# Analysis of Potent Odour-Active Volatile Thioids in Foods and Beverages with a Focus on Wine
## 3 Thiol Isolation--Extraction and Derivatisation
### Derivatisation Approaches
#### 3.2.1 Derivatisations for GC Analysis of Thiols  
As seen in Table 1 (Entries 6-14), 2,3,4,5,6-pentafluorobenzyl bromide (PFBBr), ethyl propionate (ETP), and \(o\)-methylhydroxylamine have been used as derivatisation reagents for GC-based thiol analysis. PFBBr and ETP both react with the sulfhydryl group, whereas \(o\)-methylhydroxylamine derivatises the carbonyl group in 4-MSP (forming a methoxime). After derivatisation(s), extractions can be conducted in combination with modern extraction techniques such as HS-SPME, SPE, or stir bar sorptive extraction (SBSE), in contrast to the traditional LLE or affinity chromatography normally practised in selective extractions with Hg\({}^{+}\) or Ag\({}^{+}\) (although LLE, in particular, may still feature along with derivatisation). PFBBr (Table 1, Entries 6-11) is frequently used as a derivatisation reagent for thiols, due to the bromide atom being particularly susceptible to nucleophilic substitution by thiols in the presence of base, and the obtained PFBBr thiol derivatives offer desired properties, not only by stabilising the thiol, but also with regard to electron-capturing abilities and MS detection [18]. The derivatisation of volatile thiols in wine with PFBBr has been evaluated in various formats: automated headspace on-fibre derivatisation [18], derivatisation in organic solvent system [29] or aqueous phases [32] followed by HS-SPME, in-cartridge SPE derivatisation [30], and HS-SPME coupled with SPE [31]. The SPME on-fibre derivatisation (Table 1, Entry 6) is fast, automated, and solventless. A polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fibre is exposed in sequence to the vapours of tributylamine (5 min), PFBBr solution (5 min), and then pre-incubated wine sample (containing ethylenediaminetetraacetic acid, salt, and internal standard (IS)) for extraction for 10 min at 55 \({}^{\circ}\)C [18]. This approach provided convenience and less potential interferences by using an autosampler and HS-SPME [18], but the linear ranges for the studied thiols were not very wide (and extremely narrow for 2-methyl-3-furanthiol, 2-MFT) and only two thiols (2-FT and 3-SHA) out of five were able to be analysed with this method. To improve the procedure, three conditions (two-phase liquid-liquid system, two-phase liquid-liquid system with a phase transfer catalyst, and two-phase liquid-solid system) were evaluated for wine and PFBBr derivatisation was finally conducted in a homogeneous organic solvent (benzene) system, based on relatively higher derivatisation yields and lower extraction of polar compounds from wine (Table 1, Entry 7) [29]. Apart from switching from on-fibre derivatisation to a homogeneous organic solvent system, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was used as a non-nucleophilic base to better deprotonate thiols (enhancing reactivity) and enable a decrease in the amount of PFBBr, which lessened the chance of PFBBr carryover. After comprehensive optimisation,  
Figure 2: Derivatisation reagents and reactions of volatile thiols in wine, foods, and other beverages for (**a**) gas chromatography (GC) analysis, (**b**) liquid chromatography (LC) analysis, and (**c**) LC with stable isotope labelled derivatisation reagents. PFBBr: 2,3,4,5,6-pentafluorobenzyl bromide; ETP: ethyl propionate; OPA: \(o\)-phthaldialdehydedehyde; ebesler: 2-phenyl-1,2-benzisoselenazol-3(2H)-one; DTDP: 4,4’-dithiiodipyridine; PIDP: 1-(4-(1\(H\)-phenanthro[9,10-d]imidazol-2-yl)phenyl)-1\(H\)-pyrrole-5-dione; BQB: \(\omega\)-bromoacetylquinolinium bromide; AENM: acridone-10-ethyl-\(N\)-maleimide. the method provided larger linear ranges than SPME with on-fibre derivatisation [18], but it was still unable to achieve consistent results for 2-MFT and required the use of the carcinogenic solvent benzene [29]. Subsequently, PFBBr derivatisation (after carbonyl derivatisation) in an SPE cartridge was suggested for volatile thiols in wine (Table 1, Entry 8) [30].