# 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
This protocol involved multi-step washings, derivatisation reaction, and the elution of PFBBr derivatives, but it still suffered from interferences regardless of the improved clean-up with SPE. This was ultimately compensated for by adapting to a method involving SIDA [30]. A method with methoximation and PFBBr derivatisation in combination with both SPE and HS-SPME has been developed, also using SIDA, to further eliminate matrix effects (Table 1, Entry 9) [31]. After methoximation (for 4-MSP) [30], the wine sample was adjusted to pH 7 and derivatised in-cartridge with PFBBr and DBU, and then eluates from SPE were evaporated and sampled by HS-SPME [31]. The change of pH was necessary to eliminate interferences that were not retained by SPE at higher pH [31]. The above-mentioned PFBBr derivatisation approaches were followed by GC with chemical ionisation (CI) and MS detection in negative ion mode, instead of the more routinely available electron ionisation (EI) with MS detection in positive ion mode. As such, the PFBBr derivatisation methods were investigated for thiol analysis in wine while using GC with EI-MS, again in combination with HS-SPME of the derivatives [32; 33]. Focusing on 3-SH, an extraction was proposed that involved LLE with pentane and back-extraction into cold aqueous NaOH, followed by direct PFBBr derivatisation (Table 1, Entry 11) [32]. This method showed that the analysis of a PFBBr thiol derivative could be achieved by EI-MS, although the sample volume (100 mL) was much larger than that required for NCI-MS detection, and only 3-SH was assayed (albeit at levels below its ODT) [32]. The method was improved by applying extractive arylation with PFBBr but still using GC-EI-MS (Table 1, Entry 12) [33]. The approach, which included 3-SHA and 4-MSP, along with 3-SH, employed PFBBr derivatisation of thiols in 40 mL of wine (pH adjusted to 12) with the simultaneous extraction of derivatives into pentane-diethyl ether. The extracts were dried, reconstituted, and subjected to HS-SPME [33]. The improved analytical performance, when compared to the previous method for 3-SH alone [32], was proposed to result from the removal of interferences, more optimal conditions for derivatisation or HS-SPME sampling, or having fewer steps that contribute to analyte losses during extraction [33]. In comparison to selective \(p\)-HMB extraction and the analysis of free thiols, the suggested PFBBr derivatisation-based methods for thiol extraction and analysis (either for CI-MS or EI-MS) have significantly lower sample volume and solvent consumption (especially with HS-SPME), less sample preparation steps, and no requirement for organomercurial compounds. However, the overall extraction processes are still lengthy and complicated (e.g., pH adjustment and multiple steps), and PFBBr is not entirely without safety concerns. ETP is another reagent that has been investigated for the derivatisation of thiols (Table 1, Entries 12 and 13) in wine [19], beer [34], hops [34], and work samples [34]. In the case of wine analysis, ETP rapidly reacts with thiols at basic pH (10 min), and, following an optimised SPE step, the ETP derivatives can be analysed by GC-MS (Table 1, Entry 12) [19]. The ETP-based method has further simplified the approach to thiol derivatisation when compared to \(p\)-HMB and PFBBr methods, but still requires pH adjustment of wine (not a trivial undertaking). The main shortcoming was the lack of sensitivity for 4-MSP in real wine samples as a result of poor derivatisation [19]. Nonetheless, ETP has also been evaluated in combination with SBSE for the analysis of 3-SH, 3-SHA, and 4-MSP in beer, hops, and work samples (Table 1, Entry 13) [34]. The SBSE procedure is relatively simple to conduct and it requires less solvent, but the disadvantages included the need for pH adjustment of samples, that a single SBSE of derivatives required more than 3 h, that stir bars needed to be conditioned before use and reconditioned after each use, and the need for a thermal desorption unit (TDU) [34].