# Analysis of Potent Odour-Active Volatile Thioids in Foods and Beverages with a Focus on Wine
## 3 Thiol Isolation--Extraction and Derivatisation
### Derivatisation Approaches
#### 3.2.2 Derivatisations for LC Analysis of Thiols
As a slight aside, these derivatives are claimed to enhance ionisation and improve the signal response due to the ease of ionisation and the positive charge gained by the nitrogen. However, the nitrogen in ebselen derivatives is an amide (weak acid) and not an amino nitrogen, so positive ionisation mode would rely on the protonation of the carboxamide oxygen [52]. Thiol derivatisation with DTDP has also been developed for wine analysis (Table 1, Entry 17) [7], due to its high derivatisation ability of sulfhydryl groups at acidic pH [53], whereby DTDP specifically and rapidly directly reacts with thiols in the natural wine pH range [7]. Avoiding pH adjustment throughout derivatisation and extraction is deemed to be an important point, and, when coupled with conventional SPE for clean-up and enrichment of derivatives, this procedure provides a relatively simple approach that affords the desired sensitivity for analysis of 3-SH, 3-SHA, 4-MSP, BT, and 2-FT [7]. The flexibility of the DTDP derivatisation and extraction method has been demonstrated in the chiral analysis of enantiomers of 3-SH and 3-SHA in wine [41], and in a refined form (no concentration after SPE) while using convergence chromatography for wine analysis [42]. The additional advantages of DTDP derivatisation include the formation of stable and easily ionisable derivatives (due to the pyridine moiety) that are ideal for electrospray ionisation (ESI), and, to a lesser extent, the inclusion of a chromophore, which may be useful for samples with high levels of thiols (3-SH in particular), although this was not tested. As a bonus, DTDP is a non-hazardous chemical and is safer to deal with than some other reagents, especially \(p\)-HMB or PFBBr. More recently, 1-(4-(1\(H\)-phenanthro[9,10-d]imidazol-2-yl)phenyl)-1\(H\)-pyrrole-5-dione (PIPD) has been demonstrated as a new derivatisation reagent for volatile thiols (Table 1, Entry 20). The maleimide moiety in PIPD can rapidly react with thiols to form derivatives that are stable (4 \({}^{\circ}\)C for at least three days) and detectable by HPLC-fluorescence (with atmospheric pressure chemical ionisation (APCI)-MS used for identification) [45]. Thiol extraction was performed with GP-MSE (N\({}_{2}\) at 2.5 mL/min for 30 min at 190 \({}^{\circ}\)C) in a customised apparatus prior to derivatisation (10 min at 35 \({}^{\circ}\)C in phosphate buffered saline, pH 7.5) [54]. After extraction and derivatisation, the mixtures were diluted with methanol (MeOH), filtered, and directly injected [45]. This proposed extraction and derivatisation methodology is simple and fast, and the analytical method is precise and sensitive, but the approach requires a customised gas purge chamber and the sample is kept at a high temperature for 30 min, which seems unlikely to be applicable to liquid samples, such as wine or beer. In addition, PIPD has to be synthesised, as opposed to other commercially available reagents. Whether for LC or GC, the derivatisation examples that have been mentioned so far require the use of reference standards and internal standards to establish the calibration curves for quantitative analysis. In many cases, the reference standards or internal standards (particularly deuterated internal standards) are not commercially available, or they are expensive to acquire or non-trivial to synthesise. These concerns can be somewhat simplified by the use of stable isotope labelled derivatisation-based methods [43; 44; 49], and, when considering that the derivatisation of volatile thiols appears to be essential for food and beverage analysis by LC, introducing stable isotope labelled derivatisation does not add any extra sample processing steps.