Simultaneous\nQuantification of Carboxylate Enantiomers\nin Multiple Human Matrices with the Hydrazide-Assisted Ultrahigh-Performance\nLiquid Chromatography Coupled with Tandem Mass Spectrometry

Many chiral carboxylic acids with α-amino, α-hydroxyl,\nand α-methyl groups are concurrently present in mammals establishing\nunique molecular phenotypes and multiple biological functions, especially\nhost-microbiota symbiotic interactions. Their chirality-resolved simultaneous\nquantification is essential to reveal the biochemical details of physiology\nand pathophysiology, though challenging with their low abundances\nin some biological matrices and difficulty in enantiomer resolution.\nHere, we developed a method of the chirality-resolved metabolomics\nwith sensitivity-enhanced quantitation via probe-promotion (Met-SeqPro)\nfor analyzing these chiral carboxylic acids. We designed and synthesized\na hydrazide-based novel chiral probe, (<i>S</i>)-benzoyl-proline-hydrazide\n(<i>S</i>BPH), to convert carboxylic acids into amide diastereomers\nto enhance their retention and chiral resolution on common C<sub>18</sub> columns. Using the <i>d</i><sub>5</sub>-<i>S</i>BPH-labeled enantiomers as internal standards, we then developed\nan optimized ultrahigh-performance liquid chromatography with tandem\nmass spectrometry (UHPLC-MS/MS) method for simultaneous quantification\nof 60 enantiomers of 30 chiral carboxylic acids in one run. This enantiomer-resolved\nmethod showed excellent sensitivity (LOD < 4 fmol-on-column), linearity\n(<i>R</i><sup>2</sup> > 0.992), precision (CV < 15%),\naccuracy (|RE| < 20%), and recovery (80−120%) in multiple\nbiological matrices. With the method, we then quantified 60 chiral\ncarboxylic acids in human urine, plasma, feces, and A549 cells to\ndefine their metabolomic phenotypes. This provides basic data for\nhuman phenomics and a promising tool for investigating the mammal-microbiome\nsymbiotic interactions.