## 3 Results and Discussion
### Optimization of Grape Homogeneate Preparation for IBMP Analysis  
The new SPMESH sheet-DART-MS-MS method was used to quantify IBMP in grape homogenates produced from Bordeaux cultivars (Merlot, Cabernet Sauvignon) sourced from different vineyards in California, USA. These grape cultivars were selected because they often have IBMP at concentrations at or above sensory threshold (2 ng/L in water [4], 10-16 ng/L in wine [24; 25]), and because IBMP is a useful quality marker for red wines produced from these grapes [26].  
In our previous SPMESH sheet work on IBMP, grape macerate samples were equilibrated with the internal standard for 30 min at 50 \({}^{\circ}\)C with agitation prior to extraction [18]. In our previous work, we observed good recovery of IBMP in spiked Pinot norir samples which lacked native IBMP [18; 19]. However, when we initially investigated Bordeaux cultivars with native detectable IBMP in our current work, we observed no correlation between SPMESH sheet-DART-MS-MS and the frequently-used SPME-GC-MS method (\(R^{2}\) = 0, Figure 3). We also observed high run-to-run variability for the SPMESH sheet method (CV = 63%). Using SPMESH, we also observed undetectable concentrations of IBMP in the supernatant (<4 ng/L) as compared to the homogenate (up to 24.9 ng/L, data not shown), suggesting that IBMP was not being sufficiently extracted from the skins into the liquid phase using our original conditions.  
Previous work using SPME-GC-MS for quantitation of native IBMP (as opposed to spiked IBMP) in grape samples demonstrated that the IBMP/\(d_{3}\)-IBMP ratio increases with longer SPME incubation times and/or higher incubation temperatures, eventually reaching an equilibrium [21]. These observations were reported to be due to the localization of IBMP in grape skins [27], such that native IBMP must first diffuse from the skins into the liquid phase before equilibrating with the deuterated standard and reaching the headspace SPME fiber.  
To evaluate this possibility, we used a higher temperature equilibration step (230 RPM at 70 \({}^{\circ}\)C for 1 h) prior to SPMESH sheet extraction. These conditions were previously reported by [PERSON], et al., as sufficient to reach IBMP/\(d_{3}\)-IBMP equilibrium during SPME-GC-MS [21]. The improvement in SPMESH-DART-MS-MS vs. SPME-GC-MS correlation following introduction of this equilibration step is discussed in the next section.  
One concern associated with using a high temperature incubation of grape samples is that thermal artifacts (isobaric interferences) could be generated. To evaluate this possibility, Pinot norir homogenate was incubated at 70 \({}^{\circ}\)C for 1 h while agitating at 230 RPM. We observed no detectable IBMP signal in both the control and incubated samples, indicating that thermal artifacts were unlikely (Figure 2). We also evaluated if IBMP signal measured by SPMESH-DART-MS-MS could be increased by addition of saturated NaCl solution, as is reported for SPME-GC-MS [28]. In our current work, we did not observe any increase in IBMP signal (data not shown; \(p>0.05\)). The reason for the non-effect of NaCl addition on method sensitivity is unclear. In conventional coated fiber SPME analyses, the improvement in sensitivity with NaCl addition arises from a decrease in the solubility of the volatile in the NaCl enriched solution, which favors the partitioning of the volatile into the SPME fiber coating. Because SPMESH has a much larger PDMS volume than fiber-based SPME (4 \(\times\) 4 cm square vs. 2 cm fiber),  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**-** & **IBMP (MS) 1 ** & **Linalool (MS) 2 [FOOTNOTE:2]Footnote 2: For the first time, the second time, the  
it is likely that SPMESH extraction is limited by the rate of mass transfer from the sample rather than the equilibrium partition coefficient, which would explain the lack of a \"salting-out\" effect in our current work.