## 4 Conclusions  
In this paper, a method for HS-SPME-GC\(\times\)GC-MS using hop samples was developed, applied, and demonstrated to be a powerful technique to identify metabolites in the samples. By suitable experimental design of the method, we believe this to be a \"hop-timal\" analysis strategy for total VOC composition in the headspace of the hop cone. The identification of compounds as an untargeted study through this methodology provides considerable coverage of volatile metabolite expression, and it is possible to describe both major and minor compounds, including those most likely not readily measured by single-dimension GC separations since substantially fewer compounds are reported in GC-MS analysis. Thus, the identification of 137 substances in five diverse hop samples is described, with the potential of separating 471 peaks. This of course also highlights that in terms of metabolite identification, just having the ability to separate individual compounds is not the same as being able to unambiguously identify them. In this regard, in comparison with the conventional technique of GC-MS, GC\(\times\)GC-MS improves the separation through an increase of over 300% in the number of peaks recognised as discrete components. This study reports the comprehensive study of hop through detailed chemical class assignment compounds, which can largely be separated in the 2D space of GC\(\times\)GC analysis. Multivariate statistics analysis proved a similarity between the samples ZAPP and CASC, and the samples AZAC and LORA. The use of HS-SPME-GC\(\times\)GC-MS is a bright light in understanding the metabolites present in hop.  
**Supplementary Materials:** The following supporting information (abbreviated titles here) can be downloaded at: [[https://www.mdpi.com/article/10.3390/metabo14040237/s1](https://www.mdpi.com/article/10.3390/metabo14040237/s1)]([https://www.mdpi.com/article/10.3390/metabo14040237/s1](https://www.mdpi.com/article/10.3390/metabo14040237/s1)). Table S1: Hop samples information; Table S2: Intra-day and inter-day precision of selected peaks; Figure S1: A comparison between two chromatographic settings for GC\(\times\)GC; Table S3: n-Alkanes the series C8-C21 and their respective retention times; Figure S2: Retention time vs n-alkanes series (Cn) plot; Figure S3: GC\(\times\)GC-MS chromatogram for the HS-SPME of the series of n-alkanes (C8-C15); Figure S4: GC\(\times\)GC-MS chromatogram for the HS-SPME of the series of n-alkanes (C16-C21); Figure S5: Comparison of chromatograms obtained for the Enigma (ENIG) hop by HS-SPME; Figure S6: Comparison of chromatograms obtained for the Zappa (ZAPP) hop by HS-SPME; Table S4: Information in the hop composition profile using HS-SPME-GC-MS; Figure S7: GC\(\times\)GC-MS chromatogram for the HS-SPME of Azacca (AZAC) hop; Figure S8: GC\(\times\)GC-MS chromatogram for the HS-SPME of Loral (LORA) hop.  
**Author Contributions:** Conceptualisation: [PERSON] and P.J.M.; writing--original draft preparation: [PERSON], M.S.S.A. and P.J.M.; writing--review and editing: [PERSON], M.S.S.A., P.J.M. and B.G.B.; visualisation: [PERSON] and M.S.S.A.; supervision: [PERSON] and B.G.B.; funding acquisition: P.J.M. and B.G.B. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This research received no external funding.  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The raw data supporting the conclusions of this article will be made available by the authors on request.  
**Acknowledgments:** The authors would like to thank the Brazilian government agencies CNPq, FAPEMIG, and CAPES for financial support. GAPR acknowledges CNPq for the PhD scholarship (project 155078/2019-4). This study was partially funded by Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Brasil--Finance Code 001. Carlton & United Breweries are thanked for the provision of some hop samples. The authors declare that they have no conflicts of interest.