Gas Chromatography in Metabolomics: Techniques, Instrumentation, and Applications

What is Gas Chromatography Used for Metabolomics?

In metabolomics analysis, several gas chromatography (GC) hyphenated techniques are commonly employed to enhance the sensitivity, selectivity, and information content of the analysis. Here are some commonly used GC hyphenated techniques in metabolomics:

• GC-MS (Gas Chromatography-Mass Spectrometry): GC-MS is the most commonly used and versatile hyphenated technique in metabolomics. It is suitable for both targeted and untargeted metabolite profiling, providing qualitative and quantitative analysis of a wide range of metabolites. GC-MS is typically chosen when comprehensive metabolite identification and quantification are required, and when there is a need for high-resolution mass spectrometry data for structural elucidation.
• GC-FID (Gas Chromatography-Flame Ionization Detection): Particularly when dealing with large quantities of metabolites, GC-FID is mostly employed for quantitative study of certain substances. For the target analytes, it possesses great sensitivity, linearity, and selectivity. For routine quantification, quality control analysis, and target analysis of known metabolites including fatty acids, carbohydrates, and specific volatile chemicals.
• GC-TOF-MS (Gas Chromatography-Time-of-Flight Mass Spectrometry): GC-TOF-MS combines the separation power of GC with high-resolution mass spectrometry, offering accurate mass determination and improved spectral quality. It is advantageous for untargeted metabolomics studies, where comprehensive profiling and identification of unknown metabolites are desired. GC-TOF-MS is particularly useful when dealing with complex mixtures and metabolites with similar retention times.
• GCxGC-MS (Two-Dimensional Gas Chromatography-Mass Spectrometry): GCxGC-MS is chosen when there is a need for enhanced separation power and increased peak capacity. It is particularly valuable for the analysis of complex samples containing numerous co-eluting compounds, isomeric compounds, or closely related compounds. GCxGC-MS provides improved resolution, peak shapes, and identification confidence, making it suitable for comprehensive metabolomics analysis and the discovery of novel metabolites.

According to the needs of the study, the best GC hyphenated technique should be chosen, taking into account the complexity of the sample matrix, the required amount of metabolite coverage, and the degree of identification and quantification confidence. The accessibility of tools, knowledge, and resources for data analysis and interpretation should also be taken into account. To better comprehend the intricacy of cellular metabolic networks, metabolomics researchers can collect more thorough and precise information on metabolites by combining GC with other analytical methods.

What is GC-MS?

GC-MS Instrumentation and Principles

GC-MS instrumentation consists of several key components that work together to facilitate metabolite analysis. These components include the gas chromatograph, mass spectrometer, ion source, and data acquisition system.

Gas Chromatograph (GC): The GC component is responsible for separating the complex mixture of metabolites present in a sample. It utilizes a capillary column coated with a stationary phase that selectively interacts with analytes based on their physicochemical properties, such as volatility and polarity. The sample is injected into the GC system, vaporized, and carried by an inert gas (usually helium) through the column. The metabolites are then eluted from the column at different retention times, allowing for their separation.

Learn more about the principles and structure of gas chromatographs.

Mass Spectrometer (MS): The MS component of the instrument detects and identifies the separated metabolites. It consists of three main parts: the ion source, mass analyzer, and detector. The ion source is a critical component responsible for ionizing the metabolites, converting them into charged ions for subsequent analysis. Commonly used ionization techniques in GC-MS include electron impact (EI) and chemical ionization (CI).

Electron Impact (EI) Ionization: In EI ionization, high-energy electrons bombard the sample molecules, resulting in the formation of radical cations (M+•). These ions undergo fragmentation, producing characteristic fragment ions that provide information about the molecular structure of the metabolites. EI ionization is widely used in GC-MS metabolomics due to its reproducibility and extensive spectral libraries available for metabolite identification.

Chemical Ionization (CI): In CI ionization, a reagent gas (e.g., methane, ammonia) is introduced into the ion source. The reagent gas reacts with the sample molecules, forming protonated molecular ions (M+H^+) or other adduct ions. CI ionization is particularly useful for polar and non-volatile metabolites that may not efficiently ionize using EI.

Mass Analyzer and Detector: The mass analyzer separates the ions based on their mass-to-charge ratio (m/z) and directs them towards the detector. Commonly used mass analyzers in GC-MS metabolomics include quadrupole, time-of-flight (TOF), and ion trap. The detector measures the abundance of ions at specific m/z values, generating mass spectra.

Data Acquisition and Processing: The data acquisition system records the mass spectra and associated retention times of the separated metabolites. The acquired data is processed using specialized software, which performs various tasks such as peak identification, integration, deconvolution, and alignment of chromatograms. Data processing algorithms help remove background noise, correct for instrumental variations, and generate high-quality spectra for further analysis.

