## 3 Results and Discussion
### Method Application and Shelf Life Estimation  
To confirm the applicability of the developed method, a degradation experiment with GSB specimens was performed. The deterioration process of fish includes several simultaneous alterations; therefore, different evaluation methods were applied. Besides the proposed chemical analysis, the following methods were applied: dielectric properties with the Torrymeter; sensory analysis following the QIM method; and microbiological analysis through TVCc. The results obtained with each method are presented in Figure 6. With the Torrymeter, the alterations of the muscle texture of GSB were registered throughout the storage experiment. The values started at a maximum value of 15, representing the fresh fish condition. With the progression of the days, the registered data gradually decreased until the value of 8, between 8 and 9 days, at the same time that off-odors were detected (Figure 6a). Other works also propose that when a value of 8 is achieved, the product reaches the borderline for consumption acceptability [24; 41; 42].  
The common initial values for TVCc varies between \(10^{1}\) and \(10^{4}\) (cfu), at first days of storage. At the rejection time, the TVCc values could be between \(10^{7}\) and \(10^{9}\) (cfu), depending on fish species [32; 43], as in this case, as the TVCc limit was achieved at 8-9 days (Figure 6b). For _Enterobacterreaces_ counts, the values throughout the experiment never exceeded \(10^{2}\) (cfu), indicating appropriate hygiene and handling conditions of the product. Similar results were also described in other works [44].  
Application of the QIM methodology allowed to determine altered GSB freshness levels. Through the analysis of QIM data (Figure 6c), the odor was a lead contributor to evaluate the degradation progress. During the three first days, gills and skin odor were described as being similar to seaweed or sea breeze. From the third day, the neutral descriptor was used to describe the condition in which the specimens had no clear distinctive odor. Degradation began to be perceived on days 5-6, with off-odor emergence but was only considered significant for rejection from day 7-8, associated with rancid odors (Table S12, Supplementary Material).  
According to the European Directive 91/493/EEC, TMA presence in samples is limited to 12 mg/100 g [45]. From the chromatographic results (Figure 6d), it is seen that after the slow increase of the TMA, the limit is achieved between 8 and 12 days. Applying the fitted model,  
Figure 6: Degradation results in GSB specimens. (**a**) Torrymeter analysis; (**b**) microbial analysis; (**c**) Quality index method analysis; (**d**) TMA chemical analysis with the proposed HS-SPME(DVB/CAR/PDMS)/CG-MS (BP20) method.  
the TMA limit could be achieved between 9 and 10 days. Regarding the DMA analysis, even though the methodology allows simultaneous extraction and quantification, in the present study, the DMA was detected, but its values during the storage remained residual in comparison with TMA. Table 4 compiles the information obtained from the different methods, showing the association between the different used methods to predict and estimate GSB shelf life.