Postcolumn Infusion of Labeled Racemic Chiral Selector Enables LC-Chiral MS/MS

Efforts were made here to pursue a pipeline fulfilling all three demands for chiral MS/MS coupled with LC to measure complicated matrices, such as chiral recognition, chiral selector (CS) screening, and enantiomeric excess (ee) determination. In the presence of Cu²⁺, labeled dl-Phe (d-Phe/l-Phe-<i>d</i>₅ = 1:1) was fortified to LC eluate via postcolumn infusion (PCI). Complexation enabled the conversion from enantiomers (i.e., d/l-A) to diastereomeric trimeric cluster ions, including [Cu^II(d/l-A)(d-Phe)₂ - H]⁺, [Cu^II(d/l-A)(l-Phe-<i>d</i>₅)₂ - H]⁺, and [Cu^II(d/l-A)(d-Phe)(l-Phe-<i>d</i>₅) - H]⁺, that exhibited different dissociation kinetics to dimeric ions, leading to chiral MS/MS. Chiral recognition of angular-type pyranocoumarin (AP) enantiomers in Peucedani Radix (PR) succeeded through building full collision energy ramp (FCER)-MS² spectra and correlating the maximum relative ion intensity (RII_max) values with thermal enthalpy features. For CS screening, 15 APs were captured from PR by applying two prerequisites, i.e., the desired trimeric complex ion generation and the follow-up distinct dissociation behaviors. Through deploying a selective AP as the CS, simultaneous ee determination was achieved for AAs in tea samples by linearly converting the response ratio of [Cu^II(d/l-AA)(d-Pte) - H]⁺ against [Cu^II(d/l-AA)₂ - H]⁺ to the ee feature. Chiral metabolomics and routine widely targeted metabolomics were undertaken for 13 batches of PR through simultaneous ee determination of five d/l-APs, and enantiomers instead of scalemic mixtures existed as differential variables. Together, the incorporation of a labeled racemic CS (e.g., labeled dl-Phe) and PCI-LC-MS/MS reached the "three birds with one stone" goal for enantiomeric measurements, indicating a versatile tool for chiral metabolomics even in the absence of functional moieties for metabolites.