# New Insight on the Formation of 2-Aminoacetophenone in White Wines
## 2 Materials and Methods
### UHPLC-MS/MS Measurement of 2-AAP Precursors  
Analysis was carried out by a 1290 infinity UHPLC system coupled to a 6460 triple-quadrupole mass spectrometer (Agilent Technologies). Wines were diluted in methanol (1:1, \(v/v\)), vortexed, filtered through a 0.22 \(\upmu\)m PVDF filter (Millipore, Billerica, MA, USA) and directly injected. Samples were kept at 4 \({}^{\circ}\)C during analysis, and the injection volume was 5 \(\upmu\)L. Separation was performed on a 100 mm \(\times\) 2.1 mm, 1.8 \(\upmu\)m column (Acquity HSS T3, Waters) maintained at 40 \({}^{\circ}\)C. Flow was 0.4 mL/min; mobile phase A was 0.1% formic acid and B was 0.1% formic acid in methanol. The linear gradient started at 2% B, to 55% B in 45 min, to 100% B in 1 min, at 100% B for 3 min, to 2% B in 1 min and then 5 min at 2% B. Analysis was performed in positive mode using electrospray ionization (ESI, Jet-Stream) and selected reaction monitoring (SRM) acquisition. Nitrogen was the collision gas, and the source parameters were gas temperature 250 \({}^{\circ}\)C, gas flow 6 L min\({}^{-1}\), nebulizer 241 Kpa, sheath gas heater 375 \({}^{\circ}\)C, sheath gas flow 10 L min\({}^{-1}\), capillary 4000 V. Three mass transitions were selected for each compound: one for the quantifier and two for qualifiers (Table 2). The linearity of the detector response for 2-AAP precursors was verified in synthetic wine diluted in methanol (1:1, \(v/v\)). Seven concentration levels were injected in six repetitions for each level. Linearity was determined, and the limit of detection and limit of quantification were calculated by considering signal to noise S/N = 3 and S/N = 10, respectively (Table 2). \begin{table}
\begin{tabular}{l l l l l l l l l} \hline \multirow{2}{*}{**Compounds**} & \multirow{2}{*}{**Retention Time (min)**} & \multicolumn{3}{c}{**First Transition (Quantifier)**} & \multicolumn{3}{c}{**Second Transition (Qualifier)**} \\ \cline{3-8}  & & & **Precursor Ion to** & **Collision Energy** & **Precursor Ion to** & **Collision Energy** \\  & & & **Product Ion (_un/z_)** & **(eV)** & **Product Ion (_un/z_)** & **(eV)** \\ \hline
2-AAP-d5 & 9.33 & 140 \(\rightarrow\) 122 & 12 & 140 \(\rightarrow\) 94 & 10 \\ \hline
2-AAP & 9.37 & 135 \(\rightarrow\) 120 & 12 & 135 \(\rightarrow\) 92 & 10 \\ \hline \end{tabular}
\end{table} 
Table 1: Mass spectral transitions and collision energies selected for GC-MS/MS analysis of 2-AAP-d5 and 2-AAP. \begin{table}
\begin{tabular}{l l l l l l l l l l} \hline \multirow{2}{*}{**Compound**} & \multicolumn{2}{c}{**Retention Time (min)**} & \multicolumn{2}{c}{**Parent**} & \multicolumn{2}{c}{**Q**} & \multicolumn{2}{c}{**q (_un/z_)**} & \multicolumn{2}{c}{**Concentration**} & \multirow{2}{*}{**R2**} & \multirow{2}{*}{**LOD**} & \multirow{2}{*}{**LOQ**} \\  & \multicolumn{2}{c}{**Time (min)**} & \multicolumn{1}{c}{**(_un/z_)**} & \multicolumn{1}{c}{**(_un/z_)**} & \multicolumn{1}{c}{**(_un/z_)**} & \multicolumn{1}{c}{**Range**} & & & \\ \hline \multirow{2}{*}{\begin{tabular}{l} Typtophan \\ (mg/L) \\ \end{tabular} } & \multirow{2}{*}{5.7} & \multirow{2}{*}{205.1} & \multirow{2}{*}{146.1} & \multirow{2}{*}{118.1} & \multirow{2}{*}{91.1} & \multirow{2}{*}{0.1–10} & \multirow{2}{*}{0.99974} & \multirow{2}{*}{0.01} & \multirow{2}{*}{0.04} \\  & & & & & & & & & \\ \hline \multirow{2}{*}{\
\begin{tabular}{l} Kynureine \\ (\(\upmu\)g/L) \\ \end{tabular} } & \multirow{2}{*}{3.2} & \multirow{2}{*}{209.1} & \multirow{2}{*}{146.0} & \multirow{2}{*}{94.1} & \multirow{2}{*}{65.1} & \multirow{2}{*}{0.5–10} & \multirow{2}{*}{0.99736} & \multirow{2}{*}{0.23} & \multirow{2}{*}{0.56} \\  & & & & & & & & \\ \hline \multicolumn{2}{l}{
\begin{tabular}{l} Anthranilic \\ acid \\ (\(\upmu\)g/L) \\ \end{tabular} } & \multirow{2}{*}{7.6} & \multirow{2}{*}{138.1} & \multirow{2}{*}{65.1} & \multirow{2}{*}{92.0} & \multirow{2}{*}{39.1} & \multirow{2}{*}{0.1–40} & \multirow{2}{*}{0.99955} & \multirow{2}{*}{1.20} & \multirow{2}{*}{1.33} \\  & & & & & & & & \\ \hline \end{tabular}
\end{table}