Comprehensive reversed‐phase×chiral two‐dimensional liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry with post‐first dimension flow splitting for untargeted enantioselective amino acid analysis

Abstract

This work describes a comprehensive achiral × chiral two‐dimensional liquid chromatography separation for enantioselective amino acid analysis coupled to electrospray ionization‐tandem mass spectrometry detection using data‐independent acquisition. Flow splitting after the first and second dimension separation was utilized for volumetric flow reduction and for enabling a multi‐detector approach (with ultraviolet, fluorescence, charged aerosol, and MS detection), respectively. Derivatization with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate provided a chromophore, a fluorophore, and an efficient mass tag for efficient ionization in positive electrospray ionization‐mass spectrometry. Chiral columns often have limitations in terms of their chemoselectivity, which may be a problem when complex sample mixtures with structurally related compounds need to be separated. It can be alleviated by a reversed‐phase×chiral two‐dimensional‐liquid chromatography setup, in which the first dimension provides the chemoselectivity and a chiral tandem column constituted of quinine‐carbamate derived weak anion‐exchanger and zwitterionic ion‐exchanger in the second dimension separation of D‐ and L‐amino acid enantiomers. The method was used to control the stereointegrity of the therapeutic peptide octreotide. After hydrolysis, all amino acid constituents were detected with the correct configuration and composition. Some options for flow splitting and integration of destructive detectors in the first dimension separation are outlined.