Fast, general-purpose metabolome analysis by mixed-mode liquid chromatography – mass spectrometry

Comprehensive metabolomics requires robust and efficient analytical techniques capable of addressing the chemical diver-ity, complexity, and high sample throughput demands characteristic of large-scale studies. We introduce a rapid, mixed-mode liquid chromatography method that uniquely integrates anion exchange and hydrophobic interactions within a single stationary phase. Employing an optimized ternary gradient, our method achieves comprehensive separation of diverse metabolite classes over a wide range of polarities within only 4 minutes per run. The performance was tested with standards for ca. 1000 metabolites. For two-thirds of 94 isomeric sets, we could achieve a separation of 2 or more seconds, which is sufficient for correct identification. We demonstrate robustness over 500 consecutive injections of bacterial extracts and with the analysis of complex matrices like plasma, cecum extracts, and urine. Throughout, retention time drifts were < 1 s. Our mixed-mode LC-MS approach offers a routine throughput of 360 samples/day/instrument and is ideally suited for studies that require rapid and comprehensive metabolic profiling.