# Effect of Asparaginase Treatment on Biscuit Volatile Compounds
## 2 Materials and Methods
### Determination of Levels of Asparagine in the Dough  
The extraction of asparagine was carried out as described by [PERSON] et al. [17]. One gram of dough was placed in a centrifugation tube with a cap and dissolved in 10 mL water/acetonitrile 1:1 with 0.1% (\(v/v\)) formic acid to stop the enzyme action. The tube content was mixed for 20 min by a rotary shaker, then centrifuged at 6080\(\times\)\(g\) for 5 min and the supernatant was retained. The pellet was extracted two more times with 5 mL of extracting solution and 5 min mixing, followed by centrifugation as before. Aliquots of the combined supernatants were placed in Eppendorf tubes and centrifuged at 10,000\(\times\)\(g\) for  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Sample Code**} & \multicolumn{2}{c}{**Asparaginase**} & \multirow{2}{*}{**Resting Time (min)**} & \multirow{2}{*}{**Dough T (\({}^{\circ}\)C)**} \\  & **(ASNU/kg Flour)** & & \\ \hline B500a & 500 & 0 & - \\ B500b & 500 & 15 & 20 \({}^{\circ}\)C \\ B500c & 500 & 15 & 50 \({}^{\circ}\)C \\ B750a & 750 & 0 & - \\ B750b & 750 & 15 & 20 \({}^{\circ}\)C \\ B750c & 750 & 15 & 50 \({}^{\circ}\)C \\ B1000a & 1000 & 0 & - \\ B1000b & 1000 & 15 & 20 \({}^{\circ}\)C \\ B1000c & 1000 & 15 & 50 \({}^{\circ}\)C \\ BCTrA & 0 & 0 & - \\ BCTrIB & 0 & 15 & 20 \({}^{\circ}\)C \\ \hline \hline \end{tabular}
\end{table}
Table 1: Description of asparaginase addition level and conditions for preparation of experimental biscuits.  
5 min. Then the supernatants were stored at \(-18\)\({}^{\circ}\)C until chromatography. The extraction was carried out in duplicate. On the day of the analysis, the extracts were thawed; one ml was diluted 1:1 with acetonitrile, centrifuged at 14,000\(\times\)\(g\) for 5 min and transferred to a 2 mL vial. For technical reasons, it was not possible to analyse the sample BCtrIB.  
The free asparagine content was determined using an AB Scixe 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) coupled with a PerkinElmer 200 Series liquid chromatograph (PerkinElmer, Shelton, CT, USA). The chromatograph setup included two PE 200 Series micropumps, a column oven, and a thermostatic autosampler. Separation of asparagine from other matrix components was achieved through Hydrophilic Interaction Liquid Chromatography (HILIC), with a 5 \(\upmu\)L injection onto a Luna Polar Pesticides column (100 \(\times\) 2.1 mm, 3 \(\upmu\)m, Phenomenex, VaeIosee, Denmark).  
The mobile phase consisted of 0.1% formic acid in water (Phase A) and 0.1% formic acid in acetonitrile (Phase B). Chromatographic runs were conducted at a flow rate of 0.4 mL/min with the following gradient: 0-1 min: 95% B, 1-7 min: 95% to 45% B, 7-7.1 min: return to 95% B, 7.1-14 min: maintained at 95% B. The column temperature was held constant at 25 \({}^{\circ}\)C. Detection of the analyte was performed using electrospray ionisation (ESI) in positive polarity, with data acquisition in multiple reaction monitoring (MRM) mode. The monitored transitions were 133.0 \(\rightarrow\) 74.0 (quantifier ion) and 133.0 \(\rightarrow\) 87.0 (qualifier ion), each with a dwell time of 300 ms. Mass spectrometer settings included the following: curtain gas: 10 psi, ion spray voltage: 4000 V, source temperature: 200 \({}^{\circ}\)C, nebuliser gas: 5 psi, auxiliary gas: 10 psi, declustering potential: 30 V, entrance potential: 8 V, collision energy: 15 V, collision cell exit potential: 2 V. Asparagine quantification in the extracts was performed using the external standard method. The calibration curve consisted of seven levels (0.1, 0.2, 0.5, 1, 2, 5, and 10 \(\upmu\)g/mL) of pure asparagine. To ensure optimal accuracy across all calibration levels, a linear regression model was applied, with the origin omitted and a 1/x\({}^{2}\) weighting factor.