Multimode gradient high performance liquid chromatography mass spectrometry method applicable to metabolomics and environmental monitoring

• Multimode columns used in gradient separation LC–MS method for metabolomics and environmental investigations.
• Novel combination of HILIC, anion and cation exchange, and reversed phase separation mode in a one injection one run procedure.
• Separation of model compound mixtures of endogenous metabolites and xenobiotics.
• Salt gradient for anion and cation exchange chromatography directly coupled to ESI without an ion suppression device.

Metabolomics or environmental investigations generate samples containing very large numbers of small molecular weight analytes. A single mode chromatographic separation excludes a substantial part of such complex analyte mixtures. For instance, a reversed-phase separation would not retain ionic species, resulting in a correspondingly huge front peak. To address this problem, we used two commercially available mixed-mode ion-exchange reversed-phase columns (WAX-1 and WCX-1) in sequence in a novel multimode separation method.After trapping hydrophobics on a C18-trap in loop position, hydrophilics passing the trap are separated by a simultaneous gradient for HILIC, anion and cation exchange chromatography. This gradient ends in a washout phase with a high percentage of water, the correct starting conditions for a reversed-phase gradient eluting hydrophobics from the trap in a second step of the run. Amino acids (9), organic acids (2), sugars (8), fatty acid derived compounds (11), antioxidants (4), miscellanea (6) and xenobiotics (4) were analyzed. Compounds were separated after a single sample injection during a 50 min run. Lipids derived small fatty acids up to a chain length of 12 carbons were also accessible within this run time.