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Liquid Chromatography and Mass Spectrometry Based Analytical Method Development Towards Fast and Sensitive Analysis

Bian, Juan
http://rave.ohiolink.edu/etdc/view?acc_num=osu1557242729134942

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Chemistry.
Liquid chromatography (LC) and mass spectrometry (MS) is a valuable tool in pharmaceutical analysis and is widely applied to the analysis of many real-world samples. Recent achievements in material and instrumentation have allowed LC and MS to play a critical role in the bioanalytical application. However, when dealing with a sample with high complexity, such as metabolites or proteins from biological samples, current LC and MS-based methods may not provide sufficient analytical results. As a consequence, this dissertation focuses on new LC and MS-based method development to address the challenges in small molecules and macromolecules analysis. A new matrix-free laser desorption/ionization mass spectrometry (LDI-MS) approach was developed, that takes advantage of the electrospun nanofibers with controllable size, morphology and composition. Different polymers were applied as the nanofiber support that could be adjusted to provide desirable polarity and chemistry for the surface. Nanoparticles that are good laser energy absorbers could be added to the polymer support to improve the performance of composite nanofibers. Fractal dimensional analysis was conducted to evaluate the electrospinning process for the first time, which provided useful information on the repeatability of nanofiber production. The performance of the nanofiber-assisted laser desorption/ionization method was investigated through characterization of small drug molecules and synthetic oligomers. Homogeneous sample distribution was achieved by eliminating the “sweet spot”, resulting in good reproducibility. Mass spectra features clean background, which is especially beneficial for interpretation of small molecules. On the basis of nanofibers with polymer support and nanoparticles, more efficient nanofibers were designed with polyacrylonitrile (PAN), Nafion and carbon nanotubes (CNT) via electrospinning, which combine the strong laser absorption feature of CNT with Nafion’s efficient protonating capability. The composite nanofibers PAN/Nafion/CNT were applied as the substrates for surface-assisted laser desorption/ionization (SALDI) and matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI MS) for the first time. Electrospinning produced uniform nanofibers with CNT evenly and firmly immobilized in polymeric nanofibers. Improved MS performance was achieved for both small drug molecules and high molecular weight polymers, including synthetic polymers, peptide and proteins. Markedly improved shot-to-shot reproducibility was observed compared to MALDI. Due to the composite formation between the copolymer and the CNT, no contamination by the carbon nanotubes to the mass spectrometer was observed. SALDI using nanofibers as substrate showed greatly extended signal production. The proposed SALDI approach was successfully used to quantify small drug molecules with eliminated interference in the low mass region. The results show that the limit of detection for verapamil could be detected with a surface concentration of femtomoles, indicating the high detection sensitivity of this method. For proteins analysis using ME-SALDI, the limit of detection down to several femtomoles was obtained for IgG. Both SALDI and ME-SALDI analyses displayed high reproducibility with RSD ≤ 9% for small drug molecules and RSD ≤ 14% for synthetic polymer and proteins. The combination of electrospun nanofibers with LDI-MS was proved to be a fast, versatile and sensitive approach for detection and characterization of a wide variety of analytes. In addition to the fabrication and application of electrospun nanofiber for SALDI, efforts towards exploring the desorption mechanism in the nanofiber-assisted LDI process were also made by adopting the chemical thermometer. Benzylpyridinium chloride was synthesized as a chemical thermometer to investigate the ion desorption efficiency and internal energy transfer in nanofiber-assisted laser desorption process. The ionic form of benzylpyridinium allows us to focus on the desorption process without complication of the ionization event in SALDI. The ion desorption efficiency was found to be inversely related to the amount of internal energy transfer among different nanofibers, suggesting that increasing the internal energy transfer in the SALDI process does not necessarily enhance the desorption efficiency. This phenomenon cannot be fully explained by a thermal desorption mechanism, and a non-thermal desorption, i.e. phase transition is proposed to be involved in this SALDI process. The morphological change of the nanofibrous surface after the laser irradiation suggests that phase transition of the substrates is involved in the desorption process. Moreover, it’s the first time to study the orientation and dimension of the nanofiber structure and their influence on desorption process. It reveals that crosslinked nanofiber network with small diameter and thin mat thickness could favor the nanofiber-assisted LDI process with efficient ion desorption. When dealing with complexity of practical samples, mass spectrometry becomes more powerful when it couples with liquid chromatography (LC). Efforts towards improving the separation of intact proteins and digested peptides were made by introducing enhanced fluidity liquids as the LC mobile phase. Enhanced fluidity reversed phase liquid chromatography (EFRPLC) is developed using custom instrumentation for separation and characterization of mutant proteins and tryptic peptides. The G-proteins including KRas, HRas and NRas function as GDP-GTP regulated binary switches in many signaling pathways and mutation in Ras proteins are frequently found in human cancers and represent poor prognosis markers for patients. Mutations of the KRas isoform constitute some of the most common aberrations among all human cancers and intensive drug discovery efforts have been directed toward targeting KRas protein. Separation and characterization of KRas and tryptic peptides are helpful for exploring the targeting, which has not been fully investigated using liquid chromatography-tandem mass spectrometry. The value of exploring separation selectivity and mass spectrometry sensitivity in enhanced fluidity liquid chromatography mass spectrometry (EFLC-MS) is demonstrated for characterizing of intact KRas proteins and tryptic peptides. EFLC-MS was found to provide improved chromatographic performance compared to traditional HPLC-MS in terms of shorter analysis time, higher peak capacity, increased ion intensity. The EFLC mobile phase with addition of liquefied CO2 facilitated charge state shift for intact KRas proteins, which discovered to influence the protein conformation and ionization in the electrospray process.
Susan Olesik (Advisor)
Abraham Badu-Tawiah (Committee Member)
Anne Co (Committee Member)
220 p.
Analytical Chemistry; Chemistry

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Citations

  • Bian, J. (2019). Liquid Chromatography and Mass Spectrometry Based Analytical Method Development Towards Fast and Sensitive Analysis [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1557242729134942

    APA Style (7th edition)

  • Bian, Juan. Liquid Chromatography and Mass Spectrometry Based Analytical Method Development Towards Fast and Sensitive Analysis. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1557242729134942.

    MLA Style (8th edition)

  • Bian, Juan. "Liquid Chromatography and Mass Spectrometry Based Analytical Method Development Towards Fast and Sensitive Analysis." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1557242729134942

    Chicago Manual of Style (17th edition)

osu1557242729134942
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This open access ETD is published by The Ohio State University and OhioLINK.