## 4 Discussion  
Usually, the flavor extracts of green tea infusions are prepared by steam distillation under reduced pressure followed by liquid-liquid extraction and concentration, or by using a solvent-assisted flavor evaporation (SAFE) device before the injection in the gas chromatographic system [18; 19]. Recently, solid-phase microextraction (SPME) has gained acceptance in many fields as an accurate, rapid, sensitive and solvent-free sampling method and its application is reported by many researchers as a useful approach in sample preparation of volatile compounds from tea leaves and tea infusions [7; 16; 20]. A fully validated, accurate and reproducible quantitative method based on HS-SPME combined with GC-MS for the determination of volatile flavor compounds in green tea was developed and validated. The optimized solvent-free sample extraction was shown to be of practical use and involved a limited number of handling steps with a minute amount of sample. This method is less time-consuming compared to those based on several preparation steps, such as steam distillation followed by liquid-liquid extraction and concentration. The method was successfully applied in quantitative analysis of selected odorants, and to study the effect of roasting procedure on the tea VFC composition. In particular, the changes in typical green tea odorant and pyrazine concentrations (\(\mu\)g/mL) were followed, to confirm that 160 \({}^{\circ}\)C for 30 min are the most adequate conditions to obtain the Hojicha flavor and prevent acrylamide formation, catechin degradation [8] and significant changes in the appreciated aroma. Among pyrazines, 2-ethyl-3,6-dimethylprazine was proved to be of particular interest because it is absent in the green teas we analyzed and is formed after the roasting process, so it can be considered as a marker of the treatment. A further innovative contribution of this study is the lanlool's concentration trend, as the content increases after roasting at 160 \({}^{\circ}\)C but drops at 180 and 200 \({}^{\circ}\)C, thus being a useful marker of overheating in quality control, both to verify the process itself and to monitor the roasting degree correlated to temperature and time. These potential markers can be important when considering Hojicha  
Figure 3: Effects of roasting temperature on the content of the selected pyrazines 2-ethyl-6-methyl-pyrazine (**A**), 2-ethyl-5-methyl-pyrazine (**B**), 2-ethyl-3,6-dimethyl-piraazine (**C**), 2-ethyl-3,5-dimethyl-pyrazine (**D**) and 2-acetyl-pyrazine (**E**). The roasting treatment and preparation for tea infusions were repeated three times. Each data value is expressed as mean \(\pm\) standard deviation (n = 3). green tea, which should be roasted at 160 \({}^{\circ}\)C to maintain the desired aroma characteristics and catechin content [8]. The characterization of the variation of the VCF absolute content can be an important contribution in the evaluation of roasted green tea quality, named Hojicha tea, especially with respect to the roasting conditions such as temperature and time. This is a very important aspect in the Hojicha tea trade, to obtain the typical \"roasted\" aroma, and, at the same time, to maintain the widely reported beneficial properties of green tea. The following are available online at [[https://www.mdpi.com/article/10.3390/app11178217/s1](https://www.mdpi.com/article/10.3390/app11178217/s1)]([https://www.mdpi.com/article/10.3390/app11178217/s1](https://www.mdpi.com/article/10.3390/app11178217/s1)). Table S1. List of the commercially available green teas and black teas. Figure S1. HS-SPME optimization. (A) Effects of the fibre extraction efficacy on the peak area of six representative VFCs. HS-SPME Conditions: sample 1 mL of tea infusion, extraction time 20 min, temperature 100 \({}^{\circ}\)C, stirring rate 400 rpm. The applied GC-MS conditions are described in the Experimental section. Effects of HS-SPME equilibration time (B), extraction time (C) and temperature (D) on the peak area of three representative VFCs.