Workflow for characterizing volatile oxidation compounds in walnut oil by headspace solid-phase microextraction comprehensive two-dimensional gas chromatography time of flight mass spectrometry coupled with tile-based Fisher ratio analysis and other chemometric tools.

Due to their high fat content, it is well known that walnuts are highly susceptible to oxidation and thermal degradation. These processes produce various compounds, including aromatics, aldehydes, acids, ketones, and alcohols, resulting in poor quality walnuts and shorter shelf life. The goal of this work was to demonstrate a high-level analytical workflow to follow and capture the kinetic volatile fingerprint of oxidized walnut oil through development of a method using headspace solid-phase microextraction sampling followed by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (HS-SPME GC × GC-TOFMS) coupled with chemometric data analysis. Starting with freshly cold-pressed walnut oil, vials of the walnut oil underwent accelerated oxidation at 60 °C across a span of 6 weeks. Once HS-SPME GC × GC-TOFMS data was collected for each week of oxidation, tile-based Fisher ratio analysis and ANOVA were utilized to discover 59 class-distinguishing analytes across the 6-week experiment. Density-based spatial clustering of applications with noise visualized with principal component analysis revealed three clear kinetic profile class trends within the 59 analytes, aptly named: diminishing analytes, emerging analytes, and intermediate analytes. A very minor fourth category was also observed - unique analytes - consisting of kinetic trends dissimilar from the other three trends. Of the analytes discovered, previously unreported native compounds were discovered as part of the overall volatile chemical fingerprint of the walnut oil, providing fundamental metabolic insight to the oxidative and thermal degradation processes and their influencing factors. To illuminate the significance of these results, a partial least squares regression model to predict hexanal formation - a known oxidation product - was utilized to identify analyte responses correlated to oxidation across all kinetic profiles. Overall, the analytical workflow demonstrated herein confidently studied the walnut oil oxidation process by coupling high-resolution volatile chemical fingerprints of the walnut oil samples - captured via HS-SPME GC × GC-TOFMS analysis - with rigorous chemometric data analysis, ultimately providing a powerful approach for food science studies.