Improving metabolite identification through enhanced positional isomer separation using micro-pillar array columns

Metabolite identification (MetID) workflows frequently rely on generic UHPLC/ESI-MS methods that provide a compromise between chromatographic separation and analytical throughput. However, these methods may fail to adequately resolve complex mixtures of metabolite isomers, resulting in ambiguous structural assignments and additional method development efforts. Here, we evaluate micro-pillar array columns (μPAC) as a practical strategy to improve isomer separation without changing chromatographic selectivity or developing dedicated LC methods. Using hepatocyte incubations with buspirone and verapamil as model compounds, μPAC/nano-ESI-MS was compared with conventional UHPLC/ESI-MS methods under both standard and extended-gradient conditions. For buspirone, μPAC/nano-ESI-MS substantially improved the separation of oxidative metabolite isomers and enabled detection of 14 out of 15 theoretically possible metabolites with two oxidations. For verapamil glucuronides, μPAC/nano-ESI-MS provided superior chromatographic resolution compared with both conventional UHPLC and a 90-min UHPLC gradient, demonstrating that the observed improvements extend beyond the effect of gradient length alone. In addition, μPAC/nano-ESI-MS produced more than an order-of-magnitude increase in signal response despite a 100-fold lower column load. The combined gains in chromatographic resolution and signal intensity improved confidence in metabolite annotation and facilitated the characterization of complex isomeric metabolite mixtures. These results demonstrate that μPAC/nano-ESI-MS represents a valuable workflow enhancement for metabolite identification studies and can be implemented as a second-pass analysis strategy when conventional UHPLC/ESI-MS methods provide insufficient separation power.