Katelynn A. Perrault

Mead is a fermented beverage made from honey, water, and yeast. Factors such as honey concentration and yeast strain impact the volatile organic compounds (VOCs) produced from the metabolism of sugar during fermentation. Despite the increasing consumer popularity of mead, the relation between its production variables and final aroma profile remains under‐examined. The mixture of VOCs from mead and its fermentation can be examined through analytical techniques like gas chromatography (GC). More complex samples often benefit from the higher resolution and separation capacity of comprehensive two‐dimensional gas chromatography (GC×GC), which separates analytes based on their affinity for two distinct stationary phases connected in series. This study examined mead produced from five strains of yeast and brewed with three different initial honey concentrations using comprehensive two‐dimensional gas chromatography with time‐of‐flight mass spectrometry (GC×GC–TOFMS), and assessed kinetic data collected during the fermentation process. Chemometric analysis included principal component analysis (PCA), UpSet Analysis, hierarchical cluster analysis (HCA), and partial least squares regression (PLSR). A total of 98 compounds were detected across all sample groups. There were 48 VOCs that were found in all 15 mead sample groups and 16 analytes that were found only in the honey samples. There were six compounds that were unique to specific yeast strains. Understanding the relationship between production variables and the final VOC profile of mead will contribute to consumer understanding of product contents and associated health impacts and allow brewers to target specific flavors in a more efficient manner during production.

The selection and control of carrier gas are critical factors influencing the performance of both one-dimensional gas chromatography (1D GC) and comprehensive two-dimensional gas chromatography (GC×GC). Helium is widely used due to its inertness, but increasing cost, limited supply, and reliance on fossil fuel extraction raise sustainability concerns. Hydrogen offers a viable alternative, with faster optimal linear velocity to reach comparable efficiency, and more sustainable production through generators relying on water electrolysis. While tools exist to translate 1D GC methods from helium to hydrogen, their application to GC×GC has not been well established, particularly for parameters unique to multidimensional separations. This work presents the translation of helium to hydrogen carrier gas for cryogenically modulated GC×GC-MS. Three translated methods derived from 1D tools were evaluated alongside modulation period using standard mixtures, followed by validation with authentic fingermark residue samples in a forensic application. The translate option, using a combination of flow and temperature adjustments that matched column efficiency between helium and hydrogen, delivered comparable peak capacity, as well as acceptable S/N, tailing factors, and resolution values between key peak pairs. Modulation period could be reduced by 50%, providing a practical guideline for method translation. Hydrogen reduced overall run time by up to 60%, increasing sample throughput and lowering per-sample energy consumption. Although sustainability comparisons are nuanced, hydrogen showed improved green chemistry alignment overall, supported by higher sustainability metric scores and gains in efficiency and resource use.

Background: The ketogenic diet is being explored as an adjuvant intervention in breast cancer because it lowers circulating glucose and elevates ketone bodies such as β-hydroxybutyrate (BHB), but how individual ER+ breast cancer subtypes adapt to these conditions remains poorly characterized. We examined metabolic responses to BHB supplementation under glucose restriction in two ER+ breast cancer cell lines, asking whether metabolic adaptation patterns differ between models. Methods: MCF-7 and T47D cells were cultured under high glucose, glucose-restricted (5% of standard), or glucose-restricted with 10 mM BHB conditions and profiled by comprehensive two-dimensional gas chromatography–mass spectrometry (GC×GC-MS). Pairwise Welch’s t-tests with Benjamini–Hochberg false discovery rate (FDR) correction were applied to identify treatment-responsive metabolites. Targeted assays quantified intracellular glycine, SHMT1 protein, and total branched-chain amino acid (BCAA) concentrations across a BHB dose range (2.5-15 mM). Patient tumor transcriptomic data from TCGA (n=1,084) and paired tumor-normal samples from GSE58135 (n=20) were analyzed for genes involved in one-carbon, ketone body, and BCAA metabolism. Results: MCF-7 and T47D cells exhibited markedly divergent metabolic responses to BHB. In MCF-7 cells, BHB supplementation produced a broad pattern-level metabolic shift: 75% of detected metabolites trended upward when BHB was added to glucose-restricted cultures (C vs. B comparison), with 1,4-butanediol reaching nominal significance (FC=2.35, p=0.016) and a 4.1-fold trend increase in lactic acid (p=0.11), although no individual metabolite survived FDR correction. T47D cells showed essentially no metabolic response to BHB at the global level. Targeted assays detected an elevation in glycine at 5 mM BHB in both cell lines that did not follow a monotonic dose response and was not accompanied by changes in SHMT1 protein expression. Total BCAA levels were elevated by BHB in T47D cells but remained unchanged in MCF-7 cells. In paired patient samples, OXCT1 (log2FC = −1.41), SHMT1 (log2FC = −1.31), and ACAT1 (log2FC = −1.07) were significantly downregulated in ER+ tumors relative to matched normal tissue (adjusted p < 0.001 for all three). Conclusions: ER+ breast cancer cell lines show heterogeneous metabolic responses to BHB supplementation under glucose restriction. The broad pattern of metabolite elevation in MCF-7 but not T47D cells suggests that capacity to utilize ketone bodies as metabolic substrate varies between ER+ models. The downregulation of OXCT1, ACAT1, and SHMT1 in ER+ tumors compared to normal tissue identifies these enzymes as candidate biomarkers that may help stratify which patients are likely to benefit from ketogenic interventions. Findings related to individual metabolites should be regarded as exploratory and require validation in larger, adequately powered cohorts.

