## 3 Results
### Instrumental Volatile Analysis Separates Coffees Based on Roast Level and Storage Conditions  
Figure 3 displays the principal component analysis (PCA) biplot of the HS-SPME-GC-MS analysis of coffee volatiles. Similar to the PM task, samples are again separated by both roast level and storage conditions with dark roast \"Vienna\" samples in the top two quadrants and the light roast \"City\" samples in the bottom half of the PCA plot. Larger sample differences compared to the PM are found as the 95% confidence intervals are small and do not overlap for any of the samples. Samples are also all significantly different from each other (\(p<0.05\)) by the Hotelling \(T^{2}\)-test. In contrast to the sensory evaluation where samples are separated along the first dimension due to roast level, in the volatile analysis samples are separated by storage conditions along the first principal component (PC 1), explaining 43% of the total variance. Another 36% of the total variance is explained by PC 2, which separates coffees by roast level. Similar to the PM task, differences within the roast level seem to be larger for the dark roast \"Vienna\" samples as they are positioned further apart from each other than the light roast \"City\" coffees. Especially, the newly roasted dark roast coffee VN is positioned very far from the two stored coffees, VF and VR. Looking into the individual differences of aroma compounds, it becomes apparent that storage leads to a loss of lighter volatiles, such as methanethiol, 2-acetylfuran, diacetyl, propanal, and acetaldehyde, as these compounds show higher abundance in the newly roasted samples VN and CN (Table S1). This is in agreement with other studies, such as [PERSON] et al. [5] who found a decrease in these lighter volatiles over a storage period of 21 days, with higher losses for storage at room temperature compared to 4 \({}^{\circ}\)C. Significant loss to non-detectable levels after a 9-week storage period was found for 2,2-bifuran, 2-acetylpyrrole, 2-furfurylfuran, 2-furfurylmethylsulfide, benzenemethanethiol, diacetyl, decanoic acid, furfural, and methanethiol, with the latter being present at about 50% of the original levels in the freezer-stored samples VF and CF, but not detected in the room temperature-stored CR and VR (Table S1). The stored coffees, especially the dark \"Vienna\" roast samples stored at room temperature VR, are characterized by higher levels of larger pyrazines, pyrroles, and furans, besides some phenolic compounds. Dark roast \"Vienna\" coffee stored in the freezer (VF) or at room temperature (VR) showed the highest level in phenol and 2,5-dimethylfuran, two compounds that have recently been suggested as markers for dark roast and baked roast defect [27]. As both compounds are present in all coffees (Table S1), this would imply a loss of other volatiles during storage, allowing these defects and their associated markers to become more readily detectable. Generally, dark roast coffees showed higher abundance of most compounds, except for 1-acetoxy-2-butanone, 2,3-pentanedione, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl 5-methylpyrazine, 5-methylfurfural, acetaldehyde, and acetic acid, which showed significantly higher levels in the light roast \"City\" samples (Table S1). This agrees with previous reports of overall higher volatile levels in darker roast samples due to increased Maillard reactions and lipid degradation, but lower levels of organic acids (e.g., acetic acid) [27]. Overall, the instrumental analysis of coffee aroma volatiles reflects the sensory perception captured by the PM task in that the 48 selected aroma compounds previously reported in coffee were able to capture significant changes in the roasted coffee samples due to roast level and storage conditions. The observed differences due to storage conditions were larger in the dark roast \"Vienna\" samples with dramatic losses of aroma compounds considered important for fresh coffee aroma (e.g., methanethiol, 2-acetylfuran, 1-furfurylpyrrole; Table S1).