Ultrahigh Pressure Capillary Liquid Chromatography-Mass Spectrometry for Metabolomics and Lipidomics

Abstract

Metabolites and lipids are important compounds involved in various cellular processes. The comprehensive analysis of all metabolite and lipid species, termed metabolomics and lipidomics, respectively, is challenging due to the large size, chemical diversity, and concentration range of the metabolome and lipidome. Liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful tool for metabolomics and lipidomics due to its sensitivity, selectivity, and amenability to a broad range of compounds. The goal of this thesis is to evaluate and demonstrate improvements in capillary LC-MS based metabolomics and lipidomics primarily using custom-built chromatography instrumentation capable of operating at 35 kpsi compared to what is currently commercially available. Lipid separations were evaluated from plasma extracts using one- and two-dimensional liquid chromatography coupled with mass spectrometry. Use of 50 cm columns with 1.7 µm C18 particles provided up to a 95% increase in peak capacity compared to commercial limitations. We evaluated the effect of column and gradient length on the number of lipids detected from plasma and found a roughly linear relationship between peak capacity and lipids detected, illustrating the benefits of improved separation performance in lipidomic assays. An offline two-dimensional LC-MS system was developed utilizing HILIC in the first dimension to fractionate lipids from plasma based on their class, followed by re-injection on the 50 cm capillary columns. The 2D method demonstrated high orthogonality, achieved a peak capacity of approximately 1900 in 600 min, and detected roughly double the number of lipids compared to the one-dimensional work. We evaluated the potential for fast yet high efficiency metabolite separations using porous C18 particles down to 1.1 µm and pressures up to 35 kpsi. Use of these particle sizes is possible with 35 kpsi available and are useful for high throughput metabolomics measurements. Columns were evaluated using isocratic and gradient separations of standards and metabolite extracts from plasma. Peak capacities of roughly 100 – 400 were achieved in 8 – 40 min with interfacing to MS, demonstrating relatively fast and high resolution separations. We evaluated the effect of different LC-MS variables on mass spectral feature detection. Lower flow rates (down to 700 nL/min) and larger injection volumes (up to 1 µL) increased the features detected, demonstrating practical benefits for metabolomics assays. Finally, gradient LC-MS operation up to 50 kpsi is achieved and peak capacities over 1000 are demonstrated. Gradient kinetic plots were constructed to guide choice of column length, particle size, and gradient time. Use of 100 cm capillaries packed with 1.7 µm particles achieved a peak capacity of ~1000 in about 4 h. Separations at 50 kpsi are achieved but not feasible for routine use with current hardware. Instrument modifications are evaluated and discussed for routine, leak-free operation at 50 kpsi. The work described in this thesis describes approaches for improving LC-MS based metabolomics and lipidomics through improvements in separations primarily at 35 kpsi and using 1.1 – 1.7 µm particles packed in 20 – 100 cm long columns. This work illustrates practical applications of ultrahigh pressures in liquid chromatography and discusses advantages of such instrumentation in LC-MS based assays.