Rapid enantioselective amino acid analysis by ultra-high performance liquid chromatography-mass spectrometry combining 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization with core-shell quinine carbamate anion exchanger separation

Abstract

Amino acid analysis (AAA) is of central importance for the characterization of the amino acid composition of proteins, peptides, pharmaceutical formulations, dietary supplements, in bioanalysis and metabolomics. Common methodologies are based on achiral assays and hence do not consider distinction of D and L-amino acid enantiomers. This may be misleading in many instances, because therapeutic peptides and natural peptides synthesized by non-ribosomal peptide synthetases (NRPSs) frequently contain D-amino acids to achieve proteolytic stability. Furthermore, stereochemical integrity control of peptides made from L-amino acids also need stereoselective assays, and racemization in natural products (proteins, peptides, amino acids) have shown to be useful biomarkers of disease and require assays, which can provide information about the individual stereoisomers. Hence, rapid enantioselective amino acid analysis (ReAAA) is of utmost importance. However, currently employed enantioselective assays are often only focusing on a limited number of amino acids or require long analysis times making them incompatible for high-throughput sample analysis. Here, we present an ReAAA assay, which is based on a fast UHPLC enantiomer separation using a short QN-AX core-shell particle column (2.7 µm) coupled to electrospray ionization quadrupole time of flight mass spectrometric (ESI-QTOF-MS) detection. Amino acid samples are mixed with a [u-13C15N]-labelled L- or DL-amino acid standard mixture, precolumn derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent and subjected to UHPLC-ESI-MS analysis. A number of chromatographic variables have been optimized (buffer additives and concentration, water content, flow rate and temperature) to achieve the goal of simultaneous amino acid enantiomer separation of all proteinogenic amino acids within the shortest possible analysis time. A mobile phase consisting of 50 mM NH4FA, 50 mM FA and 0.5% H2O in MeOH combined with a flow rate of 1 mL/min and a column temperature of 30 °C has been identified as optimal. With these conditions, all of the proteinogenic DL-enantiomers (except for Arg) can be separated in less than 2.5 min. If D-Arg and distinction between D-Leu/D-Ile is of concern, a second-tier method can accomplish this goal. The method can be very useful for ReAAA when high sample throughput is a major demand. Its utility was demonstrated by ReAAA of a lipopeptide hydrolysate which contained several D-amino acids and of an amino acid supplement in which minor D-amino acid impurities could be detected.