## 3 Results and Discussion
### Detection of Schiff Bases of Alanine with Sugar-Derived Aldehydes through Isotope Labeling  
The \(\upalpha\)-alanine/glucose reaction mixtures heated for 2 h at 110 \({}^{\circ}\)C were analyzed by high resolution qTOF/MS for the presence of masses corresponding to Schiff bases of \(\upalpha\)-alanine with short chain aldehydes that can be generated from glucose degradation [14]. Peaks corresponding to adducts of formaldehyde, acetaldehyde and glycolaldehyde were observed only in methanol solutions and in the presence of FeCl\({}_{2}\), as listed in Table 2. The last two aldehydes have been previously reported to be generated also from formaldehyde alone in the presence of metal ions through the formose reaction (Butlerov reaction) [21; 22; 23]. The Schiff bases of \(\upalpha\)-alanine formed with formaldehyde (_m/z_ 102), acetaldehyde (_m/z_ 116), and glycolaldehyde (_m/z_ 132) were observed only in the presence of FeCl\({}_{2}\) except the adduct formed with formaldehyde (Table 2). According to the HRMS data, the ion at _m/z_ 102 was the most prominent. As shown in Table 2, spiking the reaction mixtures with excess formaldehyde increased the intensities of all the detected Schiff bases by more than four-fold. Furthermore, the reaction mixtures were also analyzed using isotope labeling techniques with [13C-U]glucose or [13C-3]alanine, targeting ions incorporating one C-3 atom of alanine and up to two sugar carbon atoms, depending on the type of aldehydemoieties involved in the Schiff base formation. The extracted ions from the HRMS data satisfying these requirements and exhibiting highly accurate elemental composition of the Schiff bases (shown in their isomeric oxazolidin-5-one forms) and their isotopically labeled counterparts using [1\({}^{3}\)C-U]glucose or [1\({}^{3}\)C-3]alanine are shown in Table 2. The isotope labeling data together with the spiking experiments and high-resolution MS data provided sufficient evidence for the identity of these ions. The fact that Schiff bases can be hydrolyzed during the reaction and are not stable enough to survive the reaction conditions lead us to conclude that the observed ions with a Schiff base composition represented their oxazolidin-5-one isomers [10]. When the reactions were carried out in water or without FeCl\({}_{2}\), the intensities of the proposed oxazolidin-5-one were significantly decreased or disappeared altogether (Table 2). The rationale behind the divalent metal ion catalysis of oxazolidin-5-one formation and the proposed reaction pathways are shown in Figure 2. According to Figure 2, the added metal ions can stabilize the Schiff bases through complexation (see also Table 3), reducing the electrophilic character of the imine carbon and extending their half-life in the reaction mixture, thus retarding solvolysis. Table 3 provides evidence for the formation of iron complexes of some of the alanine Schiff bases with highly accurate elemental composition and isotope label incorporation data. Only when these complexes are dissociated, intramolecular cyclization will form stable oxazolidin-5-ones as shown in Figure 2.  
Further evidence for the cyclic nature of the proposed Schiff bases was provided by the observation of their interaction products with aldehydes, such as formaldehyde, and formation of their corresponding iminium ions, such as [M]\({}^{+}\) 114.0549, as shown in Figure 2 and listed in Table 4. Furthermore, the solvent methanol can trap the iminium ions as shown in Figure 2, providing additional structural information on iminium ions. Similarly, 4-methyl-5-oxoxazolidin-5-one (_m/z_ 102), shown in Figure 3, can undergo Schiff base formation with other available aldehydes and generate iminium ions at M\({}^{+}\) 128, 144, and 114 as shown in Figure 3 and listed in Table 4. The open form Schiff bases will not be able to form such iminium ions.