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This article delves into the fundamental aspects of metabolomics, highlighting the critical roles of separation and detection in this advanced scientific field. We will examine the application of techniques such as Gas Chromatography (GC), Liquid Chromatography (LC), Mass spectroscopy (MS), and Nuclear Magnetic Resonance (NMR) spectroscopy. These methods form the cornerstone of metabolomics analysis, enabling the comprehensive study of metabolic processes and biomolecular interactions. Our discussion aims to provide a detailed overview for researchers and practitioners seeking to deepen their understanding of metabolomics methodologies.
Understanding Liquid Chromatography (LC) in Metabolomics Analysis
Introduction to Mass spectroscopy Techniques in Metabolomics
Comparison of ion sources in Mass Spectrometry between LC-MS and GC-MS platform
The two core components of metabolomics analysis technology are the separation and detection of metabolites. Separation techniques primarily utilize various chromatographic methods such as Gas Chromatography (GC), Liquid Chromatography (LC), and Capillary Electrophoresis (CE). Meanwhile, detection techniques currently mainly employ Mass Spectrometry (MS) and Nuclear Magnetic Resonance Spectroscopy (NMR). The effective combination of these two components can essentially meet the demands of metabolomics detection[1].
To achieve comprehensive qualitative and quantitative analysis of metabolites in samples, it is necessary for separation and detection equipment to possess characteristics such as good stability, strong compound identification capability, high resolution and sensitivity, fast detection speed, and wide dynamic detection range.
Gas chromatography mass spectroscopy (GC-MS) is one of the earliest analytical techniques applied in metabolomics research, suitable for analyzing low polarity, low boiling point metabolites, or substances with volatility after derivatization. Volatile substances with relatively low boiling points such as some aroma related substances, volatile substances derived from high-boiling substances, such as sugars, short-chain fatty acid, organic acids, sterols etc.
On the other hand, liquid chromatography mass spectroscopy (LC-MS) has a broader application field, especially due to its wide range of detectable substances such as lipids, amino acids, flavonoids, anthocyanins, etc., along with significantly higher sensitivity compared to other detection techniques.
The advantage of Nuclear Magnetic Resonance Spectroscopy (NMR) detection technology lies in its non-destructive nature to the sample, but its limited dynamic range for detecting substance levels in the same sample makes it less applicable for qualitative and quantitative analysis of large quantities of substances.
| Platform | Sensitivity（mol） | Advantage | Defect |
| Nuclear Magnetic Resonance Spectroscopy (NMR) | 10-6 | Noninvasive detection High resolution Qualitative accuracy | Limited dynamic range Low sensitivity High investment cost |
| Gas chromatography mass spectroscopy (GC-MS) | 10-12 | High sensitivity Universal database | Complex sample preprocessing Substances type limited |
| liquid chromatography mass spectroscopy (LC-MS) | 10-15 | High sensitivity High resolution Multiple substance types can be detected | Highly dependent on databases |
Both platforms consist of three components: liquid/gas chromatography, mass spectroscopy, and workstation. The liquid/gas chromatography is primarily used for substance separation, mass spectroscopy for substance identification, and the workstation for outputting data results. Next, let's delve into the liquid chromatography, gas chromatography, and mass spectroscopy platforms.
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The function of liquid chromatography is to preliminarily separate substances in a solution. In liquid chromatography, the mobile phase under high pressure is liquid, and a very fine solid phase is used, exploiting the hydrophilic and hydrophobic properties of substances to adsorb onto the solid phase while different substances are separated out in the changing mobile phase, facilitating detection by mass spectroscopy. Traditionally, high-performance liquid chromatography can be classified into preparative and analytical types based on the size of the sample volume. Preparative liquid chromatography is commonly used for compound purification, while analytical liquid chromatography is used for qualitative and quantitative analysis of extracts from plant or animal/human sample.
In contrast to liquid chromatography, the mobile phase used in gas chromatography is mainly inert gas, with helium being commonly used in GC-MS. The chromatographic column in gas chromatography is typically much longer than in liquid chromatography, allowing for better substance separation. Substance separation in the gas phase also depends on the temperature, so appropriate temperature programming can effectively improve substance separation efficiency, optimizing analysis time.
In addition to conventional gas chromatography, there is now two-dimensional gas chromatography (GCxGC), which further enhances the ability to detect complex metabolites. GCxGC links two chromatographic columns with different and independent stationary phases in series. After separation by the first-dimensional column, each component undergoes collection and focusing, then enters the second-dimensional column in a pulsed manner, which is short and allows for rapid separation.
There are some differences in mass spectrometry when coupled with gas chromatography and liquid chromatography. Compared to the diversity of detectors in GC-MS, due to the poor universality of interfaces in LC-MS, this interface faces three issues: high flow rate in liquid chromatography requires a high vacuum environment for mass spectroscopy; the mobile phase must contain enough ions for mass spectroscopy analysis; impurities in the mobile phase need to be removed to avoid contamination of mass spectroscopy. In this context, from 1972 when Tal’roze et al. first proposed the idea of directly introducing the chromatographic column outlet into mass spectroscopy to solve the interface problem of LC-MS, until 1987 when Bruins et al. invented Atmospheric Pressure Ionization (API), the issue of flow rate limitation was resolved. API is a general term for mass spectroscopy ionization technology under atmospheric pressure conditions, mainly including Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI). Among them, ESI is suitable for most substances, while APCI is suitable for detecting carotenoids and steroid-type substances.
Unlike LC-MS, the ionization technology typically used in GC-MS is Electron Impact (EI), which is a hard ionization method. The working principle of EI ion source is mainly that under high vacuum conditions, when the current passes through the cathode, the filament temperature reaches around 2000°C, and the hot filament emits electrons. When the electron energy is higher than the ionization potential of the sample, the sample molecule or atom undergoes ionization. The ions gain kinetic energy in the electric field and enter the mass analyzer at a certain speed. Under this action, the ions entering the mass analyzer are already fragmented ions, which is different from LC-MS. In LC-MS, before entering the mass analyzer still retain the original ion state (compared to the substance itself being hydrogenated, hydroxylated, NH4, etc.).
| Platform | Ion source type | Characteristic |
| LC-MS | ESI、APCI | The substance is positively or negatively charged in the ESI/APCI ion source and does not break into fragments. |
| GC-MS | EI | The substance is broken up into fragment ions at EI ion source. |
The complexity and diversity of metabolites require robust and precise analytical approaches for effective separation, detection, and analysis. The evolution of metabolomics as a discipline depends significantly on advancements in these analytical technologies. For researchers aiming to conduct cutting-edge metabolomics analysis, collaboration with knowledgeable and specialized service providers is crucial.
MetwareBio, a premier global metabolomics service provider equipped with state-of-the-art LC-MS and GC-MS platforms, offers a breadth of services encompassing metabolomics, proteomics, lipidomics, and multi-omics. We stand poised to empower the scientific community in their quest for discovery.
[1]Souza, Lpd , et al. "Ultra-high-performance liquid chromatography high-resolution mass spectrometry variants for metabolomics research." Nature Methods.
