## 3 Results and Discussion  
### VOC Profile and Identification  
Guided by previous reports of the use of SPME for concentration of volatiles, two fibres, DVB/CAR/PDMS and DVB/PDMS, were investigated for method development [13; 14]. Based on the results of the experiment, DVB/CAR/PDMS fibres with 20 min extraction time at room temperature were chosen as best suited for the study. Further studies with DVB/CAR/PDMS fibres showed that a 20 min sampling period resulted in good signal-to-noise (S/N) ratios and after this time the sample temperature was 10 \({}^{\circ}\)C. Shorter sampling times resulted in poor S/N ratios, whilst longer sampling times allowed the sampletemperature to rise above 10 \({}^{\circ}\)C, which was deemed unacceptable as it could result in an analyte profile that was not consistent with the sample being stored in a refrigerated environment. Previous studies have shown that elevated equilibration temperatures and longer SPME sorption time (e.g., 37 \({}^{\circ}\)C for 60 min [15] and 80 \({}^{\circ}\)C for greater than 50 min [4]) result in the formation of artefact analytes. Hence, one of the key findings in this study is that cold extraction was sufficient for detailed analyte profile determination, at the same time allowing a direct correlation to be made with the analyte profile of meat samples stored at 4 \({}^{\circ}\)C. This study focused on identifying volatile compounds as they are more relevant to the freshness of meat, whereas non-volatile compounds are typically related to the flavour profile. Tentative identification of VOCs was achieved following SPME-GC-accQTOFMS analysis by a combination of several matching methods. Compounds were initially screened by matching the mass spectrum of each peak in the experimental GC trace with the list of compound mass spectra from the NIST library. The match was accepted if the differences between the experimental and literature retention index and mass accuracy were within \(\pm\)20 and \(\pm\)20 ppm, respectively. The 35 compounds tentatively identified by this method are presented in Table 1. Example GC-MS traces are shown in Figures S2-S4 and the compounds corresponding to the respective packaging systems are included in tables below the chromatograms (Tables S2-S4). The compounds of raw beef that have been previously reported are summarised in Table S1. Of the 35 compounds reported in this study (Table 1), 29 were detected with a mass accuracy of below 10 ppm with 27 previously identified in the literature as VOCs from either cooked or raw meat. For seven of the compounds in Table 1, only the mass accuracy criterion was able to be met for tentative identification. Of these, carbon disulphide (CS\({}_{2}\)) [6; 7; 16], acetic acid [6; 17; 18], ethyl acetate [19; 20], acetaldehyde [6; 21], and hexanal [6; 7; 22] have been previously identified in chemical analysis of beef and the tentative match was therefore accepted. Six compounds from this study were able to be identified only by molecular features (hydrocarbon or alcohol-containing) and five of the peaks observed in the GC experimental results were unable to be identified (Table S2). The literature-supported identification of volatile compounds released from raw beef (Table 1), as well as information about the structural features and chemical functionality of some of the compounds which were unable to be further identified (Table S5), combined with the packaging system from which these samples originated, can be useful for future applications, including freshness sensor design. Previous studies attributed many of the VOCs detected in the analysis of cooked meat to heat-induced Maillard reactions, or to the oxidation of lipids at the elevated temperature [47]. However, as we observed these VOCs from raw beef without any heat treatment, it can be concluded that heat is not the only factor responsible for the presence of these compounds. This is in agreement with the findings of [PERSON] et al.