Liquid Chromatography–Tandem Mass Spectrometry with Online, In-Source Droplet-Based Phenylboronic Acid Derivatization for Sensitive Analysis of Saccharides

The ability to identify abnormalities in the body's saccharide profile is a promising means for early disease detection but requires analytical tools capable of detecting saccharides at low concentrations and/or for volume-limited samples. The preferred analysis approach for these compounds, liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), often lacks sensitivity due to poor ionization efficiency. In this work, we employ a modified electrospray interface-termed contained-electrospray (contained-ESI) to couple accelerated droplet chemistry to conventional LC-MS for the online and automated separation, derivatization, and detection of saccharides. The chromatographic component enables complex sample and mixtures analysis with low sample volume requirements, while the enhanced reaction kinetics afforded by electrosprayed microdroplets facilitates rapid, on-the-fly derivatization to boost sensitivity. Derivatization occurs during ion formation as analytes elute from the column, eliminating the need for superfluous post-column derivatization hardware or complicated benchtop protocols. A grounded coupler was incorporated to shield the LC from the high-voltage ion source, and method conditions were optimized to accommodate the low flow rates preferred for microdroplet reactions. The new LC-contained-ESI-MS/MS platform was demonstrated for the analysis of several mono-, di-, and oligosaccharides using in-source droplet-based phenylboronic acid derivatization. Femtomole limits of detection were achieved for a 1 μL injection, representing sensitivity enhancement of 1-2 orders of magnitude over conventional LC-ESI-MS/MS without derivatization. In addition, isobaric saccharides that are difficult to differentiate by MS alone were easily distinguished. Method precision, accuracy, and linearity were established, and the ability to detect oligosaccharides at trace levels in human urine and plasma was demonstrated.