Products Cited in Publications Worldwide Small Molecule Separation, Detection, and Identification Using Liquid Chromatography and Mass Spectrometry

Small Molecule Separation, Detection, and Identification Using Liquid Chromatography and Mass Spectrometry

Tiede, Erin R

Abstract

The analysis of small molecules is an integral part of many industries and disciplines. The techniques used for small molecule analysis vary greatly depending on the purpose of analysis and the matrix of the samples being analyzed. This dissertation focuses on the combined use of liquid chromatography (LC) for the separation of complex samples and mass spectrometry (MS) for the detection of a variety of classes of compounds. Chapter 2 describes the development of a targeted LC-MS method for use in the validation of a putative biomarker for autism spectrum disorders (ASD). Previous work has indicated a depletion of stercobilin in ASD models, however the complex matrix of biological samples can lead to issues quantitating this sometimes low abundance molecule. This reversed-phase LC method was developed specifically to separate stercobilin from the species most similar to it in structure, urobilin and half-stercobilin, for improved quantitation. The method was then applied to the quantitation of stercobilin in fecal samples from a Fragile X syndrome murine model using an 18O-labeled internal standard. Chapter 3 describes the untargeted analysis of aqueous cinnamon extract, which has previously been indicated to have antiviral activity. The matrix of plant extracts is known to be extremely complex, so a reversed-phase LC-MS method was developed to separate the components as much as possible for identification. Fractions were collected from the separation to aid in narrowing down the potential species inducing antiviral activity. High-resolution MS/MS filtered through Compound Discover was used for the tentative identification of compounds in the full extract as well as the fraction that indicated the most potential for antiviral activity. Chapter 4 focuses on the untargeted analysis of bacterial spent media for the determination of a small molecule or molecules that can induce the growth of Porphyromonas gingivalis from low cell density. Both reversed-phase and HILIC separations were developed to cover the wide range of polarities that the species in this matrix contain. To help narrow down the potential active species, these methods were applied to further fractionate spent media fractions that induced Porphyromonas gingivalis growth activity from low cell density. These methods coupled to high-resolution MS/MS were also used in conjunction with Compound Discoverer for tentative identification of the matrix components. Chapter 5 continues work on bacterial spent media for the targeted, quantitative analysis of small organic acids. An LC-MS method was developed for the separation and quantitation of acetic, butyric, fumaric, lactic, propionic, succinic, and valeric acids. A variety of column chemistries were tested, with a HILIC column providing the best separation. This method was applied to wild type and lactate dehydrogenase mutant bacterial spent media to aid in determining the cause of a decreased media pH. Finally, Chapter 6 describes the use of a compendial testing method developed by the Clinical Pharmacology Quality Assurance (CPQA) program for examining the fitness of standard reference materials for proficiency testing. The standard reference materials were compared to certified reference materials and their certificates of analysis in both purity and identity tests. Purity was determined using a combination of high-performance liquid chromatography (HPLC) and thermogravimetric analysis (TGA). Identity confirmation was performed using both high-resolution MS and nuclear magnetic resonance (NMR) spectroscopy.

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