*3.3. GC*×*GC-ToFMS Analysis*

The volatiles of the *Brassica* vegetable samples were isolated using headspace solid phase microextraction (HS-SPME) and analyzed using SPME-GC×GC-TOFMS for the comparative, semiquantitive (peak area percentage) analysis only. The SPME fiber carboxen/polydimethylsiloxane (DVB/CAR/PDMS) was used for volatile extraction (Supelco, Bellefonte, PA, USA). The vegetables were cut into approximately 0.5 cm pieces with a kitchen knife and 4 g of each was placed in a separate SPME vial. The samples were pre-incubated for 5 min at 60 ◦C, then the fiber was exposed for 30 min at the same temperature to adsorb volatiles. The volatile compounds isolated by SPME were desorbed in the injector port on the GC×GC–ToF-MS system (Pegasus 4D LECO, St. Joseph, MI, USA). The GC was equipped with a DB-5 column (25 m × 0.2 mm × 0.33 μm, Agilent Technologies, Santa Clara, CA, USA) and a Supelcowax 10 (1.2 m × 0.1 mm × 0.1 μm, Supelco Bellefonte, PA, USA) as the second column. The injector temperature was set at 250 ◦C and injection was performed in a splitless mode. The gas flow was set at 0.8 mL/min. The primary oven temperature was programed as follows: 40 ◦C for 1 min, then rising at 6 ◦C/min to 200 ◦C, where it then was reduced at 25 ◦C/min to 235 ◦C, where it was held for 5 min. In the secondary oven, the following programming was used: Held at

65 ◦C for 1 min, then rising at 6 ◦C/min to 225 ◦C, where it then decreased at 25 ◦C/min to 260 ◦C, where it was held for 5 min. The transfer line temperature was 260 ◦C. The modulation time was 4 s. The time-of-flight mass spectrometer was operating at a mass range of *m/z* 38–388 and detector voltage −1700 V at 150 spectra/s. Total analysis time was 34.07 min. All analyses for any particular cultivar and preparation method were done in triplicates.

### *3.4. Data Analysis*

Data were collected using the LECO ChromaTOF v.4.44 software (St. Joseph, MI, USA). Tentative identification was accomplished using the National Institute of Standards and Technology (NIST) library (version 2.0) of mass spectra. The calculations and basic statistical analysis were performed using Excel 2010 and Microsoft Office 2010. The principal component analysis (PCA) was performed by the SIMCA software, v. 14.1.0.2047 (MKS Unimetrix AB, Umea, Sweden).

#### **4. Conclusions**

The HS-SPME-GC×GC method was proposed for the identification of a large spectrum of volatiles from selected *Brassica* vegetables. This fast and efficient technique allowed for the determination of volatile compounds in different cultivars of broccoli, cauliflower, kohlrabi and Brussels sprouts. SPME is a method that provides a relatively fast volatile profiling of numerous samples. Despite many advantages of this technique, there are some issues which need to be mentioned here. First of all, there is no possibility to store volatile extracts (isolates), thus we face the problem of vegetable storage to analyze large sets of fresh raw vegetables. As presented, the freezing process caused some changes in the volatiles profile. Moreover, this is a non-exhaustive extraction method, so components with a low partition coefficient might be undetected, even if they are present in the sample in a relatively high concentration. Additionally, non-stable, easily reactive compounds can disintegrate, mainly due to high temperatures during thermal desorption from the fiber. The technique is very good for comparative purposes, pertinent for quantitative analysis; however, in the case of an external standard method used for quantitation, the impact of the matrix cannot be ignored, or alternatively, isotopomers of analyzed compounds could be used. Components from the following chemical groups were identified in those plants: Alcohols, aldehydes, isothiocyanates, nitriles, sulfides and others (unspecified). The proportions between the different groups were dependent on the vegetable type/species. The volatiles analyzed in raw and cooked vegetables showed changes occurring in the volatile fraction in cooked vegetables in comparison with raw ones. One of the most important differences in the volatile compounds concerned was a marked decrease in the contents of aldehydes and alcohols. The total amounts of sulfides decreased after cooking in all vegetables, except for broccoli. Isothiocyanate concentrations unexpectedly increased in most analyzed vegetables, while in the case of nitriles, a decrement was observed. Because all vegetables were frozen before analysis, the influence of freezing on the volatile fraction composition was also investigated. It was found that postharvest freezing of the *Brassica* vegetables induces biochemical changes that give rise to a significant modification of the compounds responsible for the aroma properties of those plants. Most of the changes are closely related to the activity of the LOX pathway enzymes. They are characterized by a high reduction in alcohol contents and an increase in aldehyde contents. Moreover, important changes were noted in the concentrations of bioactive components, e.g., isothiocyanates. The comparison of volatiles between fresh and thawed raw and cooked Brussels sprouts showed that after freezing, it is suggested to cook vegetables before consumption to maximize their beneficial properties.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1420-3049/24/3/391/s1, Table S1. Main volatile compounds identified using GC×GC in raw and cooked broccoli varieties; Table S2. Main volatile compounds identified using GC×GC in raw and cooked cauliflower varieties; Table S3. Main volatile compounds identified using GC×GC in raw and cooked kohlrabi varieties; Table S4. Main volatile compounds identified using GC×GC in raw and cooked Brussels sprouts varieties; Table S5. Compounds and their corresponding numbers used in PCA analysis (Figure 1).

**Author Contributions:** Conceptualization, M.N.W. and H.H.J.; Methodology, H.H.J. and M.N. Wieczorek; Software, M.N.W.; Investigation, M.N.W.; Resources, H.H.J.; Data curation, M.N.W.; Writing—original draft preparation, H.H.J. and M.N.W.; Writing—review and editing, H.H.J.; Visualization, M.N.W.; Supervision, H.H.J.; Project administration, H.H.J.; Funding acquisition, H.H.J.

**Funding:** This work was supported by the National Science Centre (Poland) under project Harmonia 7, 2015/18/M/NZ9/00372.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of the compounds are not available from the authors.

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*Article*
