# Analysis of Potent Odour-Active Volatile Thioids in Foods and Beverages with a Focus on Wine
## 4 Analytical Instrumentation
### Analysis by LC  
In recent years, LC-based analytical methods have emerged as promising and novel alternatives for volatile thiol analysis (both screening and quantitation) in foods and beverages (Table 2, Entries 15, 16, 21, 22, 26, 28, 29). Although LC has been used to assess non-volatile thiols, such as cysteine and glutathione, in biological samples for some time [70; 71; 72], the LC analysis of volatile thiols in foods and beverages has arisen more recently. The high volatility and low abundance of the analytes meant that they were more compatible with GC separations rather than LC with liquid-phase MS detection. Even when bypassing the chromatographic system, underivatised 3-SH was undetectable by direct liquid infusion MS in positive or negative ion mode [43], so to facilitate the potential application of LC to the analysis of volatile thiols, the analytes first have to be converted into non-volatile derivatives with suitable reagents. Indeed, with promising derivatisation reagents having been specifically suggested (Section 3.2.2 and Table 1), complementary LC methods have necessarily been developed in tandem (Table 2, Entries 16, 21, 22, 26, 28), some of which even rival the analytical performance of the GC methods. RP-LC separations with C18 stationary phases are commonly used for derivatised volatile thiols. The injection volumes and flow rates (0.20-0.40 mL/min) varied slightly, depending on the column and instrument used (Table 2). For example, slightly higher flow rates (0.35 mL/min and 0.40 mL/min) were utilised in ultra-performance (UP) LC with a column containing 1.7 \(\upmu\)m particles [36]. Mobile phases are the same as commonly used for RP-LC, for instance, aqueous MeOH [37; 43] and aqueous acetonitrile (MeCN) [7; 41; 44]. Proposed RP-LC methods have been able to resolve the analytes in a relatively shorter period of time (e.g., 17 min with UPLC [36]) compared to GC methods (e.g., 55 min [33]). MS is still the preferred detection technique for LC methods, even when derivatisation introduces a chromophore into volatile thiols (which may facilitate UV [41] or fluorescence detection [36; 45]) (Table 2). In fact, detection is typically undertaken with MS/MS, and instruments, including QqQ, Q-TOF, and Orbitrap operated in ESI mode have been described for identifying or quantitating volatile thiols, with superb sensitivity and selectivity. More specifically, with a QqQ, MRM [7; 36; 41] is the main mode that is utilised for quantitation and double precursor ion scan (DPIS) mode has been applied more for qualitative purposes [43; 49]. The selection of mass transition pairs for MRM typically includes one quantifier pair and at least one qualifier pair as the minimum requirement, with the transitions being chosen from direct infusion product ion MS experiments of the reference standard derivatives [7]. MRM of unique mass transition pairs provides a cleaner chromatographic background, which directly results in greater sensitivity [44]. QqQ with MRM mode has been employed for thiol derivatives that were obtained either with conventional derivatisation reagents [7; 33; 36; 41] or stable isotope derivatisation reagent pairs [44]. DPIS combined with stable isotope labelled derivatisation has been investigated for thiol profiling in beers (Table 2, Entry 16) [43; 49]. Admittedly, the thiols that were tentatively identified in beer with this method were biological thiols, but this could be of potential use for volatile thiols analysis. Light- and heavy-labelled thiol derivatives have characteristic ions with a fixed mass shift that can be distinguished by DPIS with QqQ by employing stable isotope labelled derivatisation, and identities can then elucidated by product ion scan and Q-TOF [43]. Only peak pairs from extracted DPIS chromatograms with the same retention time and peak intensity are considered and relative quantitation can be readily achieved by varying the ratios of light- and heavy-labelled samples being mixed [43].