# Chemical Changes in the Broccoli Volatilome Depending on the Tissue Treatment
## 2 Results  
### Aspects Related to SPME Extraction  
SPME is a predominant extraction method used for the isolation of flavour compounds and profiling volatiles. The initial step in the elaboration of the SPME extraction parameters was fiber coating selection, as this parameter significantly influences the extraction efficiency. The four most popular fibers (DVB/PDMS, CAR/PDMS, PDMS, and CAR/DVB/PDMS) were tested for isothiamate extraction (allyl isothianate, benzyl isothianate, and isobutyl isothianate) and both Carboxene-containing fibers showed the highest extraction efficiency (Figure S5A--Supplementary Materials). Then, the same fibers were used to extract volatiles from blended broccoli florets to evaluate their usefulness for extraction of other important groups of broccoli volatiles. The results are shown in Figure S5B (supplementary materials). Four groups of metabolites were taken into consideration in experiments involving broccoli florets: alcohols, aldehydes, sulphides, and isothiates. For esters, aldehydes, and alcohols the peak areas of detected compounds were the highest for the CAR/DVB/PDMS fiber. Only the peak area of sulphides was higher for the CAR/PDMS fiber. Based on this finding, the CAR/DVB/PDMS fiber was selected for subsequent experiments.  
To elaborate the optimal temperature for SPME extraction, blended broccoli samples were extracted at 20 \({}^{\circ}\)C, 4 \({}^{\circ}\)C, 60 \({}^{\circ}\)C, and 80 \({}^{\circ}\)C for 30 min (Figure S5C, Supplementary Materials). The alcohol concentrations increased with increasing temperature from 20 \({}^{\circ}\)C to 60 \({}^{\circ}\)C. The peak area increases for aldehydes paralleled that for alcohols, and for sulphides the increment related to temperature was much less prominent than for the remaining two groups of metabolites. This suggests that sampling at 20 \({}^{\circ}\)C could be sufficient for the extraction of compounds belonging to these groups which would result in minimising thermal changes in the matrix. However, the only group of metabolites that required a higher extraction temperature was isothiances; they were detected only at 60 \({}^{\circ}\)C and 80 \({}^{\circ}\)C. The peak area of isothiates increased by 20% when the extraction temperature was changed from 60 \({}^{\circ}\)C to 80 \({}^{\circ}\)C. The increase of extraction temperature can increase the migration of isothiances to the headspace, however, it can also promote the formation of isothi cyanates by thermal decomposition of glucosinolates. The increasing concentration of isothiances with increasing temperature was not likely to be related to a higher myrosinase activity because higher temperatures, such as 80 \({}^{\circ}\)C, are too high for the enzymatic reaction. This reaction occurred before the extraction process; thus, the high temperature apparently increased the volatility of compounds occurring after the enzymatic process. The isothiocyanates considered in this experiment were isobutyl-, n-pentyl- and, hexyl isothiamate). The highest total peak area was achieved after extraction at 60 \({}^{\circ}\)C; therefore, this temperature was applied in the subsequent trials.  
The next examined parameter was the extraction time. Different extraction times from 5 min to 30 min were compared. The highest peak areas of all compounds of interest were achieved after 10 min of fiber exposition, as presented in Figure S5D (supplementary materials). Moreover, the standard deviation was also at the lowest level after this short extraction time. This was probably caused by the high volatility of compounds and their rapid transfer from the matrix to the gas phase. These results showed the importance of optimising this parameter. An additional 10 min of extraction also significantly reduced the extraction time, which made it possible to reduce the total analysis time.