Project Summary/Abstract There is an increasing need to improve the characterization of lipids and saccharides for clinical and biomedical research purposes. NMR is the method of choice for obtaining detailed structural information about saccharides. However, NMR typically requires milligram (micromole) quantities of analyte; this often exceeds biologically relevant levels. For lipids, mass spectrometry (MS) provides an efficient avenue for rapid profiling, but quantitative analysis is challenged by difficulty in isolating species of interest due to wide structural diversity. A new MS approach is proposed that fundamentally addresses challenges in quantitative and qualitative MS by utilizing online accelerated droplet chemistry. Since ion suppression effects in electrospray ionization (ESI) MS occur during droplet formation, our method is designed to tackle this intellectual challenge exactly at the point of droplet formation – not before by adding reagents in solution, and not after by performing gas-phase reactions. This strategy simplifies instrumentation requirements and allows effective coupling to liquid chromatography (LC). Selected droplet-based reactions improve signal-to-noise ratios to enable femtomole sensitivity using <1 µL sample volume. Gas-phase ion intensities generated by our platform reflect the corresponding analyte concentration in solution. Importantly, selected droplet-based reactions allow isomers of lipids and saccharides to be differentiated. We propose to couple online droplet reactions with LC to enable high throughput quantification of lipids and saccharides in complex mixtures. The specific research aims are: Aim 1: To develop a functional contained-electrospray platform for coupling accelerated droplet chemistry on LC-MS for saccharide analysis. A novel contained-ESI source is proposed to couple droplet chemistry with LC-MS. Our method will enable LC mobile phase and ESI spray solvent to be independently optimized. This orthogonal feature is expected to allow effective separation of isomeric saccharides (linkage, anomeric, and position isomers). Selected droplet reactions will improve detectability of saccharides and provide a second layer of identification for isomers that co-elute. The LC-contained-ESI-MS/MS platform will be validated via high throughput combinatorial studies. Aim 2: To develop a plasma-droplet fusing contained-electrospray source for coupling LC-MS for lipid analysis. We propose to include etched silica capillaries on our LC-contained-ESI-MS/MS platform for accurate quantification of all types of lipids, including triglycerides. The device is expected to enable instantaneous determination of degree of unsaturation, C=C bond position, and bond orientation (cis/trans). The tandem development of quantitative analytical methods for lipids and saccharides will result in concomitant creation of versatile platforms for applications in diseases diagnosis and high throughput analysis of rare sugars t...