Pseudomonas aeruginosa is a notorious pathogen that is a leading cause of hospital-acquired infections, for which there are few treatment options. The quorum sensing (QS) pathway governs many pathogenic behaviors that allow for P. aeruginosa to stage infections. Within the QS pathway, there is a key protein-protein interaction between an enzyme, PqsE, and one of the master QS regulators, RhlR. Although its catalytic function is dispensable for its interaction with RhlR, previous mutagenic work characterizing the active site of PqsE identified active site mutations that induce a conformational change in PqsE, preventing it from forming a complex with RhlR. These active site mutations, when introduced stably into the genome of P. aeruginosa, also lead to a significant decrease in production of a key toxin, pyocyanin, and prevent colonization in the lungs of a murine host. Here, we performed a fluorescence polarization screen of an FDA-approved drug library to identify molecules that bind in the active site of PqsE. Three molecules were identified, two of which showed inhibitory activity consistent with a competitive mode of inhibition. One hit molecule, Apomorphine, had a distinctly different inhibitory profile and is potentially binding outside of the active site to allosterically inhibit enzyme activity of PqsE. All three hit molecules were tested in a cellular enzyme assay, and one of the competitive inhibitors, Vorinostat, was found to inhibit intracellular PqsE. Vorinostat is now being explored as a candidate for synthetic derivatization to inhibit the PqsE-RhlR protein-protein interaction via binding in the PqsE active site.

Kombucha is a fermented beverage made from the addition of a symbiotic community of bacteria and yeast (SCOBY) to a sugary base tea. Despite the odors associated with kombucha, few studies have examined its chemical aroma profile. The mixture of volatile organic compounds (VOCs) from tea and its fermentation is typically examined using one-dimensional gas chromatography with time-of-flight mass spectrometry (GC-TOFMS). More complex samples benefit from the higher resolution and separation capacity of comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS), which separates analytes based on their affinity for two distinct stationary phases. This study aimed to compare the efficacy of GC-TOFMS to GC×GC-TOFMS, to use VOC data to differentiate between kombucha products, and to establish an understanding of the VOCs originating from tea versus microorganisms. Samples were taken from 12 sources including two SCOBYs, one starter tea, and eight kombucha teas and analyzed using GC-TOFMS and GC×GC-TOFMS. GC-TOFMS detected 127 analytes across 15 compound classes while GC×GC-TOFMS detected 182 analytes across 17 compound classes. The enhanced separation power of GC×GC-TOFMS allowed for the resolution of coelutions that occurred in GC-TOFMS data. Statistical techniques including principal component analysis (PCA), principal coordinate analysis (PCoA), hierarchical cluster analysis (HCA), Fisher ratio, and fold change were applied to the VOC data. Locally produced products contained simple esters and acids while commercial brands contained terpenes and aromatic compounds. This work builds on the overall aim of linking VOC data from microbes and tea with the flavor profile of fermented foods as examined by advanced analytical techniques. Graphical abstract

Fingerprints are routinely used as evidence in forensic investigations. Fingermarks, any mark left by a donor whether a complete print or not, include sweat and oil excreted by the donor. The chemical components of fingermarks are typically analyzed by gas chromatography-mass spectrometry (GC-MS). Complexity from the number of endogenous and exogenous components associated with fingermarks tends to cause challenging coelutions in resulting chromatograms. In these scenarios, nontargeted analysis can provide substantial benefits over traditional targeted methods that exist in the literature. In this proof-of-concept study, a nontargeted method for analyzing fingermarks was developed and optimized using comprehensive two-dimensional gas chromatography (GC×GC-TOFMS). Two different methods for extracting fingermarks off a microscope slide were evaluated for reproducibility and the quantity of extracted analytes, and a cotton swab collection with solvent extraction was chosen. Instrumental parameters were experimentally optimized to produce a final workflow. The optimized extraction and instrument methods together identified 70 fingermark analytes. Exogenous components within the deposited residue were resolved from endogenous fingermark compounds and used to differentiate donors based on personal care products used by the donor. The extra chromatographic space from GC×GC-TOFMS analysis was beneficial for resolving cosmetic compounds from endogenous fingermark compounds, some of which have been shown to coelute in GC-MS studies previously. The potential for a nontargeted screening of fingermarks for exogenous compounds in a forensic setting is demonstrated as an analysis of trace evidence.

