A validated chiral LC-MS/MS method for enantioselective quantification of aromatic lactic acids in human faeces

Bifidobacterium, a key genus of the infant gut microbiome, produces d- and l- enantiomers of aromatic lactic acids that may influence early-life immune development through stereochemistry-dependent biological activity. No validated analytical methods currently enable their accurate enantio‑separation and quantification in human biological samples. We report the first validated, targeted, liquid chromatography-mass spectrometry method using a chiral column for the enantioselective separation and quantification of d- and l- forms of phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (4OH-PLA), and indolelactic acid (ILA) in faecal samples. The method achieves baseline separation of all enantiomers within 10 min, with resolution values of 2.66 (PLA), 1.77 (4OH-PLA), and 2.42 (ILA). Solid-phase extraction reduces matrix effects (>80 %) and improves analyte recovery (>80 %). Limits of quantification range from 2.9 to 6.7 ng mL^-1, and calibration curves show excellent linearity (R² > 0.99). Inter- and intra-day precision expressed as %RSD are < 15 % for most analytes. The method was successfully applied to infant faecal samples, enabling sensitive and stereospecific quantification of aromatic lactic acids, thus establishing a foundation for exploring the biological relevance of these enantiomers in infant health.