The combination of GC separation with MS detection in GC-MS metabolomics enables the accurate identification and quantification of metabolites within complex biological samples. It provides valuable information about the structure, abundance, and dynamics of metabolites, contributing to a comprehensive understanding of cellular metabolism.

GC-MS Metabolomics Pipeline

The process of GC-MS-based metabolomics typically involves the following steps:

1. Sample Collection and Preparation: Samples, such as tissues, biofluids, or cell extracts, are collected and processed to extract metabolites. Various extraction methods, including liquid-liquid extraction, solid-phase microextraction, or derivatization techniques, may be employed to enhance the detection of metabolites.

2. GC-MS Instrumentation: The extracted metabolites are then subjected to GC-MS analysis. The GC system separates the metabolites based on their volatility and physicochemical properties, while the MS component ionizes and detects the separated analytes.

3. Data Acquisition and Processing: The GC-MS instrument generates complex raw data, consisting of retention times, mass spectra, and peak intensities. This data is processed using specialized software to align chromatograms, remove background noise, deconvolute overlapping peaks, and match mass spectra with metabolite databases.

4. Metabolite Identification: After data processing, metabolite identification is performed by comparing the obtained mass spectra with reference libraries or in-house databases. The identification process can be further enhanced by incorporating additional analytical techniques, such as high-resolution MS or tandem MS (MS/MS) experiments.

5. Statistical Analysis and Data Interpretation: Statistical analysis methods, including multivariate analysis, such as principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA), are applied to reveal patterns, correlations, and significant differences between experimental groups. This step aids in identifying potential biomarkers or understanding metabolic alterations associated with different conditions.

6. Metabolic Pathway Analysis: The identified metabolites are mapped onto metabolic pathways using various bioinformatics tools and databases. This analysis provides insights into the biological significance of the identified metabolites and their role in specific cellular processes.

Overview on GC-MS metabolomics workflow (Rey et al., 2022).

Commonly Used GC-MS Instruments in Metabolomics Research

What is GC-FID?

GC-FID is an analytical technique used for the separation and quantification of organic compounds, including volatile and semi-volatile compounds. GC-FID is commonly employed in various fields, including environmental analysis, forensic science, pharmaceuticals, and food analysis.

In GC-FID, the sample is injected into a gas chromatograph, where it is vaporized and carried by an inert gas through a capillary column. The capillary column separates the mixture of compounds based on their different physicochemical properties, such as boiling points and polarities. As the compounds elute from the column, they enter a flame ionization detector (FID) for detection.

8 Scheme of a GC-FID equipment (Soria et al., 2014).

The FID operates by combusting the organic compounds eluted from the column in a hydrogen/air flame. During combustion, carbon-containing compounds produce ions and electrons. These ions and electrons are collected and measured by a detector, generating an electrical signal. The magnitude of the electrical signal is proportional to the concentration of the compound present in the sample.

One of the significant advantages of GC-FID is its high sensitivity, allowing for the detection of compounds at low concentrations. It also offers a wide linear dynamic range, enabling accurate quantification over a broad concentration range. Additionally, the FID is highly selective for organic compounds, making it suitable for the analysis of complex mixtures.

GC-FID is often used for the analysis of fatty acids, hydrocarbons, pesticides, flavors, fragrances, and other volatile or semi-volatile compounds. It is a versatile and widely utilized technique that provides valuable information about the composition and concentration of target compounds in various samples.

What is GCxGC-MS?

Gas chromatography (GC) separation in two stages combined with mass spectrometry (MS) detection is known as GCxGC-MS, a sophisticated analytical technology. Compared to conventional one-dimensional GC-MS, it offers greater separation capabilities and improved analytical resolution.

In GCxGC-MS, two different stationary phases with complementary selectivities are used in a series, creating two stages of separation. The sample is first injected into the primary column, where the compounds are separated based on their volatility and interaction with the stationary phase. The effluent from the primary column is then transferred to a modulator, which traps and releases small portions of the sample onto a secondary column.

The secondary column has a different stationary phase, providing a second dimension of separation. As the sample is released from the modulator onto the secondary column, compounds are further separated based on their chemical properties, such as polarity or molecular weight. This comprehensive two-dimensional separation results in improved peak capacity, increased resolution, and enhanced peak shapes compared to one-dimensional GC.

GCxGC-MS makes it easier to analyze materials with high complexity, such as biological samples, environmental samples and food matrices, and its improved chemical separation of co-mixes allows the detection and identification of low-abundance metabolites in complex mixtures. The improved resolution and peak capacity also allow for better chemical characterization, especially in the case of structural isomers or closely related compounds.

References

1. Rey-Stolle, Fernanda, et al. "Low and high resolution gas chromatography-mass spectrometry for untargeted metabolomics: A tutorial." Analytica Chimica Acta 1210 (2022): 339043.
2. Soria, Ana Cristina, et al. "Gas Chromatographic Analysis of Food Bioactive Oligosaccharides." Food Oligosaccharides: Production, Analysis and Bioactivity (2014): 370-398.