Pseudomonas aeruginosa is a notorious pathogen that is a leading cause of hospital-acquired infections. Due to its heightened resistance to a broad range of antibiotics, there is great need for new antimicrobial agents that are effective in treating P. aeruginosa infections. One attractive option for exploring new antibiotic targets is the quorum sensing (QS) pathway, as it governs many of the pathogenic behaviors that allow P. aeruginosa to stage and maintain infections. Within the QS pathway, there is a key protein-protein interaction between an enzyme, PqsE, and one of the master QS regulators, RhlR. Although its catalytic function is dispensable for its interaction with RhlR, previous mutagenic work characterizing the active site of PqsE identified active site mutations that induce a conformational change in PqsE, preventing it from forming a complex with RhlR. These active site mutations, when introduced stably into the genome of P. aeruginosa , also lead to a significant decrease in production of a key toxin, pyocyanin, and prevent colonization in the lungs of a murine host. These findings encourage drug discovery efforts to identify small molecules that bind in the active site of PqsE and induce a conformational change similar to the ones induced by particular amino acid substitutions in the active site. Here, we performed a fluorescence polarization screen to identify molecules that bind in the active site of PqsE. We screened a library of FDA-approved drugs, with the intention of potentially repurposing a known molecule for the treatment of P. aeruginosa infections. Three molecules were identified, two of which showed inhibitory activity that was consistent with a competitive mode of inhibition. One hit molecule, Apomorphine, had a distinctly different inhibitory profile, and is potentially binding outside of the active site to allosterically inhibit enzyme activity of PqsE. All three hit molecules were tested in an in vivo enzyme activity assay, and one of the competitive inhibitors, Vorinostat, was found to inhibit intracellular PqsE. Vorinostat is now being explored as a candidate for synthetic derivatization to inhibit the PqsE-RhlR protein-protein interaction via binding in the PqsE active site.

Taro (Colocasia esculenta L.) is a plant originating from Southeast Asia now prevalent throughout the Pacific Islands. Growing taro was and remains an important facet of the Hawaiian culture. Poi is a food product prepared from steamed taro that has been macerated and allowed to ferment. While poi is commonly prepared at home, it can also be found commercially from select Hawaiian brands. The general fermentation process of poi produces volatile organic compounds (VOCs) such as alcohols, aldehydes, ketones, organic acids, esters, and heterocycles, among others, through microbial and enzymatic reactions. The VOCs found in poi are a class of compounds traditionally analyzed through gas chromatography (GC)–mass spectrometry (GC–MS) but have not previously been characterized in the literature. This study aimed to identify a core VOC profile of commercial poi products to better understand sample composition. Changes in fresh to aged poi were observed as well as its brand specific profiles. Analysis was conducted using comprehensive two-dimensional GC-quadrupole mass spectrometry with flame ionization detection (GC×GC–qMS). Samples were prepared according to package instructions, extracted via headspace SPME Arrow, and analyzed in replicates of 10. A total of 56 analytes were identified across three brands of commercial poi, ranging from 11 different compound classes. All brands showed a clear distinction between fresh and aged samples. Fresh samples across all brands contained 4,4-dimethoxy-3-methylbutan-2-one while aged samples all contained 2,5-dimethylfuran. Within the fresh state, the VOC profiles from each brand appeared to be distinct from one another. Taro brand poi was the least similar to Hanalei brand poi and He Mea Ono brand poi. He Mea Ono and Hanalei had some overlap in VOCs, but a substantial portion of their VOC profile was unique to the individual brand. In the aged state, there was a significant overlap among all three brands, which indicates that fermentation caused the VOC profiles to become more similar to one another. Visual distinctions in chromatographic plots were supplemented by principal component analysis and box plots, identifying distinguishing compounds. Investigating the profile of poi via GC×GC–qMS enhanced our understanding of its unique qualities, historical context, and contemporary use.

Comprehensive two‐dimensional gas chromatography (GC×GC) has been explored in forensic research to provide advanced chromatographic separation for forensic evidence, including illicit drugs, fingerprint residue, chemical, biological, nuclear, and radioactive (CBNR) substances, toxicological evidence, odor decomposition, and petroleum analysis for arson investigations and oil spill tracing. In GC×GC, the separation and analysis of analytes is similar to one‐dimensional GC, but the primary column is connected to a secondary column via a modulator to provide two independent separation mechanisms, thus increasing the peak capacity of the analysis. The goal of implementing GC×GC in forensic studies is often to increase the separation and detectability of analytes and has most often been applied in nontargeted forensic applications where a wide range of analytes must be analyzed simultaneously. To date, there has been no summary of the current state of forensic research that evaluates both analytical and legal readiness for routine use. For these analytical methods to be adopted into forensic laboratories and be used in evidence analysis, they must meet rigorous analytical standards. In addition, new analytical methods for evidence analysis must adhere to standards laid out by the legal system, including the Frye Standard, Daubert Standard, and Federal Rule of Evidence 702 in the United States and the Mohan Criteria in Canada. Current research on GC×GC use for forensic applications was summarized and reviewed for analytical advances and technology readiness to provide a comprehensive view of GC×GC use for future routine implementation. A technology readiness scale, with levels from 1 to 4, was used to characterize the advancement of research in each individual application area. Seven forensic chemistry applications are discussed related to courtroom criteria and categorized into technology readiness levels based on current literature as of 2024. Future directions for all applications should place a focus on increased intra‐ and inter‐laboratory validation, error rate analysis, and standardization.