# Detection of Sub-Aroma Threshold Concentrations of Wine Methoxyprazines by Multidimensional GCMS
## 3 Results and Discussion
### Method Validation  
Following optimization a validation procedure occurred taking into account: linearity, repeatability, detection and quantitation limits in model wine solutions, while recoveries were performed in spiked model wines and wine.  
The analytes were analyzed over the range of 0.260--9.96 ng/L (IPMP), 0.130--9.99 ng/L (SBMP), and 0.267--41.16 ng/L (IBMP). Each was found to be linear (R\({}^{2}\) > 0.99), however a quadratic fit the datapoints, providing better accuracy over the examined range without requiring multiple analyses (Table 3). The quadratic regression coefficients for each analyte were greater than 0.999. Additionally, the residuals for the lower concentrations were smaller for the quadratic regression, thereby validating its use [21].  
The limits of blank, detection, and quantitation were established over multiple injections with the findings reported in Table 3. The LOQ is reported as the low concentration in the concentration range, 0.260 (IPMP), 0.130 (SBMP), and 0.267 ng/L (IBMP). The upper end of the concentration was established as the highest concentration run in the standard curve, as to not over saturate the mass spectral detector. Higher concentrations could be obtained, but this would need to be verified by running the appropriate standards. In this study it was also unnecessary to examine a concentration maximum given the low levels commonly found in wines and the pursuit of a low detection limit to determine concentrations that have the potential to contribute to the aromatic profile of a wine. The coefficient of variation was established for each analyte at a concentration of 0.66 (IPMP), 0.66 (SBMP), and 2.73 ng/L (IBMP), and found to be extremely low: 1.4, 2.8, and 3.5%, respectively. These values are well below the accepted \"functional sensitivity\" level and demonstrate the precision of sample analysis at the low concentrations that were obtained [22].  
Recovery was performed in multiple solutions (Sauvignon blanc, Rose, Pinot noir, and Model wine) with Sauvignon blanc and Model wine being spiked at two concentrations (Table 4). Recoveries ranged from 95 to 102% (IPMP), 94.3 to 101.3% (SBMP), and 95.7 to 106.3% (IBMP). Additionally, these spikes were evaluated over three weeks to establish the method reproducibility. The samples had a % CV of 0.30 to 5.47% (IPMP), 1.15 to 5.21% (SBMP) and 0.57 to 6.57% (IBMP), which can be considered satisfactory for the low concentrations examined in this study.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \multirow{2}{*}{**Compound**} & \multirow{2}{*}{**Concentration Range (ng/L)**} & \multirow{2}{*}{**LOB (ng/L)**} & \multirow{2}{*}{**LOD (ng/L)**} & \multirow{2}{*}{**\%CV b**} & \multirow{2}{*}{**Equation**} & \multirow{2}{*}{**R2 c c**} \\  & & & & & & & \\ \hline IPMP & 0.260–9.96 & 0.159 & 0.192 & 1.4 & \(\text{y}=-0.9383\text{x}^{2}+5.401\text{x}+0.0334\) & 0.9993 \\ SBMP & 0.130–9.99 & 0.016 & 0.061 & 2.8 & \(\text{y}=-0.1479\text{x}^{2}+13.188\text{x}-0.0216\) & 0.9998 \\ IBMP & 0.267–41.16 & 0.110 & 0.175 & 3.5 & \(\text{y}=-0.0615\text{x}^{2}+3.533\text{x}-0.0238\) & 0.9996 \\ \hline \end{tabular}  
* LOQ is determined as the lowest concentration of the concentration range. b Data was measured in wine spiked at 0.66, 0.66, and 2.73 ng/L levels for IPMP, SBMP, and IBMP, respectively. c Measurements were made in deionized water. IBMP–2-Isobutyl-3-methoxyrazine, \(\text{d}_{3}\)-IBMP—deuterated 2-Isobutyl-3-methoxypyrazine, IPMP—2-Isopropyl-3-methoxypyrazine, \(\text{d}_{3}\)-IPMP—deuterated 2-Isopropyl-3-methoxypyrazine, SBMP—2-sec-Butyl-3-methoxypyrazine, LOB—limit of blank, LOD—limit of detection, LOQ—limit of quantitation.  
\end{table}
Table 3: Compound concentration range, limit of blank, limit of detection, and limit of quantitation a for the optimized assay conditions.