# Detection of Sub-Aroma Threshold Concentrations of Wine Methoxyprazines by Multidimensional GCMS
## 3 Results and Discussion  
### Extraction Optimization  
The goal of the method development was to determine the concentration of MPs in wines below the ng/L level and thus some parameters needed further optimization for the heart-cut multidimensional GC-MS method. Much research already exists for the selection of SPME fibers and pH of the samples, therefore these parameters were considered fixed for this study as they have been established in this laboratory and others [10, 11, 15]. Consequently, a DVB-CAR-PDMS fiber was selected due to its ability to absorb a wide range of volatile organic compounds, rapid equilibrium, and reproducibility of injection [16]. Likewise, [PERSON], et al. [11] established that the optimum pH for the volatilization of MPs is greater than pH 12, which also suppressed the volatile acids. While dilutions have been examined in the past, it was reevaluated due to the low analytical concentrations sought. The analytes were diluted for a factor of: 1.15, 2, 3, 4, 5, 7, and 10. Results for each MP are displayed in Figure 1. While the IBMP and SMBP had an initial drop and then plateaued for dilution factors 2 and 3 in the response, the IPMP increased over the same range before all three declined. The corresponding deuterated internal standards increased in response across all dilutions. Consequently, a dilution factor of three was chosen since the analytes were at the upper range of their linear response and the internal standards were within the mid-range of their response. At the SPME fiber compounds compete for the adsorption sites, therefore it is vital to increase the amount of the target analytes in the headspace. The pH adjustment provided optimum conditions for the MPs to be volatilized; however it was expected that high concentration compounds, such as ethanol, could still volatilize in sufficient concentration to out compete the target molecules. Dilution of these compounds would allow for decreased competition and increased adsorption of the target molecules given that the samples were optimized for MPs volatilization. [PERSON] and [PERSON] [17] investigated the partition coefficients of volatile compounds in wines and grapes. In their study they were able to identify compounds that were more easily detected in wines upon dilution. The study demonstrates the complex nature of the sample-headspace-fiber equilibrium. [PERSON] and [PERSON] [17] demonstrated the importance of the equilibrium between the three phases and how it impacts compound detection. [PERSON] et al. [18] further demonstrated this principle of dilution increasing the adsorption onto SPME fibers in their study of  
Figure 1: Effect of the dilution factor on methoxyprazine recovery. IBMP—2-Isobutyl-3-methoxypyrazine, d\({}_{3}\)-IBMP—deuterated 2-Isobutyl-3-methoxypyrazine, IPMP—2-Isopropyl-3-methoxypyrazine, d\({}_{3}\)-IPMP—deuterated 2-Isopropyl-3-methoxypyrazine, and SBMP—2-sec-Butyl-3-methoxypyrazine. volatile by-products in industrial ethanol. Their study showed that ethanol concentrations between 10 and 40% showed increases in the other volatile compounds, low molecular weight, and that higher molecular weight compounds were not detected at concentrations above 20% ethanol. The importance of understanding the volatile equilibrium in HS-SPME for the target analytes is demonstrated in these two studies and they further highlight the limitations associated with dilution to maximize response. In the current study, focusing on three similar compounds, there was minimal impact, as any reduction in other compounds was not examined in the assay; in this way we achieved obtaining a true representation of the MPs present in wine. Heart-cuts were established to optimize the number of peaks being sent to the second dimension (Figure 2). Two heart-cut regions were established. The analyte standards and internal standards were run in the first dimension using an FID and compared to the literature for retention indices (RIs) (Table 1).