# Non-Enzymatic Formation of \(N\)-acetylated Amino Acid Conjugates in Urine
## 3 Results  
### Determining the Enantiomeric Composition of N-AAA Conjugates Observed in the Case Study  
The efficacy of our method for separating and hydrolysing \(N\)-AAA conjugates is illustrated in Figure 4. Figure 4a shows the organic acid analysis result of \(N\)-Ac-L-Ala before hydrolysis, which indicates an adequate quantity of this conjugate for the procedure. Figure 4c confirms the absence of detectable amounts of Ala contamination in the same sample through amino acid analysis before hydrolysis. Thus, the organic acid extraction approach successfully provided sufficient quantities of \(N\)-AAA conjugates without extracting native amino acids.  
Moreover, Figure 4b displays the organic acid analysis result of \(N\)-Ac-L-Ala after hydrolysis, indicating complete hydrolysis of \(N\)-Ac-L-Ala as only a trace amount of the \(N\)-Ac-L-Ala mono-TMS derivative was detected. Additionally, amino acid analyses of Ala were performed after hydrolysis, as shown in Figure 4d, confirming the presence of sufficient amounts of Ala and the procedure's minimal decomposition of the desired end product.  
The method was also effective with all available \(N\)-AAA conjugates (results not shown). Figure 4e demonstrates the enantiomeric separation of L- and D-Ala after \(N\)-Ac-L-Ala hydrolysis, indicating an acceptably low level of D-Ala. However, the origin of the detected D-Ala remains unclear, potentially stemming from a slightly enantio-impure \(N\)-Ac-L-Ala standard, racemisation induced during hydrolysis and derivatisation, the opposing L-Ala diastereomer resulting from an enantio-impure R-2-butanoic HCl derivatisation reagent, or a combination of these factors. Nevertheless, the intensity of the second peak is low enough to determine whether the original sample had a racemic or enantiomeric excess of the original \(N\)-AAA conjugate. Thus, hydrolysis was successfully completed without significant hydrolysis-induced racemisation.  
The GC-MS separation of enantiomeric \(N\)-AAA conjugates is shown in Figure 5, including DL-Ala, -Nva, and -BCAAs. While baseline separation of DL-Leu was not achieved, the opposite enantiomer remained distinguishable when there was an enantiomeric excess of either enantiomer (e.g., if L-Leu was significantly elevated compared to D-Leu, D-Leu could still be differentiated from L-Leu).  
Based on these results, it is evident that amino acid enantiomers can be distinguished through GC-MS analysis, although limitations were observed with l-isoleucineine and D-alloisoleucine. After method standardisation, this chiral strategy was applied to investigate a specific case of MSUD that contained racemic \(N\)-AAA conjugates (Figure 6). More detailed chromatograms, which confirm significant amounts of various 2-keto acids, \(N\)-AAA conjugates, and trace amounts of hydantoin conjugates in the case study after organic acid extraction, are available in the Supplementary Material (Figures S1-S7).  
Figure 4: GC–MS results of extracted and hydrolysed \(N\)-AAA conjugates. **Organic acid analysis of N-Ac-Ala (a) before hydrolysis and (b) after hydrolysis.** Compound A is the mono-TMS derivative of \(N\)-Ac-Ala, B is the di-TMS derivative of \(N\)-Ac-Ala, and C is the internal standard, 3-phenyl butyric acid. The y-axis (abundance) is scaled to 100% C, and the x-axis (retention time in minutes) is cropped to between 13.8 and 18.4 min to show only the section of interest. **Amino acid analysis of free Ala (c) before hydrolysis and (d) after hydrolysis.** Compound A is Ala, and B is the internal standard, Nva. The y-axis is scaled to 100% B, and the x-axis is cropped to between 3.5 and 4.5 min. In (d), the graph overlays (blue and red lines) the ions used during amino acid single-ion monitoring. (e) **Hydrolysis- and/or derivatisation-induced racemisation of L-Ala.** Compound A is L-Ala, and B is D-Ala and/or the opposing diastereomeric derivative of L-Ala. The y-axis is scaled to 100% of the di-iBDMS ester of Ala (not shown), and the x-axis is cropped to between 15.7 and 16.7 min.