Since its introduction as derivatization reagent, AQC has shown interesting features. Reaction of AQC with primary and secondary amino acids is a simple, straightforward process that occurs within seconds and produces stable derivatives; in contrast the hydrolysis of the excess reagent is a much slower reaction [30,41]. The only disadvantages reported in the

literature are related to the use of HPLC separation with fluorescence or UV detection: long analysis time (25–65 min), low sensitivity (UV only), peak interference Inhibitors,research,lifescience,medical by AQC hydrolysis product and intramolecular quenching [41,42,43,44,45]. An analytical platform that exploits the greater chromatographic capacity and throughput of UPLC and the sensitivity and selectivity of MS/MS would overcome those drawbacks. The applicability of a UPLC-MS/MS Inhibitors,research,lifescience,medical method coupled with AQC precolumn derivatization for targeted amino acid analysis in large-scale metabolomics studies

is demonstrated. 2. Results and Discussion 2.1. Development of an Infusion Protocol for ESI-MS/MS Parameter Selleckchem Galunisertib optimization of AQC Amino Acid Derivatives Derivatization with AQC offers a simple and reproducible conversion of amino acids into their stable adducts amenable Inhibitors,research,lifescience,medical for RPLC [41]. Although the superior throughput and resolution of the UPLC technology can now be combined with UV, fluorescence (FL), or photodiode array (PDA) detection of AQC amino acid derivatives thanks to the commercial Inhibitors,research,lifescience,medical availability of the AccQ•Tag Ultra Chemistry package

(Waters Corp.) [46,47], the possibility of using UPLC-MS technology has not received enough attention even though the eluents used for AccQ•Tag UPLC amino acid analysis and the AQC adducts are amenable for MS. Armstrong et al. [20] pointed out that although the preparation of samples for LC-MS analysis using amino acid kits simplifies the derivatization step, the non-volatile buffers included in those kits (such as Inhibitors,research,lifescience,medical the Waters AccQ•Tag) are not readily compatible with ESI-MS, bringing disadvantages to the LC-MS approach. Our preliminary studies in the optimization of MS parameters for the analysis of amino acids derivatized with the AccQ•Tag kit proved that signal suppression was particularly problematic during direct infusion of the adducts into the mass spectrometer. As indicated by Armstrong et al. [20], this problem is attributed to the non-volatile second borate buffer provided with the AccQ•Tag derivatization kit, which is used for optimum pH adjustment of the reaction solution in order to obtain maximum product yields [41]. To overcome the drawback presented by the borate buffer in direct infusion experiments, an alternative buffer for the AQC derivatization of amino acids is needed in order to facilitate the optimization of critical MS parameters (cone voltage and collision energy) that affect the selectivity and sensitivity of LC-MS/MS amino acid analysis. 2.1.1.