Search

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 nano-Splitter provides the high sensitivity of nanoelectrospray MS while allowing 98% 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 2% and sample recovery on the order of 93%. When applied to a cyanobacterial extract showing antibacterial activity, the platform recognized several previously known metabolites, down to the 1% level, in a single 30-[micro]g injection, and prioritized one unknown for further study.

The structural elucidation of antillatoxin, an ichthyotoxic cyclic lipopeptide from the tropical cyanobacterium Lyngbya majuscula, reveals the spectacular adaptive and biosynthetic capabilities of cyanobacteria. Antillatoxin is isolated as an amorphous powder from the ichthyotoxic crude extract by successive chromatography on silica and RP-18 gels, followed by final purification using RP-18 HPLC.