Plants and microbes accumulate a diverse assortment of bioactive lipid metabolites, yet the genetic and environmental factors that influence lipid composition remain uncertain. Discoveries of these factors will rely on rapid methodologies to detect, identify, and quantify these important compounds. In this dissertation, new mass spectrometry based protocols were developed and applied to serve as platforms for future studies of the role of genetics in lipid composition. Methodology for identification, detection and quantification of diterpene glycoside sweeteners in Stevia leaf extracts using ultrahigh performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) is presented and evaluated. This approach employed a QTRAP mass analyzer in multiple reaction monitoring (MRM) mode for selective and sensitive identification of sweeteners. Prior to this selective MS/MS detection, a rapid ultra-high performance liquid chromatographic separation was performed using a fused-core C18 column to elute and resolve sweeteners and their isomers. In some cases, the presence of unexpected diterpene glycoside isomers was revealed. Levels of stevioside, different Rebaudiosides and related natural sweeteners differed significantly across a population of more than 1200 Stevia leaf extracts. The detection of multiple isomers of Stevia glycosides suggested a need to distinguish these isomers using mass spectrometry. Using flow injection and selective collision induced dissociation (CID) on different anionic forms of the various sweeteners, breakdown curves were generated at various collision energies. These breakdown curves exhibited dramatic differences in ion abundances as a function of collision energies for isomeric sweeteners differing in position of sugar attachment. A combination of ultrahigh performance liquid chromatography and quasi-simultaneous acquisition of mass spectra at multiple collision energies yields a technique that provides fast spectrum acquisition using a time-flight mass analyzer. Non-selective collision induced dissociation facilitated identification of lipids based on their accurate molecular and fragment ion masses. This approach, called multiplexed collision induced dissociation (mux-CID), generates molecular and fragmentation mass spectra at different collision energies for abundant and low-abundance lipids in a single analysis. This methodology coupled to fast chromatography (less than 5 minutes per sample) was used for high-throughput screening of lipid species. Using gradients as long as 26 minutes with appropriate mobile phases allowed partial resolution of lipids that coeluted in the fast 5-minute screens. Different classes of lipids including sulfo-, galacto-, and phospholipids were resolved and identified using mux-CID approach. The research presented in this dissertation has developed mass spectrometry-based protocols that greatly enhance the rate at which new bioactive lipids can be detected, identified, and quantified. Such methodologies can be applied for analysis of lipid species in a wide range of biological tissues including plants and microbes.
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