## 3 Results and Discussion
The intra-day precision (\(n=5\) injections on the same day) displayed RSD % of retention time of 0.00-0.23% for \({}^{1}\)\({}_{R}\), 0.05-4.30% for \({}^{2}\)\({}_{R}\), and between 1.51% and 9.63% for peak areas. The inter-day precision was analysed with \(n=9\), where three replicates were injected daily over 3 different days with results of 0.22-0.58% for \({}^{1}\)\({}_{R}\), 1.21-8.77% for \({}^{2}\)\({}_{R}\), and 1.65-12.81% for the area. These results indicate an acceptable repeatability for the proposed method, as compared with the values of [PERSON] et al. [34], which means a low value of RSD even when considering the SPME reproducibility for peak area. Compared to previous studies (Table 1), the present methodology resulted in the highest number of peaks tentatively identified in the hop by using GC\(\times\)GC-MS. The profile of the metabolites present in the five different hops identified numerous peaks by GC\(\times\)GC-MS, totalling 205 for AZAC, 258 for CASC, 421 for LORA, 472 for ZAPP, and 413 for ENIG. Comparing the number of compounds reported with the GC-MS for the samples (Table 2) proved the improvement in metabolic coverage by GC\(\times\)GC-MS with increases of 140.2% for CASC, 273.5% for ENIG, and 316.8% for ZAPP. Figure 2 (see Figures S6 and S7 for the other samples) represents aligned peaks with seven metabolites that have significant characteristics for the flavour and odour of hop: (a) \(\beta\)-pinene, (b) \(\beta\)-myrcene, (c) 2-methylbutyl isobutyrate, (d) linalool, (e) geraniol, (f) carophyllene, and (g) humulene. All these components are now apparently free from interference, although  
Figure 2: A comparison between two chromatograms of (**A**) GC–MS and (**B**) GC\(\times\)GC–MS applying HS–SPME for Cascade hop, where the GC–MS and \({}^{1}\)D column of GC\(\times\)GC-MS is a DB-5 ms UI (30 m \(\times\) 0.25 mm I.D. \(\times\) 0.25 \(\upmu\)m \(d_{l}\)), the \({}^{2}\)D column is SUPERLOWAX 10 (1.0 m \(\times\) 0.10 mm I.D. \(\times\) 0.10 \(\upmu\)m \(d_{l}\)), and the structure of the selected compounds (**C**) is represented by a–g indicated in (**A**,**B**). the suite of non-polar compounds could potentially lead to overlapping compounds. Better separation should correspond to improved MS matching with databases. The overall results demonstrate evidence of a better separation, clear distinction of compounds and, consequently, an increase in identified peaks. The tentative identification was performed using the NIST 11 database, and the highest probability compound identity was determined, considering the MS RMatch (>700) and retention indices (\(\pm 20\) units) based on the van den Dool and Kratz equation (Table S3 and Figures S3-S5). A total of 137 compounds were identified with GC\(\times\)GC-MS analysis of five hops and a total of 73 compounds for GC-MS analysis of three hops (\(n=3\), Table S4). The final list of compounds tentatively identified by GC\(\times\)GC-MS (Table 3) includes their molecular formula, CAS identifier, retention time, retention indices from literature and experiment, the relative chromatographic area of the peaks in each sample, and compound chemical class. Analysis of selected standards was performed for specific compound identification confirmation. The main classes of volatiles identified and the number of compounds were as follows: alcohols (3), aldehydes (4), esters (47), hydrocarbons (7), ketones (9), monoterepne hydrocarbons (17), oxygenated monoterepenes (12), sesquiterpene hydrocarbons (32), and oxygenated sesquiterpenes (3). The total amount of volatiles is expressed in the last line of the table, demonstrating that there remains a small fraction of unknown compounds, from 3.1% to 12.4% of the total area, which could not be identified using the reported criteria. However, this is an improvement in the identified or tentatively identified molecules over previous studies of essential oils since studies such as [PERSON] et al. (2017) reported 50.8-90.0% of the total area of the sample by GC\(\times\)GC.