Microscale LC-MS-NMR Platform Applied to the Identification of Active Cyanobacterial Metabolites

摘要

An LC-MS-NMR platform is demonstrated, which combines two innovations in microscale analysis, nanoSplitter LC-MS and microdroplet NMR, for the identification of unknown compounds found at low concentrations in complex sample matrixes as frequently encountered in metabolomics or natural products discovery. The nanoSplitter provides the high sensitivity of nanoelectrospray MS while allowing 98percent of the HPLC effluent from a large-bore LC column to be collected and concentrated for NMR. Microdroplet NMR is a droplet microfluidic NMR loading method providing severalfold higher sample efficiency than conventional flow injection methods. Performing NMR offline from LC-UV-MS accommodates the disparity between MS and NMR in their sample mass and time requirements, as well as allowing NMR spectra to be requested retrospectively, after review of the LC-MS data. Interpretable 1D NMR spectra were obtained from analytes at the 200-ng level, in 1 h/well automated NMR data acquisitions. The system also showed excellent intra-and interdetector reproducibility with retention time RSD values less than 2percent and sample recovery on the order of 93percent. When applied to a cyanobacterial extract showing antibacterial activity, the platform recognized several previously known metabolites, down to the 1percent level, in a single 30-(mu)g injection, and prioritized one unknown for further study.