiling of central carbon metabolism. Journal of Chromatography A. 2023;1697:463994. doi:10.1016/j.chroma.2023.463994
10. Yang SO, Lee SW, Kim YO, et al. HPLC-based metabolic profiling and quality control of leaves of different Panax species. J Ginseng Res. 2013;37(2):248-253. doi:10.5142/jgr.2013.37.248