Simultaneous chirality separation of amino acids and their derivative by natamycin based on mobility measurements

The separation of chiral amino acids (AAs) and their derivatives has always been a research difficulty in the field of biochemistry due to the high similarity of enantiomeric structures. In this work, a simple and quick method using natamycin (Nat) as chiral selector has been developed to simultaneously separate chiral AAs and their derivatives of carbobenzoxy/benzyl-AAs (Cbz/Bzl-AAs) by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). Specifically, 12 groups of the Cbz-AAs and Bzl-AAs can get baseline mobility separation by simple mixing with Nat to form binary diastereomeric complex ions [Nat+(Cbz-D/L-AA)+H] + and [Nat+(Bzl-D/L-AA)+H] + . While for the remained 5 groups of Bzl-D/L-AAs and 16 groups of D/L-AAs with unsatisfying separation, by further adding P-toluenesulfonic acid (PTS), the formed ternary complexes can allow their baseline chiral separation . Specifically, Bzl-D-AAs and Bzl-L-AAs get much improved separation effect by the formed diastereomeric complexes of [Nat+(Bzl-AA) 2 +PTS 2 +H] + , which the R p-p was improved from 0 to 2.40; while the D/L-AAs can get baseline separation by the formed diastereomeric complexes of [Nat + AA + PTS + H] + , [Nat + AA+(PTS) 2 +H] + , and [Nat+(AA) 2 +(PTS) 2 +H] + , with the R p-p ranged from 0.44 to 3.53. Definitely, PTS is the first time reported as the ligand to improve the separation effect for the enantiomers, and with the higher assembly of chiral analyte, Nat, and PTS might enable better chiral separation for the chiral amino acid and their derivatives. Moreover, method validation of relative quantification and accuracy for the D/L-AA and their derivatives were measured in a mixture, yielding R 2 greater than 0.99 and RSD% ≤ 2.68%. Overall, Nat and PTS as chiral selector and ligand can be widely used for chiral AAs and their derivatives mobility separation, and potentially for the separation of other AA-related chiral molecules.