Powerful Steroid-Based Chiral Selector for High-Throughput Enantiomeric Separation of α-Amino Acids Utilizing Ion Mobility-Mass Spectrometry

Stereospecific recognition of amino acids (AAs) plays a crucial role in chiral biomarker-based diagnosis and prognosis. Separation of AA enantiomers is a long and tedious task due to the requirement of AA derivatization prior to the chromatographic or electrophoretic steps which are also timeconsuming. Here, a mass-tagged chiral selector named [d(0)]/[d(5)]-estradiol-3-benzoate-17 beta-chloroformate ([d(0)]/[d(5)]-17 beta-EBC) with high reactivity and good enantiomeric resolution in regard to AAs was developed. After a quick and easy chemical derivatization step of AAs using 17 beta-EBC as the single chiral selector before ion mobility-mass spectrometry analysis, good enantiomer separation was achieved for 19 chiral proteinogenic AAs in a single analytical run (similar to 2 s). A linear calibration curve of enantiomeric excess was also established using [d(0)]/[d(5)]-17 beta-EBC. It was demonstrated to be capable of determining enantiomeric ratios down to 0.5% in the nanomolar range. 17 beta-EBC was successfully applied to investigate the absolute configuration of AAs among peptide drugs and detect trace levels of D-AAs in complex biological samples. These results indicated that [d(0)]/[d(5)]-17 beta-EBC may contribute to entail a valuable step forward in peptide drug quality control and discovering chiral disease biomarkers.

Automated summary

The mass-tagged chiral selector [d(0)]/[d(5)]-17 beta-EBC was successfully developed for the separation and identification of amino acid enantiomers, achieving good enantiomeric resolution for 19 chiral proteinogenic AAs in a quick analytical run. The linear calibration curve of enantiomeric excess using [d(0)]/[d(5)]-17 beta-EBC showed promising results, demonstrating its potential in peptide drug quality control and discovery of chiral disease biomarkers.