[CuII(l-Phe)2–H]+ Complex: A Versatile Selector for Wide Enantiomeric Excess Determination and Chiral Recognition in Complicated Matrices by Post-column Infusion LC–Energy-resolved MS/MS

Abstract

Enantiomeric measurement of complicated matrices always challenges LC–MS/MS. Efforts were made here to pursue a fit-for-purpose chiral selector (CS) for LC–MS/MS and to clarify the enantioselective mechanisms. After assaying diverse candidates for 22 pairs of enantiomers, differentiation was successful for 15 pairs when employing [Cu^II(l-Phe)₂–H]⁺ as the CS. With d- vs l-proline (Pro) as representatives, diastereomeric complex ions such as [Cu^II(d-Pro)(l-Phe)₂–H]⁺ and [Cu^II(l-Pro)(l-Phe)₂–H]⁺ were formed and their featured fragment ions such as [Cu^II(d/l-Pro)(l-Phe)–H]⁺ and [Cu^II(l-Phe)₂–H]⁺ exhibited different abundance ratios (R). R was linearly correlated with enantiomeric excess (ee %), and R^max, termed as the R value at optimal collision energy, enabled chiral recognition. Crystallization was successful for both [Cu^II(d-Pro)(l-Phe)–2H] and [Cu^II(l-Phe)₂–2H] complexes, and X-ray spectroscopy revealed that the distorted square planar configuration by tetrahedrally coordinating Cu²⁺ to two nitrogen atoms and two oxygen atoms from (Phe)₂ or (d-Pro)(l-Phe) led to enantioselectivity. The diastereomeric complex was constructed by an octahedron core consisting of six spatially orthometric coordination bonds around nuclear Cu^II. ΔG differences for the generations of [Cu^II(d/l-Pro)(l-Phe)–H]⁺ and [Cu^II(l-Phe)₂–H]⁺ contributed to enantioselective MS/MS behaviors. Because of mirror images between [Cu^II(d-Pro)(l-Phe)₂–H]⁺ and [Cu^II(l-Pro)(d-Phe)₂–H]⁺, l-Pro solely completed ee % determination. By correlating R to ee %, simultaneous ee % determination for nine pairs of amino acid (AA) enantiomers was reached in cell medium extracts. Through correlating R^max to ΔG, chiral recognition, even configurational determination, was achieved for AA enantiomers and pyranocoumarin enantiomers in the complicated matrices. Together, [Cu^II(l-Phe)₂–H]⁺ is an eligible CS to facilitate post-column infusion LC–energy-resolved MS/MS allowing wide ee % determination and chiral recognition in complicated samples.