Metabolomics is a mainstream approach for investigating the metabolic underpinnings of complex biological phenomena and is increasingly being applied to large-scale studies involving hundreds or thousands of samples. Although metabolomics methods are robust in smaller-scale studies, they can be challenging to apply to larger cohorts due to the inherent variability of liquid chromatography mass spectrometry (LC-MS). Much of this difficulty results from the time-dependent changes in the LC-MS system, which affects both the qualitative and quantitative performances of the instrument. Herein, we introduce an analytical strategy for addressing this problem in large-scale microbial studies. Our approach quantifies microbial boundary fluxes using two zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) columns that are plumbed to enable offline column equilibration. Using this strategy, we show that over 397 common metabolites can be resolved in 4.5 min per sample and that metabolites can be quantified with a median coefficient of variation of 0.127 across 1100 technical replicates. We illustrate the utility of this strategy via an analysis of 960 strains of Staphylococcus aureus isolated from bloodstream infections. These data capture the diversity of metabolic phenotypes observed in clinical isolates and provide an example of how large-scale investigations can leverage our novel analytical strategy.