Comparison of two azobenzene-based amino acid derivatization reagents for LC-MS/MS analysis in positive and negative ESI modes

Derivatization reagents based on azobenzene containing different N-hydroxysuccinimidyl moieties- AzoB (carbamate) and AzoC (ester) - are proposed for the LC-ESI-MS analysis of free amino acids in fermented beverages and juices. A dual comparison between LC-MS/MS in positive and negative ESI modes in dynamic Multiple Reaction Monitoring (dMRM) and Neutral Loss Scan was investigated. The results indicate that the studied carbamate derivatization reagent, AzoB, can be employed for targeted analysis (MS/MS) but also for non-targeted analysis of derivatized amino acids thanks to its constant neutral loss (223 Da) that is the same in both ionisation modes. For amines, precursor ion scan can be used as identification tool. The derivatization properties of AzoB and AzoC were compared against other derivatization reagents, and they showed advantages such as fast derivatization reaction and good reactivity with secondary amines. AzoC also displayed a disadvantage -side products were formed that affect the quantitation. Free amino acids profile of Kvass (a fermented beverage from eastern Europe) was determined for first time, proline was found to be the most abundant amino acid.