2D Gas Chromatography and TOFMS to Study Moisture-Damaged Cacao Beans

Scientists from the University of Washington in Seattle, Washington used comprehensive two-dimensional (2D) gas chromatography (GC×GC) paired with time-of-flight mass spectrometry (TOFMS) to study moisture-damaged cacao beans. Their findings were published in the Journal of Chromatography A (1).

Cocoa beans and cocoa fruit on the cocoa concept with raw materials Aromatic cocoa beans as background | Image Credit: © somchai20162516 - stock.adobe.com

Comprehensive two-dimensional (2D) gas chromatography (GC×GC) paired with time-of-flight mass spectrometry (TOFMS) is becoming widely used in various application areas, including the analysis of food, fuel, and monitoring health. With the growing use of GC×GC-TOFMS, there is also a growing need for developing chemometric techniques, both targeted and nontargeted, to analyze these large and complex datasets.

For this study, two “orthogonal” characteristics of moisture damaged cacao beans (temporally dependent molding kinetics versus the time-independent geographical region of origin) are simultaneously analyzed in a comprehensive GC×GC-TOFMS dataset using tile-based Fisher ratio (F-ratio) analysis. Fisher-ratio (F-ratio) analysis applied to the supervised comparison of sample classes algorithmically reduces complex GC × GC–TOFMS data sets to find class distinguishing chemical features. F-ratio analysis, using a tile-based algorithm, significantly reduces the adverse effects of chromatographic misalignment and spurious covariance of the detected signal, enhancing the discovery of true positives while simultaneously reducing the likelihood of detecting false positives (2).

As part of this experiment, cacao beans from six geographical regions were analyzed once a day for six days following the initiation of moisture damage to trigger the molding process. According to the scientists, geographical origin of beans, be it cacao or coffee, can have an impact on the volatile profile of beans. The team developed a computational approach to simultaneously tease apart these two sample class structures using the tile-based F-ratio analysis software platform with suitable modifications. There were two “extremes” for the experimental sample class design: six time points for the molding kinetics versus the six geographical regions of origin from around the world, resulting in a 6×6 element signal array from the GC×GC-TOFMS data referred to as a composite chemical fingerprint (CCF) for each analyte.

Usually, this study would involve initial generation of two separate hit lists using F-ratio analysis, the scientists wrote, one hit list from inputting the data with the six time point classes, then another hit list from inputting the dataset from the perspective of geographic region of origin. However, analysis of two separate hit lists with the intent to distill them down to one hit list is extremely time-consuming and can pose challenges associated with attempting to match analytes across two hit lists. To address these issues, tile-based F-ratio analysis is “orthogonally applied” to each analyte CCF to simultaneously determine two F-ratios at the chromatographic 2D location (F-ratio_kinetic and F-ratio_region) for each hit, by ranking a single hit list using the higher of the two F-ratios resulting in the discovery of 591 analytes. 458 of the initial 591 compounds on the hit list passed the 99% null threshold, indicating that they possessed a “reasonable” amount of statistically significant chemical information to belong to one of these two classes; however, the CCF signal pattern of most of these analytes could not be readily interpreted by the human eye. At the 99.999% null threshold, all 127 analytes expressed a CCF signal pattern that was readily interpreted by the human eye as being either kinetic dominated or region dominated.

References

(1) Mikaliunaite, L.; Synovec, R. E. Simultaneous Discovery of Compounds Dominated by Either Molding Kinetics or Geographical Region of Origin for Moisture Damaged Cacao Beans Using Orthogonally Applied Tile-Based Fisher Ratio Analysis of GC×GC-TOFMS Data. J. Chromatogr. A 2024, 1730, 465093. DOI: 10.1016/j.chroma.2024.465093

(2) Parsons, B. A.; Marney, L. C.; Siegler, W. C.; et al. Tile-Based Fisher Ratio Analysis of Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry (GC×GC–TOFMS) Data Using a Null Distribution Approach. Anal. Chem. 2015, 87 (7), 3812–3819. DOI: 10.1021/ac504472s