3 ureidopropionic acid

Molecule identification was done using the average exact mass for each molecule. Samples were prepared as described above and analyzed using the MS/MS feature of the Agilent Technologies model 6520 Q-TOF LC/MS. MS/MS spectral analysis was performed using Agilent Technologies Qualitative analysis program and confirmed by Agilent Molecular Structure Correlator software and Metlin and HMDB databases. Molecule identification was further confirmed by comparing MS/MS spectra of each molecule with purchased pure chemicals as standards: 3-ureidopropionic acid (product # 94295-1G, Sigma-Aldrich), 3-hydroxy-DL-kynurenine (product # H1771, Sigma-Aldrich), D-neopterin (product # N3386 Sigma-Aldrich), N1,N12-diacetylspermine (item # 17918, Cayman Chem), 7-methylguanine (product # 67073, Sigma-Aldrich), 3’-N-acetylneuraminyl-N-acetyllactosamine (product # A6936, Sigma-Aldrich), N-acetylneuraminic acid (product # 19023, Sigma-Aldrich), N-acetyl-β-D-mannosamine (product # sc-295642A, Santa Cruz Biotechnology), and N-acetylglucosamine (product # A4106, Sigma-Aldrich). Standards were analyzed alone, in a 1:1 solution of 0.2% formic acid in methanol and MilliQ H₂O at a final concentration of 50 nM and in urine samples spiked with each respective standard to ensure accurate retention time and m/z value.