# Detection of Sub-Aroma Threshold Concentrations of Wine Methoxyprazines by Multidimensional GCMS
## 2 Materials and Methods
### Proposed Method  
Capped sample vials containing standards or samples were placed in a chilled auto-sampler trap set to 8 \({}^{\circ}\)C prior to sampling by a PAL LHX-xt robotic auto-sampler (CTC Analytics AG, Zwingen, Switzerland). The 2 cm DVB/CAR/PDMS Stableflex SPME fiber was conditioned at 270 \({}^{\circ}\)C for 10 min prior to each sample vial incubation. Sample vials were stirred and incubated at 50 \({}^{\circ}\)C for 10 min prior to static sampling with the SPME fiber at the \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Analyte**} & \multirow{2}{*}{\
\begin{tabular}{c} **Molecular** \\ **Weight** \\ \end{tabular} } & \multicolumn{2}{c}{**Retention Time (min)**} & \multicolumn{2}{c}{**Retention Indices**} & \multirow{2}{*}{
\begin{tabular}{c} **Monitored** \\ **Ions (\(m/z\)) \({}^{*}\)** \\ \end{tabular} } \\ \cline{3-6}  & & **Dimension 1** & **Dimension 2** & **Calculated** & **Literature** & **Ions (\(m/z\)) \({}^{*}\)** \\ \hline IBMP & 166.22 & 37.20 & 48.38 & 1517 & 1516 & **124**, 151 \\ IPMP & 152.19 & 31.14 & 43.78 & 1422 & 1423 & **152**, 137 \\ SBMP & 166.22 & 35.66 & 47.74 & 1494 & 1494 & **124**, 138 \\ d\({}_{3}\)-IBMP & 169.24 & 37.14 & 48.29 & 1515 & N/A & **127**, 154 \\ d\({}_{3}\)-IPMP & 155.21 & 31.08 & 43.69 & 1421 & N/A & **140**, 155 \\ \hline \hline \end{tabular}  
* N/A—Not available, \({}^{*}\)**Bolded** ion is the target ion, 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-methoxyrazine, SBMP—2-sec-Butyl-3-methoxyrazine.  
\end{table} 
Table 1: Retention times and indices of the methoxypyrazines and the corresponding deuterated internal standards. same temperature for 40 min. The SPME fiber was desorbed in the GC injection port fitted with a 0.75 mm I.D. SPME liner at a temperature of 270 \({}^{\circ}\)C for 5 min in splitless mode with an initial flow rate of 1.46 mL/min, and a linear velocity of 19.9 cm/s. Outflow was to a SH-Rtx-wax column, 60 m \(\times\) 0.25 mm ID \(\times\) 0.25 \(\upmu\)m film thickness, installed in a Shimadzu 2010 GC-FID. The column oven was initially held at 35 \({}^{\circ}\)C for 3 min and then increased to 101 \({}^{\circ}\)C at a rate of 7.33 \({}^{\circ}\)C/min followed by an increase to 148 \({}^{\circ}\)C at 1.50 \({}^{\circ}\)C/min before rising to 250 \({}^{\circ}\)C at 40 \({}^{\circ}\)C/min and held for 16.11 min for a total runtime of 62 min. During the oven ramp the column flow rate was controlled using a pressure program where the pressure was increased from an initial pressure of 218.7 kPa to a final pressure of 328.1 kPa. Two heart-cuts were made to transfer the three MPs to the second dimension, the first occurred at 30.50-32.00 min and the second at 35.25-38.40 min. The heart-cut flowed onto a Rtx-5 ms (5% diphenyl 95% dimethyl polysiloxane), 30 m \(\times\) 0.25 mm ID \(\times\) 0.25 \(\upmu\)m film thickness, column connected to the outflow of the Dean's style switch in the first dimension GC oven via a short transfer line held at 62 \({}^{\circ}\)C, which extended into the oven of a Shimadzu QP2010 Ultra GCMS where it was connected to the mass spectrometer detector. The ion source and transfer line in the mass spectrometer were held at 200 and 250 \({}^{\circ}\)C, respectively. The column oven was programmed to increase the initial temperature of 35 \({}^{\circ}\)C (30 min hold) to 90 \({}^{\circ}\)C at 3 \({}^{\circ}\)C/min and held for 4.67 min before a final increase to 250 \({}^{\circ}\)C at 50 \({}^{\circ}\)C/min with a final hold time of 5.8 min for a total run time of 62 min. The column flow rate in the second dimension was initially 1.69 mL/min, however as the pressure increased this slowed to a rate of 1.3 mL/min at 25 min and was maintained at this flow rate throughout the remaining run time. The mass spectrometer was operated in selective ion monitoring (SIM) mode and monitored two selected ions for each MP standard (Table 1).