Unraveling the Cyclization of l-Argininosuccinic Acid in Biological Samples: A Study via Mass Spectrometry and NMR Spectroscopy.

Since l-argininosuccinic acid (ASA) is the characteristic biomarker for the diagnosis of certain diseases, its reliable detection in complex biological samples is necessary to obtain a complete evaluation with greater specificity and accuracy. ASA can undergo intramolecular cyclization, yielding an equilibrium with the resulting cyclic forms, which can predominate under different analytical conditions. In this work, the appearance and transformation of the different forms of ASA have been studied and a strategy for targeted screening analysis of ASA and its cyclic forms using capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry (CE-ESI-TOF-MS) has been developed. The data and spectra obtained allowed us to gain further insight into accurate identification, concluding that there is a dynamic equilibrium depending on the pH. Moreover, one- and two-dimensional NMR spectroscopy experiments have allowed us to determine the predominant tautomeric structure for the major cyclic ASA derivative, confirming the importance of intramolecular hydrogen bonds.