## 2 Results and Discussion
### Comparison of Current Method to Existing GC-MS Methods for Wine Polyfunctional Thiol Analysis  
Literature methods that rely on a low selectivity extraction step (e.g., SPE, SPME) to pre-concentrate underivatized wine polyfunctional thiols (3-MH, 3-MHA, 4-MMP) prior to GC-EI-MS have not achieved detection limits below sensory thresholds [6, 7]. Quantification of these thiols below sensory threshold can be achieved using organomercuric reagents and anion exchanges for selective pre-concentration [4], but the approach has been criticized for needing large sample volumes and hazardous reagents [10, 11]. More recent reports have achieved detection limits below sensory thresholds by preparing PFBBr derivatives of the thiols prior to GC-NICI-MS detection. The detection limits of these methods are generally better than what we have achieved with our current report: for example [PERSON], _et al._ reported LODs of 0.1 ng/L, 2 ng/L, and 0.3 ng/L, for 4-MMP, 3-MH, and 3-MHA, respectively, as compared to 0.9 ng/L, 1 ng/L, and 17 ng/L in our work. Correcting for the fact that this earlier report used a 6 mL wine sample compared to 40 mL in our work, NICI-MS provides about a 1-3 order of magnitude improvement in detection limits compared to EI-MS for PFB derivatives, comparable to values reported for other compound classes [25]. However, an advantage of our current method is that EI-MS is more widespread than NICI-MS in laboratories. One other group has utilized GC-EI-MS for quantification of the 3-MH-PFB derivative, although they did not report characterization of 3-MHA and 4-MMP [13]. In that report, 3-MH was initially extracted into pentane and back extracted into aqueous alkaline buffer prior to derivatization. Similar to our approach, the 3-MH-PFB derivative was extracted by SPME prior to GC-EI-MS. We achieved a detection limit of 1 ng/L for a 40 mL wine sample, as compared to 30 ng/L for a 200 mL wine sample in the previous report. The most notable difference between the two reports is that our current work utilized a dry-down of the organic layer step prior to reconstitution and HS-SPME, as opposed to the back-extraction step in the other report. Potentially, this dry-down step removed lower boiling volatiles that could compete with the thiol-PFB derivatives for absorptive sites on the SPME fiber. While we observed a considerable enhancement in S/N when aqueous NaCl solution was added to our dried-down \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Analytic**} & \multicolumn{2}{c}{**Recovery (\%)**} & \multicolumn{2}{c}{**RSD (\%)**} \\ \cline{2-5}  & **Low Level** & **High Level** & **Low Level** & **High Level** \\ \hline
4-MMP & 104.9 & 108.7 & 9.8 & 6.6 \\
3-MH & 102.6 & 90.5 & 6.9 & 5.4 \\
3-MHA & 90.2 & 100.5 & 11.1 & 5.6 \\ \hline \hline \end{tabular} Recovery: samples spiked at low level (60 ng/L 4-MMP, 3-MH, 3-MHA); high level (500 ng/L 4-MMP, 3-MH, 3-MHA). RSD: relative standard deviation of 5 samples spiked at low level (10 ng/L 4-MMP; 25 ng/L 3-MH; 25 ng/L 3-MHA); high level (100 ng/L 4-MMP; 200 ng/L 3-MH; 100 ng/L 3-MHA).  
\end{table} 
Table 2: Recovery and precision experiments. SPME vials, this should have been comparable to the conditions in the other report for alkaline back-extraction, and is thus unlikely to account for the differences. Other potential explanations for the enhanced sensitivity of our current report are more optimal derivatization or HS-SPME conditions, or decreased losses during sample preparation due to fewer work-up steps. GC-EI-MS has also been reported for quantification of ethyl propoidate derivatives of thiols, but detection limits were well above sensory thresholds for 4-MMP and 3-MH [14]. Beyond using EI-MS, another advantage of our extractive alkylation method is that it requires fewer clean-up steps than many other methods that have analyzed PFB derivatives.