Gas chromatography (GC) is a popular tool for metabolomics research. For discovery work in the areas of volatile and semi-volatile analysis for applications in foods, plant metabolomics, breath, and environmental exposures in metabolomics, comprehensive two-dimensional gas chromatography (GC×GC) with a mass spectrometer is an ideal platform as it allows for the complete separation of mixtures of thousands of compounds. The GC×GC separation ensures that pure compounds are provided to the high-speed mass spectrometer, which can then provide high-quality spectra for compound identification due to the lack of interference from other compounds in the sample.
Dr. James Harynuk has been studying and applying GC×GC to a variety of sample types for >20 years. His laboratory is a TMIC node based at the University of Alberta that is equipped with a variety of GC×GC and GC×GC‑MS instruments. With the available sample preparation and introduction options in the laboratory, they are equipped to handle a wide variety of sample types. Automated sample introduction options include simple liquid injections, solid-phase microextraction (SPME), thermal desorption of sorbent tubes commonly used for air sampling, and both static and dynamic headspace analysis. In addition to generating data, they are also leading efforts to more efficiently process GC×GC-MS and GC-MS data.
One of their recent publications in Phytochemistry, Global metabolome analysis of Dunaliella teriolecta, Phaeobacter italicus R11 co-cultures using thermal desorption-comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (TD-GC×GC-TOFMS) demonstrates a sample preparation approach for metabolomics studies of algae that comprises an innovative injection method. Using thermal desorption technology (usually used for air sampling sorbent tubes), an innovative sample preparation and injection technique was devised. Briefly, derivatized sample extracts were automatically transferred as 9-μL aliquots into micro-vials placed inside of thermal desorption tubes. These were then heated gently in solvent vent mode to allow excess solvent and derivatization reagents to escape, before being heated more aggressively to transfer the derivatized metabolites to the GC×GC system for analysis. The biggest advantage of this approach is that the heavy, non-volatile fraction of the samples was contained in the micro-vials. These components never enter the GC×GC system, so they do not collect in the injector or the analytical column, as would happen with conventional injections. The approach maintains a cleaner analytical system, leading to improved reproducibility of results even across large studies.
In the area of human metabolomics, second-year Ph.D. student, Ryland Giebelhaus presented Fluids three-ways: Comparison of dynamic headspace, solid phase microextraction, and derivatization for the untargeted GC×GC-TOFMS metabolomics of urine and human breastmilk. at the 4th Annual Conference of the Metabolomics Association of North America (MANA). These results will be written up shortly, once a few analyses are performed to help positively identify some of the compounds after some new instrumentation comes online in late 2022 or early 2023.
The field of fecal metabolomics is garnering much attention recently; however, standard protocols for sample preparation and analysis have yet to be established. Fecal samples provide a direct insight into human health, allowing researchers to better understand the complex interactions between the gut microbiome and the host. However, fecal sample preparation remains a challenge given the heterogeneous matrix and microbial-rich composition. Bacteria and enzymes present within feces remain active and continue to alter the metabolite profile after the sample leaves the body. Proper controls of pre-analytical conditions including storage and handling must be established so as to ensure results are representative of samples at the time of collection. Another recent paper from the group, Evaluation of fresh, frozen, and lyophilized fecal samples by SPME and derivatization methods using GC×GC-TOFMS highlights the impacts of sample preservation steps on fecal metabolomics profiles.
Another student from Harynuk’s team, Ewenet Mesfin, compared SPME with active sampling onto sorbent tubes for collecting biogenic volatiles from plants. Her poster, Analysis of Plant Volatiles Using TD-GC×GC-TOFMS, won the Early Career Member Best Poster award for undergraduate students during the MANA Conference.
Data normalization for metabolomics studies is another challenge. In Towards Standardization of Data Normalization Strategies to Improve Urinary Metabolomics Studies by GC×GC-TOFMS, the group introduced the concept of total useful peak area (TUPA), a modification of MS total useful signal (MSTUS) concept. TUPA is applicable to chromatographic data from any one- or multi-dimensional separation with any kind of detector, not only a mass spectrometer. Briefly, it accounts for variations in sample concentration by scaling responses for all analytes based on the response of those analytes which are present in all samples. This approach allows for robust data normalization even when the profiles of the samples are highly variable between and/or within sample classes.
Untargeted Metabolomics by GC×GC TOF MS is an ideal platform for discovery studies involving volatile and semi-volatile compounds, as well as compounds that can be derivatized with standard chemistries (amino acids, simple sugars, sterols, etc). Aside from simple liquid injections of extracts, a wide range of automated injection methods are available in the laboratory including solid-phase microextraction, static headspace, dynamic headspace, and thermal desorption. This allows us to easily handle a wide variety of sample types including soil, water, oil/petroleum, air samples, plants, food and beverages, breath, tissue samples, and biofluids (i.e. urine, plasma, etc). This service typically allows for the detection of 2000-9000 compounds (depending on sample complexity). Relative quantification is provided based on peak areas, normalized to appropriate internal standards. For some sample injection methods, sample types, and compounds, absolute quantification is available.
Thanks to a successful CFI-IF grant, with contributions from the Government of Alberta, LECO, and GERSTEL, the Harynuk Lab is adding two new instruments to their laboratory including a GC×GC-BT and a GC×GC-HRT+. This will greatly expand their capacity to support research studies and add the capacity for high-resolution accurate-mass spectrometric detection. The new technology is expected to be fully operational in January 2023.
Summarized by Harynuk’s group