A two-dimensional liquid chromatography approach for simultaneous separation and quantification of structural and...

Oolong tea contains diverse isomers, such as amino acids. D-amino acids, compared with their L-enantiomers, exhibit distinct properties, influencing both the flavor and bioactivity of the tea. However, the analysis of these isomers remains challenging, especially the simultaneous determination of structural and chiral isomers. This study introduced a stepwise two-dimensional liquid chromatography heart-cut (LC-LC) method for improving resolution, followed by exploration of selective comprehensive (sLC × LC) for precise quantification by quadrupole time-of-flight mass spectrometry (QTOF/MS), demonstrated using D/L-Leu and D/L-Ile. LC-LC improved the resolution of L-Leu and L-Ile isomers from 0.5 to 1.5, while sLC × LC further improved the precision and robustness using optimized loop filling. D/L-Leu and D/L-Ile were successfully quantified, ranging from 0.08 μg/g for D-Ile and 22.34 μg/g for L-Leu with RSD% below 5 %. This study demonstrated the potential of sLC × LC in addressing complex isomer challenges in food analysis, enabling deeper insights into food composition and authenticity.